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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。
Objective To analyze the number of B cells and the expression of inflammatory factors and sirtuin-1 (SIRT1) in spleen of CD38 knockout mice, and to explore the effect of CD38 knockout on inflammatory cytokines in B cells Potential mechanism. Methods The DNA expression levels of CD38 and Neo genes were detected by polymerase chain reaction (PCR). Wild type (WT) C57BL / 6 mice and CD38 - / - mice B cells in the spleen and B cells were identified by flow cytometry. The mRNA expression of CD38 and inflammatory cytokines TNF-α and IL-1β were detected by real-time quantitative PCR. The protein level of CD38 and SIRT1 was detected by Western blot. Results CD38 - / - mice were identified and B cells (purity> 95%) in the spleens of WT and CD38 - / - mice were successfully sorted. Compared with WT mice, CD38 - / - mice had decreased spleen development, total number of spleen cells and number of B cells in them (P <0.01), and decreased expression of inflammatory cytokines TNF-α and IL-1β <0.01), SIRT1 protein expression increased (P <0.05). Conclusions CD38 knockdown can reduce the number of spleen B cells and inhibit the expression of inflammatory cytokines TNF-α and IL-1β in spleen B cells by activating SIRT1 pathway.