慢病毒介导短发夹状RNA抑制survivin基因治疗子宫内膜异位症的实验研究
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摘要
研究背景
子宫内膜异位症(Endometriosis,简称内异症)是生育年龄妇女最常见的疾病之一,发病率高达10%～15%。内异症主要引起不孕、痛经、性交痛及慢性盆腔痛等症状。内异症虽是一种良性疾病,却具有类似恶性肿瘤的种植、侵袭、扩散、远处转移及复发等行为。本病难以根治,复发率高,严重影响患者的身心健康。目前内异症的治疗以手术与药物治疗为主。手术治疗通过去除异位病灶,恢复盆腔解剖结构,缓解症状,增加受孕机会。但手术存在不彻底性,不能避免或减少复发。根治性手术(切除子宫和双附件)能彻底治疗内异症,但对于正值生育期的广大患者极不适用。而药物治疗是采用抑制卵巢激素分泌的方法,导致内膜萎缩,从而起到抑制异位灶的作用。药物治疗常见的副作用较大,不能长时间连续使用。此外,停药后复发率高,费用昂贵等问题近年凸显。由此可见,目前内异症治疗总的效果并不满意。因此,迫切需要进一步探求内异症的发病机理,并寻求高效、安全、持久的治疗措施,以期达到治疗彻底、妊娠率高、副作用低的目的。
内异症的病因复杂,现多数学者认可Sampson的经血逆流子宫内膜种植学说,但经血逆流现象可高达76%～90%,却只有少数妇女患病,说明随经血逆流的内膜碎片可能只是诱因或基础,而异位子宫内膜组织是否具备粘附、种植、生长等生物学行为才可能是内异症发病的关键。研究表明,凋亡是子宫内膜保持周期性结构和功能稳定的关键因素。异位内膜细胞得以在宫腔外种植并继续存活,与其对凋亡的抵抗力增强有关,子宫内膜细胞凋亡特性的改变是内膜细胞具有异位种植和生长能力的重要因素。除此之外,子宫内膜在腹腔种植及其进一步发展形成病灶需要新生血管供给血液。也就是说,异位内膜细胞抗凋亡能力的增强及新生血管的形成是内异症发生发展的基本条件。因此,促进细胞凋亡和阻断新生血管的形成是防治内异症的有效策略。
生存素(survivin)是凋亡抑制蛋白家族成员,是迄今发现的作用最强的凋亡抑制因子,在凋亡抑制中具有关键性作用。Survivin表达具有明显的组织细胞选择性,其特异性表达于人和鼠的胚胎组织以及多数人类肿瘤细胞,在正常成人组织的表达仅见于胸腺、睾丸和分泌期子宫内膜。Survivin不仅抑制细胞凋亡,还参与血管形成,其表达可能调节生理性血管修复或病理性血管形成,对肿瘤细胞的浸润、迁徙起重要作用。
近年研究发现,内异症患者异位内膜中survivin高表达,使得其能逃避多种环境刺激诱导的细胞凋亡,survivin在内异症的发生和发展中起着重要的作用。据此推测,如果阻断survivin表达及抑制其作用,可促进异位内膜细胞凋亡,抑制新生血管形成,从而阻断内异症的发生和发展。因此,survivin基因可能成为治疗内异症的理想靶点。
RNA干扰技术是目前最有效的转录后基因抑制的方法之一,因其可以特异性剔除或关闭基因的表达,且具备经济、快捷、高效、高稳定性及可扩散性等诸多优势,已广泛应用于探索基因功能,以及肿瘤和传染性疾病等的治疗。而基因治疗成功的关键之一在于载体的选择,目前慢病毒载体以其特有的优势已逐渐成为广为关注的热点。慢病毒载体系统不同于质粒、脂质体和腺病毒等载体,具有能感染分裂和非分裂细胞、免疫反应小、可重复应用、转移基因片段容量大、能长期稳定地表达、生物安全性高等优点,适于作为体内外基因治疗的载体。
目的
本课题拟构建携带survivin基因特异性短发夹状RNA(shRNA)的慢病毒表达载体并包装慢病毒,感染至人异位内膜细胞、鸡胚绒毛尿囊膜(CAM)内异症血管生成模型以及裸鼠内异症皮下种植模型中,观察其在体外、体内抑制survivin基因表达、诱导异位内膜细胞凋亡、影响异位内膜细胞生物学行为、抑制新生血管形成、控制内异症发生发展的效果以及可能出现的毒副反应,探讨慢病毒介导shRNA抑制survivin基因靶向治疗内异症的有效性、安全性及相关机制,为内异症的防治策略提供新的思路和方法,并为内异症的新药开发提供实验依据及技术平台。
方法
1.构建靶向survivin基因的shRNA慢病毒载体及包装病毒：根据GenBank数据库提供的人survivin基因核苷酸序列,选择最佳干扰位点,无同源编码序列。针对靶位点设计并合成两条互补的寡核苷酸序列。退火形成双链寡核苷酸后克隆到pGCL-GFP慢病毒载体,PCR鉴定、测序分析及阳性克隆质粒抽提,在脂质体的介导下将慢病毒包装辅助质粒和survivin-shRNA重组慢病毒载体导入293T细胞包装病毒,测定病毒滴度。
2.观察慢病毒介导shRNA在体外抑制survivin基因对人异位内膜细胞增殖和凋亡的影响：通过胰蛋白酶消化、贴壁纯化等技术原代培养人异位内膜细胞,免疫细胞化学法鉴定细胞类型。将重组慢病毒在体外感染人异位内膜细胞,分别以不加病毒液及空载慢病毒感染的异位内膜细胞作为空白对照及阴性对照。采用RT-PCR、蛋白质免疫印迹(Western-blot)法分别检测各组异位内膜细胞中survivin mRNA和蛋白表达的变化,用四甲基偶氮唑蓝法(MMT)检测细胞的增殖,Hoechst染色及Cy5-Annexin V/PI双染标记流式细胞仪测定细胞凋亡。
3.观察靶向survivin基因的shRNA重组慢病毒对CAM内异症模型血管生成的影响：选择第8d鸡胚,建立CAM内异症血管生成模型,随机分成单纯接种组、shRNA慢病毒组、空载慢病毒组和空白载体组,每组30只。单纯接种组将子宫内膜组织接种至CAM相对无血管区,用无菌贴膜封口；后3组均先将子宫内膜碎片接种至CAM相对无血管区,24 h后再以无菌明胶海绵碎片为载体,shRNA慢病毒组、空载慢病毒组分别选用浓度为8×108TU/ml的survivin-shRNA慢病毒4μ1和2×109TU/ml的空载慢病毒1.6μ1用无血清DMEM培养液稀释至20μ1,滴加于无菌明胶海绵上,贴附于鸡胚种植内膜处。空白载体组于明胶海绵上滴加无血清DMEM培养液20μ1。转染7d后取出鸡胚,通过Image-proplus软件测量以载体为中心直径15mm区域内新生血管面积与该区域鸡胚尿囊膜面积比(VA/CAM),评价抑制survivin基因对CAM血管生成的影响,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测种植内膜细胞凋亡,组织病理学观察种植内膜病灶生长行为。
4.观察慢病毒介导shRNA抑制survivin基因表达后对裸鼠内异症模型中异位病灶生长的影响：选择6周龄的BALB/c雌性裸鼠50只,在裸鼠皮下注射内异症患者子宫内膜碎屑,建立裸鼠内异症皮下种植模型。选择建模成功的裸鼠模型45只,随机分为3组(空白对照组、阴性对照组、治疗组),每组15只,3组裸鼠皮下异位结节灶内分别注射磷酸盐缓冲液(BPS)、空载体慢病毒BPS稀释液和shRNA慢病毒BPS稀释液各50μl。各组裸鼠实验前后,测量其体重、皮下异位病灶体积的变化,观察裸鼠活动、皮色、食欲等情况。注射后15 d,断颈法处死裸鼠,剥离出异位内膜病灶,比较各组病灶的体积与重量。分别采用RT-PCR及免疫组化检测异位内膜组织survivin和caspase-3 mRNA及蛋白的表达,TUNEL法检测各组异位组织凋亡,光镜观察病灶、肝脏、肾脏、卵巢和子宫组织病理变化。放射免疫法检测各组裸鼠血清E2、FSH水平。
结果
1.经阳性克隆PCR鉴定及测序分析,结果显示慢病毒重组质粒中含有所设计的寡核苷酸单链,表明合成的survivin-shRNA序列插入正确。慢病毒重组质粒及包装质粒共转染293T包装细胞,能产生重组慢病毒LV-survivin-shRNA,经纯化浓缩后产生的慢病毒滴度为8×108TU/ml。
2. LV-survivin-shRNA在体外对人异位内膜细胞中survivin基因表达及细胞增殖和凋亡的影响
(1)原代培养的异位内膜细胞均有腺上皮细胞和间质细胞两种形态细胞。腺上皮细胞和间质细胞分别经角蛋白单抗和波形蛋白单抗免疫组化染色为阳性,腺上皮细胞平均生长时间为5周,间质细胞平均生长时间为14周。
(2)重组慢病毒感染人异位内膜细胞后24 h即可见到有绿色荧光蛋白表达,以后逐渐增强,至72 h后其绿色荧光的表达呈高峰。病毒感染复数(MOI)为100时,细胞病理现象明显,细胞变圆,漂浮,细胞的正常增殖受影响。MOI为50时,细胞的形态和生长不受影响,且感染效率较高,其在异位内膜细胞中的阳性率达80%。
(3)与对照组细胞相比,shRNA慢病毒感染人异位内膜细胞后72 h survivin mRNA及蛋白表达水平均明显下降,survivin mRNA及蛋白表达抑制率分别为64.20%和58.79%,感染后3周,实验组异位内膜细胞中survivin基因表达仍显著降低,survivin mRNA表达的抑制率达70.93%。
(4)MTT法分别检测感染后第1d～5d细胞的OD值,结果显示,除感染后第1d外,实验组OD值均显著低于阴性对照组和空白对照组(P值均<0.001),而后两组细胞的增殖速度差异无统计学意义(P值均>0.05)。感染后不同时间点之间OD值有显著差异(P<0.001), shRNA慢病毒感染后异位内膜细胞增殖抑制率有时间依赖性(rs=1,P<0.001),感染后第1 d-5 d的异位内膜细胞增殖抑制率分别为2.97%、36.19%、52.22%、58.45%、65.96%。
(5)流式细胞仪分析显示,实验组细胞凋亡率为(41.61±3.64)%,显著高于阴性对照组的(9.14±1.88)%和空白对照组的(7.07±1.16)%(P<0.001),而后两组间比较,差别则无统计学意义(P=0.087)。
3. LV-survivin-shRNA对CAM内异症模型子宫内膜种植及血管生成的影响
(1)成功建立CAM内异症血管生成模型,单纯种植组、空载慢病毒组及空白载体组的内膜种植灶周围见丰富的血管网生成,以病灶为中心呈放射状生长,形成血管“辐辏”现象。
(2)shRNA慢病毒组的种植灶周围的CAM血管生成明显受抑,新生血管稀疏,分布无规律,可见无血管的苍白区,shRNA慢病毒组的VA/CAM显著低于其余3组(P<0.001),血管生成抑制率达75.89%。而其余3组之间比较,VA/CAM差异均无统计学意义(P值均>0.05)。
(3)各组鸡胚中子宫内膜的种植成功率差异显著(P=0.003), shRNA慢病毒组种植内膜存活率最低。
(4)shRNA慢病毒组的细胞凋亡率显著高于单纯种植组、空载慢病毒组及空白载体组,而后3组间细胞凋亡率两两比较,差异均无统计学意义(P值均>0.05)。
(5)shRNA慢病毒组病灶在光镜下可见腺体结构不完整,间质伴有不同程度的坏死。
4. LV-survivin-shRNA对裸鼠内异症皮下种植模型中异位病灶生长的影响
(1)成功建立裸鼠内异症皮下种植模型,裸鼠皮下在注入内膜后第10 d可见5mm～10mm的结节,50只裸鼠中有45只皮下种植成功,成功率为90%。治疗组、阴性对照组及空白对照组异位病灶体积在注射前无统计学差异(P=0.827)。
(2)各组裸鼠在注射前后的活动、皮色、食欲等未见明显的异常变化,各组裸鼠注射前后的体重变化差异均无显著性意义(P值均>0.05)。
(3)在注射后第15 d,肉眼观察阴性对照组及空白对照组的异位病灶生长良好,呈隆起的小囊状,表面血管可见,而治疗组的异位病灶体积明显缩小、变硬,萎缩成斑块状。
(4)在注射后第15 d,治疗组、阴性对照组及空白对照组病灶的重量分别为(0.21±0.07)g、(0.79±0.12)g、(0.83±0.07)g,治疗组病灶的重量显著低于后两组(P值均<0.001),而后两组间比较,差异无统计学意义(P=0.719),治疗组的抑瘤率为74.69%。治疗组、阴性对照组及空白对照组病灶的相对体积分别为0.350±0.059、1.466±0.119、1.548±0.135,治疗组异位病灶的相对体积显著低于后两组(P值均<0.001),而后两组间比较,差异无统计学意义(P=0.064)。生长体积-时间曲线显示治疗组异位病灶生长速度显著低于阴性对照组及空白对照组。
(5)光镜下治疗组可见异位内膜腺体萎缩明显,部分结构不完整,可见腺上皮细胞空泡变性,间质伴有不同程度的坏死。
(6) Survivin、caspase-3 mRNA及蛋白在各组标本中均有表达,治疗组survivin mRNA及蛋白表达量均显著低于阴性对照组和空白对照组(P值均<0.001),而后两组间比较,survivin mRNA及蛋白表达量差异均无统计学意义(P值分别为0.263、0.611),治疗组survivin mRNA及蛋白表达的抑制率分别为70.88%和66.38%。治疗组caspase-3 mRNA及蛋白表达量均显著高于阴性对照组和空白对照组(P值均<0.001),而后两组比较均无统计学差异(P值分别为0.918、0.604)。3组异位病灶中survivin的表达与caspase-3的表达之间均存在显著负相关关系(P值均<0.001)。
(7) TUNEL法检测结果显示,治疗组、阴性对照组及空白对照组异位病灶中细胞凋亡率分别为(37.94±4.18)%、(10.29±2.25)%、(9.92±2.10)%,治疗组的细胞凋亡率显著高于后两组(P值均<0.001),而后两组相比差异不显著(P=0.952)。
(8)治疗组裸鼠肝、肾、子宫、卵巢等重要脏器行常规病理检查未见明显异常。
(9)治疗后15 d,治疗组、阴性对照组及空白对照组3组间比较,裸鼠血清E2、FSH水平均无显著性差异(P值分别为0.273、0.364)。
结论
1.成功构建了携带survivin基因特异性shRNA的重组慢病毒载体,其携带的绿色荧光蛋白便于观察追踪；慢病毒重组质粒及包装质粒共转染293T细胞,成功地包装和生产了高滴度的重组慢病毒LV-survivin-shRNA。
2.通过胰蛋白酶消化、贴壁纯化等技术原代培养人异位内膜细胞的方法经济、简单、高效,适用于异位内膜腺上皮细胞和间质细胞的混合培养。
3. LV-survivin-shRNA在体外可高效地感染异位内膜细胞,其可在转录及翻译两个水平上高效、特异、稳定地抑制异位内膜细胞中survivin基因的表达,显著促进异位内膜细胞的凋亡,明显抑制异位内膜细胞的增殖,并随着时间延长,其抑制效应呈增强趋势,而空载慢病毒本身对细胞生长无明显影响。
4. LV-survivin-shRNA能够显著抑制人子宫内膜异位种植CAM模型的新生血管形成,促进异位种植内膜细胞的凋亡,从而抑制子宫内膜在CAM上的种植生长。
5.裸鼠内异症皮下种植模型建模操作简单,便于观察,成模率高。
6. LV-survivin-shRNA能通过高效抑制裸鼠内异症模型体内异位病灶中survivin的表达,解除对凋亡通路下游基因caspase-3的抑制,有效地激活caspase-3的表达,促进异位内膜细胞的凋亡,从而显著地抑制裸鼠体内异位病灶的生长。这可能是其有效控制裸鼠内异症的作用机制之一。
7.经LV-survivin-shRNA治疗后,裸鼠体重、皮色、活动及食欲无明显异常变化,肝、肾、子宫及卵巢标本未见组织学异常,血清E2及FSH水平与对照组无差别,提示该治疗方法无明显的毒副作用,但其远期影响仍需进一步观察。
Backgroud
Endometriosis is one of the most common gynecologic diseases with an increasing prevalence. Endometriosis presents in approximately 10 to 15 percent of reproductive age women. It often causes infertility, dysmenorrhea, dyspareunia and chronic pelvic pain. Although endometriosis is a kind of benign disease, it has many similar characters to malignant disease, such as cellular proliferation, infiltration and recidivation. Endometriosis is hard to be cured because of its recrudescence and has heavy impacts on women's body and mind health.
The treatments for endometriosis include surgery and medication. Operation is good for cleaning the lesion, restoration of pelvic anatomy, releasing symptom, and increasing the chances of conceiving. But its recurrence rate is still up to nearly 50% except radical surgery which would result in the loss of reproductive and endocrine abilty. Currently available medical therapies are designed to inhibit ectopic lesions by depressing the ovarial function to redcuse the level of estrogen directly or indirectly. However, none of these drugs can eradicate the disease and the substantial side effects. So far, recurrence is the key point to treatment of endometriosis because the true cause of the disease is ambiguous. All of the therapies are lack of the exact target. It is very urgent to find out the pathogenesis of endometriosis and get effective, safe, long-lasting treatment measures for endometriosis.
The transplantation theory that Sampson proposed in 1927 is still recognized widely. However, the incidence of reflux of shed endometrial fragments is up to 76%～90% which is not to accord with the incidence of endometriosis. It is suggested that the refluent endometrial fragments is only the inducement. Recent studies indicated that the biological behavior of cell adhesion, invasion, planting, growth and transfer is the key to the pathogenesis of endometriosis. There are more and more researches indicated:Spontaneous apoptosis is decreased and proliferation is inereased in women with endomertiosis. This showed that apoptosis is the key to maintain stability of endometrial structure and function. The change of apoptosis characteristics is important for ectopic endometrial cells to have the ability to implant and grow. At the same time, many studies indicated that angiogenesis play a important role in formation and development of endometriosis, the formation and growth of ectopic lesions is depend on the new blood vessels. Therefore, promoting apoptosis and blocking angiogenesis are effective strategy for prevention and control endometriosis.
Survivin is the strongest member of inhibitor of apoptosis proteins family and play an important role in the regulation of apoptosis. It expresses selectively in human embryonic tissues and tumor cells but almost not in normal differeniiated human tissues except for thymus, testes and secretory phase endometrium. survivin not only inhibits cell apoptosis, but also involves in angiogenesis. Therefore, survivin has been paid significant attention as a new target for anti-tumor and anti-vessel therapy.
Recent studies have shown that survivin expression in ectopic endometrium was much higher than that in normal endometrium and eutopic endometrium. Those results indicate that survivin gene inhibits cell apoptosis in ectopic endometrium, destroies the balance between proliferative and apoptosis, favor abnormal proliferative, so it may contribute to the formation and progression of endometriosis. As above, If survivin expression have been suppressed, the occurrence and development of endometriosis would be blocked result from promotion of ectopic endometrial cells apoptosis and inhibition of angiogenesis. Therefore, survivin gene may be an ideal target for treatment of endometriosis.
The important aim of gene therapy is the continually and steadily expression of obtained genetic material in transferred cell. Because the inhibition effect caused by RNA interference was fast, efficient and high stable, RNA interference is the most effective method of the sequence-specific post-transcriptional gene silencing in biology. It has become a powerful tool in exploring gene function and gene therapy for tumor and infectious diseases. Nevertheless, the key to the success of gene therapy is selecting suitable vector. Lentiviral vector become the focus of current attention widely due to its unique advantage. Lentiviral vector system have many advantages such as infection of non-dividing cells and divided cells, small immune response, reusable applications, huge gene fragments carried, long-term stable expression and high biological safety.
Objective
The feasibility and effectiveness of lentivirus mediated short hairpin RNA (shRNA) to repress the expression of survivin and promote apoptosis in ectopic endometrium cells were investigated. Furthermore, the effectiveness and security of lentivirus mediated shRNA targeting survivin gene to inhibit the establishment and growth of ectopic lesion in nude mice were studied. The main object is to seek a novel treatment strategy for endometriosis.
Methods
1. Construct a lentiviral expression vector mediated shRNA targeting human survivin gene and package lentivirus particles. Human survivin gene shRNA sequence was designed using software available on-line. After synthesis and annealing, the shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin-shRNA, which was subsequently confirmed by PCR and DNA sequencing analysis. The recombinant plasmid, packaging plasmid pHelper 1.0 and pHelper 2.0 were co-transfected into human embryonic kidney epithelial cell line (293T cells) and high titer replication defective lentivirus particles were collected and determined.
2. Study the effect of lentiviral vector-mediated shRNA on the expression of survivin mRNA and protein and its influence on the proliferation and apoptosis in human ectopic endometrial cells. The ectopic endometrial cells from patients with endometriosis were isolated and purified by trypsin digestion and pasted wall purification. The cellular morphology was observed by optical microscope. Endometrial cells were identified by immunocytochemistry. Lentiviral vector mediated shRNA targeting survivin gene (LV-survivin-shRNA) was transfected into human ectopic endometrial cells, the empty vector and culture media as control, respectively. The survivin mRNA and protein expression were detected by RT-PCR and Western-blot. The proliferation and apoptotic rate of cells after transfection were assayed by methy-thiazoly-tetrazolium and flow cytometry.
3. Investigate the effect of LV-survivin-shRNA on angiogenesis and growth of endometriosis like lesions in the chick embryo chorioallantocic membrane (CAM). Eutopic endometria from women with endometriosis were transplanted onto the non-vascular region of CAM. After successful established, the human endometriosis CAM angiogenesis models were randomly divided into 4 groups (n=30). It include inoculated alone group, LV-survivin-shRNA group, LV-NC-GFP group and empty vector group. Another 7 days later, CAMs were photographed under the anatomical microscope after entirely exposure. Graph software automatically counted vascular area and the area of CAM. Transplant specimens were analyzed by histology. Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method was used to detect cells apoptosis of endometriotic lesions.
4. Animal endometriosis models of nude mouse were established by inoculating human endometrial fragments. Forty five female nude endometriosis models were randomized into three groups,15 mice each group, including LV-survivin-shRNA group, LV-NC-GFP control group and blank control group when ectopic lesion diameter reached 5mm-10mm. Endometriosis models of nude mouse were administrated with LV-survivin-shRNA, LV-NC-GFP and PBS into the transplants. The activity and appetite of those mice were monitored each day, and the body weight of each mouse was weighing on the first day and the last day. The ectopic lesion sizes were measured every three days. All of these mice were killed at 15th day after treatment. The mRNA and protein expression of survivin and caspase-3 in ectopic lesion after transfer was detected by RT-PCR and immunohistochemistry method, respectively. TUNEL method was used to detect endometriosis like lesions cells apoptosis. Meanwhile we detected the level of steroid hormone and histological change in mice uterus, ovary, liver and kidney.
Results
1. PCR analysis and DNA sequencing confirmed that the shRNA sequence was successfully inserted into the lentiviral vector. The titer of concentrated virus was 8×108TU/ml.
2. The cell culture of ectopic endometrium was successfully established. Both endometrial epithelial cell (EEC) and endometrial stromal cell (ESC) were existed in the culture and stained positively by cytokeratin and vimentin monoclonal antibody respectively. The life span of EEC and ESC can last 5 weeks and 14 weeks, respectively. We could see the expression of green fluorescent protein after lentivirus infecting primary ectopic endometrial cells for 24h, the strongest expression of EGFP could be seen after 72h. Lentivirus of Multiplicity of Infection (MOI) less than 10 had low transfection efficiency, but when viral MOI was equal to 50, the transfection efficiency is high, up to 80%, and cell morphology and growth of cells are the same as the proliferation of normal cells. When MOI is equal to 100, the cells became obviously pathological cells, such as round and floating cells with normal proliferation affected. After transfected with LV-survivin-shRNA for 72 h, the expression of survivin mRNA and protein in human ectopic endometrial cells was inhibited by 64.20% and 58.79% respectively. After transfected with LV-survivin-shRNA for 3 weeks, the expression of survivin mRNA in human ectopic endometrial cells was still inhibited by 70.93%. The growth speed of the human ectopic endometrial cells transfected with LV-survivin-shRNA was significantly slowed than other two groups. There was a significant difference between them (P<0.001) except at the first day after being transfected. There was no difference between the growth speed of non-transfected cells and that of empty vector transfected cells (P>0.05). The apoptosis rate of survivin-shRNA group reached (41.61±3.64)%, which was significantly higher than those of non-transfected group (7.07±1.16)% and empty vector transfected group (9.14±1.88)% (P<0.001). There was no difference between the apoptosis rate of non-transfected cells and that of empty vector transfected cells (P=0.087).
3. The human endometriosis chick chorioallantoic membrane angiogenesis models(hEMCAM) were established successfully. Transplantation of endometrium onto the CAM led to a strong angiogenic response in the chicken tissue. The vessels grew radially around focus and the density of newborn blood vessels increased significantly in the hEMCAM. The angiogenesis and endometriosis-like lesions formation were significantly suppressed after treatment with LV-survivin-shRNA in comparison with the control groups. The transplantation achievement ratio of LV-survivin-shRNA group was significantly lower than those of other groups (P<0.001). Quantitative analysis shows that the ratio of vascular area (VA)/CAM of LV-survivin-shRNA group is significantly reduced (P<0.001). There is no significant difference between the other three groups (P>0.05). The apoptosis rate of endometriosis-like lesions after treatment with LV-survivin-shRNA was significantly higher than those of other three groups(P<0.001), and accomponied by various degree of necrosis in the endometriosis-like lesions under microscopic observation.
4.50 nude mice were modeled by subcutaneous implantation by injection with the endometria from endometriosis patients, there are 45 animal endometriosis models were established successfully. After endometrial fragment being inoculated for 10 days, lesion nodes (5mm-10mm in diameter) were found. There were no significant difference in volume of the endometrial lesion and weight of the nude mice before treatment among blank control group, negative control group and LV-survivin-shRNA group. At the 15th days after treatment, compared with the other two control groups, the volume and weight of the lesion nodes were decreased obviously (P<0.001). Microscopic examination showed that the glandular epithelium of LV-survivin-shRNA group had partially degenerated, and necrotic debris was present in the endometrial stroma. The difference was not statistically significant between the two control groups. The results of RT-PCR and immunohistochemisty staining showed that the expression of survivin mRNA and protein in ectopic lesions of the LV-survivin-shRNA group were significantly less than the two control groups (P<0.001). The expression of survivin mRNA and protein in ectopic lesions were inhibited by 70.88% and 66.38%, respectively. Nevertheless, the mRNA and protein expression of caspase-3 in ectopic lesions of the LV-survivin-shRNA group were significantly higher than the two control groups (P<0.001). The apoptosis rate of LV-survivin-shRNA group was significantly higher than that of blank control group and negative control group (P<0.001). There was no difference between the apoptosis rate of blank control group and that of negative control group (P=0.952). There were no obvious changes in the nude mouse liver, kidney, uterus and ovary were observed through routine pathological check after the LV-survivin-shRNA treatment. At the 15th days after treatment, there was no significant difference in concentration of E2 and FSH among three groups. During administrating, side effects were not found in the LV-survivin-shRNA group.
Conclusions
1. The recombinant lentiviral vector mediated shRNA targeting survivin gene have been constructed successfully. The recombinant lentiviral vector is effective, safe and convenient, which is convenient to observed because of GFP and can be packaged high-titer, high-purity lentivirus.
2. The primary culture method for ectopic endometrial cells by trypsin digestion and pasted wall purification was convenient and of high performance. It can be applied to culture EEC and ESC incorporately.
3. LV-survivin-shRNA can infect ectopic endometrial cells effectively in vitro and can inhibit the expression of survivin gene, which resulted in suppressing the proliferation and promoting the apoptosis in human ectopic endometrial cells.
4. The CAM endometriosis models have been constructed successfully. LV-survivin-shRNA can effectively inhibit angiogenesis induced by eutopic endometrium onto the CAM, obviously promote the apoptosis in ectopic endometrial cells and remarkably suppress endometriosis-like lesions formation.
5. The endometriosis mice models of subcutaneous implantation were established successfully. The model of subcutaneous implantation is more convenient to observe. The modeling method is simple and accomponied by high achievement ratio.
6. Lentiviral vector-mediated shRNA can be transferred into mice ectopic lesion and can effectively inhibit the expression of survivin gene and activate the expression of caspase-3, which leaded to obviously promote the apoptosis in ectopic endometrial cells and remarkably suppress the growth of endometriosis like lesions in nude mice.
7. There were no significant side effects were observed. The therapy of endometriosis with lentiviral vector-mediated shRNA was effective and safe. It lays the foundation for the experimental study and future clinical application.
子宫内膜异位症(Endometriosis,简称内异症)是生育年龄妇女最常见的疾病之一,发病率高达10%～15%。内异症主要引起不孕、痛经、性交痛及慢性盆腔痛等症状。内异症虽是一种良性疾病,却具有类似恶性肿瘤的种植、侵袭、扩散、远处转移及复发等行为。本病难以根治,复发率高,严重影响患者的身心健康。目前内异症的治疗以手术与药物治疗为主。手术治疗通过去除异位病灶,恢复盆腔解剖结构,缓解症状,增加受孕机会。但手术存在不彻底性,不能避免或减少复发。根治性手术(切除子宫和双附件)能彻底治疗内异症,但对于正值生育期的广大患者极不适用。而药物治疗是采用抑制卵巢激素分泌的方法,导致内膜萎缩,从而起到抑制异位灶的作用。药物治疗常见的副作用较大,不能长时间连续使用。此外,停药后复发率高,费用昂贵等问题近年凸显。由此可见,目前内异症治疗总的效果并不满意。因此,迫切需要进一步探求内异症的发病机理,并寻求高效、安全、持久的治疗措施,以期达到治疗彻底、妊娠率高、副作用低的目的。
内异症的病因复杂,现多数学者认可Sampson的经血逆流子宫内膜种植学说,但经血逆流现象可高达76%～90%,却只有少数妇女患病,说明随经血逆流的内膜碎片可能只是诱因或基础,而异位子宫内膜组织是否具备粘附、种植、生长等生物学行为才可能是内异症发病的关键。研究表明,凋亡是子宫内膜保持周期性结构和功能稳定的关键因素。异位内膜细胞得以在宫腔外种植并继续存活,与其对凋亡的抵抗力增强有关,子宫内膜细胞凋亡特性的改变是内膜细胞具有异位种植和生长能力的重要因素。除此之外,子宫内膜在腹腔种植及其进一步发展形成病灶需要新生血管供给血液。也就是说,异位内膜细胞抗凋亡能力的增强及新生血管的形成是内异症发生发展的基本条件。因此,促进细胞凋亡和阻断新生血管的形成是防治内异症的有效策略。
生存素(survivin)是凋亡抑制蛋白家族成员,是迄今发现的作用最强的凋亡抑制因子,在凋亡抑制中具有关键性作用。Survivin表达具有明显的组织细胞选择性,其特异性表达于人和鼠的胚胎组织以及多数人类肿瘤细胞,在正常成人组织的表达仅见于胸腺、睾丸和分泌期子宫内膜。Survivin不仅抑制细胞凋亡,还参与血管形成,其表达可能调节生理性血管修复或病理性血管形成,对肿瘤细胞的浸润、迁徙起重要作用。
近年研究发现,内异症患者异位内膜中survivin高表达,使得其能逃避多种环境刺激诱导的细胞凋亡,survivin在内异症的发生和发展中起着重要的作用。据此推测,如果阻断survivin表达及抑制其作用,可促进异位内膜细胞凋亡,抑制新生血管形成,从而阻断内异症的发生和发展。因此,survivin基因可能成为治疗内异症的理想靶点。
RNA干扰技术是目前最有效的转录后基因抑制的方法之一,因其可以特异性剔除或关闭基因的表达,且具备经济、快捷、高效、高稳定性及可扩散性等诸多优势,已广泛应用于探索基因功能,以及肿瘤和传染性疾病等的治疗。而基因治疗成功的关键之一在于载体的选择,目前慢病毒载体以其特有的优势已逐渐成为广为关注的热点。慢病毒载体系统不同于质粒、脂质体和腺病毒等载体,具有能感染分裂和非分裂细胞、免疫反应小、可重复应用、转移基因片段容量大、能长期稳定地表达、生物安全性高等优点,适于作为体内外基因治疗的载体。
目的
本课题拟构建携带survivin基因特异性短发夹状RNA(shRNA)的慢病毒表达载体并包装慢病毒,感染至人异位内膜细胞、鸡胚绒毛尿囊膜(CAM)内异症血管生成模型以及裸鼠内异症皮下种植模型中,观察其在体外、体内抑制survivin基因表达、诱导异位内膜细胞凋亡、影响异位内膜细胞生物学行为、抑制新生血管形成、控制内异症发生发展的效果以及可能出现的毒副反应,探讨慢病毒介导shRNA抑制survivin基因靶向治疗内异症的有效性、安全性及相关机制,为内异症的防治策略提供新的思路和方法,并为内异症的新药开发提供实验依据及技术平台。
方法
1.构建靶向survivin基因的shRNA慢病毒载体及包装病毒：根据GenBank数据库提供的人survivin基因核苷酸序列,选择最佳干扰位点,无同源编码序列。针对靶位点设计并合成两条互补的寡核苷酸序列。退火形成双链寡核苷酸后克隆到pGCL-GFP慢病毒载体,PCR鉴定、测序分析及阳性克隆质粒抽提,在脂质体的介导下将慢病毒包装辅助质粒和survivin-shRNA重组慢病毒载体导入293T细胞包装病毒,测定病毒滴度。
2.观察慢病毒介导shRNA在体外抑制survivin基因对人异位内膜细胞增殖和凋亡的影响：通过胰蛋白酶消化、贴壁纯化等技术原代培养人异位内膜细胞,免疫细胞化学法鉴定细胞类型。将重组慢病毒在体外感染人异位内膜细胞,分别以不加病毒液及空载慢病毒感染的异位内膜细胞作为空白对照及阴性对照。采用RT-PCR、蛋白质免疫印迹(Western-blot)法分别检测各组异位内膜细胞中survivin mRNA和蛋白表达的变化,用四甲基偶氮唑蓝法(MMT)检测细胞的增殖,Hoechst染色及Cy5-Annexin V/PI双染标记流式细胞仪测定细胞凋亡。
3.观察靶向survivin基因的shRNA重组慢病毒对CAM内异症模型血管生成的影响：选择第8d鸡胚,建立CAM内异症血管生成模型,随机分成单纯接种组、shRNA慢病毒组、空载慢病毒组和空白载体组,每组30只。单纯接种组将子宫内膜组织接种至CAM相对无血管区,用无菌贴膜封口；后3组均先将子宫内膜碎片接种至CAM相对无血管区,24 h后再以无菌明胶海绵碎片为载体,shRNA慢病毒组、空载慢病毒组分别选用浓度为8×108TU/ml的survivin-shRNA慢病毒4μ1和2×109TU/ml的空载慢病毒1.6μ1用无血清DMEM培养液稀释至20μ1,滴加于无菌明胶海绵上,贴附于鸡胚种植内膜处。空白载体组于明胶海绵上滴加无血清DMEM培养液20μ1。转染7d后取出鸡胚,通过Image-proplus软件测量以载体为中心直径15mm区域内新生血管面积与该区域鸡胚尿囊膜面积比(VA/CAM),评价抑制survivin基因对CAM血管生成的影响,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测种植内膜细胞凋亡,组织病理学观察种植内膜病灶生长行为。
4.观察慢病毒介导shRNA抑制survivin基因表达后对裸鼠内异症模型中异位病灶生长的影响：选择6周龄的BALB/c雌性裸鼠50只,在裸鼠皮下注射内异症患者子宫内膜碎屑,建立裸鼠内异症皮下种植模型。选择建模成功的裸鼠模型45只,随机分为3组(空白对照组、阴性对照组、治疗组),每组15只,3组裸鼠皮下异位结节灶内分别注射磷酸盐缓冲液(BPS)、空载体慢病毒BPS稀释液和shRNA慢病毒BPS稀释液各50μl。各组裸鼠实验前后,测量其体重、皮下异位病灶体积的变化,观察裸鼠活动、皮色、食欲等情况。注射后15 d,断颈法处死裸鼠,剥离出异位内膜病灶,比较各组病灶的体积与重量。分别采用RT-PCR及免疫组化检测异位内膜组织survivin和caspase-3 mRNA及蛋白的表达,TUNEL法检测各组异位组织凋亡,光镜观察病灶、肝脏、肾脏、卵巢和子宫组织病理变化。放射免疫法检测各组裸鼠血清E2、FSH水平。
结果
1.经阳性克隆PCR鉴定及测序分析,结果显示慢病毒重组质粒中含有所设计的寡核苷酸单链,表明合成的survivin-shRNA序列插入正确。慢病毒重组质粒及包装质粒共转染293T包装细胞,能产生重组慢病毒LV-survivin-shRNA,经纯化浓缩后产生的慢病毒滴度为8×108TU/ml。
2. LV-survivin-shRNA在体外对人异位内膜细胞中survivin基因表达及细胞增殖和凋亡的影响
(1)原代培养的异位内膜细胞均有腺上皮细胞和间质细胞两种形态细胞。腺上皮细胞和间质细胞分别经角蛋白单抗和波形蛋白单抗免疫组化染色为阳性,腺上皮细胞平均生长时间为5周,间质细胞平均生长时间为14周。
(2)重组慢病毒感染人异位内膜细胞后24 h即可见到有绿色荧光蛋白表达,以后逐渐增强,至72 h后其绿色荧光的表达呈高峰。病毒感染复数(MOI)为100时,细胞病理现象明显,细胞变圆,漂浮,细胞的正常增殖受影响。MOI为50时,细胞的形态和生长不受影响,且感染效率较高,其在异位内膜细胞中的阳性率达80%。
(3)与对照组细胞相比,shRNA慢病毒感染人异位内膜细胞后72 h survivin mRNA及蛋白表达水平均明显下降,survivin mRNA及蛋白表达抑制率分别为64.20%和58.79%,感染后3周,实验组异位内膜细胞中survivin基因表达仍显著降低,survivin mRNA表达的抑制率达70.93%。
(4)MTT法分别检测感染后第1d～5d细胞的OD值,结果显示,除感染后第1d外,实验组OD值均显著低于阴性对照组和空白对照组(P值均<0.001),而后两组细胞的增殖速度差异无统计学意义(P值均>0.05)。感染后不同时间点之间OD值有显著差异(P<0.001), shRNA慢病毒感染后异位内膜细胞增殖抑制率有时间依赖性(rs=1,P<0.001),感染后第1 d-5 d的异位内膜细胞增殖抑制率分别为2.97%、36.19%、52.22%、58.45%、65.96%。
(5)流式细胞仪分析显示,实验组细胞凋亡率为(41.61±3.64)%,显著高于阴性对照组的(9.14±1.88)%和空白对照组的(7.07±1.16)%(P<0.001),而后两组间比较,差别则无统计学意义(P=0.087)。
3. LV-survivin-shRNA对CAM内异症模型子宫内膜种植及血管生成的影响
(1)成功建立CAM内异症血管生成模型,单纯种植组、空载慢病毒组及空白载体组的内膜种植灶周围见丰富的血管网生成,以病灶为中心呈放射状生长,形成血管“辐辏”现象。
(2)shRNA慢病毒组的种植灶周围的CAM血管生成明显受抑,新生血管稀疏,分布无规律,可见无血管的苍白区,shRNA慢病毒组的VA/CAM显著低于其余3组(P<0.001),血管生成抑制率达75.89%。而其余3组之间比较,VA/CAM差异均无统计学意义(P值均>0.05)。
(3)各组鸡胚中子宫内膜的种植成功率差异显著(P=0.003), shRNA慢病毒组种植内膜存活率最低。
(4)shRNA慢病毒组的细胞凋亡率显著高于单纯种植组、空载慢病毒组及空白载体组,而后3组间细胞凋亡率两两比较,差异均无统计学意义(P值均>0.05)。
(5)shRNA慢病毒组病灶在光镜下可见腺体结构不完整,间质伴有不同程度的坏死。
4. LV-survivin-shRNA对裸鼠内异症皮下种植模型中异位病灶生长的影响
(1)成功建立裸鼠内异症皮下种植模型,裸鼠皮下在注入内膜后第10 d可见5mm～10mm的结节,50只裸鼠中有45只皮下种植成功,成功率为90%。治疗组、阴性对照组及空白对照组异位病灶体积在注射前无统计学差异(P=0.827)。
(2)各组裸鼠在注射前后的活动、皮色、食欲等未见明显的异常变化,各组裸鼠注射前后的体重变化差异均无显著性意义(P值均>0.05)。
(3)在注射后第15 d,肉眼观察阴性对照组及空白对照组的异位病灶生长良好,呈隆起的小囊状,表面血管可见,而治疗组的异位病灶体积明显缩小、变硬,萎缩成斑块状。
(4)在注射后第15 d,治疗组、阴性对照组及空白对照组病灶的重量分别为(0.21±0.07)g、(0.79±0.12)g、(0.83±0.07)g,治疗组病灶的重量显著低于后两组(P值均<0.001),而后两组间比较,差异无统计学意义(P=0.719),治疗组的抑瘤率为74.69%。治疗组、阴性对照组及空白对照组病灶的相对体积分别为0.350±0.059、1.466±0.119、1.548±0.135,治疗组异位病灶的相对体积显著低于后两组(P值均<0.001),而后两组间比较,差异无统计学意义(P=0.064)。生长体积-时间曲线显示治疗组异位病灶生长速度显著低于阴性对照组及空白对照组。
(5)光镜下治疗组可见异位内膜腺体萎缩明显,部分结构不完整,可见腺上皮细胞空泡变性,间质伴有不同程度的坏死。
(6) Survivin、caspase-3 mRNA及蛋白在各组标本中均有表达,治疗组survivin mRNA及蛋白表达量均显著低于阴性对照组和空白对照组(P值均<0.001),而后两组间比较,survivin mRNA及蛋白表达量差异均无统计学意义(P值分别为0.263、0.611),治疗组survivin mRNA及蛋白表达的抑制率分别为70.88%和66.38%。治疗组caspase-3 mRNA及蛋白表达量均显著高于阴性对照组和空白对照组(P值均<0.001),而后两组比较均无统计学差异(P值分别为0.918、0.604)。3组异位病灶中survivin的表达与caspase-3的表达之间均存在显著负相关关系(P值均<0.001)。
(7) TUNEL法检测结果显示,治疗组、阴性对照组及空白对照组异位病灶中细胞凋亡率分别为(37.94±4.18)%、(10.29±2.25)%、(9.92±2.10)%,治疗组的细胞凋亡率显著高于后两组(P值均<0.001),而后两组相比差异不显著(P=0.952)。
(8)治疗组裸鼠肝、肾、子宫、卵巢等重要脏器行常规病理检查未见明显异常。
(9)治疗后15 d,治疗组、阴性对照组及空白对照组3组间比较,裸鼠血清E2、FSH水平均无显著性差异(P值分别为0.273、0.364)。
结论
1.成功构建了携带survivin基因特异性shRNA的重组慢病毒载体,其携带的绿色荧光蛋白便于观察追踪；慢病毒重组质粒及包装质粒共转染293T细胞,成功地包装和生产了高滴度的重组慢病毒LV-survivin-shRNA。
2.通过胰蛋白酶消化、贴壁纯化等技术原代培养人异位内膜细胞的方法经济、简单、高效,适用于异位内膜腺上皮细胞和间质细胞的混合培养。
3. LV-survivin-shRNA在体外可高效地感染异位内膜细胞,其可在转录及翻译两个水平上高效、特异、稳定地抑制异位内膜细胞中survivin基因的表达,显著促进异位内膜细胞的凋亡,明显抑制异位内膜细胞的增殖,并随着时间延长,其抑制效应呈增强趋势,而空载慢病毒本身对细胞生长无明显影响。
4. LV-survivin-shRNA能够显著抑制人子宫内膜异位种植CAM模型的新生血管形成,促进异位种植内膜细胞的凋亡,从而抑制子宫内膜在CAM上的种植生长。
5.裸鼠内异症皮下种植模型建模操作简单,便于观察,成模率高。
6. LV-survivin-shRNA能通过高效抑制裸鼠内异症模型体内异位病灶中survivin的表达,解除对凋亡通路下游基因caspase-3的抑制,有效地激活caspase-3的表达,促进异位内膜细胞的凋亡,从而显著地抑制裸鼠体内异位病灶的生长。这可能是其有效控制裸鼠内异症的作用机制之一。
7.经LV-survivin-shRNA治疗后,裸鼠体重、皮色、活动及食欲无明显异常变化,肝、肾、子宫及卵巢标本未见组织学异常,血清E2及FSH水平与对照组无差别,提示该治疗方法无明显的毒副作用,但其远期影响仍需进一步观察。
Backgroud
Endometriosis is one of the most common gynecologic diseases with an increasing prevalence. Endometriosis presents in approximately 10 to 15 percent of reproductive age women. It often causes infertility, dysmenorrhea, dyspareunia and chronic pelvic pain. Although endometriosis is a kind of benign disease, it has many similar characters to malignant disease, such as cellular proliferation, infiltration and recidivation. Endometriosis is hard to be cured because of its recrudescence and has heavy impacts on women's body and mind health.
The treatments for endometriosis include surgery and medication. Operation is good for cleaning the lesion, restoration of pelvic anatomy, releasing symptom, and increasing the chances of conceiving. But its recurrence rate is still up to nearly 50% except radical surgery which would result in the loss of reproductive and endocrine abilty. Currently available medical therapies are designed to inhibit ectopic lesions by depressing the ovarial function to redcuse the level of estrogen directly or indirectly. However, none of these drugs can eradicate the disease and the substantial side effects. So far, recurrence is the key point to treatment of endometriosis because the true cause of the disease is ambiguous. All of the therapies are lack of the exact target. It is very urgent to find out the pathogenesis of endometriosis and get effective, safe, long-lasting treatment measures for endometriosis.
The transplantation theory that Sampson proposed in 1927 is still recognized widely. However, the incidence of reflux of shed endometrial fragments is up to 76%～90% which is not to accord with the incidence of endometriosis. It is suggested that the refluent endometrial fragments is only the inducement. Recent studies indicated that the biological behavior of cell adhesion, invasion, planting, growth and transfer is the key to the pathogenesis of endometriosis. There are more and more researches indicated:Spontaneous apoptosis is decreased and proliferation is inereased in women with endomertiosis. This showed that apoptosis is the key to maintain stability of endometrial structure and function. The change of apoptosis characteristics is important for ectopic endometrial cells to have the ability to implant and grow. At the same time, many studies indicated that angiogenesis play a important role in formation and development of endometriosis, the formation and growth of ectopic lesions is depend on the new blood vessels. Therefore, promoting apoptosis and blocking angiogenesis are effective strategy for prevention and control endometriosis.
Survivin is the strongest member of inhibitor of apoptosis proteins family and play an important role in the regulation of apoptosis. It expresses selectively in human embryonic tissues and tumor cells but almost not in normal differeniiated human tissues except for thymus, testes and secretory phase endometrium. survivin not only inhibits cell apoptosis, but also involves in angiogenesis. Therefore, survivin has been paid significant attention as a new target for anti-tumor and anti-vessel therapy.
Recent studies have shown that survivin expression in ectopic endometrium was much higher than that in normal endometrium and eutopic endometrium. Those results indicate that survivin gene inhibits cell apoptosis in ectopic endometrium, destroies the balance between proliferative and apoptosis, favor abnormal proliferative, so it may contribute to the formation and progression of endometriosis. As above, If survivin expression have been suppressed, the occurrence and development of endometriosis would be blocked result from promotion of ectopic endometrial cells apoptosis and inhibition of angiogenesis. Therefore, survivin gene may be an ideal target for treatment of endometriosis.
The important aim of gene therapy is the continually and steadily expression of obtained genetic material in transferred cell. Because the inhibition effect caused by RNA interference was fast, efficient and high stable, RNA interference is the most effective method of the sequence-specific post-transcriptional gene silencing in biology. It has become a powerful tool in exploring gene function and gene therapy for tumor and infectious diseases. Nevertheless, the key to the success of gene therapy is selecting suitable vector. Lentiviral vector become the focus of current attention widely due to its unique advantage. Lentiviral vector system have many advantages such as infection of non-dividing cells and divided cells, small immune response, reusable applications, huge gene fragments carried, long-term stable expression and high biological safety.
Objective
The feasibility and effectiveness of lentivirus mediated short hairpin RNA (shRNA) to repress the expression of survivin and promote apoptosis in ectopic endometrium cells were investigated. Furthermore, the effectiveness and security of lentivirus mediated shRNA targeting survivin gene to inhibit the establishment and growth of ectopic lesion in nude mice were studied. The main object is to seek a novel treatment strategy for endometriosis.
Methods
1. Construct a lentiviral expression vector mediated shRNA targeting human survivin gene and package lentivirus particles. Human survivin gene shRNA sequence was designed using software available on-line. After synthesis and annealing, the shRNA sequence was cloned into the pGCL-GFP vector to construct LV-survivin-shRNA, which was subsequently confirmed by PCR and DNA sequencing analysis. The recombinant plasmid, packaging plasmid pHelper 1.0 and pHelper 2.0 were co-transfected into human embryonic kidney epithelial cell line (293T cells) and high titer replication defective lentivirus particles were collected and determined.
2. Study the effect of lentiviral vector-mediated shRNA on the expression of survivin mRNA and protein and its influence on the proliferation and apoptosis in human ectopic endometrial cells. The ectopic endometrial cells from patients with endometriosis were isolated and purified by trypsin digestion and pasted wall purification. The cellular morphology was observed by optical microscope. Endometrial cells were identified by immunocytochemistry. Lentiviral vector mediated shRNA targeting survivin gene (LV-survivin-shRNA) was transfected into human ectopic endometrial cells, the empty vector and culture media as control, respectively. The survivin mRNA and protein expression were detected by RT-PCR and Western-blot. The proliferation and apoptotic rate of cells after transfection were assayed by methy-thiazoly-tetrazolium and flow cytometry.
3. Investigate the effect of LV-survivin-shRNA on angiogenesis and growth of endometriosis like lesions in the chick embryo chorioallantocic membrane (CAM). Eutopic endometria from women with endometriosis were transplanted onto the non-vascular region of CAM. After successful established, the human endometriosis CAM angiogenesis models were randomly divided into 4 groups (n=30). It include inoculated alone group, LV-survivin-shRNA group, LV-NC-GFP group and empty vector group. Another 7 days later, CAMs were photographed under the anatomical microscope after entirely exposure. Graph software automatically counted vascular area and the area of CAM. Transplant specimens were analyzed by histology. Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method was used to detect cells apoptosis of endometriotic lesions.
4. Animal endometriosis models of nude mouse were established by inoculating human endometrial fragments. Forty five female nude endometriosis models were randomized into three groups,15 mice each group, including LV-survivin-shRNA group, LV-NC-GFP control group and blank control group when ectopic lesion diameter reached 5mm-10mm. Endometriosis models of nude mouse were administrated with LV-survivin-shRNA, LV-NC-GFP and PBS into the transplants. The activity and appetite of those mice were monitored each day, and the body weight of each mouse was weighing on the first day and the last day. The ectopic lesion sizes were measured every three days. All of these mice were killed at 15th day after treatment. The mRNA and protein expression of survivin and caspase-3 in ectopic lesion after transfer was detected by RT-PCR and immunohistochemistry method, respectively. TUNEL method was used to detect endometriosis like lesions cells apoptosis. Meanwhile we detected the level of steroid hormone and histological change in mice uterus, ovary, liver and kidney.
Results
1. PCR analysis and DNA sequencing confirmed that the shRNA sequence was successfully inserted into the lentiviral vector. The titer of concentrated virus was 8×108TU/ml.
2. The cell culture of ectopic endometrium was successfully established. Both endometrial epithelial cell (EEC) and endometrial stromal cell (ESC) were existed in the culture and stained positively by cytokeratin and vimentin monoclonal antibody respectively. The life span of EEC and ESC can last 5 weeks and 14 weeks, respectively. We could see the expression of green fluorescent protein after lentivirus infecting primary ectopic endometrial cells for 24h, the strongest expression of EGFP could be seen after 72h. Lentivirus of Multiplicity of Infection (MOI) less than 10 had low transfection efficiency, but when viral MOI was equal to 50, the transfection efficiency is high, up to 80%, and cell morphology and growth of cells are the same as the proliferation of normal cells. When MOI is equal to 100, the cells became obviously pathological cells, such as round and floating cells with normal proliferation affected. After transfected with LV-survivin-shRNA for 72 h, the expression of survivin mRNA and protein in human ectopic endometrial cells was inhibited by 64.20% and 58.79% respectively. After transfected with LV-survivin-shRNA for 3 weeks, the expression of survivin mRNA in human ectopic endometrial cells was still inhibited by 70.93%. The growth speed of the human ectopic endometrial cells transfected with LV-survivin-shRNA was significantly slowed than other two groups. There was a significant difference between them (P<0.001) except at the first day after being transfected. There was no difference between the growth speed of non-transfected cells and that of empty vector transfected cells (P>0.05). The apoptosis rate of survivin-shRNA group reached (41.61±3.64)%, which was significantly higher than those of non-transfected group (7.07±1.16)% and empty vector transfected group (9.14±1.88)% (P<0.001). There was no difference between the apoptosis rate of non-transfected cells and that of empty vector transfected cells (P=0.087).
3. The human endometriosis chick chorioallantoic membrane angiogenesis models(hEMCAM) were established successfully. Transplantation of endometrium onto the CAM led to a strong angiogenic response in the chicken tissue. The vessels grew radially around focus and the density of newborn blood vessels increased significantly in the hEMCAM. The angiogenesis and endometriosis-like lesions formation were significantly suppressed after treatment with LV-survivin-shRNA in comparison with the control groups. The transplantation achievement ratio of LV-survivin-shRNA group was significantly lower than those of other groups (P<0.001). Quantitative analysis shows that the ratio of vascular area (VA)/CAM of LV-survivin-shRNA group is significantly reduced (P<0.001). There is no significant difference between the other three groups (P>0.05). The apoptosis rate of endometriosis-like lesions after treatment with LV-survivin-shRNA was significantly higher than those of other three groups(P<0.001), and accomponied by various degree of necrosis in the endometriosis-like lesions under microscopic observation.
4.50 nude mice were modeled by subcutaneous implantation by injection with the endometria from endometriosis patients, there are 45 animal endometriosis models were established successfully. After endometrial fragment being inoculated for 10 days, lesion nodes (5mm-10mm in diameter) were found. There were no significant difference in volume of the endometrial lesion and weight of the nude mice before treatment among blank control group, negative control group and LV-survivin-shRNA group. At the 15th days after treatment, compared with the other two control groups, the volume and weight of the lesion nodes were decreased obviously (P<0.001). Microscopic examination showed that the glandular epithelium of LV-survivin-shRNA group had partially degenerated, and necrotic debris was present in the endometrial stroma. The difference was not statistically significant between the two control groups. The results of RT-PCR and immunohistochemisty staining showed that the expression of survivin mRNA and protein in ectopic lesions of the LV-survivin-shRNA group were significantly less than the two control groups (P<0.001). The expression of survivin mRNA and protein in ectopic lesions were inhibited by 70.88% and 66.38%, respectively. Nevertheless, the mRNA and protein expression of caspase-3 in ectopic lesions of the LV-survivin-shRNA group were significantly higher than the two control groups (P<0.001). The apoptosis rate of LV-survivin-shRNA group was significantly higher than that of blank control group and negative control group (P<0.001). There was no difference between the apoptosis rate of blank control group and that of negative control group (P=0.952). There were no obvious changes in the nude mouse liver, kidney, uterus and ovary were observed through routine pathological check after the LV-survivin-shRNA treatment. At the 15th days after treatment, there was no significant difference in concentration of E2 and FSH among three groups. During administrating, side effects were not found in the LV-survivin-shRNA group.
Conclusions
1. The recombinant lentiviral vector mediated shRNA targeting survivin gene have been constructed successfully. The recombinant lentiviral vector is effective, safe and convenient, which is convenient to observed because of GFP and can be packaged high-titer, high-purity lentivirus.
2. The primary culture method for ectopic endometrial cells by trypsin digestion and pasted wall purification was convenient and of high performance. It can be applied to culture EEC and ESC incorporately.
3. LV-survivin-shRNA can infect ectopic endometrial cells effectively in vitro and can inhibit the expression of survivin gene, which resulted in suppressing the proliferation and promoting the apoptosis in human ectopic endometrial cells.
4. The CAM endometriosis models have been constructed successfully. LV-survivin-shRNA can effectively inhibit angiogenesis induced by eutopic endometrium onto the CAM, obviously promote the apoptosis in ectopic endometrial cells and remarkably suppress endometriosis-like lesions formation.
5. The endometriosis mice models of subcutaneous implantation were established successfully. The model of subcutaneous implantation is more convenient to observe. The modeling method is simple and accomponied by high achievement ratio.
6. Lentiviral vector-mediated shRNA can be transferred into mice ectopic lesion and can effectively inhibit the expression of survivin gene and activate the expression of caspase-3, which leaded to obviously promote the apoptosis in ectopic endometrial cells and remarkably suppress the growth of endometriosis like lesions in nude mice.
7. There were no significant side effects were observed. The therapy of endometriosis with lentiviral vector-mediated shRNA was effective and safe. It lays the foundation for the experimental study and future clinical application.
引文
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