【摘要】：目的研究自然衰老小鼠與年輕小鼠脾組織中CD8+CD28+T細(xì)胞和CD4+CD25+T細(xì)胞培養(yǎng)48小時后比例的差異。進(jìn)一步研究探索：(1)小鼠骨髓間充質(zhì)干細(xì)胞對自然衰老小鼠CD8+CD28+T細(xì)胞、CD4+CD25+T細(xì)胞的影響作用,從而證明骨髓間充質(zhì)干細(xì)胞對小鼠T細(xì)胞衰老的調(diào)節(jié)作用。(2)小鼠骨髓間充質(zhì)干細(xì)胞對自然衰老小鼠CD8+CD28+T細(xì)胞的調(diào)節(jié)作用是否具有劑量依賴性,為下一步研究機(jī)制提供實驗基礎(chǔ)。 方法第一部分：C57BL/6N小鼠12-14月齡和6-8周齡各五只,分別分成年老組和年輕組。用小鼠淋巴細(xì)胞分離液將這些小鼠脾細(xì)胞中的淋巴細(xì)胞分離計數(shù),每只小鼠的淋巴細(xì)胞分成兩份,在細(xì)胞培養(yǎng)箱里用含有10%特級胎牛血清的RPMI1640培養(yǎng)基培養(yǎng),48小時后用熒光抗體PE-anti-mouse CD8、FITC-anti-mouse CD28, PE-anti-mouse CD25、FITC-anti-mouse CD4分別標(biāo)記這些細(xì)胞,最后用流式細(xì)胞儀檢測CD8+CD28+T細(xì)胞、CD4+CD25+T細(xì)胞在年老組和年輕組的比例差異。第二部分：將從賽業(yè)(廣州)公司購買的取自6-8周小鼠的骨髓間充質(zhì)干細(xì)胞從第六代擴(kuò)增到第八代,胰酶消化后計數(shù)備用。首先檢測年老小鼠CD8+CD28+T細(xì)胞經(jīng)過與骨髓間充質(zhì)干細(xì)胞共培養(yǎng)后的比例變化情況,同時研究這種改變是否有劑量依賴性,此作為實驗一。實驗一分為三組：隔離共培養(yǎng)組：骨髓間充質(zhì)干細(xì)胞與淋巴細(xì)胞以1：1、1：2、2：1比例放入Transwell系統(tǒng),骨髓間充質(zhì)干細(xì)胞放在底下,而淋巴細(xì)胞放在小室里,每個比例的淋巴細(xì)胞取自不同的五只小鼠,加入淋巴細(xì)胞完全培養(yǎng)基共培養(yǎng)。混合共培養(yǎng)組：骨髓間充質(zhì)干細(xì)胞與五只不同小鼠的淋巴細(xì)胞以1：1比例放入6孔板的小室內(nèi),同時加入淋巴細(xì)胞完全培養(yǎng)基共培養(yǎng)。對照組：五只不同小鼠的淋巴細(xì)胞放入培養(yǎng)皿加入淋巴細(xì)胞完全培養(yǎng)基培養(yǎng)。48小時后加入PE-anti-mouse CD8、FITC-anti-mouse CD28抗體,上流式細(xì)胞儀進(jìn)行檢測。檢測衰老小鼠CD4+ CD25+ T細(xì)胞經(jīng)過與骨髓間充質(zhì)干細(xì)胞共培養(yǎng)后的比例變化情況,此作為實驗二,實驗二分為兩個組,共培養(yǎng)組：骨髓間充質(zhì)干細(xì)胞與淋巴細(xì)胞以1：1比例放入Transwell系統(tǒng),每個比例的淋巴細(xì)胞取自不同的五只小鼠,加入淋巴細(xì)胞完全培養(yǎng)基共培養(yǎng)。對照組：五只不同小鼠的淋巴細(xì)胞放入培養(yǎng)皿加入淋巴細(xì)胞完全培養(yǎng)基培養(yǎng)。培養(yǎng)48小時后加入PE-anti-mouse CD25、FITC-anti-mouse CD4抗體,上流式細(xì)胞儀進(jìn)行檢測。數(shù)據(jù)用SPSS 13.0統(tǒng)計軟件分析,p0.05有統(tǒng)計學(xué)意義。 結(jié)果(1)經(jīng)過淋巴細(xì)胞分離液分離,年老鼠組中淋巴細(xì)胞數(shù)量高于年輕鼠淋巴細(xì)胞數(shù)量。(2)單純培養(yǎng)48小時后T細(xì)胞CD8+ CD28+的共表達(dá)率年老組低于年輕組；但T細(xì)胞的CD4+ CD25+的共表達(dá)率年老組卻高于年輕組。(3)實驗一隔離共培養(yǎng)組中的CD8+ CD28+ T細(xì)胞比例與對照組相比有顯著提高,并且骨髓間充質(zhì)干細(xì)胞提高T細(xì)胞表面CD8+ CD28+的共表達(dá)具有劑量依賴性�；旌瞎才囵B(yǎng)組的CD8+ CD28+T細(xì)胞比例與對照組相比升高,而與隔離共培養(yǎng)組相比降低。(4)實驗二中共培養(yǎng)組的CD4+CD25+T細(xì)胞的表達(dá)率低于對照組,有統(tǒng)計學(xué)差異。 結(jié)論(1)單純培養(yǎng)48小時后T細(xì)胞CD8+ CD28+的共表達(dá)率年老鼠組低于年輕組；但是年老鼠組的CD4+ CD25+的共表達(dá)率卻高于年輕鼠組。 (2)骨髓間充質(zhì)干細(xì)胞可以使衰老T細(xì)胞變的傾向于“年輕”狀態(tài),平衡原理可能起到了至關(guān)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，

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