摘要
研究背景及目的
瘢痕疙瘩是皮肤损伤如创伤、烧伤或手术后引发的纤维化病理改变,以成纤维细胞(fibroblast)的过度增生和胶原等细胞外基质(extracellular matrix, ECM)的过度沉积为特征的纤维代谢性疾病。瘢痕疙瘩的形成会破坏人的体表完美,导致功能障碍,给患者带来极大的生理和心理上的痛苦,对瘢痕疙瘩的防治一直是临床研究的重要课题。具有独特生长特性以及临床表现的瘢痕疙瘩,被众多学者视为一种创伤愈合后形成的良性肿瘤,成纤维细胞是瘢痕形成过程中的关键效应细胞。近年来研究认为成纤维细胞的异常增殖是瘢痕疙瘩发病的重要机制,其增殖与凋亡的失衡是导致瘢痕疙瘩不断增生而且难已退化的细胞学基础。而survivin基因作为迄今为止发现的最强的凋亡抑制因子之一,和瘢痕疙瘩的形成有密切关系。研究表明：survivin基因可抑制成纤维细胞的程序化死亡,导致细胞的异常增殖和恶性转化,与瘢痕疙瘩形成密切相关。因此,抗survivin基因的治疗可能成为治疗瘢痕疙瘩的有效途径。
目前,对瘢痕疙瘩的大量研究主要集中在明确病因方面,治疗还局限在单纯的药物注射和病灶的手术切除,均无法从根本上彻底治愈,治疗后常复发,尤其瘢痕疙瘩手术后增生更明显。临床上迫切需要一种可以在瘢痕形成早期抑制瘢痕疙瘩成纤维细胞增殖的方法。随着基因工程技术的出现和迅猛发展,基因治疗将成为今后治疗瘢痕疙瘩理想的治疗方法。目前,许多学者尝试应用反义寡核苷酸、核酶的方法抑制目的基因的表达,但是存在转染效率低下,作用时间短暂等缺点。随着RNA干扰(RNA interference)现象被发现,为瘢痕疙瘩的基因治疗提供了广阔的空间,RNA干扰目前已经在这一领域取得了令人瞩目的进展和成果。RNA干扰是利用与目的mRNA具有同源性的siRNA诱发序列特异性的转录后基因沉默的现象,它产生类似于“基因敲除”的生物表型,能够高效、特异地抑制靶基因,从而产生相应功能的缺失,这个过程属于转录后的基因沉默(posttranscriptional gene silencing, PTGS)。RNA干扰具有特异性、高效性和持久性而得到广泛应用,目前已成功地应用RNAi技术敲除疾病相关基因,表明该技术开启了新的治疗途径的大门。RNAi目前已经在广泛的领域包括医学领域里取得了令人瞩目的进展,并广泛应用于医疗各种疾病尤其是肿瘤相关方面的治疗。虽然有整形外科学者开始应用RNAi技术特异性抑制瘢痕形成的相关因子基因的表达,但是应用体外合成siRNA对瘢痕疙瘩成纤维细胞survivin基因表达抑制的研究目前国内外未见相关报道。由此设想,如果瘢痕疙瘩成纤维细胞中survivin存在着高表达,并抑制了成纤维细胞的程序化死亡,那么应用RNAi技术阻止或抑制survivin基因的表达,减少survivin蛋白合成,从而抑制成纤维细胞的异常增殖和恶性转化,就有可能达到治疗瘢痕疙瘩增生的目的。因此,运用生物信息学方法,针对人survivin基因设计并合成相应siRNA分子,转染原代培养的瘢痕疙瘩成纤维细胞,下调survivin表达,抑制survivin的有效合成,观察siRNA分子对人瘢痕疙瘩成纤维细胞survivin表达和成纤维细胞功能的影响,并探讨其机制,为瘢痕疙瘩的基因治疗提供初步的理论依据。
材料和方法
1、标本
取自昆明医学院第一附属医院整形外科手术切除的瘢痕组织,所有患者均无合并皮肤疾病、结缔组织病和其他重要脏器疾病；未使用过类固醇激素、青霉胺和抗肿瘤药物；所有的标本切取前均未行放疗、激光治疗及免疫治疗；所有标本均经病理证实。采用消化法来进行成纤维细胞的原代培养。正常皮肤中均取自上述患者供皮区,为手术取皮修剪后残余皮片。
2、方法
(1)免疫组织化学染色检测survivin基因在正常皮肤组织、增生性瘢痕和瘢痕疙瘩组织中的表达,并计数瘢痕疙瘩组织的微血管密度(MVD),分析瘢痕疙瘩中survivin的表达与血管生成的关系；
(2)免疫细胞化学染色检测survivin基因在瘢痕疙瘩和正常皮肤原代培养的成纤维细胞中的表达；
(3) siRNA-survivin分子的设计与合成；
(4)实验分组及脂质体转染siRNA-survivin分子;
(5)RT-PCR法检测转染后瘢痕疙瘩成纤维细胞中survivin mRNA表达；
(6)Western-blot法检测转染后瘢痕疙瘩成纤维细胞中survivin蛋白的表达；
(7)流式细胞术分析细胞周期；
(8)MTT法检测成纤维细胞增殖；
(9)Tunel检测细胞凋亡；
(10)数据处理：应用SPSS12.0进行数据统计分析,计量资料以平均数±标准差(X±S),进行T检验和方差分析,细胞生长曲线采用重复测量方差分析进行比较,P<0.05为有显著性差异。
结果
1、survivin在正常皮肤、增生性瘢痕和瘢痕疙瘩中表达的阳性率分别为0(0/10)、34.78%(8/23)和66.67%(20/30)；瘢痕疙瘩中survivin阳性表达明显高于增生性瘢痕；瘢痕疙瘩中随survivin表达增强,MVD逐渐增高。survivin基因在瘢痕疙瘩原代培养的成纤维细胞中的表达阳性,而正常皮肤组织原代培养的成纤维细胞未见阳性表达。
2、成功构建了重组质粒pSIREN-siRNA/survivin,经基因测序显示重组质粒shRNA编码序列与我们设计的靶向survivin的核苷酸序列完全一致。
3、构建的真核表达质粒瞬时转染成纤维细胞,荧光显微镜观察转染效率达67%。RT-PCR检测成纤维细胞survivin mRNA表达,发现转染后的24h、48hsurvivin mRNA受到明显抑制,抑制率分别为52.2%和75.7%；Western-blot分析survivin蛋白表达,转染后的24h、48hsurvivin蛋白表达分别下调至54.8%和76.7%。
4、流式细胞仪检测细胞周期发现干扰质粒组S期细胞减少；G_2/M期所占比例增高,出现了G_2/M期的阻滞现象。干扰质粒组24h、48h成纤维细胞的凋亡率分别为8.8%、19.6%,显著高于阴性对照组和未处理组,随干扰时间的延长,凋亡细胞逐渐增多。
5、流式细胞仪Tunel法检测细胞凋亡：转染siRNA-survivin后,24h和48h的凋亡率分别为11.9±1.96%、21.2±2.14%,与阴性对照组(3.60±0.83%)和处理组(2.40±0.63%)相比,统计学上具有显著性差异(P<0.05)。
6、MTT测定显示转染重组质粒pSIREN-siRNA/survivin后瘢痕疙瘩成纤维细胞的增殖受到明显抑制。
结论
1、survivin蛋白在瘢痕疙瘩组织中表达上调,并与微血管生成关系密切,提示survivin与病理性瘢痕的发生发展过程有关。
2、针对survivin基因的siRNA干扰质粒转染瘢痕疙瘩成纤维细胞后能明显抑制survivin mRNA表达,降低survivin蛋白含量,提示该基因可作为基因治疗的靶点。
3、siRNA-survivin分子对靶基因survivin抑制效果明显,抑制成纤维细胞从G_1期进入S期,而阻滞于G_2/M期,随干扰时间的延长,细胞凋亡增加。
4、siRNA-survivin转染瘢痕疙瘩成纤维细胞,生长曲线平缓,明显抑制瘢痕疙瘩成纤维细胞的生长。
5、本研究证实siRNA-survivin分子能够特异性抑制survivin基因表达,进而抑制瘢痕疙瘩成纤维细胞异常增殖,诱导细胞凋亡。
Background and objective
Keloids are skin lesions such as wounds, burns or surgery-induced fibrosis changes, a fiber metabolic disease characterized by excessive proliferation of fibroblasts and excessive collagen of extracellular matrix (extracellular matrix, ECM) excessive deposition. Keloid destructs human body's surface, leads to dysfunction, and gives patients tremendous physical and heart pain, therefore, the prevention of keloid has been an important issue in clinical studies. The unique keloid growth characteristics and clinical performance are considered to be benign for tumor formed after wound healing by many scholars. Fibroblasts are key effector cells in the-process of forming scar. Researches in recent years have found that the abnormal proliferation of fibroblasts is an important mechanism of keloid disease, also the imbalance of proliferation and apoptosis lead to continued proliferation of keloid and difficult degrading of cellular. The survivin gene as the strongest ever found apoptosis inhibitory factor is closely related to the formation of keloids. The results show that survivin gene could inhibit fibroblasts’death, cause cells abnormal proliferation and malignant transformation. Because survivin is closely related with keloid formation, anti-survivin therapy is likely to be an effective way to treat keloids.
Currently, a large number of studies on keloid are mainly focusing on the diagnoses of etiology, and treatments are also limited to drug injection and the surgical removal of lesion, but they can't completely cure from the root, relapse often occurs after treatment, especially after the proliferation of keloid surgery. Clinics urgently need a method that could inhibit proliferation of keloid fibroblasts during the early stage of wound healing. With the emergence and rapid development of genetic engineering, gene therapy will become an ideal treatment for keloid treatment. Currently, many scholars try to use antisense oligonucleotide, ribozyme gene to inhibit the expression of gene, but these methods have shortcomings such as low transfection efficiency and short duration of action. The discovery of RNA interference (RNA interference) phenomenon provides a new era for the gene therapy of keloid, and RNA interference has already made remarkable progress. RNA interference uses the targeted mRNA that has homologous sequence-specific siRNA to induce transcriptional gene silencing, which resulted in a. similar "knock out" phenotypes, can be effective and specifically inhibit the target gene, and then causes corresponded lack of functionality. This process is post-transcriptional gene silencing (post transcriptional gene silencing, PTGS). Because of its specificity, efficiency and durability it has been widely used. The successful application of RNAi technology knockout disease genes indicates that the technique has opened the door to new therapeutic approaches. RNAi has made great progress in a wide range of areas such as medicine, and been widely used in medical treatment of various diseases, especially relevant aspects of tumor treatment. Although plastic surgery academics and scholars began to apply RNAi technology to inhibit formation of scar-related gene, but none of the research about application of vitro synthesized siRNA on keloid fibroblasts to inhibit survivin expression has been reported domestically and internationally. Thus we assume that if the keloid fibroblasts has high survivin expression and inhibits fibroblast programmed death, then that using RNAi technology to block or inhibit the expression of survivin gene, reduce survivin protein synthesis, and inhibit fibroblast abnormal cell proliferation and malignant transformation, could be able to treat hypertrophic scar. Therefore, we could use this bio-informatics method to design and synthesize siRNA molecules according to a person's survivin Gene, transfect of primary cultured keloid fibroblasts, reduce survivin expression and inhibitthe effective synthesis of survivin, observe its effect to human's survivin expression in keloid fibroblasts and fibroblast cell function, and provide the preliminary theory for the therapy of keloid.
Materials and Methods
1. Samples
Samples are taken from the scar tissue that removed through Plastic Surgery by the first affilated hospital of kunming medical college. None of the patients had skin diseases, connective tissue disease or any other major organ diseases; and had used steroids, or anti-penicillamine oncology; none of the specimens were treated by radiotherapy, laser therapy or immunotherapy; all samples were confirmed by pathology. Primary fibroblast culture is carried out by digestion. Normal skin was taken from the donor site of patients for surgery after taking the residual skin leather trim.
2. Methods
(1) Immunohistochemical detection of survivin gene in normal skin, the differences in the expression among hypertrophic scar, keloid tissue of survivin and CD34, microvessel density (MVD), analysis the correlation of survivin expression in abnormal scars and angiogenesis;
(2) The expression of immunocytochemical detection between survivin gene in keloid and normal skin fibroblasts in primary culture cells;
(3) The molecular design and synthesis of siRNA-survivin;
(4) Group testing and transfected siRNA-survivin molecular;
(5) Use RT-PCR to assay the survivin mRNA expression after transfection in keloid fibroblasts;
(6) Use Westernblot to assay the expression of survivin protein after transfection in keloid fibroblasts
(7) Use flow cytometry to analyze cell cycle;
(8) Use MTT to assay fibroblasts;
(9) Use Tunel to detect apoptotic cells;
(10) Data processing:using SPSS12.0 to statistically analyze data, measure mean±standard deviation (X±S), perform T test and variance analysis, use RMANOVA (repeated-measures anova) to analyze cell growth curve, P<0.05 to have significant differences.
3. Results
(1) In normal skin and keloid survivin expresses positive rate of 0 (0/10) and 66.67%(20/30); keloid survivin expression is significantly higher than in that of hypertrophic scars; with survivin expression in keloid enhanced, MVD increased gradually, survivin gene in primary culture of keloid fibroblasts expresses as positive, but primary cultured normal skin fibroblasts have no significant expression.
(2) Plasmid pSIREN-siRNA/survivin has been successfully recombined. The coding sequence of recombinant plasmid shRNA tested by gene sequencing and the nucleotide sequences that we designed shRNA targeting survivin are identical.
(3) Construction of eukaryotic expression vector transiently transfected cells stained fibers, fluorescence microscopy up to 67% transfection rate. Through RT-PCR detection of survivin mRNA expression in fibroblasts, survivin mRNA was inhibited 24.48 hours after transfection, the inhibitory rates were 54% and 79.3%; Westerm-blot analyzed survivin protein expression that survivin protein expression were reduced to 50.34% and 82.30% after 24 and 28 hours accordingly.
(4) Cell cycle by flow cytometry:the interference plasmid S phase cells decreased; the proportions during the G2/M phase increased, and stagnate phenomenon appeared in of G2/M phase. The apoptosis rates of interfering plasmid 24h,48h fibroblast were 8.8%,19.6%, significantly higher than the negative and the untreated groups, with time prolonged, apoptotic cells gradually increased.
(5) Detected by flow cytometry Tunel Apoptosis:after transfection of survivin-shRNA, the apoptosis rates of 24h and 28h were 11.9±1.96%, and 21.2±2.14% according, having significant difference which is (P<0.05) compared with the negative (3.60±0.83%) and treatment group (2.40±0.63%)
(6) MTT tests showed that after transfection of plasmid pSIREN-siRNA/survivin keloid fibroblasts proliferation was inhibited.
Conclusions
1. survivin protein expression increased in keloid, and is closely related with angiogenesis, suggesting that survivin is relevant to the hypertrophic scar development process.
2. That siRNA interference plasmids transfecting Keloid fibroblasts can inhabit survivin mRNA expression, reduce survivin protein, suggests that the gene can be used as targets for gene therapy.
3. siRNA-survivin molecule greatly inhibits the target gene survivin, inhibiting of fibroblast cells from Gl phase to S phase, but staying in the G2/M phase. Cells underwent apoptosis over time, and the number of fibroblasts is reduced.
4. The growth curve of fibroblast become mild after survivin—hRNA transfer fibroblast of keloid. Survivin—hRNA obviously restrain growth of fibroblast of keloid. 5. This study demonstrated that siRNA-survivin molecules can interfere with survivin gene expression, thereby inhibiting abnormal proliferation of early keloid.
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