本文選題：骨髓間充質(zhì)干細(xì)胞　切入點：分離培養(yǎng)　出處：《山東農(nóng)業(yè)大學(xué)》2008年碩士論文

【摘要】： 近年來的研究發(fā)現(xiàn),骨髓間充質(zhì)干細(xì)胞具有可塑性,在特定的條件下可分化為各胚層來源的組織細(xì)胞,稱為橫向分化(transdifferentiation),這一發(fā)現(xiàn)不僅對于細(xì)胞生物學(xué)理論的發(fā)展有重大意義,對于各種因素導(dǎo)致的組織和器官病變以及遺傳缺陷性疾病的細(xì)胞替代治療也具有深遠(yuǎn)的影響。由于骨髓MSCs易于分離培養(yǎng)和體外擴增,可自體移植無免疫排斥反應(yīng)等特點,因此利用骨髓MSCs橫向分化潛能,可以根據(jù)需要將其誘導(dǎo)分化為特定的組織細(xì)胞,用以修復(fù)受損的組織或器官,這也將為免疫功能紊亂的治療提供了另一條途徑。 在骨髓MSCs應(yīng)用過程中,它與血管內(nèi)皮細(xì)胞或組織細(xì)胞及細(xì)胞外基質(zhì)(extracellular matrix, ECM)的粘附是發(fā)揮修復(fù)治療作用的第一步。研究證明,粘附分子在細(xì)胞與細(xì)胞、細(xì)胞與細(xì)胞外基質(zhì)間粘附過程中起著重要的作用。因此,探討骨髓MSCs的黏附分子的生物學(xué)及分子學(xué)基礎(chǔ)就顯得尤為重要。 近年來愈來愈多的研究還顯示了MSCs具有免疫調(diào)節(jié)特性,使其在免疫抑制方面具有可預(yù)期的理論意義和使用價值。 就此,本課題第一部分系統(tǒng)研究了骨髓MSCs的分離培養(yǎng)、體外擴增及黏附分子測定,第二部分重點研究了骨髓間充質(zhì)干細(xì)胞對T淋巴細(xì)胞免疫功能的影響。 試驗一:大鼠骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及黏附分子測定 目的:探討體外分離培養(yǎng)和擴增大鼠間充質(zhì)干細(xì)胞(MSCs)的最佳條件,并對其進行表型鑒定及黏附分子測定。 方法:采用全骨髓直接貼壁法獲得原代大鼠骨髓MSCs,差速貼壁結(jié)合消化控制法純化細(xì)胞,Ⅰ型膠原作為細(xì)胞外基質(zhì)擴增MSCs;鏡下觀察細(xì)胞生物學(xué)形態(tài)及生長狀態(tài),流式細(xì)胞儀檢測第4代MSCs的細(xì)胞周期,免疫細(xì)胞化學(xué)方法對其進行表型鑒定與細(xì)胞黏附分子檢測。 結(jié)果:原代培養(yǎng)的MSCs呈圓形、梭形、多角形等,24 h內(nèi)即可見少量細(xì)胞體壁伸展,7～8 d左右可達(dá)90%融合,純化擴增后的MSCs呈均勻一致的長梭形,24 h內(nèi)細(xì)胞已基本貼壁,傳代周期為5 d左右。流式細(xì)胞儀檢測顯示,第4代MSCs約有80%處于G0/G1期。免疫細(xì)胞化學(xué)顯示,MSCs CD34、CD45表達(dá)陰性,CD29、CD105、CD166、VLA-4、P-selectin表達(dá)陽性。 結(jié)論:本實驗方法能很容易地獲得大量高純度的MSCs,并表達(dá)一定的黏附分子,為MSCs進一步臨床應(yīng)用打下了良好的基礎(chǔ)。 試驗二:骨髓間充質(zhì)干細(xì)胞對T淋巴細(xì)胞免疫功能的影響 目的:探討大鼠骨髓間充質(zhì)干細(xì)胞對T淋巴細(xì)胞免疫功能的影響 方法:采用試驗一的方法分離獲得MSCs,尼龍棉柱法從外周血分選出T淋巴細(xì)胞; MTT法測定MSCs對T淋巴細(xì)胞增殖的影響。 結(jié)果:MSCs形態(tài)均勻一致,生長曲線顯示增殖能力強;尼龍棉柱法分離的單個核細(xì)胞其CD3陽性細(xì)胞高表達(dá)。MSCs各組及其上清與T淋巴細(xì)胞共培養(yǎng),其增殖百分率呈明顯下降趨勢。 結(jié)論:實驗?zāi)塬@得性能可靠的MSCs及T淋巴細(xì)胞;MSCs及其上清對T淋巴細(xì)胞增殖表現(xiàn)為抑制效應(yīng)并呈劑量及濃度依賴性。
[Abstract]:Recent studies have found that bone marrow mesenchymal stem cells have plasticity, differentiation of the endoderm derived tissue cells in specific condition, called transdifferentiation (transdifferentiation), this discovery not only has great significance for the development of the theory of cell biology, cell replacement therapy for a variety of factors that lead to tissue and organ disease and genetic defect disease also has far-reaching implications. Because the bone marrow MSCs easy to be isolated and amplified in vitro, autologous transplantation without immune rejection and other characteristics, so the use of bone marrow MSCs horizontal differentiation potential can be induced to differentiate into specific tissue cells to repair damaged tissues or organs, the the treatment of immune dysfunction provides another way.
In the bone marrow in the process of MSCs application, its cells and vascular endothelial cells or tissue and extracellular matrix (extracellular matrix, ECM) adhesion is the first step to play a role in the treatment of repair. Studies have shown that adhesion molecules in cells and cells, cells and extracellular matrix adhesion plays an important role in the process. Therefore, it is very important to investigate the biological and molecular basis of adhesion molecule MSCs in bone marrow.
In recent years, more and more studies have shown that MSCs has the characteristics of immunomodulatory, which has the expected theoretical significance and the use value in the area of immunosuppression.
In this regard, the first part of the project studied the isolation and culture of bone marrow MSCs, expansion and adhesion molecules in vitro. The second part focused on the effect of bone marrow mesenchymal stem cells on T lymphocyte immune function.
Experiment 1: isolation and culture of rat bone marrow mesenchymal stem cells and determination of adhesion molecules
Objective: To explore the best conditions for the isolation, culture and amplification of rat mesenchymal stem cells (MSCs) in vitro, and to identify the phenotypic and adhesion molecules of the rat mesenchymal stem cells.
Methods: using the whole bone marrow adherent primary MSCs rat bone marrow, differential adhesion purified by digestion control cells, collagen as extracellular matrix MSCs amplification; observation of cell biology, morphology and growth status under microscope, detection of the fourth generation of MSCs cell cycle by flow cytometry, immunohistochemistry methods detect the phenotype and cell adhesion molecules on it.
Results: the cultured MSCs were round, fusiform, polygon, 24 h within the small amount of cell wall extension, about 7~8 d up to 90% MSCs after amplification and purification of fusion were uniform long spindle, 24 h cells have basic adherent passage cycle was about 5 d. The results of flow cytometry detection, the fourth generation of MSCs is about 80% in the G0/G1 phase. The expression of MSCs, CD34, CD29, CD45 negative expression of CD105, CD166, VLA-4, P-selectin positive expression.
Conclusion: this method can easily get a lot of high purity MSCs and express some adhesion molecules, which lays a good foundation for further clinical application of MSCs.
Test two: the effect of bone marrow mesenchymal stem cells on the immune function of T lymphocyte
Objective: To investigate the effect of bone marrow mesenchymal stem cells (MSCs) on the immune function of T lymphocytes in rats
Methods: MSCs was obtained by the method of experiment one. T lymphocytes were separated from peripheral blood by nylon cotton column method, and the effect of MSCs on the proliferation of T lymphocytes was measured by MTT.
Results: the morphology of MSCs was uniform and the growth curve showed a strong proliferation ability. The CD3 positive cells isolated from nylon monocytic cells were highly expressed.MSCs, and the proliferation percentage of each group and its supernatants and T lymphocytes co cultured showed a significant downward trend.
Conclusion: the reliable performance of MSCs and T lymphocytes can be obtained, and MSCs and its supernatant can inhibit the proliferation of T lymphocytes in a dose and concentration dependent manner.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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