可调控表达脑啡肽的永生化大鼠星形胶质细胞株的构建及其镇痛效应研究
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摘要
研究背景
慢性疼痛治疗是现代医学中最富有挑战性的难题之一。慢性疼痛的形成机理极其复杂,现有的治疗手段如药物治疗、针灸治疗和交感神经切除术的远期效果差,并且长期应用阿片类药物还易产生依赖和耐受等副作用。因此寻找一种安全有效、作用持久的镇痛方法已成为疼痛研究的重要方向。
上世纪80年代后期兴起的嗜铬细胞镇痛是慢性疼痛治疗领域的新突破,但由于供体不足、免疫原性高及镇痛效应不稳定等问题限制了这一方法的广泛应用。转基因细胞移植镇痛是近年来疼痛研究的新领域,有望为疼痛治疗提供一种全新的技术和方法。转基因细胞移植镇痛是利用基因工程技术将外源性镇痛基因导入靶细胞内,制造出可以分泌镇痛物质的工程化细胞,再将这种基因修饰细胞回输入机体内,通过细胞持续分泌镇痛物质,产生长期镇痛作用。人前脑啡肽原基因(Human preproenkephalin gene,hPPE)可编码加工出蛋氨酸脑啡肽和亮氨酸脑啡肽,通过激活脊髓背角初级传入神经纤维突触前膜的阿片受体,产生吗啡样的镇痛作用,人前脑啡肽原基因是较为理想的外源性镇痛基因选择。研究表明,转前脑啡肽原基因细胞移植后,能产生明显的镇痛效应。然而,目前所用的基因工程细胞多来源于肿瘤细胞,存在致瘤性,故其安全性受到质疑。星形胶质细胞的组织相容性好、免疫原性低、还可与其它神经细胞整合完成解剖学或生理学上的细胞替换,故是中枢神经系统基因治疗载体细胞的良好选择,但原代培养的星形胶质细胞增殖速度较慢,体外存活时间短,难以满足科研和临床研究的需要。而最近本研究室以猿肾病毒40大T抗原(SV40Tag)转染原代培养大鼠星形胶质细胞,获得永生化大鼠星形胶质细胞株(Immortalized astrocyte strain, IAST),其在体外培养中能够自我更新和无限扩增,可以提供大量表型、基因型稳定的神经胶质细胞,研究证实永生化星形胶质细胞作为载体细胞可以安全有效地用于慢性疼痛的基因治疗。
虽然解决了上述问题,然而转基因细胞移植镇痛仍面临着很多挑战,其中之一就是如何在基因工程细胞中精确调节镇痛物质的表达和分泌,即根据疼痛程度按需产生一定数量和浓度的镇痛物质以满足机体的需求,避免外源基因持续表达带来的不良后果。四环素基因表达调控系统可在基因转录水平定时、定量地调节基因表达,是在分子水平精确调控外源基因表达的有力工具。本研究拟将四环素基因表达调控系统和永生化细胞技术结合起来,构建可调控表达脑啡肽的永生化大鼠星形胶质细胞株,并将该细胞株植入慢性神经病理性疼痛大鼠蛛网膜下腔内,通过口服强力霉素调控脑啡肽的表达并观察其镇痛效应,以评价其用于疼痛治疗的可控性和有效性。
研究方法与结果
1、可调控表达前脑啡肽原基因的逆转录病毒载体的构建和病毒包装方法:采用PCR法扩增人前脑啡肽原基因(human preproenkephalin,hPPE),定向克隆入逆转录病毒四环素反应质粒(pRevTRE)中,酶切反应、PCR及DNA测序鉴定重组逆转录病毒载体pRevTRE/hPPE;在脂质体介导下分别将pRevTRE/hPPE和调节质粒pRevTet-On转入PT67包装细胞,经相应的抗生素稳定筛选,直至出现抗性克隆。RT-PCR鉴定得到阳性产病毒细胞系PT67/Tet-On和PT67/TRE-hPPE。用NIH3T3细胞测定病毒滴度。
结果:应用基因重组技术成功构建了携带可调控hPPE镇痛基因的逆转录病毒载体,对重组质粒pRevTRE/hPPE DNA进行正、反测序及分析表明插入片段的方向及全部编码序列,且开放阅读框正确,没有发生移码突变,并明确其启动密码子ATG在hPPE上端的位置。RT-PCR表明克隆的目的基因序列正确。转染了pRevTet-On的PT67细胞RT-PCR可以扩增出约237bp的条带;转染了pRevTRE/hPPE的PT67可以扩增出约950bp条带。转染pRevTet-On的包装细胞病毒滴度为2.6×105CFU/ml ,转染pRevTRE/hPPE的包装细胞病毒滴度为3.2×105CFU/ml。
2、开关基因(Tet-On)修饰的永生化大鼠星形胶质细胞株的构建
方法:将RevTet-On病毒上清感染永生化大鼠星形胶质细胞株(Immortalized rat astrocyte strain, IAST),采用有限稀释法挑选G418抗性单克隆;通过瞬时转染报告质粒pRevTRE-Luc,检测荧光素酶活性,挑选出强力霉素(Doxycycline,Dox)诱导表达高,背景表达低IAST/Tet-On细胞株,并检测Dox诱导与荧光素酶基因表达的时效、量效关系。
结果:经过有限稀释法挑选,共形成48个G418抗性克隆细胞。挑选6号克隆细胞作为高表达、低背景细胞株,其荧光素酶诱导表达值为876.1 RLU,背景表达值为42.5 RLU,诱导倍数为20.6,命名为IAST/Tet-On细胞;该细胞株在加入诱导因子Dox 1h后荧光素酶基因即开始表达,48h时达到高峰,Dox浓度在100～2 000 ng/ml的范围内,荧光素酶的诱导表达活性与药物浓度呈剂量依赖性。
3、强力霉素诱导表达脑啡肽的永生化大鼠星形胶质细胞株的构建和鉴定
方法:将RevTRE/hPPE病毒上清感染IAST/Tet-On细胞株,通过G418(500μg/ml)及潮霉素B(400μg/ml)共筛选21天后得到可诱导表达脑啡肽的IAST/Tet-On/hPPE细胞株,通过放射免疫测定法、RT-PCR、免疫细胞化学、间接免疫荧光染色检测该细胞株中Dox定量调控脑啡肽的表达情况。并通过放射免疫测定法检测Dox诱导IAST/Tet-On/hPPE细胞分泌脑啡肽的动力学变化。
结果:RT-PCR、免疫细胞化学、间接免疫荧光染色、放射免疫测定法显示,Dox以剂量依赖方式调节hPPE基因的表达。当培养液中加入0.1μg/ml Dox后,hPPE基因开始表达。hPPE基因表达量随着Dox浓度的增加而增加。不同剂量Dox诱导组与对照组比较,hPPE基因表达量差异具有统计学意义(P<0.05)。放射免疫测定法显示,培养液中加入诱导剂Dox 1μg/ml后脑啡肽分泌量逐渐增高,48h后达高峰,为初始量的12倍;撤药后脑啡肽分泌量迅速下降,24h时恢复到初始水平。重复加入或撤除Dox,脑啡肽表达呈现剂量依赖的“开关”现象。
4、鞘内移植可诱导表达脑啡肽的永生化大鼠星形胶质细胞株的镇痛效应
方法:雄性成年SD大鼠50只(体重150～200g)随机分为5组:Naiive组,大鼠不进行任何处理;SNI组,大鼠右侧下肢行保留性神经损伤(spared nerve injury, SNI)手术; SNI/control组、SNI/hPPE/Dox(-)组和SNI/hPPE/Dox(+)组分别于SNI术后1周鞘内移植对照细胞IAST或基因修饰细胞IAST/Tet-On/hPPE,而SNI/hPPE/Dox(+)组大鼠每日饮用含有2mg/ml强力霉素的水,SNI/hPPE/Dox(-)组大鼠每日饮用生理盐水。分别于SNI术前、SNI术后1周至移植后6周每周测定各组大鼠的机械缩爪阈值(Mechanical withdrawal threshold, MWT)和辐射热抬脚时间(Thermal withdrawal duration, TWD)。于移植后6周取各组大鼠L4-6脊髓组织,免疫组织化学法检测移植细胞BrdU的表达,借此判断细胞存活的情况。实时定量PCR测定各组大鼠hPPE基因的表达情况;放射免疫法及免疫荧光化学检测脊髓内L-EK表达情况;免疫组织化学检测c-Fos蛋白的表达。
结果:SNI术后1周大鼠结扎侧后足出现痛觉异常现象,表现为不同程度的MWT降低和TWD延长(P<0.05)。鞘内植入转前脑啡肽原基因细胞后1周即出现大鼠疼痛行为学症状明显减轻,MWT增加和TWD缩短(P<0.05),其中饮用强力霉素组(SNI/hPPE/Dox(+)组),其MWT和TWD变化最为显著(P<0.05)。细胞移植后6周,脊髓背角软脑膜表面移植细胞的BrdU免疫染色阳性,表明移植后6周细胞仍然存活。免疫细胞化学法、RT-PCR和放射免疫法结果均表明SNI/hPPE/Dox(+)组hPPE的表达和L-EK的分泌高于其它四组(P<0.05),c-Fos蛋白表达低于其它四组(P<0.05)。
5、统计学处理
采用SPSS11.0统计软件进行处理。计量资料以均数±标准差(±s)表示,行为学测定的组间比较采用重复变量数据的方差分析;其余计量资料采用单因素方差分析。P<0.05为差异有统计学意义。
研究总结
一、主要研究结果
1.本研究采用逆转录病毒转染技术,成功构建了可诱导表达脑啡肽的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE),并通过RT-PCR,免疫组织化学、放射免疫法等对该细胞分泌脑啡肽的特性进行了鉴定。
2.体外实验证实IAST/Tet-On/hPPE细胞中,脑啡肽的分泌随着培养液中的Dox浓度的增加而增加,并呈明显的剂量依赖关系。培养液中加入诱导剂Dox时,脑啡肽分泌量逐渐增高,48h达高峰,为初始量的12倍;撤药后脑啡肽分泌量迅速下降,24h时恢复到初始水平。加入或撤除Dox,脑啡肽的分泌呈现剂量依赖的“开关”现象。
3.体内实验结果表明,鞘内移植IAST/Tet-On/hPPE细胞后,可明显减轻慢性神经病理痛大鼠的疼痛行为反应,同时其镇痛效应和脊髓背角脑啡肽的表达受强力霉素调控。
二、研究结论
可调控表达脑啡肽的永生化大鼠星形胶质细胞的建立,为细胞移植镇痛提供了理想的细胞载体,是一种安全、有效、可控性的镇痛方法。
三、创新之处
本研究首次结合了永生化细胞与Tet-On四环素基因表达系统的优势,成功构建了可诱导表达脑啡肽的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE),旨在克服以往转基因载体细胞来源有限和镇痛物质分泌无法调控的缺点。通过体外强力霉素诱导脑啡肽分泌的动力学研究以及慢性神经病理性疼痛大鼠鞘内移植IAST/Tet-On/hPPE后,饮用强力霉素大鼠与未饮用强力霉素大鼠疼痛行为学变化的比较和脊髓背角脑啡肽表达的不同,证实在体内、外,强力霉素均可调节IAST/Tet-On/hPPE细胞株中脑啡肽的表达。该细胞株可作为细胞载体实现可控性镇痛的目的。
四、展望
1.鞘内移植可诱导表达脑啡肽的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE)有着传统转基因镇痛不可比拟的优势,它不仅可以作为一种“生物微泵”分泌大量的脑啡肽;而且还可根据疼痛的状态,人为地增减强力霉素来调节脑啡肽的表达量,对慢性神经病理痛的治疗可达到长效、稳定、安全、可控性的镇痛效果,有望成为一种新的转基因镇痛方式。
2.实验中需要关注的主要问题是基因表达的泄漏现象。泄漏的原因可能是因为Tet-On系统的某些不足和缺陷。近年了针对Tet-On系统的严密性和特异性进行一系列改进和发展,今后的研究可选用更为完善的Tet-On系统,实现更严密高效的基因表达调控。
3.尽管本研究室以往的研究结果表明永生化细胞是安全的,无致瘤性,但仍需要更长期的观察和评价。
4.由于技术手段的限制,本研究没能对体内脑脊液中脑啡肽的表达进行实时监测,以后的研究可采用微透析方法,动态地观察不同浓度的强力霉素对脑脊液中脑啡肽表达的调控作用。
Background
Chronic neuropathic pain is a common symptom in clinical practice and patients with chronic pain are subject to a greatly impaired quality of life. The treatment of chronic pain with traditional methods such as drug, acupuncture therapy or sympathetectomy has many unresolvable side effects and complications, especially the tolerance and dependence for long-period drug delivery that could cause the ineffectiveness of the treatment. An important issue of pain research is to find a novel method of analgesia that is safe and effective. Grafted genetically-modified cells secreting enkephalin have been considered an encouraging treatment for chronic pain. Importantly, the transplanted cell as a therapeutic agent should be reproducible, safe, and controllable. Astrocytes, as components of the brain, have served as grafted cells in gene therapy research because of their histocompatibility and lower immunogenicity. However, it is difficult to obtain primary astrocytes in large quantities and the life span of primary cells in culture is limited. Immortalized astrocytes are easy to manipulate and can be produced unlimitedly, which makes it possible for primary astrocytes to serve as the vehicle of therapeutic genes. Moreover, our previous work has shown that immortalized astrocytes are reproducible and nontumorigenic in vitro and in vivo.
However, excessive and constant release of opioid peptides from implanted cells is not necessary and is potentially toxic. Therefore, it may be necessary to engineer a cell that enables tight regulation of the levels and duration of expression of transgene products. The recent development of tetracycline-regulated transactivation systems serve as gene switches to control gene expression promptly and appear to be well-suited for regulating long-term protein secretion.
In this study, by taking advantage of both the tight regulation provided by the tetracycline-regulated ystem and the reproducibility of immortalized astrocyte cells, we aimed to develop an immortalized rat astrocyte cell whose enkephalin production can be controlled by doxycycline in a dose-responsive and reversible manner in vitro or in vivo. The human preproenkephalin (hPPE) gene encodes production of preproenkephalin which can be converted to bioactive enkephalin, which can ameliorate nociception by activating peripheral opioid receptors. Thus, we expected that the hPPE gene, introduced into modified cells and delivered into spinal cords, would be a good candidate to alleviate nociception. The regulatory analgesic effects of doxycycline on spared nerve injury (SNI) -induced nociceptive behavior in rats were investigated after these cells were transplanted into their subarachnoid space. The availability of regulatable grafted cells to treat chronic neuropathic pain was assessed.
Methods and Results
1. Retroviral vector constructs and virus production
Methods Human proenkephalin gene was amplificated by polymerase chain reaction (PCR) and was directionally cloned into the pRevTRE vector to obtain the recombinant vector pRevTRE/hPPE which was further verified by restriction endonuclease analysis and DNA sequencing. Then this recombinant plasmid and regulatory plasmid pRevTet-On were packed by packing cell line PT67 to yield two classes of viruses. The transfected PT67/Tet-On and PT67/TREhPPE cells were selected by the corresponding antibiotics and examined by using RT-PCR. Then selected cell were enriched to assay virus titer using NIH3T3 cell.
Results Restriction endonuclease analysis, PCR analysis and DNA sequencing analysis confirmed that the recombinant RevTRE/hPPE retroviral vector was constructed successfully. The virus titer of recombinant was 3.2×105CFU/ml and the virus titer of pRevTet-On was 2.6×105CFU/ml.
2. Construction of an immortalized rat astrocyte strain genetically modified with Tet-on gene
Methods The RevTet-On virus supernatants was used to infect immortalized rat astrocyte strain (IAST). Forty-eight hours post-infection, cells were screened in medium containing 400μg/ml G418. Then the G418-resistant cell clones were rescreened by transient transfection with the reporter plasmid pRevTRE-Luc. One IAST/Tet-On clone with high inducibility and low background was selected by detecting Luciferase activity. Results A total of 48 IAST/Tet-on positive clones were assessed by luciferase assay. Finally, clone 6 was picked for its high induction of luciferase activity in response to doxycycline and low leakiness (activity in the absence of doxycycline); its inducible efficiency was 20.1-fold. The expression of luciferase was induced in dose-dependent manner by doxycycline at the concentrations between 100 and 2 000ng/ml .The expression of luciferase began 1 h after doxycycline administration (1 000ng/ml) and reached the maximum level 48 h later.
3. Construction of an immortalized rat astrocyte strain with inducible enkephalin expression
Methods RevTRE/hPPE virus was used to infect the IAST/Tet-On strain, after 48 h, transfected cells were cultured for 21 days in the presence of G418 (500μg/ml) and hygromycin (400μg/ml). A single clone named IAST/Tet-On/hPPE was isolated. hPPE gene expression level of IAST/Tet-On/hPPE cells at different concentrations of doxycycline were detected by RT-PCR, immunocytochemistry and indirect immunofluorescence staining. The kinetics of Leu-enkephalin protein expression level secreted by IAST/Tet-On/hPPE cells under different concentrations of doxycycline administration and incubation for various times was assayed using a Leu-enkephalin radioimmunoassay.
Results The regulatable expression of the hPPE gene by doxycycline in IAST/Tet-On/hPPE cells was confirmed by RT-PCR immunocytochemistry, indirect immunofluorescence staining and radioimmunoassay. Treatment of doxycycline at concentrations greater than 0.1μg/ml reliably induced enkephalin expression and its expression was increased following increasing concentrations of doxycycline in the culture medium. When doxycycline was added to the culture media (1μg/ml), enkephalin secretion increased gradually, then rapidly to 12-fold after 48 h, and returned to basal levels after doxycycline removal for at least 24 h. Comparable results were obtained during the second on-off cycle. These results suggest that the expression of the hPPE gene can be controlled by doxycycline in a dose-dependent manner.
4. Analgesic effect of intrathecal transplantation of immortalized
enkephalin expressing astrocytes on chronic neuropathic pain in rats Methods 50 adult female Sprague-Dawley rats (150~200g) were randomly divided into five groups: Na?ve group, SNI group, SNI/control, SNI/hPPE/Dox(-)and SNI/hPPE/Dox(+). One week after right side spared nerve injury (SNI), IAST or IAST/Tet-On/hPPE cells were transplanted in the subarachnoid space of lumbar 4 to 6 of SNI rats and rats grafted with IAST/Tet-On/hPPE were administered saline or doxycycline in drinking water ad libitum for different predetermined times. One week before SNI and once a week for seven weeks following SNI and cell transplantation, all animals were retested on nociceptive tests. The spinal cord of L4-6 was removed eight weeks after cell transplantation, Leu-enkephalin expression using immunohistochemistry or real-time PCR, then the expression of hPPE was assessed in the dorsal horn, the content of Leu-enkephalin in the spinal cord was measured by radioimmunoassay.
Results Allodynia-like behavior and the decrease of MWT and TWD levels were observed one week after SNI. The tactile allodynia induced by SNI was alleviated in IAST/Tet-On/hPPE group at the one week after cell transplantation. Administration of 2 mg/ml doxycycline in the drinking water increased the analgesia effect in animals grafted with IAST/Tet-On/hPPE cells (P<0.05). The expression of hPPE and the level of L-EK were higher in IAST/ hPPE /Dox(+) group than other groups (P<0.05) and the expression of Fos protein was lower in IAST/ hPPE /Dox(+) group than other groups (P<0.05).
5. Statistical analysis
All of the analyses were performed by SPSS 11.0 software package. All data were expressed as the mean±standard deviation (SD). Group comparisons were made using repeated measures analysis of variance (behavioral testing data) or one-way analysis of variance (the level of enkephalin). A P-value of < 0.05 was considered statistically significant.
Conclusion
An immortalized rat astrocyte strain secreting enkephalin under the control of doxycycline was established successfully in our study. Enkephalin production from IAST/Tet-On/hPPE cells can be controlled by doxycycline in a dose-responsive and reversible manner in vitro or in vivo. The tactile allodynia induced by SNI was alleviated in IAST/Tet-On/hPPE group and the analgesic effect was also able to be regulated by Dox. Our study provides a new strategy for regulatable gene therapy for chronic pain in the future.
Summary
In this study, Tet-on system and the reproducibility of immortalized astrocyte cells were in a novel combination to achieve the useful goal of regulated release of a transgene product in vivo to treat pain. An immortalized astrocyte cell line has been engineered which releases enkephalin in a doxycycline-dependent fashion. Both in vitro and in vivo data are presented to characterize regulation of the hPPE gene expression and these enkephalin-producing cells significantly suppressed nociceptive responses when grafted into the spinal cord of SNI rats. Although improvements in the Tet-on system are necessary, this study suggests transgenic hPPE gene cell transplantation regulated by a Tet-on system may provide an alternative approach for ex vivo cell transplantation to treat chronic pain.
慢性疼痛治疗是现代医学中最富有挑战性的难题之一。慢性疼痛的形成机理极其复杂,现有的治疗手段如药物治疗、针灸治疗和交感神经切除术的远期效果差,并且长期应用阿片类药物还易产生依赖和耐受等副作用。因此寻找一种安全有效、作用持久的镇痛方法已成为疼痛研究的重要方向。
上世纪80年代后期兴起的嗜铬细胞镇痛是慢性疼痛治疗领域的新突破,但由于供体不足、免疫原性高及镇痛效应不稳定等问题限制了这一方法的广泛应用。转基因细胞移植镇痛是近年来疼痛研究的新领域,有望为疼痛治疗提供一种全新的技术和方法。转基因细胞移植镇痛是利用基因工程技术将外源性镇痛基因导入靶细胞内,制造出可以分泌镇痛物质的工程化细胞,再将这种基因修饰细胞回输入机体内,通过细胞持续分泌镇痛物质,产生长期镇痛作用。人前脑啡肽原基因(Human preproenkephalin gene,hPPE)可编码加工出蛋氨酸脑啡肽和亮氨酸脑啡肽,通过激活脊髓背角初级传入神经纤维突触前膜的阿片受体,产生吗啡样的镇痛作用,人前脑啡肽原基因是较为理想的外源性镇痛基因选择。研究表明,转前脑啡肽原基因细胞移植后,能产生明显的镇痛效应。然而,目前所用的基因工程细胞多来源于肿瘤细胞,存在致瘤性,故其安全性受到质疑。星形胶质细胞的组织相容性好、免疫原性低、还可与其它神经细胞整合完成解剖学或生理学上的细胞替换,故是中枢神经系统基因治疗载体细胞的良好选择,但原代培养的星形胶质细胞增殖速度较慢,体外存活时间短,难以满足科研和临床研究的需要。而最近本研究室以猿肾病毒40大T抗原(SV40Tag)转染原代培养大鼠星形胶质细胞,获得永生化大鼠星形胶质细胞株(Immortalized astrocyte strain, IAST),其在体外培养中能够自我更新和无限扩增,可以提供大量表型、基因型稳定的神经胶质细胞,研究证实永生化星形胶质细胞作为载体细胞可以安全有效地用于慢性疼痛的基因治疗。
虽然解决了上述问题,然而转基因细胞移植镇痛仍面临着很多挑战,其中之一就是如何在基因工程细胞中精确调节镇痛物质的表达和分泌,即根据疼痛程度按需产生一定数量和浓度的镇痛物质以满足机体的需求,避免外源基因持续表达带来的不良后果。四环素基因表达调控系统可在基因转录水平定时、定量地调节基因表达,是在分子水平精确调控外源基因表达的有力工具。本研究拟将四环素基因表达调控系统和永生化细胞技术结合起来,构建可调控表达脑啡肽的永生化大鼠星形胶质细胞株,并将该细胞株植入慢性神经病理性疼痛大鼠蛛网膜下腔内,通过口服强力霉素调控脑啡肽的表达并观察其镇痛效应,以评价其用于疼痛治疗的可控性和有效性。
研究方法与结果
1、可调控表达前脑啡肽原基因的逆转录病毒载体的构建和病毒包装方法:采用PCR法扩增人前脑啡肽原基因(human preproenkephalin,hPPE),定向克隆入逆转录病毒四环素反应质粒(pRevTRE)中,酶切反应、PCR及DNA测序鉴定重组逆转录病毒载体pRevTRE/hPPE;在脂质体介导下分别将pRevTRE/hPPE和调节质粒pRevTet-On转入PT67包装细胞,经相应的抗生素稳定筛选,直至出现抗性克隆。RT-PCR鉴定得到阳性产病毒细胞系PT67/Tet-On和PT67/TRE-hPPE。用NIH3T3细胞测定病毒滴度。
结果:应用基因重组技术成功构建了携带可调控hPPE镇痛基因的逆转录病毒载体,对重组质粒pRevTRE/hPPE DNA进行正、反测序及分析表明插入片段的方向及全部编码序列,且开放阅读框正确,没有发生移码突变,并明确其启动密码子ATG在hPPE上端的位置。RT-PCR表明克隆的目的基因序列正确。转染了pRevTet-On的PT67细胞RT-PCR可以扩增出约237bp的条带;转染了pRevTRE/hPPE的PT67可以扩增出约950bp条带。转染pRevTet-On的包装细胞病毒滴度为2.6×105CFU/ml ,转染pRevTRE/hPPE的包装细胞病毒滴度为3.2×105CFU/ml。
2、开关基因(Tet-On)修饰的永生化大鼠星形胶质细胞株的构建
方法:将RevTet-On病毒上清感染永生化大鼠星形胶质细胞株(Immortalized rat astrocyte strain, IAST),采用有限稀释法挑选G418抗性单克隆;通过瞬时转染报告质粒pRevTRE-Luc,检测荧光素酶活性,挑选出强力霉素(Doxycycline,Dox)诱导表达高,背景表达低IAST/Tet-On细胞株,并检测Dox诱导与荧光素酶基因表达的时效、量效关系。
结果:经过有限稀释法挑选,共形成48个G418抗性克隆细胞。挑选6号克隆细胞作为高表达、低背景细胞株,其荧光素酶诱导表达值为876.1 RLU,背景表达值为42.5 RLU,诱导倍数为20.6,命名为IAST/Tet-On细胞;该细胞株在加入诱导因子Dox 1h后荧光素酶基因即开始表达,48h时达到高峰,Dox浓度在100～2 000 ng/ml的范围内,荧光素酶的诱导表达活性与药物浓度呈剂量依赖性。
3、强力霉素诱导表达脑啡肽的永生化大鼠星形胶质细胞株的构建和鉴定
方法:将RevTRE/hPPE病毒上清感染IAST/Tet-On细胞株,通过G418(500μg/ml)及潮霉素B(400μg/ml)共筛选21天后得到可诱导表达脑啡肽的IAST/Tet-On/hPPE细胞株,通过放射免疫测定法、RT-PCR、免疫细胞化学、间接免疫荧光染色检测该细胞株中Dox定量调控脑啡肽的表达情况。并通过放射免疫测定法检测Dox诱导IAST/Tet-On/hPPE细胞分泌脑啡肽的动力学变化。
结果:RT-PCR、免疫细胞化学、间接免疫荧光染色、放射免疫测定法显示,Dox以剂量依赖方式调节hPPE基因的表达。当培养液中加入0.1μg/ml Dox后,hPPE基因开始表达。hPPE基因表达量随着Dox浓度的增加而增加。不同剂量Dox诱导组与对照组比较,hPPE基因表达量差异具有统计学意义(P<0.05)。放射免疫测定法显示,培养液中加入诱导剂Dox 1μg/ml后脑啡肽分泌量逐渐增高,48h后达高峰,为初始量的12倍;撤药后脑啡肽分泌量迅速下降,24h时恢复到初始水平。重复加入或撤除Dox,脑啡肽表达呈现剂量依赖的“开关”现象。
4、鞘内移植可诱导表达脑啡肽的永生化大鼠星形胶质细胞株的镇痛效应
方法:雄性成年SD大鼠50只(体重150～200g)随机分为5组:Naiive组,大鼠不进行任何处理;SNI组,大鼠右侧下肢行保留性神经损伤(spared nerve injury, SNI)手术; SNI/control组、SNI/hPPE/Dox(-)组和SNI/hPPE/Dox(+)组分别于SNI术后1周鞘内移植对照细胞IAST或基因修饰细胞IAST/Tet-On/hPPE,而SNI/hPPE/Dox(+)组大鼠每日饮用含有2mg/ml强力霉素的水,SNI/hPPE/Dox(-)组大鼠每日饮用生理盐水。分别于SNI术前、SNI术后1周至移植后6周每周测定各组大鼠的机械缩爪阈值(Mechanical withdrawal threshold, MWT)和辐射热抬脚时间(Thermal withdrawal duration, TWD)。于移植后6周取各组大鼠L4-6脊髓组织,免疫组织化学法检测移植细胞BrdU的表达,借此判断细胞存活的情况。实时定量PCR测定各组大鼠hPPE基因的表达情况;放射免疫法及免疫荧光化学检测脊髓内L-EK表达情况;免疫组织化学检测c-Fos蛋白的表达。
结果:SNI术后1周大鼠结扎侧后足出现痛觉异常现象,表现为不同程度的MWT降低和TWD延长(P<0.05)。鞘内植入转前脑啡肽原基因细胞后1周即出现大鼠疼痛行为学症状明显减轻,MWT增加和TWD缩短(P<0.05),其中饮用强力霉素组(SNI/hPPE/Dox(+)组),其MWT和TWD变化最为显著(P<0.05)。细胞移植后6周,脊髓背角软脑膜表面移植细胞的BrdU免疫染色阳性,表明移植后6周细胞仍然存活。免疫细胞化学法、RT-PCR和放射免疫法结果均表明SNI/hPPE/Dox(+)组hPPE的表达和L-EK的分泌高于其它四组(P<0.05),c-Fos蛋白表达低于其它四组(P<0.05)。
5、统计学处理
采用SPSS11.0统计软件进行处理。计量资料以均数±标准差(±s)表示,行为学测定的组间比较采用重复变量数据的方差分析;其余计量资料采用单因素方差分析。P<0.05为差异有统计学意义。
研究总结
一、主要研究结果
1.本研究采用逆转录病毒转染技术,成功构建了可诱导表达脑啡肽的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE),并通过RT-PCR,免疫组织化学、放射免疫法等对该细胞分泌脑啡肽的特性进行了鉴定。
2.体外实验证实IAST/Tet-On/hPPE细胞中,脑啡肽的分泌随着培养液中的Dox浓度的增加而增加,并呈明显的剂量依赖关系。培养液中加入诱导剂Dox时,脑啡肽分泌量逐渐增高,48h达高峰,为初始量的12倍;撤药后脑啡肽分泌量迅速下降,24h时恢复到初始水平。加入或撤除Dox,脑啡肽的分泌呈现剂量依赖的“开关”现象。
3.体内实验结果表明,鞘内移植IAST/Tet-On/hPPE细胞后,可明显减轻慢性神经病理痛大鼠的疼痛行为反应,同时其镇痛效应和脊髓背角脑啡肽的表达受强力霉素调控。
二、研究结论
可调控表达脑啡肽的永生化大鼠星形胶质细胞的建立,为细胞移植镇痛提供了理想的细胞载体,是一种安全、有效、可控性的镇痛方法。
三、创新之处
本研究首次结合了永生化细胞与Tet-On四环素基因表达系统的优势,成功构建了可诱导表达脑啡肽的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE),旨在克服以往转基因载体细胞来源有限和镇痛物质分泌无法调控的缺点。通过体外强力霉素诱导脑啡肽分泌的动力学研究以及慢性神经病理性疼痛大鼠鞘内移植IAST/Tet-On/hPPE后,饮用强力霉素大鼠与未饮用强力霉素大鼠疼痛行为学变化的比较和脊髓背角脑啡肽表达的不同,证实在体内、外,强力霉素均可调节IAST/Tet-On/hPPE细胞株中脑啡肽的表达。该细胞株可作为细胞载体实现可控性镇痛的目的。
四、展望
1.鞘内移植可诱导表达脑啡肽的永生化大鼠星形胶质细胞株(IAST/Tet-On/hPPE)有着传统转基因镇痛不可比拟的优势,它不仅可以作为一种“生物微泵”分泌大量的脑啡肽;而且还可根据疼痛的状态,人为地增减强力霉素来调节脑啡肽的表达量,对慢性神经病理痛的治疗可达到长效、稳定、安全、可控性的镇痛效果,有望成为一种新的转基因镇痛方式。
2.实验中需要关注的主要问题是基因表达的泄漏现象。泄漏的原因可能是因为Tet-On系统的某些不足和缺陷。近年了针对Tet-On系统的严密性和特异性进行一系列改进和发展,今后的研究可选用更为完善的Tet-On系统,实现更严密高效的基因表达调控。
3.尽管本研究室以往的研究结果表明永生化细胞是安全的,无致瘤性,但仍需要更长期的观察和评价。
4.由于技术手段的限制,本研究没能对体内脑脊液中脑啡肽的表达进行实时监测,以后的研究可采用微透析方法,动态地观察不同浓度的强力霉素对脑脊液中脑啡肽表达的调控作用。
Background
Chronic neuropathic pain is a common symptom in clinical practice and patients with chronic pain are subject to a greatly impaired quality of life. The treatment of chronic pain with traditional methods such as drug, acupuncture therapy or sympathetectomy has many unresolvable side effects and complications, especially the tolerance and dependence for long-period drug delivery that could cause the ineffectiveness of the treatment. An important issue of pain research is to find a novel method of analgesia that is safe and effective. Grafted genetically-modified cells secreting enkephalin have been considered an encouraging treatment for chronic pain. Importantly, the transplanted cell as a therapeutic agent should be reproducible, safe, and controllable. Astrocytes, as components of the brain, have served as grafted cells in gene therapy research because of their histocompatibility and lower immunogenicity. However, it is difficult to obtain primary astrocytes in large quantities and the life span of primary cells in culture is limited. Immortalized astrocytes are easy to manipulate and can be produced unlimitedly, which makes it possible for primary astrocytes to serve as the vehicle of therapeutic genes. Moreover, our previous work has shown that immortalized astrocytes are reproducible and nontumorigenic in vitro and in vivo.
However, excessive and constant release of opioid peptides from implanted cells is not necessary and is potentially toxic. Therefore, it may be necessary to engineer a cell that enables tight regulation of the levels and duration of expression of transgene products. The recent development of tetracycline-regulated transactivation systems serve as gene switches to control gene expression promptly and appear to be well-suited for regulating long-term protein secretion.
In this study, by taking advantage of both the tight regulation provided by the tetracycline-regulated ystem and the reproducibility of immortalized astrocyte cells, we aimed to develop an immortalized rat astrocyte cell whose enkephalin production can be controlled by doxycycline in a dose-responsive and reversible manner in vitro or in vivo. The human preproenkephalin (hPPE) gene encodes production of preproenkephalin which can be converted to bioactive enkephalin, which can ameliorate nociception by activating peripheral opioid receptors. Thus, we expected that the hPPE gene, introduced into modified cells and delivered into spinal cords, would be a good candidate to alleviate nociception. The regulatory analgesic effects of doxycycline on spared nerve injury (SNI) -induced nociceptive behavior in rats were investigated after these cells were transplanted into their subarachnoid space. The availability of regulatable grafted cells to treat chronic neuropathic pain was assessed.
Methods and Results
1. Retroviral vector constructs and virus production
Methods Human proenkephalin gene was amplificated by polymerase chain reaction (PCR) and was directionally cloned into the pRevTRE vector to obtain the recombinant vector pRevTRE/hPPE which was further verified by restriction endonuclease analysis and DNA sequencing. Then this recombinant plasmid and regulatory plasmid pRevTet-On were packed by packing cell line PT67 to yield two classes of viruses. The transfected PT67/Tet-On and PT67/TREhPPE cells were selected by the corresponding antibiotics and examined by using RT-PCR. Then selected cell were enriched to assay virus titer using NIH3T3 cell.
Results Restriction endonuclease analysis, PCR analysis and DNA sequencing analysis confirmed that the recombinant RevTRE/hPPE retroviral vector was constructed successfully. The virus titer of recombinant was 3.2×105CFU/ml and the virus titer of pRevTet-On was 2.6×105CFU/ml.
2. Construction of an immortalized rat astrocyte strain genetically modified with Tet-on gene
Methods The RevTet-On virus supernatants was used to infect immortalized rat astrocyte strain (IAST). Forty-eight hours post-infection, cells were screened in medium containing 400μg/ml G418. Then the G418-resistant cell clones were rescreened by transient transfection with the reporter plasmid pRevTRE-Luc. One IAST/Tet-On clone with high inducibility and low background was selected by detecting Luciferase activity. Results A total of 48 IAST/Tet-on positive clones were assessed by luciferase assay. Finally, clone 6 was picked for its high induction of luciferase activity in response to doxycycline and low leakiness (activity in the absence of doxycycline); its inducible efficiency was 20.1-fold. The expression of luciferase was induced in dose-dependent manner by doxycycline at the concentrations between 100 and 2 000ng/ml .The expression of luciferase began 1 h after doxycycline administration (1 000ng/ml) and reached the maximum level 48 h later.
3. Construction of an immortalized rat astrocyte strain with inducible enkephalin expression
Methods RevTRE/hPPE virus was used to infect the IAST/Tet-On strain, after 48 h, transfected cells were cultured for 21 days in the presence of G418 (500μg/ml) and hygromycin (400μg/ml). A single clone named IAST/Tet-On/hPPE was isolated. hPPE gene expression level of IAST/Tet-On/hPPE cells at different concentrations of doxycycline were detected by RT-PCR, immunocytochemistry and indirect immunofluorescence staining. The kinetics of Leu-enkephalin protein expression level secreted by IAST/Tet-On/hPPE cells under different concentrations of doxycycline administration and incubation for various times was assayed using a Leu-enkephalin radioimmunoassay.
Results The regulatable expression of the hPPE gene by doxycycline in IAST/Tet-On/hPPE cells was confirmed by RT-PCR immunocytochemistry, indirect immunofluorescence staining and radioimmunoassay. Treatment of doxycycline at concentrations greater than 0.1μg/ml reliably induced enkephalin expression and its expression was increased following increasing concentrations of doxycycline in the culture medium. When doxycycline was added to the culture media (1μg/ml), enkephalin secretion increased gradually, then rapidly to 12-fold after 48 h, and returned to basal levels after doxycycline removal for at least 24 h. Comparable results were obtained during the second on-off cycle. These results suggest that the expression of the hPPE gene can be controlled by doxycycline in a dose-dependent manner.
4. Analgesic effect of intrathecal transplantation of immortalized
enkephalin expressing astrocytes on chronic neuropathic pain in rats Methods 50 adult female Sprague-Dawley rats (150~200g) were randomly divided into five groups: Na?ve group, SNI group, SNI/control, SNI/hPPE/Dox(-)and SNI/hPPE/Dox(+). One week after right side spared nerve injury (SNI), IAST or IAST/Tet-On/hPPE cells were transplanted in the subarachnoid space of lumbar 4 to 6 of SNI rats and rats grafted with IAST/Tet-On/hPPE were administered saline or doxycycline in drinking water ad libitum for different predetermined times. One week before SNI and once a week for seven weeks following SNI and cell transplantation, all animals were retested on nociceptive tests. The spinal cord of L4-6 was removed eight weeks after cell transplantation, Leu-enkephalin expression using immunohistochemistry or real-time PCR, then the expression of hPPE was assessed in the dorsal horn, the content of Leu-enkephalin in the spinal cord was measured by radioimmunoassay.
Results Allodynia-like behavior and the decrease of MWT and TWD levels were observed one week after SNI. The tactile allodynia induced by SNI was alleviated in IAST/Tet-On/hPPE group at the one week after cell transplantation. Administration of 2 mg/ml doxycycline in the drinking water increased the analgesia effect in animals grafted with IAST/Tet-On/hPPE cells (P<0.05). The expression of hPPE and the level of L-EK were higher in IAST/ hPPE /Dox(+) group than other groups (P<0.05) and the expression of Fos protein was lower in IAST/ hPPE /Dox(+) group than other groups (P<0.05).
5. Statistical analysis
All of the analyses were performed by SPSS 11.0 software package. All data were expressed as the mean±standard deviation (SD). Group comparisons were made using repeated measures analysis of variance (behavioral testing data) or one-way analysis of variance (the level of enkephalin). A P-value of < 0.05 was considered statistically significant.
Conclusion
An immortalized rat astrocyte strain secreting enkephalin under the control of doxycycline was established successfully in our study. Enkephalin production from IAST/Tet-On/hPPE cells can be controlled by doxycycline in a dose-responsive and reversible manner in vitro or in vivo. The tactile allodynia induced by SNI was alleviated in IAST/Tet-On/hPPE group and the analgesic effect was also able to be regulated by Dox. Our study provides a new strategy for regulatable gene therapy for chronic pain in the future.
Summary
In this study, Tet-on system and the reproducibility of immortalized astrocyte cells were in a novel combination to achieve the useful goal of regulated release of a transgene product in vivo to treat pain. An immortalized astrocyte cell line has been engineered which releases enkephalin in a doxycycline-dependent fashion. Both in vitro and in vivo data are presented to characterize regulation of the hPPE gene expression and these enkephalin-producing cells significantly suppressed nociceptive responses when grafted into the spinal cord of SNI rats. Although improvements in the Tet-on system are necessary, this study suggests transgenic hPPE gene cell transplantation regulated by a Tet-on system may provide an alternative approach for ex vivo cell transplantation to treat chronic pain.
引文
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1 Toniatti , Bujard H, Cortese R, et al. Gene therapy progress and prospects: transcription regulatory systems. Gene Ther, 2004, 11(8):649-657.
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2 Ducharme J.The future of pain management in emergency medicine.Emerg Med Clin North Am, 2005, 23(2):467-475.
3 Wu CL, Garry MG, Zollo RA, et al. Gene therapy for the management of pain: Part I: Methods and strategies. Anesthesiology, 2001, 94(6):1119-1132.
4 Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline responsive promoters. Proc Natl Acad Sci USA, 1992, 89:5547-5551.
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6 Meunier A, Latremoliere A, Mauborgne A, et al.Attenuation of pain-related behavior in a rat model of trigeminal neuropathic pain by viral-driven enkephalin overproduction in trigeminal ganglion neurons.Mol Ther, 2005, 11(4):608-616.
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10 Shockett P, Schatz D. Switching on gene expression. Nature Biotech, 1997, 15:219.
11 Toniatti, Bujard H, Cortese R, et al.Gene therapy progress and prospects: transcription regulatory systems. Gene Ther, 2004, 11(8):649-657.
12 Baron U, Bujard H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol, 2000, 327: 401–421
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14 Miller AD. Cell-surface receptors for retroviruses and implications for gene transfer. Proc.Natl. Acad. Sci. USA,1996, 93(21):11407-11413.
15 Miller AD, Chen F. Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry. J. Virol, 1996, 70(8):5564–5571.
1 Toniatti , Bujard H, Cortese R, et al. Gene therapy progress and prospects: transcription regulatory systems. Gene Ther, 2004, 11(8):649-657.
2 Shockett P, Schatz D. Switching on gene expression. Nature Biotechnology, 1997, 15: 219-221.
3 Gossen M, Freundlieb S, Bender G, et al. Transcriptional activation by tetracyclines in mammalian cells. Science, 1995, 268(5218):1766-1769.
4安珂,田玉科,杨辉等.猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株的构建.中华麻醉学杂志, 2004,24(11): 838-841.
5薛庆善,肖渝平.神经胶质细胞的体外培养方法.解剖科学进展, 1999,5:111-117
6 Fink D, Mata M, Glorioso JC. Cell and gene therapy in the treatment of pain. Adv Drug Deliv Rev, 2003,55(8):1055-1064.
7 Saitoh Y, Eguchi Y, Hagihara Y, et al. Dose-dependent doxycycline-mediated adrenocorticotropic hormone secretion from encapsulated Tet-on proopiomelanocortin Neuro2A cells in the subarachnoid space.Hum Gene Ther. 1998,9(7):997-1002.
8 Parllard F.”Tet-on”: a gene switch for exogenous regulation of transgene expression. Human Gene Ther, 1998, 9:981-985.
9 Baron U, Bujard H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol ,2000, 327: 401–421
10 Paulus W, Baur I, Keyvani K, et al. Variability of transcriptional regulation after gene transfer with the retroviral tetracycline system. J Biotechnol, 2000; 81(2-3):159-165.
11 Nahreini P, Hanson AJ, Prasad KN. Enrichment of cells exhibiting tetracycline regulated gene expression. Biotechniques, 2003,34:958-962.
12 F.奥斯伯等著(美),颜子颖,王海林译.“精编分子生物学实验指南”.北京:科学出版社,1998:290-307.
13 Sambrook J,Russell D.分子克隆试验指南.黄培堂,等,译.第3版.北京:科学出版社,2002.1362-1366.
14 Bohl D, Heard JM. Modulation of erythropoietin delivery from engineered muscles in mice. Hum Gene Ther, 1997, 8:195-204.
1 Duplan H, Li RY, Vue C, et al.Grafts of immortalized chromaffin cells bio-engineered to improve met-enkephalin release also reduce formalin-evoked c-fos expression in rat spinal cord. Neurosci Lett, 2004, 370(1):1-6.
2 An K, Tian Y, Yang H, Gao F, et al. Immortalized rat astrocyte strain genetically modified by rat preprogalanin gene. J Huazhong Univ Sci Technolog Med Sci, 2005, 25(2):144-6, 197.
3安珂,田玉科,杨辉等.猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株的构建.中华麻醉学杂志,2004,24(11): 838-841.
4田学愎,田玉科,徐颖等.四环素调控系统修饰永生化大鼠星形胶质细胞株的构建及鉴定.中华麻醉学杂志, 2006,26(3):265-268.
5 Fink D, Mata M, Glorioso JC. Cell and gene therapy in the treatment of pain. Adv Drug Deliv Rev, 2003,55(8):1055-1064.
6 Wilson SP, Yeomans DC, Bender MA, et al. Antihyperalagesic effects of infection with a preproenkephalin-encoding herpes virus. Pro Natl Acad Sci USA, 1999 ,96:3211-3216.
7 Hao S, Mata M, Goins W, et al. Transgene-mediated enkephalin release enhances the effect of morphine and evades tolerance to produce a sustained antiallodynic effect in neuropathic pain. Pain, 2003, 102(1-2):135-142.
8 Ridet JL, Sarkis C, Serguera C, et al. Transplantation of human adult astrocytes: efficiency and safety requirements for an autologous gene therapy. J Neurosci Res, 2003; 15; 72(6):704-708.
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