| Objective:1.To reveale the regulation of Gaq protein on CD4+T cells homeostatic proliferation.2. To elucidate the mechanism of Gaq protein affecting homeostatic proliferation of CD4+T cells. Methods:1. Establishing bone marrow chimericmouse. Gaq knock-out mice and wild C57BL/6 mice both with same age and gender were sacrificed by cervical dislocation, the intact femur and tibia were taken and soaked in 70% alcohol for 3min, and washed with PBS for 3 times and then transferred to sterile culture dish,with lmL syringe blowing out the bone marrow, the marrow mononuclear cells were counted after the RBC were lysed with red blood lysis buffe. The wildC-57BL/6 mice with same age and gender were exposed to X-rays at a lethal dose of 1000cGy,then they were injected intravenously 4x106 cells coming from C-57BL/6 mice and Gaq knock-out mice to establish bone marrow chimeric mouse. The spleens were taken out for experiment after Two months of the feed under the specific-pathogen-free (SPF) environment.2.Isolation and purification of CD4+T cells. Spleens taken from the bone marrow chimeric mice and Gaq knock-out mice and wild C57BL/6 mice were made into single-cell suspension by polishing and filtration, and red blood cells were removed with RBC lysis buffer. At this time, magnetic positive selection method was applied for CD4+T cells isolation. By doing this, The spleen mononuclear cells and the anti-CD4 were firstly incubated on ice for 30min, added with magnetic beads for ice incubation of 30min after unconjugated antibodies removed Through the centrifugal, Mini-MACS separator was adopted to purify CD4+T cells. The purified cells need undergoing purity identification with FCM (flow cytometry), if purity was failed to reach 95%, new separation columns might be used for re-purification.3. Inspection of homeostatic proliferation of CD4+T cells as well as its affecting factors. By applying above steps, CD4+T cells were sorted from Gaq knock-out mice and wild C57BL/6 mice. Each mouse, after CFSE labeling in vitro, was injected intravenously 4x106 cells, The wildC-57BL/6 mice with same age and gender were exposed to X-rays at a lethal dose of 300cGy were given with intravenous infusion.And their spleens were taken out at different time for testing. According to the CFSE fluorescence degree, flow cytometry was applied to test cell homeostatic proliferation.The experimental design was as follows:20 C57BL/6 mice (8-12 weeks old) of the same sex were classified as two groups,10 for each group, and were exposed to 300 cGy sub-lethal dose irradiation. The first Group was injected intravenously with doses of 4x106 CFSE-labelled CD4+T cells taking from the spleens of Gaq knock-out mice; the second Group was injected intravenously with doses of 4x106 CFSE-labelled CD4+T cells from the spleens of wild mice; The spleen cell of five mice from each group extracted on the 2nd and 5th day were analyzed for their CFSE positive cells proliferation with FCM, Comparing the difference between homeostatic proliferation respectively. Purified CD4+T cells which undergo BTLA, CD62L surface markers were tested before homeostatic proliferation and on the 2nd and 5th days of homeostatic proliferation with FCM and their expression difference was compared. Results:1. Before homeostatic proliferation, according to FCM detection, The expressions levels of BTLA and CD62Lon CD4+T cells in wild-type and Gaq knock-out mice showed no statistically significant difference (P> 0.05).2. After CFSE staining, the difference of CD4+T cell proliferations between wild-type and Gaq knock-out mice on the 2nd day of proliferation wasn't statistically significant(P> 0.05).By analyzing and comparing with flow cytometer. but for the 5th day of proliferation, there were more CD4+T cell proliferations in Gaq knock-out mice than wild-type mice(P<0.01).3. BTLA expression on CFSE-labelled CD4+T cells in wild-type and Gaq knock-out mice on the 2nd day of proliferation presented no significant difference (P> 0.05), yet the CD62L expressions of wild-type mice was significantly higher than Gaq knock-out mice on 2nd day of proliferation (P< 0.01). And on the 5th day of proliferation, BTLA and CD62L expressions levels on CFSE-labelled CD4+T cells in Gaq knock-out mice were significantly lower than wild-type mice (P<0.01). Conclusion:1. Gaq protein can prompt CD4+T cells greater and faster homeostatic proliferation.2. Gaq protein may regulate the homeostatic proliferation of CD4+T cell by regulating expressions of BTLA and CD62L... |
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