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

【摘要】： 中樞神經(jīng)系統(tǒng)損害的干細(xì)胞干預(yù)是目前國(guó)內(nèi)外廣泛關(guān)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(fù)治療帶來(lái)新的曙光。然而,干細(xì)胞治療的安全性和有效性仍是目前制約其進(jìn)一步發(fā)展的瓶頸。如何在利用干細(xì)胞修復(fù)神經(jīng)損害的同時(shí)避免其迅速衰老、向腫瘤細(xì)胞分化等不良后果,這是目前此類(lèi)研究亟待解決的問(wèn)題。 與神經(jīng)干細(xì)胞(NSCs)相比,骨髓間充質(zhì)干細(xì)胞(BMSCs)因其不受倫理學(xué)方面的限制,來(lái)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(dǎo)條件下可向神經(jīng)細(xì)胞分化,還能分泌多種神經(jīng)營(yíng)養(yǎng)因子,可作為中樞神經(jīng)系統(tǒng)移植治療的種子細(xì)胞。然而一直有研究發(fā)現(xiàn)BMSCs在長(zhǎng)期體外培養(yǎng)中經(jīng)常出現(xiàn)增殖,分化能力喪失,過(guò)早老化等現(xiàn)象,使其生存時(shí)間受限的同時(shí),影響移植細(xì)胞的數(shù)量和質(zhì)量,制約干細(xì)胞移植治療的開(kāi)展。為了克服這個(gè)限制,我們通過(guò)向rBMSCs轉(zhuǎn)染陽(yáng)離子脂質(zhì)體介導(dǎo)的人端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptasehTERT)表達(dá)載體來(lái)增強(qiáng)端粒酶的活性。并因此建立了rBMSCs永生化細(xì)胞系。由于端粒酶的增強(qiáng)表達(dá)經(jīng)常是許多腫瘤的特征,我們通過(guò)米非司酮調(diào)控的pGeneSwitch-V5-His-hTERT系統(tǒng)進(jìn)一步上調(diào)rBMSCs中端粒酶的活性,在體內(nèi)外,對(duì)端粒酶永生化的rBMSCs從形態(tài)學(xué),生物學(xué)特性、成瘤可能性以及神經(jīng)細(xì)胞方向分化等幾個(gè)方面進(jìn)行了研究。進(jìn)一步評(píng)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(yīng)用奠定基礎(chǔ)。 本研究分三部分。第一部分我們使用當(dāng)前較為成熟的原代貼壁培養(yǎng)法獲得rBMSCs,以流式細(xì)胞術(shù)鑒定其表面標(biāo)記抗原予,結(jié)果符合BMSCs特征,證實(shí)rBMSCs培養(yǎng)成功。同時(shí)我們?cè)隗w外對(duì)rBMSCs的成骨、脂肪及神經(jīng)細(xì)胞方向的分化潛能進(jìn)行了初步研究。結(jié)果顯示rBMSCs易于提取、純化和擴(kuò)增,其在體外能自發(fā)表達(dá)神經(jīng)干細(xì)胞相關(guān)蛋白并可通過(guò)誘導(dǎo)向脂肪,成骨及神經(jīng)細(xì)胞分化。提示其具有多向分化潛能的同時(shí)可能具有自發(fā)向神經(jīng)干細(xì)胞分化的特性。 第二部分,在體外建立端粒酶永生化rBMSCs細(xì)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(guò)米非司酮調(diào)控的重組表達(dá)質(zhì)粒pGene/V5-His-hTERT和調(diào)控質(zhì)粒pSwitch。應(yīng)用RT-PCR、Western Blot等技術(shù)檢測(cè)轉(zhuǎn)染結(jié)果及端粒酶活性。使用流式細(xì)胞儀、MTT等方法檢測(cè)實(shí)驗(yàn)組細(xì)胞生物學(xué)特性及生長(zhǎng)情況。通過(guò)血清依賴(lài)實(shí)驗(yàn)、軟瓊脂克隆試驗(yàn)、流式細(xì)胞儀細(xì)胞周期檢測(cè)、Western Blot體外檢測(cè)實(shí)驗(yàn)組細(xì)胞的成瘤傾向。結(jié)果轉(zhuǎn)染組細(xì)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(xì)胞術(shù)鑒定顯示轉(zhuǎn)染組細(xì)胞符合BMSCs特征。MTT比色實(shí)驗(yàn)顯示實(shí)驗(yàn)組細(xì)胞保持了良好的增殖能力。血清依賴(lài)實(shí)驗(yàn)、軟瓊脂克隆試驗(yàn)、細(xì)胞周期檢測(cè)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(xì)胞中的表達(dá)無(wú)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(xì)胞系的安全手段。 第三部分,在體內(nèi)進(jìn)一步對(duì)端粒酶永生化rBMSCs的成瘤可能性及其神經(jīng)細(xì)胞方向自發(fā)分化潛能進(jìn)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(dǎo)pGeneSwtch-V5-His-hTER-rBMSCs組細(xì)胞植入正常及腦損傷SD大鼠項(xiàng)葉皮質(zhì),同時(shí)種植于裸鼠左側(cè)腹股溝皮下組織內(nèi),通過(guò)對(duì)移植區(qū)腦組織HE染色及對(duì)裸鼠的細(xì)胞移植區(qū)皮膚的觀察檢測(cè)其成瘤性。同時(shí)將Hoechst33258標(biāo)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(cè)移植部位神經(jīng)標(biāo)志蛋白表達(dá),計(jì)數(shù)移植存活細(xì)胞并進(jìn)行統(tǒng)計(jì)分析。結(jié)果顯示:大鼠腦內(nèi)移植部位及裸鼠皮下均未見(jiàn)腫瘤生長(zhǎng)。大鼠腦內(nèi)移植區(qū)免疫熒光檢測(cè)可見(jiàn)部分Hoechst 33258標(biāo)記細(xì)胞神經(jīng)標(biāo)志蛋白表達(dá)呈陽(yáng)性。無(wú)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(biāo)記的存活細(xì)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(dá)神經(jīng)細(xì)胞標(biāo)志蛋白,同時(shí)細(xì)胞在植區(qū)的存活數(shù)量明顯高于單純r(jià)BMSCs。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(dá)會(huì)增加移植細(xì)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(yàn)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(duì)BMSCs的增殖起促進(jìn)作用。轉(zhuǎn)染正義hTERT載體可作為建立BMSCs永生化細(xì)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(xì)胞治療的安全而有效的種子細(xì)胞或工具細(xì)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 路曉淼;王恩群;;神經(jīng)生長(zhǎng)因子對(duì)骨髓基質(zhì)細(xì)胞成骨分化影響的研究進(jìn)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(hào)：1619222