本文選題：骨髓間充質干細胞　切入點：神經元樣細胞　出處：《南華大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的：通過體外培養(yǎng)骨髓間充質干細胞（BMSCs），初步探討不同劑量的甲鈷胺體外定向誘導BMSCs向神經元樣細胞分化的可行性，以及觀察分化后的細胞的生長和增殖情況，尋求一種較合理的甲鈷胺誘導濃度，為臨床上利用甲鈷胺誘導后的BMSCs進行細胞移植更好更有效的治療脊髓損傷提供前期研究依據(jù)。 方法：1、采用密度梯度離心和貼壁培養(yǎng)法分離、培養(yǎng)和純化大鼠骨髓間充質干細胞，倒置顯微鏡下連續(xù)觀察細胞生長情況、生物活性及形態(tài)學變化，免疫熒光細胞化學檢測BMSCs相對特異性表面標志物CD44，證實實驗所需的細胞為BMSCs。 2、取生長狀態(tài)良好的第4-5代BMSCs，制成單細胞懸液，調整細胞濃度，實驗分為對照組（control組）和實驗組（分25ug/ml、50ug/ml和100ug/ml三個濃度組），實驗組分別加入不同濃度甲鈷胺的10%FBS L-DMEM培養(yǎng)基，對照組加入10%FBS L-DMEM，不加任何誘導劑，各組以24h、48h、72h為觀察記錄的時間坐標軸，在倒置顯微鏡下連續(xù)觀察細胞形態(tài)學變化和生長狀態(tài)，MTT法檢測誘導后細胞的生長和增殖情況，RT-PCR和western blotting方法鑒定分化的神經前體細胞的特異性標志物神經巢蛋白（neuroepithelial stem cell protein，Nestin）和神經細胞的特異性標志物神經元特異性烯醇化酶（neon-specific enolase，NSE）的表達。 結果：1、原代細胞接種24-48小時后可見貼壁細胞，貼壁細胞單個散在分布，多數(shù)細胞呈小圓形或橢圓形。72小時后可見散在的梭形細胞，一周左右細胞形成集落，細胞呈梭形、三角形或星形。原代培養(yǎng)約兩周，細胞增殖明顯加快，集落中的細胞增殖成“漩渦狀”，具有典型的成纖維細胞樣形態(tài)，免疫熒光細胞化學鑒定顯示BMSCs細胞表面標志物CD44表達陽性，造血干細胞表面抗原CD34表達陰性。2、BMSCs經25ug/ml、50ug/ml和100ug/ml誘導24h、48h和72h后，各實驗組細胞折光性增強，部分細胞呈現(xiàn)神經元樣變化，，具有神經元樣形態(tài)：細胞向周圍長出較長的突起，有立體感，折光性增強，倒置顯微鏡下觀察以100ug/ml組細胞折光性及突觸延長最為明顯，并且相鄰細胞間的突觸出現(xiàn)連接，而對照組細胞形態(tài)無明顯變化，仍然呈長梭形。MTT結果顯示24h、48h和72h各實驗組與對照組無顯著性差異（P＞0.05），RT-PCR與western blotting結果顯示不同劑量甲鈷胺誘導48h后，Nestin和NSE在mRNA和蛋白水平表達均上調，其中100ug/ml組表達上調最明顯，與control組比較，差異具有統(tǒng)計學意義（P＜0.05）；同樣，100ug/ml甲鈷胺誘導24h、48h和72h后，Nestin和NSE在mRNA和蛋白水平表達均上調，其中72h表達上調最明顯，與control組比較，差異具有統(tǒng)計學意義（P＜0.05）。 結論：1、甲鈷胺可定向誘導BMSCs向神經元樣細胞分化，分化后的細胞具有一定的增殖能力；甲鈷胺對分化后的細胞生長增殖沒有明顯的抑制作用。2、體外實驗表明，實驗組中100ug/ml甲鈷胺為較佳誘導濃度，能有效快捷的誘導BMSCs向神經元樣細胞分化。
[Abstract]:Objective: to investigate the feasibility of BMSCs differentiation into neuron-like cells induced by different doses of mecobalamin in vitro, and to observe the growth and proliferation of differentiated cells by cultured bone marrow mesenchymal stem cells (BMSCs) in vitro. To seek a more reasonable concentration of mecobalamin induction for clinical use of mecobalamin induced BMSCs for cell transplantation better and more effective treatment of spinal cord injury to provide a preliminary study basis. Methods the bone marrow mesenchymal stem cells of rats were isolated and purified by density gradient centrifugation and adherent culture. The cell growth, biological activity and morphological changes were observed under inverted microscope. CD44, a relative specific surface marker of BMSCs, was detected by immunofluorescence cytochemistry, and it was confirmed that the cells needed for the experiment were BMSCs. 2. BMSCs, which had good growth condition, were prepared into single cell suspension and adjusted cell concentration. The experiment was divided into control group (control group) and experimental group (25ugr / ml 50ugrml and 100ugrml). The experimental group was supplemented with 10s L-DMEM medium with different concentrations of mecobalamin. In the control group, 10s L-DMEM was added without any inducer, and 24 h, 48 h and 72 h was taken as the observation time axis. The morphological changes and growth state of cells were continuously observed under inverted microscope. MTT assay was used to detect the cell growth and proliferation after induction. RT-PCR and western blotting were used to identify the specific marker of neural precursor cells, neuroepithelial nestin. The expression of stem cell protein nestin and neuron-specific enolase (NSE). Results the adherent cells were found 24 to 48 hours after inoculation. Most of the cells were small round or ellipse. After 72 hours, the cells formed colony, and the cells were fusiform. Triangle or star. After about two weeks of primary culture, cell proliferation was significantly accelerated, and the cells in the colony proliferated into a "whirlpool" with typical fibroblast-like morphology. Immunofluorescence cytochemical analysis showed that BMSCs cell surface marker CD44 was positive, and hematopoietic stem cell surface antigen CD34 expression was negative. After 24 h and 72 h after induction of 25ugmml / ml 50ugrml and 100ugrml respectively, the refractive index of the cells in each experimental group was enhanced, and some cells showed neuronal changes. It has neuron-like morphology: the cells have long protuberance, stereosensory and enhanced refraction. Under the inverted microscope, the refraction and synaptic prolongation of the cells in the 100ug-ml group are most obvious, and the synapses of the adjacent cells are connected. However, there was no significant change in cell morphology in the control group. The results showed that there was no significant difference in the expression of nestin and NSE at 24 h and 72 h between the two groups (P > 0.05). The results of RT-PCR and western blotting showed that the expression of nestin and NSE were up-regulated at the level of mRNA and protein after 48h induction with different doses of mecobalamin. The expression of nestin and NSE in 100ugr / ml group was significantly higher than that in control group (P < 0.05), and the expression of nestin and NSE were up-regulated in mRNA and protein levels at 24 h and 72 h after induction by 100ugr / ml mecobalamin, the expression of nestin and NSE was the most obvious at 72 h, which was significantly higher than that in control group. The difference was statistically significant (P < 0.05). Conclusion Mecobalamin can induce the differentiation of BMSCs into neuron-like cells, and the differentiated cells have the ability to proliferate, while mecobalamin has no obvious inhibitory effect on the growth and proliferation of differentiated cells. In the experimental group, 100ugr / ml mecobalamin was the best concentration, which could induce the differentiation of BMSCs into neuron-like cells effectively and quickly.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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