端粒酶永生化大鼠骨髓间充质干细胞的体内外研究

发布时间：2018-03-16 09:02

本文选题：骨髓间充质干细胞　切入点：端粒酶逆转录酶催化亚单位　出处：《天津医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 中枢神经系统损害的干细胞干预是目前国内外广泛关注的创伤治疗新策略,它极有可能为颅脑创伤后的神经修复治疗带来新的曙光。然而,干细胞治疗的安全性和有效性仍是目前制约其进一步发展的瓶颈。如何在利用干细胞修复神经损害的同时避免其迅速衰老、向肿瘤细胞分化等不良后果,这是目前此类研究亟待解决的问题。与神经干细胞(NSCs)相比,骨髓间充质干细胞(BMSCs)因其不受伦理学方面的限制,来源方便,近几年更多地被研究人员采纳。研究显示,BMSCs具有自我更新能力和多向分化潜能。BMSCs在体内或体外诱导条件下可向神经细胞分化,还能分泌多种神经营养因子,可作为中枢神经系统移植治疗的种子细胞。然而一直有研究发现BMSCs在长期体外培养中经常出现增殖,分化能力丧失,过早老化等现象,使其生存时间受限的同时,影响移植细胞的数量和质量,制约干细胞移植治疗的开展。为了克服这个限制,我们通过向rBMSCs转染阳离子脂质体介导的人端粒酶逆转录酶(human telomerase reverse transcriptasehTERT)表达载体来增强端粒酶的活性。并因此建立了rBMSCs永生化细胞系。由于端粒酶的增强表达经常是许多肿瘤的特征,我们通过米非司酮调控的pGeneSwitch-V5-His-hTERT系统进一步上调rBMSCs中端粒酶的活性,在体内外,对端粒酶永生化的rBMSCs从形态学,生物学特性、成瘤可能性以及神经细胞方向分化等几个方面进行了研究。进一步评估端粒酶永生化BMSCs的安全性及可靠性,为端粒酶永生化BMSCs的临床应用奠定基础。本研究分三部分。第一部分我们使用当前较为成熟的原代贴壁培养法获得rBMSCs,以流式细胞术鉴定其表面标记抗原予,结果符合BMSCs特征,证实rBMSCs培养成功。同时我们在体外对rBMSCs的成骨、脂肪及神经细胞方向的分化潜能进行了初步研究。结果显示rBMSCs易于提取、纯化和扩增,其在体外能自发表达神经干细胞相关蛋白并可通过诱导向脂肪,成骨及神经细胞分化。提示其具有多向分化潜能的同时可能具有自发向神经干细胞分化的特性。第二部分,在体外建立端粒酶永生化rBMSCs细胞系,并研究端粒酶永生化rBMSCs成瘤的可能性。我们分别向第五代rBMSCs转染pLXSN-S-hTERT以及共转染通过米非司酮调控的重组表达质粒pGene/V5-His-hTERT和调控质粒pSwitch。应用RT-PCR、Western Blot等技术检测转染结果及端粒酶活性。使用流式细胞仪、MTT等方法检测实验组细胞生物学特性及生长情况。通过血清依赖实验、软琼脂克隆试验、流式细胞仪细胞周期检测、Western Blot体外检测实验组细胞的成瘤倾向。结果转染组细胞端粒酶活性明显增高,并表现出良好的永生化特征。流式细胞术鉴定显示转染组细胞符合BMSCs特征。MTT比色实验显示实验组细胞保持了良好的增殖能力。血清依赖实验、软琼脂克隆试验、细胞周期检测结果均未发现成瘤倾向。Western Blot分析发现c-Myc在各组细胞中的表达无明显差异。结果提示端粒酶永生化rBMSCs在体外尚不具备成瘤性,转染正义hTERT载体可作为建立BMSCs永生化细胞系的安全手段。第三部分,在体内进一步对端粒酶永生化rBMSCs的成瘤可能性及其神经细胞方向自发分化潜能进行了研究。我们先成功的制备了大鼠中度液压冲击脑损伤模型,然后我们分别将rBMSCs、pLXSN-hTERT-rBMSCs、米非司酮持续诱导pGeneSwtch-V5-His-hTER-rBMSCs组细胞植入正常及脑损伤SD大鼠项叶皮质,同时种植于裸鼠左侧腹股沟皮下组织内,通过对移植区脑组织HE染色及对裸鼠的细胞移植区皮肤的观察检测其成瘤性。同时将Hoechst33258标记的rBMSCs和永生化pLXSN-hTERT-rBMSCs植入正常及脑损伤SD大鼠顶叶皮质,用免疫荧光的方法检测移植部位神经标志蛋白表达,计数移植存活细胞并进行统计分析。结果显示:大鼠脑内移植部位及裸鼠皮下均未见肿瘤生长。大鼠脑内移植区免疫荧光检测可见部分Hoechst 33258标记细胞神经标志蛋白表达呈阳性。无论在正常还是脑损大鼠的顶叶移植区内,端粒酶永生化rBMSCs Hoechst33258标记的存活细胞数明显高于单纯BMSCs(F=57.888,P＜0.05)。结果提示,端粒酶永生化大鼠BMSCs移植到正常及脑损伤大鼠脑内后存活良好,并可表达神经细胞标志蛋白,同时细胞在植区的存活数量明显高于单纯rBMSCs。即rBMSCs的分化方向受体内环境的影响而且外源性hTERT表达会增加移植细胞的存活率。端粒酶永生化rBMSCs在体内不具有成瘤倾向。上述试验结果提示:端粒酶永生化rBMSCs不具备成瘤倾向,提高端粒酶活性对BMSCs的增殖起促进作用。转染正义hTERT载体可作为建立BMSCs永生化细胞系的安全手段。端粒酶永生化的BMSCs有希望成为创伤脑组织干细胞治疗的安全而有效的种子细胞或工具细胞。
[Abstract]:Stem cells on central nervous system damage is widespread concern at home and abroad trauma treatment new strategy, it is likely to bring a new dawn for nerve repair after traumatic brain injury treatment. However, stem cell therapy is safe and effective is still the bottleneck of its further development. At the same time how to use stem cells repair of nerve damage to avoid the rapid aging, tumor cell differentiation and other adverse consequences, this is the research problem to be solved.
With neural stem cells (NSCs) in bone marrow mesenchymal stem cells (BMSCs) because they are not subject to the ethical limit, convenient source, in recent years, more and more researchers are adopted. Research shows that BMSCs has the ability to self renew and multilineage differentiation potential induced by.BMSCs in vivo or in vitro conditions to nerve cells differentiation can secrete various neurotrophic factors, can be used as seed cells for central nervous system transplantation. However studies have found that often appear in the long-term proliferation of BMSCs in vitro, lost the ability to differentiate, premature aging phenomenon, the survival time is limited at the same time, affects the quality and quantity of transplanted cells, stem cells to carry out control the transplantation. In order to overcome this limitation, we by human telomerase reverse transcriptase to rBMSCs transfection mediated by cationic liposome (human telomerase reverse transcriptasehTERT) expression The carrier to enhance the activity of telomerase. Thus established immortalized cell line rBMSCs. The telomerase expression is often a feature of many tumors, we further up-regulated rBMSCs pGeneSwitch-V5-His-hTERT telomerase activity in the regulation system of mifepristone, in vivo, on telomerase immortalized rBMSCs from morphology, biological characteristics, and the possibility of tumor several neural cell differentiation and other aspects of the study. Further evaluation of telomerase immortalized BMSCs safety and reliability, and lay the foundation for the clinical application of telomerase immortalized BMSCs.
This study is divided into three parts. The first part we use the mature primary rBMSCs adherent culture method, flow cytometry was used to identify the surface marker antigen to results in line with the characteristics of BMSCs, rBMSCs. At the same time we confirmed the successful culture in vitro osteogenic differentiation potential of rBMSCs, fat and nerve cells in the direction of a preliminary study. The results show that rBMSCs is easy to extract, purification and amplification, the spontaneous expression of neural stem cell related protein and can be induced to adipose in vitro, osteoblasts and neural cell differentiation. At the same time, suggesting that the multilineage differentiation potential may be spontaneously differentiated into neural stem cells.
The second part, the establishment of telomerase immortalized rBMSCs cell lines in vitro, and to study the telomerase immortalized rBMSCs into tumor possibility. We respectively to the fifth generation of rBMSCs transfected pLXSN-S-hTERT and co transfected with expression plasmid pGene/V5-His-hTERT and control plasmid pSwitch. RT-PCR by application of mifepristone regulatory reorganization, the activity of Western Blot technique to detect the transfection results and flow cytometry using telomerase. Cells were detected in experimental group, MTT cell biological characteristics and growth. By serum dependence experiment, soft agar test, flow cytometry to detect the cell cycle of Western, Blot in vitro experimental group cell tumorigenicity. Results the transfection of telomerase activity increased significantly, and showed good characteristics of immortalization. Flow cytometry analysis showed that transfected cells with the characteristics of BMSCs.MTT assay showed that the experimental group cells maintain Good proliferation ability. The serum dependence experiment, soft agar test, cell cycle test results were not found in tumor prone.Western Blot analysis found no significant difference in the expression of c-Myc in cells of each group. The results suggest that telomerase immortalized rBMSCs in vitro does not have tumorigenicity, transfected with hTERT vector can be used as a safe means of establishing BMSCs immortalized cell lines.
The third part, further in vivo tumor cells into the possibility and direction of nerve telomerase immortalized rBMSCs spontaneous differentiation were studied. The rat moderate fluid percussion brain injury model was prepared successfully for us first, then we will be rBMSCs, pLXSN-hTERT-rBMSCs, pGeneSwtch-V5-His-hTER-rBMSCs group of mifepristone induced by continuous cell implantation and normal brain injury in SD rats a leaf cortex, and left inguinal subcutaneous tissue implanted in nude mice, through the observation of skin detection brain tissue HE staining and cell transplantation on nude mice transplanted area their tumorigenicity. The Hoechst33258 labeled rBMSCs and immortalized pLXSN-hTERT-rBMSCs implantation in normal and brain injury SD rat parietal cortex, we detected the nerve transplantation site marker protein expression, cell count and graft survival were analyzed. The results showed that: Rat Intracerebral transplantation site and no tumor growth in nude mice by subcutaneous transplantation in the rat brain region. Immunofluorescence staining showed that Hoechst 33258 cells labeled neural marker protein expression was positive. In both normal and brain damaged rats transplanted parietal region, the number of survival Cells Telomerase immortalized rBMSCs Hoechst33258 marker was obviously higher than that of pure BMSCs (F=57.888, P < 0.05). The results suggest that telomerase immortalized rat BMSCs transplanted into normal and injured rat brain after survived well, and the expression of neural cell marker protein, while the number of survival cells in the planting area was significantly higher than that of pure rBMSCs. differentiation of rBMSCs is affected by the internal environment and exogenous hTERT expression will increase the survival rate of transplanted cells. Telomerase immortalized rBMSCs have no tumorigenicity in vivo.
The test results suggest that telomerase immortalized rBMSCs have no tumorigenicity, increase telomerase activity on the proliferation of BMSCs and promote the carrier transfected with hTERT can be used as a safe means of establishing immortalized cell line BMSCs. Telomerase immortalized BMSCs may be traumatic brain stem cell therapy is safe and effective to seed cells or tool cells.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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