發(fā)布時(shí)間：2018-12-27 18:20

【摘要】： 目的在建立人骨髓基質(zhì)細(xì)胞培養(yǎng)體系基礎(chǔ)上,觀察人骨髓基質(zhì)細(xì)胞在裸鼠體內(nèi)的分布情況和對(duì)臟器的影響,為人骨髓基質(zhì)細(xì)胞作為干細(xì)胞移植的前期研究和安全性提供實(shí)驗(yàn)依據(jù)。方法采用密度梯度離心結(jié)合差速貼壁法分離培養(yǎng)人骨髓基質(zhì)細(xì)胞,通過細(xì)胞形態(tài)學(xué)、表型特征對(duì)成人骨髓來(lái)源的基質(zhì)細(xì)胞進(jìn)行鑒定;采用細(xì)胞生長(zhǎng)曲線分析和成骨誘導(dǎo)分化分析其體外增殖特性和分化潛能。而后建立人骨髓基質(zhì)細(xì)胞靜脈移植的裸鼠模型,觀察受體裸鼠的一般情況,通過組織學(xué)分析、RT-PCR檢測(cè)人Alu基因和CD45基因等技術(shù)手段,研究移植的骨髓基質(zhì)細(xì)胞體內(nèi)的分布情況和對(duì)臟器的影響。此外,還進(jìn)行安全性檢測(cè),包括長(zhǎng)期傳代培養(yǎng)的觀察和成瘤性實(shí)驗(yàn)檢測(cè)。結(jié)果體外培養(yǎng)的人骨髓基質(zhì)細(xì)胞為成纖維樣細(xì)胞,細(xì)胞緊密排列呈漩渦狀生長(zhǎng);流式細(xì)胞術(shù)檢測(cè)CD34、CD45為陰性表達(dá);細(xì)胞生長(zhǎng)曲線提示第1至第5代人骨髓基質(zhì)細(xì)胞增殖速度無(wú)顯著性差異(P＞0.05);成骨誘導(dǎo)18天后,茜素紅染色可見橙紅色鈣結(jié)節(jié)。在裸鼠移植模型中,輸注第4代體外培養(yǎng)的人骨髓基質(zhì)細(xì)胞后,受體裸鼠一般狀況良好,解剖大體觀、HE染色、瑞氏染色觀察受體組織結(jié)構(gòu)無(wú)異常。RT-PCR檢測(cè)人Alu基因、CD45基因在不同移植時(shí)間相應(yīng)臟器均有表達(dá)。此外,細(xì)胞長(zhǎng)期傳代后出現(xiàn)衰老征象,而無(wú)明顯的成瘤特性。結(jié)論采用密度梯度離心結(jié)合差速貼壁法分離、DMEM-LG-10%FBS培養(yǎng)液可成功分離培養(yǎng)含有間充質(zhì)干細(xì)胞的人骨髓基質(zhì)細(xì)胞群。經(jīng)靜脈輸注后,在不同移植時(shí)間檢測(cè),發(fā)現(xiàn)受體內(nèi)各臟器均有分布,但進(jìn)一步作安全檢測(cè)發(fā)現(xiàn)移植的骨髓基質(zhì)細(xì)胞尚無(wú)明顯的惡變趨勢(shì)和現(xiàn)象發(fā)生。
[Abstract]:Objective To establish a human bone marrow stromal cell culture system, observe the distribution of human bone marrow stromal cells in nude mice and the effect on the organs, and provide experimental basis for the early research and safety of human bone marrow stromal cells as stem cell transplantation. Methods The human bone marrow stromal cells were isolated and cultured by density gradient centrifugation and differential adhesion method. The matrix cells of adult bone marrow were identified by cell morphology and phenotypic characteristics. The proliferation and differentiation potential of human bone marrow stromal cells were analyzed by cell growth curve and bone-induced differentiation. Then, a nude mouse model of human bone marrow stromal cell vein transplantation was established, and the general situation of the receptor naked mice was observed. By means of histological analysis, RT-PCR, human Alu gene and CD45 gene were used to study the distribution of bone marrow stromal cells and the effect on the organs. In addition, safety detection is carried out, including the observation of long-term subculture and the test of tumorigenicity. Results The human bone marrow stromal cells cultured in vitro were fibroblast-like cells, and the cells were closely arranged in a vortex-like growth. The expression of CD34 and CD45 was detected by flow cytometry. The growth curve of the cells showed no significant difference in the proliferation of bone marrow stromal cells from the 1st to 5th generation (P> 0.05). After 18 days of bone-induced bone induction, the red-red calcium nodules were seen in the red-red staining. In the nude mouse transplantation model, after the fourth generation of the human bone marrow stromal cells cultured in vitro, the receptor naked mice were generally in good condition, the general view of the anatomy, the HE staining, and the Rechsler's staining showed no abnormality in the structure of the receptor. The expression of the human Alu gene and the CD45 gene in the corresponding organs of the human Alu gene was detected by RT-PCR. In addition, the cells showed signs of aging after long-term passage without obvious tumor characteristics. Conclusion The cell population of human bone marrow stromal cells containing mesenchymal stem cells can be isolated and cultured in DMEM-LG-10% FBS culture medium by means of density gradient centrifugation and differential adhesion. After intravenous infusion, the internal organs of the transplanted bone marrow were found to be distributed in different time, but there was no obvious tendency and phenomenon of malignant transformation in the bone marrow stromal cells of the transplanted bone marrow.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王忠;高毅;汪艷;潘明新;;人骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)與鑒定[J];中華神經(jīng)醫(yī)學(xué)雜志;2006年10期

2 馮善偉,姚曉黎,李中,柳太云,黃文,張成;BrdU體外標(biāo)記大鼠骨髓間充質(zhì)干細(xì)胞的研究[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2005年02期

3 李經(jīng)倫,劉長(zhǎng)征;干細(xì)胞移植治療中的示蹤術(shù)研究現(xiàn)狀(文獻(xiàn)綜述)[J];放射免疫學(xué)雜志;2004年06期

4 張麗蓉,項(xiàng)鵬,夏文杰,陳振光,張秀明,李艷,李樹濃;人骨髓間質(zhì)干細(xì)胞分離和培養(yǎng)擴(kuò)增方法的研究[J];廣東藥學(xué)院學(xué)報(bào);2002年02期

5 徐，

本文編號(hào)：2393428