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。
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 B-cells 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/6 and 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 SIRT1 were 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-α and IL-1β） in spleen B-cells by activating SIRT1 pathway.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2016年第11期1373-1377,共5页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(81302600)~~
