【摘要】：問充質(zhì)干細胞(^4esenchvmal Stem Cells，MSCs)是一多能干細胞，具有定向誘導分化為多種組織的潛力。最近研究表明誘導后的MSCs可表達視網(wǎng)膜光感受器細胞特異性標志物。因此，研究MSCs定向誘導分化為光感受器樣細胞為干細胞移植治療視網(wǎng)膜變性疾病奠定基礎。目前，對MSCs的特征及其誘導分化為光感受器樣細胞的蛋白調(diào)控機制還不清楚。本研究模擬視網(wǎng)膜體內(nèi)生存的微環(huán)境，將人骨髓問充質(zhì)干細胞(BoneⅣIesench’'mal Stem Cells，BMSCs)誘導分化-成視網(wǎng)膜光感受器樣細胞，建立BMSCs誘導分化為光感受器樣細胞的差異蛋白質(zhì)庫，，探討B(tài)MSCs誘導分化為光感受器樣細胞的分化標記和蛋白質(zhì)調(diào)控機制。 第一部分骨髓間充質(zhì)干細胞體外誘導分化為光感受器樣細胞 目的：研究BMSCs在體外定向分化為光感受器樣細胞及光感受器細胞特異性標志物視紫紅質(zhì)、恢復蛋白的表達情況。 方法：取第2～4代的人BMSCs細胞株及第3代RPE細胞株進行實驗。實驗分兩組：誘導組和對照組。誘導組：BMSCs與20ng／~mlbl’GF、20ng／'mltEGF及20ng／'mlBI)NF的MSCM及RPE細胞培養(yǎng)液培養(yǎng)。對照組：單純使用配制好的～IS(：M培養(yǎng)。應用免疫細胞化學檢測不同誘導時間(3d、5d、7d)視紫紅質(zhì)蛋白的陽性表達率。：Realtime：一PCR方法檢測誘導7d后細胞視紫紅質(zhì)、恢復蛋白mRNA的表達情況。結果：免疫細胞化學方法檢測第3d即有視紫紅質(zhì)表達，第7d視紫紅質(zhì)陽性率為(29．8±2．3‰，對照組均未檢測到視紫紅質(zhì)表達。誘導組均較對照組有顯著性差異，p0．01。在誘導7d后采用Realtime—PCR方法檢測到誘導細胞有視紫紅質(zhì)、恢復蛋白mRNA的表達，對照組未見視紫紅質(zhì)、恢復蛋白mRNA表達。 結論：本研究證實采用含有細胞因子的培養(yǎng)液及RPE細胞培養(yǎng)液培養(yǎng)，BMscs在體外能夠誘導分化成光感受器樣細胞，表達其特異性標志物視紫紅質(zhì)、恢復蛋白。 第二部分骨髓間充質(zhì)干細胞體外誘導分化為光感受器樣細胞的蛋白質(zhì)組學研究目的：研究體外B^／IS(：s向光感受器樣細胞分化過程中的差異蛋白質(zhì)，探討B(tài)ⅣIS(：s定向分化的標記和蛋白調(diào)控機制。 方法：取第4～6代的人B^4S(：s細胞株進行實驗。細胞培養(yǎng)誘導分化同前。實驗分兩組：ⅣIS(：s組和誘導組(誘導后7d的細胞)。通過2D—DIGE雙向電泳技術及MALDI—TOF—MS分忻，共鑒定出32個差異表達蛋白。Westem—Not進一步驗證了zyxin、p-opomyOSl’n的差異表達。 結果：共鑒定出32個差異表達蛋白，其中11個蛋白質(zhì)表達上調(diào)，21個蛋白質(zhì)表達下調(diào)，建立了B～IS(：s向光感受器樣細胞誘導分化7d后的差異表達蛋白譜。鑒定出的蛋白質(zhì)分為7類：細胞骨架蛋白、分子伴侶、能量代謝的激酶、信號轉(zhuǎn)導通路相關蛋白、細胞增殖、分化和凋亡相關蛋白、鈣結合蛋白和離子通道及其他蛋白。研究發(fā)現(xiàn)p一訂opomyOSl’P、p38、人類增殖細胞核抗原C鏈表達下調(diào)，肌球蛋白、線粒體的熱休克蛋白60 kD蛋白質(zhì)1變異體1、含硫氧還蛋白域蛋白質(zhì)5亞型3表達上調(diào)，表明在誘導過程中發(fā)揮著重要的調(diào)控作用。’Westem—blot驗證了誘導后細胞Zxy’in和B-opom)，OSl’n表達呈下調(diào)趨勢。 結論：應用差異蛋白質(zhì)組學的方法，建立了BⅣIS(：s誘導分化為光感受器樣細胞的差異蛋白質(zhì)組圖譜，初步分忻了B～IS(：s誘導分化為光感受器樣細胞潛在的分子機制。
[Abstract]:Q & A stem cell (MSCs) is a multipotent stem cell with the potential of directional induction to differentiate into multiple tissues. Recent studies have shown that the induced MSCs can express the specific markers of the retinal photoreceptors. Therefore, it is necessary to study the orientation and differentiation of MSCs as the foundation of stem cell transplantation for the treatment of the degenerative diseases of the retina. At present, it is not clear that the characteristics of MSCs and their induction and differentiation are the protein regulation mechanisms of photoreceptors-like cells. In this study, the micro-environment for the survival of the retina was simulated, and the human bone marrow was induced to differentiate into the retinal photoreceptor-like cells, and the BMSCs were established to induce the differential protein library of the photoreceptors-like cells. The differentiation of BMSCs into photoreceptor-like cells and the mechanism of protein regulation were discussed. In vitro induction and differentiation of the first part of bone marrow mesenchymal stem cells into photoreceptors The purpose of the study is to study the expression of BMSCs in vitro, in which the specific markers of the photoreceptors-like cells and the photoreceptors-like cells and the photoreceptor-like cell-specific markers are as follows: The method comprises the following steps of: taking the human BMSCs and the third generation RPE cells on the second to fourth generations; The experiment was carried out. The experiment was divided into two groups: induction Group and control group. Induction group: BMSCs and 20ng/ ~ mlbl 'GF, 20ng/' mltEGF and MSCM and RPE cells of 20ng/ 'mlBI) NF Culture of culture medium. Control group: simple use of prepared ~ IS (: M). Using immunocytochemistry to detect the different induction time (3d, 5d, 7d) of the viologen protein. Positive rate of expression.: Realtime: A PCR method for detecting the red and red blood of the cells after the induction of 7d, and the mRNA of the recovery protein. Results: The expression of red and red in the third day was detected by the method of immunocytochemistry. The positive rate of the red and red samples in the 7th day was (2.9. 8). in that induction group, there was a significant difference between the control group and the control group, p0.01. After the induction of 7d, the expression of the viologen and the recovery protein mRNA in the induction cells was detected by the Realtime polymerase chain reaction (RT-PCR) method. The control group did not see the viologen and the recovery protein m. Conclusion: In this study, the culture medium containing cytokines and the culture medium of RPE cells were used to culture, and the BMscs could induce differentiation into photoreceptor-like cells in vitro, and expressed their specific markers. In vitro induction and differentiation of the bone marrow mesenchymal stem cells of the second part into the photoreceptor-like cells: the study of the differential protein in the differentiation of B-I/ IS (: s) to the photoreceptor-like cells in vitro On the Quality of B-鈪

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