骨髓间充质干细胞体外诱导分化为光感受器样细胞的蛋白质组学研究
发布时间：2018-12-25 08:09
【摘要】：问充质干细胞(^4esenchvmal Stem Cells，MSCs)是一多能干细胞，具有定向诱导分化为多种组织的潜力。最近研究表明诱导后的MSCs可表达视网膜光感受器细胞特异性标志物。因此，研究MSCs定向诱导分化为光感受器样细胞为干细胞移植治疗视网膜变性疾病奠定基础。目前，对MSCs的特征及其诱导分化为光感受器样细胞的蛋白调控机制还不清楚。本研究模拟视网膜体内生存的微环境，将人骨髓问充质干细胞(BoneⅣIesench’'mal Stem Cells，BMSCs)诱导分化-成视网膜光感受器样细胞，建立BMSCs诱导分化为光感受器样细胞的差异蛋白质库，，探讨BMSCs诱导分化为光感受器样细胞的分化标记和蛋白质调控机制。 第一部分骨髓间充质干细胞体外诱导分化为光感受器样细胞 目的：研究BMSCs在体外定向分化为光感受器样细胞及光感受器细胞特异性标志物视紫红质、恢复蛋白的表达情况。 方法：取第2～4代的人BMSCs细胞株及第3代RPE细胞株进行实验。实验分两组：诱导组和对照组。诱导组：BMSCs与20ng／~mlbl’GF、20ng／'mltEGF及20ng／'mlBI)NF的MSCM及RPE细胞培养液培养。对照组：单纯使用配制好的～IS(：M培养。应用免疫细胞化学检测不同诱导时间(3d、5d、7d)视紫红质蛋白的阳性表达率。：Realtime：一PCR方法检测诱导7d后细胞视紫红质、恢复蛋白mRNA的表达情况。结果：免疫细胞化学方法检测第3d即有视紫红质表达，第7d视紫红质阳性率为(29．8±2．3‰，对照组均未检测到视紫红质表达。诱导组均较对照组有显著性差异，p0．01。在诱导7d后采用Realtime—PCR方法检测到诱导细胞有视紫红质、恢复蛋白mRNA的表达，对照组未见视紫红质、恢复蛋白mRNA表达。 结论：本研究证实采用含有细胞因子的培养液及RPE细胞培养液培养，BMscs在体外能够诱导分化成光感受器样细胞，表达其特异性标志物视紫红质、恢复蛋白。 第二部分骨髓间充质干细胞体外诱导分化为光感受器样细胞的蛋白质组学研究目的：研究体外B^／IS(：s向光感受器样细胞分化过程中的差异蛋白质，探讨BⅣIS(：s定向分化的标记和蛋白调控机制。 方法：取第4～6代的人B^4S(：s细胞株进行实验。细胞培养诱导分化同前。实验分两组：ⅣIS(：s组和诱导组(诱导后7d的细胞)。通过2D—DIGE双向电泳技术及MALDI—TOF—MS分忻，共鉴定出32个差异表达蛋白。Westem—Not进一步验证了zyxin、p-opomyOSl’n的差异表达。 结果：共鉴定出32个差异表达蛋白，其中11个蛋白质表达上调，21个蛋白质表达下调，建立了B～IS(：s向光感受器样细胞诱导分化7d后的差异表达蛋白谱。鉴定出的蛋白质分为7类：细胞骨架蛋白、分子伴侣、能量代谢的激酶、信号转导通路相关蛋白、细胞增殖、分化和凋亡相关蛋白、钙结合蛋白和离子通道及其他蛋白。研究发现p一订opomyOSl’P、p38、人类增殖细胞核抗原C链表达下调，肌球蛋白、线粒体的热休克蛋白60 kD蛋白质1变异体1、含硫氧还蛋白域蛋白质5亚型3表达上调，表明在诱导过程中发挥着重要的调控作用。’Westem—blot验证了诱导后细胞Zxy’in和B-opom)，OSl’n表达呈下调趋势。 结论：应用差异蛋白质组学的方法，建立了BⅣIS(：s诱导分化为光感受器样细胞的差异蛋白质组图谱，初步分忻了B～IS(：s诱导分化为光感受器样细胞潜在的分子机制。
[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
本文编号：2390907
[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
本文编号：2390907
本文链接：https://www.wllwen.com/xiyixuelunwen/2390907.html
最近更新
教材专著
