發(fā)布時間：2018-03-25 09:40

  本文選題：骨髓間充質干細胞　切入點：分離培養(yǎng)　出處：《山東農業(yè)大學》2008年碩士論文

【摘要】： 近年來的研究發(fā)現(xiàn),骨髓間充質干細胞具有可塑性,在特定的條件下可分化為各胚層來源的組織細胞,稱為橫向分化(transdifferentiation),這一發(fā)現(xiàn)不僅對于細胞生物學理論的發(fā)展有重大意義,對于各種因素導致的組織和器官病變以及遺傳缺陷性疾病的細胞替代治療也具有深遠的影響。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等特點,因此利用骨髓MSCs橫向分化潛能,可以根據需要將其誘導分化為特定的組織細胞,用以修復受損的組織或器官,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應用過程中,它與血管內皮細胞或組織細胞及細胞外基質(extracellular matrix, ECM)的粘附是發(fā)揮修復治療作用的第一步。研究證明,粘附分子在細胞與細胞、細胞與細胞外基質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及分子學基礎就顯得尤為重要。 近年來愈來愈多的研究還顯示了MSCs具有免疫調節(jié)特性,使其在免疫抑制方面具有可預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干細胞對T淋巴細胞免疫功能的影響。 試驗一:大鼠骨髓間充質干細胞的分離培養(yǎng)及黏附分子測定 目的:探討體外分離培養(yǎng)和擴增大鼠間充質干細胞(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,差速貼壁結合消化控制法純化細胞,Ⅰ型膠原作為細胞外基質擴增MSCs;鏡下觀察細胞生物學形態(tài)及生長狀態(tài),流式細胞儀檢測第4代MSCs的細胞周期,免疫細胞化學方法對其進行表型鑒定與細胞黏附分子檢測。 結果:原代培養(yǎng)的MSCs呈圓形、梭形、多角形等,24 h內即可見少量細胞體壁伸展,7～8 d左右可達90%融合,純化擴增后的MSCs呈均勻一致的長梭形,24 h內細胞已基本貼壁,傳代周期為5 d左右。流式細胞儀檢測顯示,第4代MSCs約有80%處于G0/G1期。免疫細胞化學顯示,MSCs CD34、CD45表達陰性,CD29、CD105、CD166、VLA-4、P-selectin表達陽性。 結論:本實驗方法能很容易地獲得大量高純度的MSCs,并表達一定的黏附分子,為MSCs進一步臨床應用打下了良好的基礎。 試驗二:骨髓間充質干細胞對T淋巴細胞免疫功能的影響 目的:探討大鼠骨髓間充質干細胞對T淋巴細胞免疫功能的影響 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淋巴細胞; MTT法測定MSCs對T淋巴細胞增殖的影響。 結果: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胞其CD3陽性細胞高表達。MSCs各組及其上清與T淋巴細胞共培養(yǎng),其增殖百分率呈明顯下降趨勢。 結論:實驗能獲得性能可靠的MSCs及T淋巴細胞;MSCs及其上清對T淋巴細胞增殖表現(xiàn)為抑制效應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.

【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

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本文編號：1662563