骨髓间充质干细胞对自然衰老小鼠T细胞的调节作用研究
发布时间：2018-12-18 09:53
【摘要】：目的研究自然衰老小鼠与年轻小鼠脾组织中CD8+CD28+T细胞和CD4+CD25+T细胞培养48小时后比例的差异。进一步研究探索：(1)小鼠骨髓间充质干细胞对自然衰老小鼠CD8+CD28+T细胞、CD4+CD25+T细胞的影响作用,从而证明骨髓间充质干细胞对小鼠T细胞衰老的调节作用。(2)小鼠骨髓间充质干细胞对自然衰老小鼠CD8+CD28+T细胞的调节作用是否具有剂量依赖性,为下一步研究机制提供实验基础。 方法第一部分：C57BL/6N小鼠12-14月龄和6-8周龄各五只,分别分成年老组和年轻组。用小鼠淋巴细胞分离液将这些小鼠脾细胞中的淋巴细胞分离计数,每只小鼠的淋巴细胞分成两份,在细胞培养箱里用含有10%特级胎牛血清的RPMI1640培养基培养,48小时后用荧光抗体PE-anti-mouse CD8、FITC-anti-mouse CD28, PE-anti-mouse CD25、FITC-anti-mouse CD4分别标记这些细胞,最后用流式细胞仪检测CD8+CD28+T细胞、CD4+CD25+T细胞在年老组和年轻组的比例差异。第二部分：将从赛业(广州)公司购买的取自6-8周小鼠的骨髓间充质干细胞从第六代扩增到第八代,胰酶消化后计数备用。首先检测年老小鼠CD8+CD28+T细胞经过与骨髓间充质干细胞共培养后的比例变化情况,同时研究这种改变是否有剂量依赖性,此作为实验一。实验一分为三组：隔离共培养组：骨髓间充质干细胞与淋巴细胞以1：1、1：2、2：1比例放入Transwell系统,骨髓间充质干细胞放在底下,而淋巴细胞放在小室里,每个比例的淋巴细胞取自不同的五只小鼠,加入淋巴细胞完全培养基共培养。混合共培养组：骨髓间充质干细胞与五只不同小鼠的淋巴细胞以1：1比例放入6孔板的小室内,同时加入淋巴细胞完全培养基共培养。对照组：五只不同小鼠的淋巴细胞放入培养皿加入淋巴细胞完全培养基培养。48小时后加入PE-anti-mouse CD8、FITC-anti-mouse CD28抗体,上流式细胞仪进行检测。检测衰老小鼠CD4+ CD25+ T细胞经过与骨髓间充质干细胞共培养后的比例变化情况,此作为实验二,实验二分为两个组,共培养组：骨髓间充质干细胞与淋巴细胞以1：1比例放入Transwell系统,每个比例的淋巴细胞取自不同的五只小鼠,加入淋巴细胞完全培养基共培养。对照组：五只不同小鼠的淋巴细胞放入培养皿加入淋巴细胞完全培养基培养。培养48小时后加入PE-anti-mouse CD25、FITC-anti-mouse CD4抗体,上流式细胞仪进行检测。数据用SPSS 13.0统计软件分析,p0.05有统计学意义。 结果(1)经过淋巴细胞分离液分离,年老鼠组中淋巴细胞数量高于年轻鼠淋巴细胞数量。(2)单纯培养48小时后T细胞CD8+ CD28+的共表达率年老组低于年轻组；但T细胞的CD4+ CD25+的共表达率年老组却高于年轻组。(3)实验一隔离共培养组中的CD8+ CD28+ T细胞比例与对照组相比有显著提高,并且骨髓间充质干细胞提高T细胞表面CD8+ CD28+的共表达具有剂量依赖性。混合共培养组的CD8+ CD28+T细胞比例与对照组相比升高,而与隔离共培养组相比降低。(4)实验二中共培养组的CD4+CD25+T细胞的表达率低于对照组,有统计学差异。 结论(1)单纯培养48小时后T细胞CD8+ CD28+的共表达率年老鼠组低于年轻组；但是年老鼠组的CD4+ CD25+的共表达率却高于年轻鼠组。 (2)骨髓间充质干细胞可以使衰老T细胞变的倾向于“年轻”状态,平衡原理可能起到了至关重要的作用。
[Abstract]:Objective To study the difference of the ratio of CD8 + CD28 + T cells and CD4 + CD25 + T cells in the spleen tissue of natural aging mice and young mice. The effects of (1) mouse bone marrow mesenchymal stem cells on CD8 + CD28 + T cells and CD4 + CD25 + T cells in natural aging mice were studied. (2) The effect of mouse bone marrow mesenchymal stem cells on CD8 + CD28 + T cells in natural aging mice was dose-dependent and provided an experimental basis for the next study. Methods The first part: C57BL/ 6N mice from 12 to 14 months and from 6 to 8 weeks old were divided into the old group and the old group. Light group. The lymphocytes in the spleen cells of these mice were separated by a mouse lymphocyte separation solution, and the lymphocytes of each mouse were divided into two, cultured in a cell incubator with RPMI1640 medium containing 10% of the super-grade fetal bovine serum, and after 48 hours, with a fluorescent antibody PE-anti-mouse CD8, FITC-anti-mouse CD28, PE-anti-mouse CD, 25, FITC-anti-mouse CD4 labeled these cells, respectively, and finally, the ratio of CD8 + CD28 + T cells, CD4 + CD25 + T cells in the old and young groups was detected by flow cytometry. The second part: from the sixth generation to the eighth generation, the bone marrow mesenchymal stem cells from the 6-8-week mouse purchased from the competition (Guangzhou) company are counted and counted after the pancreatin is digested. The ratio of CD8 + CD28 + T cells of the aged mouse to the co-culture of the mesenchymal stem cells of the bone marrow was first tested, and the dose-dependence of the change was also studied. The experiment was divided into three groups: isolated co-culture group: the bone marrow mesenchymal stem cells and the lymphocytes were put into the Transwell system at a ratio of 1: 1, 1: 2 and 2: 1, and the bone marrow mesenchymal stem cells were placed under the bottom, and the lymphocytes were placed in the cell, and the lymphocytes of each proportion were taken from five different cells In mice, the complete culture medium of lymphocytes was added. Culture. Mixed co-culture group: the lymphocytes of the bone marrow mesenchymal stem cells and the five different mice were placed in the small chamber of the 6-well plate at a ratio of 1: 1, and the complete culture medium of the lymphocytes was added. Culture. Control group: the lymphocytes of five different mice were placed in a culture dish and the lymphocytes were cultured in full medium. After 48 hours, PE-anti-mouse CD8, FITC-anti-mouse CD28 antibody and flow cytometry were added. The ratio of CD4 + CD25 + T cells in aging mice after co-culture with bone marrow mesenchymal stem cells was detected. The system, each proportion of the lymphocytes were taken from the different five mice, and the lymphocyte complete culture medium was added. Culture. Control group: the lymphocytes of five different mice were placed in a culture dish and the lymphocyte complete culture medium was added. Culturing. After 48 hours of culture, PE-anti-mouse CD25, FITC-anti-mouse CD4 antibody was added, and the flow cytometry was performed. The data was analyzed by SPSS 13.0. The data was statistically analyzed by SPSS 13.0. Significance. The results (1) were isolated by lymphocyte separation, and the number of lymphocytes in the rat group was higher than that of the young rats. (2) The co-expression rate of T-cell CD8 + CD28 + was lower than that in the younger group after 48 hours of culture alone, but the co-expression rate of CD4 + CD25 + in T-cells was higher than that in the old group. (3) The ratio of CD8 + CD28 + T cells in the isolated co-culture group was significantly higher than that of the control group, and the co-expression of CD8 + CD28 + on the surface of T cell was enhanced by the mesenchymal stem cells. Dose-dependent. The ratio of CD8 + CD28 + T cells in the mixed co-culture group was higher than that of the control group and co-cultured with isolation (4) The expression rate of CD4 + CD25 + T cells in the experimental group was lower than that of the control group. Conclusion (1) The co-expression of CD8 + CD28 + in T-cells after 48-hour culture alone is lower than that in the younger group; however, the co-expression of CD4 + CD25 + in the rat group The rate is higher than that of the young mouse group. (2) The mesenchymal stem cells of the bone marrow can change the aging T cell to the 鈥測oung鈥，
本文编号：2385677
[Abstract]:Objective To study the difference of the ratio of CD8 + CD28 + T cells and CD4 + CD25 + T cells in the spleen tissue of natural aging mice and young mice. The effects of (1) mouse bone marrow mesenchymal stem cells on CD8 + CD28 + T cells and CD4 + CD25 + T cells in natural aging mice were studied. (2) The effect of mouse bone marrow mesenchymal stem cells on CD8 + CD28 + T cells in natural aging mice was dose-dependent and provided an experimental basis for the next study. Methods The first part: C57BL/ 6N mice from 12 to 14 months and from 6 to 8 weeks old were divided into the old group and the old group. Light group. The lymphocytes in the spleen cells of these mice were separated by a mouse lymphocyte separation solution, and the lymphocytes of each mouse were divided into two, cultured in a cell incubator with RPMI1640 medium containing 10% of the super-grade fetal bovine serum, and after 48 hours, with a fluorescent antibody PE-anti-mouse CD8, FITC-anti-mouse CD28, PE-anti-mouse CD, 25, FITC-anti-mouse CD4 labeled these cells, respectively, and finally, the ratio of CD8 + CD28 + T cells, CD4 + CD25 + T cells in the old and young groups was detected by flow cytometry. The second part: from the sixth generation to the eighth generation, the bone marrow mesenchymal stem cells from the 6-8-week mouse purchased from the competition (Guangzhou) company are counted and counted after the pancreatin is digested. The ratio of CD8 + CD28 + T cells of the aged mouse to the co-culture of the mesenchymal stem cells of the bone marrow was first tested, and the dose-dependence of the change was also studied. The experiment was divided into three groups: isolated co-culture group: the bone marrow mesenchymal stem cells and the lymphocytes were put into the Transwell system at a ratio of 1: 1, 1: 2 and 2: 1, and the bone marrow mesenchymal stem cells were placed under the bottom, and the lymphocytes were placed in the cell, and the lymphocytes of each proportion were taken from five different cells In mice, the complete culture medium of lymphocytes was added. Culture. Mixed co-culture group: the lymphocytes of the bone marrow mesenchymal stem cells and the five different mice were placed in the small chamber of the 6-well plate at a ratio of 1: 1, and the complete culture medium of the lymphocytes was added. Culture. Control group: the lymphocytes of five different mice were placed in a culture dish and the lymphocytes were cultured in full medium. After 48 hours, PE-anti-mouse CD8, FITC-anti-mouse CD28 antibody and flow cytometry were added. The ratio of CD4 + CD25 + T cells in aging mice after co-culture with bone marrow mesenchymal stem cells was detected. The system, each proportion of the lymphocytes were taken from the different five mice, and the lymphocyte complete culture medium was added. Culture. Control group: the lymphocytes of five different mice were placed in a culture dish and the lymphocyte complete culture medium was added. Culturing. After 48 hours of culture, PE-anti-mouse CD25, FITC-anti-mouse CD4 antibody was added, and the flow cytometry was performed. The data was analyzed by SPSS 13.0. The data was statistically analyzed by SPSS 13.0. Significance. The results (1) were isolated by lymphocyte separation, and the number of lymphocytes in the rat group was higher than that of the young rats. (2) The co-expression rate of T-cell CD8 + CD28 + was lower than that in the younger group after 48 hours of culture alone, but the co-expression rate of CD4 + CD25 + in T-cells was higher than that in the old group. (3) The ratio of CD8 + CD28 + T cells in the isolated co-culture group was significantly higher than that of the control group, and the co-expression of CD8 + CD28 + on the surface of T cell was enhanced by the mesenchymal stem cells. Dose-dependent. The ratio of CD8 + CD28 + T cells in the mixed co-culture group was higher than that of the control group and co-cultured with isolation (4) The expression rate of CD4 + CD25 + T cells in the experimental group was lower than that of the control group. Conclusion (1) The co-expression of CD8 + CD28 + in T-cells after 48-hour culture alone is lower than that in the younger group; however, the co-expression of CD4 + CD25 + in the rat group The rate is higher than that of the young mouse group. (2) The mesenchymal stem cells of the bone marrow can change the aging T cell to the 鈥測oung鈥，
本文编号：2385677
本文链接：https://www.wllwen.com/xiyixuelunwen/2385677.html
最近更新
教材专著
