本文選題：骨髓　切入點：間充質(zhì)干細(xì)胞　出處：《山西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：第一章小鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)的體外分離擴(kuò)增及鑒定 目的建立一種簡單易行的小鼠骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cellsBMscs)分離培養(yǎng)方法，并對其表面標(biāo)志進(jìn)行鑒定。方法通過全骨髓貼壁分離法體外分離、擴(kuò)增小鼠BMSCs。觀察原代及傳代小鼠BMSCs形態(tài)變化，對第3代小鼠BMSCs表面抗原CD34、CD45、CDl05和CDl06進(jìn)行流式細(xì)胞儀檢測，并對小鼠BMscs進(jìn)行純度測定。 結(jié)果用此法進(jìn)行小鼠BMscs的原代接種培養(yǎng)24 h后可見大量懸浮細(xì)胞，少許貼壁細(xì)胞，呈小圓形；72h后貼壁細(xì)胞逐漸增多，培養(yǎng)至第7d細(xì)胞伸展成長梭形，呈集落生長。經(jīng)反復(fù)換液后未貼壁的造血細(xì)胞被棄去。BMscs在培養(yǎng)的2～6d細(xì)胞增殖較慢，7～10d細(xì)胞增殖較快。細(xì)胞培養(yǎng)約10—12d左右，可接近75％～80％融合。傳代后的小鼠BMscs 24b基本全部貼壁，細(xì)胞形態(tài)較為均一，呈均勻性分布生長。分離純化后所得細(xì)胞活力為92.6±1.8％。流式細(xì)胞術(shù)結(jié)果顯示第3代小鼠BMscs細(xì)胞均一性較好，在90%以上。CD34、CD45表達(dá)陰性，cDl05、cDl06表達(dá)陽性。 結(jié)論采用全骨髓貼壁分離法可獲得：BMscs，這種方法簡單、便捷、實用。所得小鼠BMscs活力及純度均較高，流式細(xì)胞技術(shù)可以鑒定體外分離培養(yǎng)的小鼠BMscs。 第二章TGF-β1表達(dá)載體的構(gòu)建及其在小鼠骨髓間充質(zhì)干細(xì)胞中的表達(dá) 目的將transforming growth factor beta 1(TGF-β1)序列構(gòu)建到帶GFP的表達(dá)載體pcDHl-Mcsl-EFl-copGFP中，并將重組質(zhì)粒轉(zhuǎn)染入BMscs，使其在BMsc8中表達(dá)。 方法以小鼠肺組織cDNA為模板，PcR擴(kuò)增出TGF-β1基因，并將其插入pcDHl-Mcsl-EFl-copGFP載體質(zhì)粒中，轉(zhuǎn)化至感受態(tài)菌DH5a，抽提質(zhì)粒，經(jīng)PcR擴(kuò)增和測序鑒定后轉(zhuǎn)染入BMscs細(xì)胞，利用激光共聚焦顯微鏡和Real-time PCR方法對其表達(dá)位置和表達(dá)量進(jìn)行檢測。 結(jié)果經(jīng)PcR及測序鑒定，構(gòu)建入載體質(zhì)粒的基因為TGF-β1基因，pcDHl-TGFβ1-EFl-copGFP重組質(zhì)粒成功構(gòu)建，，且它能在BMSCs細(xì)胞中成功表達(dá)。 結(jié)論TGF-β1基因在BMSCs細(xì)胞成功表達(dá)，為進(jìn)一步研究TGFβ1影響B(tài)MSCs細(xì)胞的生理功能奠定了實驗和理論基礎(chǔ)。
[Abstract]:In vitro isolation, amplification and identification of mouse bone marrow mesenchymal stem cells (BMSCs)Objective to establish a simple method for isolation and culture of marrow mesenchymal stem cells BMscs from mouse bone marrow mesenchymal stem cells, and to identify its surface markers.Methods BMSCs were isolated by whole bone marrow adherent method in vitro.The morphological changes of BMSCs in primary and subculture mice were observed. The surface antigens CD34, CD45, CDl05 and CDl06 were detected by flow cytometry, and the purity of BMscs in mice was determined by flow cytometry.Results after 24 hours of primary inoculation and culture of mouse BMscs, a large number of suspension cells were observed, a few adherent cells were observed, and the adherent cells increased gradually after 72 hours of small circle. After 7 days of culture, the cells expanded and spindle-shaped, and showed colony growth.After repeated fluid exchange, the unadherent hematopoietic cells were abandoned. BMscs proliferated more slowly on the 6th day of culture than on the 7th day after 10 days.Cell culture was about 10-12 days, close to 75% fusion.After passage, the BMscs 24b of mice was almost adherent to the wall, and the cell morphology was uniform, and the cells grew homogeneously.The cell viability after purification was 92.6 鹵1.8.Conclusion the whole bone marrow adherent separation method can be used to obtain bone marrow BMscs. This method is simple, convenient and practical.The BMscs activity and purity of the obtained mice were high. Flow cytometry could be used to identify the mouse BMSCs isolated and cultured in vitro.Construction of TGF- 尾 1 expression Vector and its expression in Mouse Bone Marrow Mesenchymal Stem cellsObjective to construct the transforming growth factor beta 1hTGF- 尾 1) sequence into the expression vector pcDHl-Mcsl-EFl-copGFP with GFP, and to transfect the recombinant plasmid into BMscs and make it express in BMsc8.Methods the TGF- 尾 1 gene was amplified from mouse lung tissue cDNA and inserted into the plasmid of pcDHl-Mcsl-EFl-copGFP vector. The plasmid was extracted from the plasmid DH 5a. The plasmid was transfected into BMscs cells by PcR amplification and sequencing.Laser confocal microscopy and Real-time PCR were used to detect its expression position and quantity.Results the recombinant plasmid of TGF- 尾 1 gene, pcDHl-TGF 尾 1-EFl-copGFP, was successfully constructed by PcR and sequencing, and it was successfully expressed in BMSCs cells.Conclusion TGF- 尾 1 gene was successfully expressed in BMSCs cells, which laid an experimental and theoretical foundation for further study on the effect of TGF 尾 1 on the physiological function of BMSCs cells.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 樊建玲;來曉瑜;黃黎;王艷;曹軍麗;黃河;;人骨髓間充質(zhì)干細(xì)胞腦內(nèi)移植對大鼠缺氧缺血性腦損傷的保護(hù)作用[J];第二軍醫(yī)大學(xué)學(xué)報;2008年09期

2 劉凡鳳;邱慧穎;解琳娜;章衛(wèi)平;鄭曉麗;高磊;王健民;;骨髓間充質(zhì)干細(xì)胞移植重建極重度放射損傷小鼠造血功能[J];第二軍醫(yī)大學(xué)學(xué)報;2008年09期

3 曹明媚;基因治療載體的研究進(jìn)展[J];國外醫(yī)學(xué)(腫瘤學(xué)分冊);2004年01期

4 趙峰;李圣青;張宇飛;陳衛(wèi)強(qiáng);侯志峰;吳昌歸;戚好文;;骨髓間充質(zhì)干細(xì)胞在肺損傷大鼠肺組織的分化[J];解放軍醫(yī)學(xué)雜志;2007年02期

5 常立文;李文斌;;關(guān)注早產(chǎn)兒支氣管肺發(fā)育不良[J];中國新生兒科雜志;2011年01期

6 陳瑩,董靜,陳杰;γ-干擾素對矽肺大鼠肺臟白介素-4和轉(zhuǎn)化生長因子-β_1蛋白表達(dá)的影響[J];中華勞動衛(wèi)生職業(yè)病雜志;2004年05期

7 胡永斌;宗豫蓉;馮德云;金中元;蔣海鷹;彭勁武;;p38/ERK激酶調(diào)控TGF-β_1誘導(dǎo)的HLF-02細(xì)胞Ⅰ型膠原表達(dá)及MMP-2活力[J];中華勞動衛(wèi)生職業(yè)病雜志;2006年02期

8 王彤;黃子通;;骨髓間充質(zhì)干細(xì)胞和心血管疾病[J];中國急救醫(yī)學(xué);2008年07期

9 ;Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma[J];World Journal of Gastroenterology;2008年29期

本文編號：1691514