本文選題：骨髓間充質(zhì)干細胞　切入點：端粒酶逆轉(zhuǎn)錄酶催化亞單位　出處：《天津醫(yī)科大學》2008年博士論文　論文類型：學位論文

【摘要】： 中樞神經(jīng)系統(tǒng)損害的干細胞干預是目前國內(nèi)外廣泛關注的創(chuàng)傷治療新策略,它極有可能為顱腦創(chuàng)傷后的神經(jīng)修復治療帶來新的曙光。然而,干細胞治療的安全性和有效性仍是目前制約其進一步發(fā)展的瓶頸。如何在利用干細胞修復神經(jīng)損害的同時避免其迅速衰老、向腫瘤細胞分化等不良后果,這是目前此類研究亟待解決的問題。 與神經(jīng)干細胞(NSCs)相比,骨髓間充質(zhì)干細胞(BMSCs)因其不受倫理學方面的限制,來源方便,近幾年更多地被研究人員采納。研究顯示,BMSCs具有自我更新能力和多向分化潛能。BMSCs在體內(nèi)或體外誘導條件下可向神經(jīng)細胞分化,還能分泌多種神經(jīng)營養(yǎng)因子,可作為中樞神經(jīng)系統(tǒng)移植治療的種子細胞。然而一直有研究發(fā)現(xiàn)BMSCs在長期體外培養(yǎng)中經(jīng)常出現(xiàn)增殖,分化能力喪失,過早老化等現(xiàn)象,使其生存時間受限的同時,影響移植細胞的數(shù)量和質(zhì)量,制約干細胞移植治療的開展。為了克服這個限制,我們通過向rBMSCs轉(zhuǎn)染陽離子脂質(zhì)體介導的人端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptasehTERT)表達載體來增強端粒酶的活性。并因此建立了rBMSCs永生化細胞系。由于端粒酶的增強表達經(jīng)常是許多腫瘤的特征,我們通過米非司酮調(diào)控的pGeneSwitch-V5-His-hTERT系統(tǒng)進一步上調(diào)rBMSCs中端粒酶的活性,在體內(nèi)外,對端粒酶永生化的rBMSCs從形態(tài)學,生物學特性、成瘤可能性以及神經(jīng)細胞方向分化等幾個方面進行了研究。進一步評估端粒酶永生化BMSCs的安全性及可靠性,為端粒酶永生化BMSCs的臨床應用奠定基礎。 本研究分三部分。第一部分我們使用當前較為成熟的原代貼壁培養(yǎng)法獲得rBMSCs,以流式細胞術鑒定其表面標記抗原予,結(jié)果符合BMSCs特征,證實rBMSCs培養(yǎng)成功。同時我們在體外對rBMSCs的成骨、脂肪及神經(jīng)細胞方向的分化潛能進行了初步研究。結(jié)果顯示rBMSCs易于提取、純化和擴增,其在體外能自發(fā)表達神經(jīng)干細胞相關蛋白并可通過誘導向脂肪,成骨及神經(jīng)細胞分化。提示其具有多向分化潛能的同時可能具有自發(fā)向神經(jīng)干細胞分化的特性。 第二部分,在體外建立端粒酶永生化rBMSCs細胞系,并研究端粒酶永生化rBMSCs成瘤的可能性。我們分別向第五代rBMSCs轉(zhuǎn)染pLXSN-S-hTERT以及共轉(zhuǎn)染通過米非司酮調(diào)控的重組表達質(zhì)粒pGene/V5-His-hTERT和調(diào)控質(zhì)粒pSwitch。應用RT-PCR、Western Blot等技術檢測轉(zhuǎn)染結(jié)果及端粒酶活性。使用流式細胞儀、MTT等方法檢測實驗組細胞生物學特性及生長情況。通過血清依賴實驗、軟瓊脂克隆試驗、流式細胞儀細胞周期檢測、Western Blot體外檢測實驗組細胞的成瘤傾向。結(jié)果轉(zhuǎn)染組細胞端粒酶活性明顯增高,并表現(xiàn)出良好的永生化特征。流式細胞術鑒定顯示轉(zhuǎn)染組細胞符合BMSCs特征。MTT比色實驗顯示實驗組細胞保持了良好的增殖能力。血清依賴實驗、軟瓊脂克隆試驗、細胞周期檢測結(jié)果均未發(fā)現(xiàn)成瘤傾向。Western Blot分析發(fā)現(xiàn)c-Myc在各組細胞中的表達無明顯差異。結(jié)果提示端粒酶永生化rBMSCs在體外尚不具備成瘤性,轉(zhuǎn)染正義hTERT載體可作為建立BMSCs永生化細胞系的安全手段。 第三部分,在體內(nèi)進一步對端粒酶永生化rBMSCs的成瘤可能性及其神經(jīng)細胞方向自發(fā)分化潛能進行了研究。我們先成功的制備了大鼠中度液壓沖擊腦損傷模型,然后我們分別將rBMSCs、pLXSN-hTERT-rBMSCs、米非司酮持續(xù)誘導pGeneSwtch-V5-His-hTER-rBMSCs組細胞植入正常及腦損傷SD大鼠項葉皮質(zhì),同時種植于裸鼠左側(cè)腹股溝皮下組織內(nèi),通過對移植區(qū)腦組織HE染色及對裸鼠的細胞移植區(qū)皮膚的觀察檢測其成瘤性。同時將Hoechst33258標記的rBMSCs和永生化pLXSN-hTERT-rBMSCs植入正常及腦損傷SD大鼠頂葉皮質(zhì),用免疫熒光的方法檢測移植部位神經(jīng)標志蛋白表達,計數(shù)移植存活細胞并進行統(tǒng)計分析。結(jié)果顯示:大鼠腦內(nèi)移植部位及裸鼠皮下均未見腫瘤生長。大鼠腦內(nèi)移植區(qū)免疫熒光檢測可見部分Hoechst 33258標記細胞神經(jīng)標志蛋白表達呈陽性。無論在正常還是腦損大鼠的頂葉移植區(qū)內(nèi),端粒酶永生化rBMSCs Hoechst33258標記的存活細胞數(shù)明顯高于單純BMSCs(F=57.888,P＜0.05)。結(jié)果提示,端粒酶永生化大鼠BMSCs移植到正常及腦損傷大鼠腦內(nèi)后存活良好,并可表達神經(jīng)細胞標志蛋白,同時細胞在植區(qū)的存活數(shù)量明顯高于單純rBMSCs。即rBMSCs的分化方向受體內(nèi)環(huán)境的影響而且外源性hTERT表達會增加移植細胞的存活率。端粒酶永生化rBMSCs在體內(nèi)不具有成瘤傾向。 上述試驗結(jié)果提示:端粒酶永生化rBMSCs不具備成瘤傾向,提高端粒酶活性對BMSCs的增殖起促進作用。轉(zhuǎn)染正義hTERT載體可作為建立BMSCs永生化細胞系的安全手段。端粒酶永生化的BMSCs有希望成為創(chuàng)傷腦組織干細胞治療的安全而有效的種子細胞或工具細胞。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R329

【共引文獻】

相關期刊論文 前10條

1 路曉淼;王恩群;;神經(jīng)生長因子對骨髓基質(zhì)細胞成骨分化影響的研究進展[J];安徽醫(yī)藥;2009年02期

2 張文鵬;葉發(fā)剛;y囇澡，

本文編號：1619222