【摘要】 目的分析CD38基因敲除小鼠脾脏中B细胞的数量及B细胞中炎性因子和去乙酰化酶1(SIRT1)的表达水平,探讨CD38基因敲除对B细胞中炎性因子的影响及其潜在机制。方法采用聚合酶链反应(PCR)检测小鼠尾巴CD38和新霉素(Neo)基因的DNA表达水平;磁珠阴选法分选出野生型(WT)C57BL/6和CD38-/-小鼠脾脏中的B细胞,经流式细胞仪鉴定B细胞分选纯度;实时定量PCR检测CD38和炎性因子肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)基因的mRNA表达水平;蛋白质印迹法检测CD38及SIRT1的蛋白表达水平。结果鉴定CD38-/-小鼠,并成功分选出WT和CD38-/-小鼠脾脏中的B细胞(纯度>95%)。与WT小鼠相比,CD38-/-小鼠脾脏发育障碍,脾细胞总数及其中的B细胞数量均减少(P<0.01),伴炎性因子TNF-α和IL-1βmRNA表达水平下降(P<0.01),SIRT1蛋白表达水平上升(P<0.05)。结论 CD38基因敲除可引起脾脏B细胞数量减少;并可能通过激活SIRT1通路,抑制脾脏B细胞中炎性因子TNF-α和IL-1β的表达。更多还原

【Abstract】 Objective To analyze the number of spleen B-cells and the expression of inflammatory factors and sirtuin 1(SIRT1)in spleen B-cells of CD38-/-mice,so as to explore the effects of CD38 gene knockout on inflammatory factors in Bcells and its potential mechanism.Methods The DNA levels of CD38 and Neo gene in mouse tail tissues were detected by polymerase chain reaction(PCR).Spleen B-cells from wide-type(WT)C57BL/6and CD38-/-mice were sorted by magnetic activated cell sorting(MACS),and the purity of sorting B-cells were identified by flow cytometry.The mRNA levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and CD38 gene were detected by real-time PCR,the protein expressions of CD38 and SIRT1were detected by Western blotting analysis.Results We confirmed the successful establishment of CD38-/-mice and sorted spleen B-cells from WT and CD38-/-mice(purity>95%).Compared with WT mice,the development of spleen was hampered in the CD38-/-mice,the number of spleen cells and spleen B-cells were significantly reduced(P<0.01),the mRNA levels of TNF-α and IL-1β were significantly decreased(P <0.01),and the expression level of SIRT1 was significantly increased in CD38-/-mice(P<0.05).Conclusion CD38 gene knockout can reduce the number of B-cells in the spleen;and it can inhibit the expression of inflammatory factors(TNF-αand IL-1β)in spleen B-cells by activating SIRT1 pathway.更多还原