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目的:探讨微小RNA-19b(miR-19b)对强毒力结核分枝杆菌(H37Rv)诱导的大鼠肺泡巨噬细胞凋亡的影响。方法:以强毒力H37Rv感染miR-19b模拟物或其抑制剂转染的大鼠肺泡巨噬细胞,流式细胞术检测大鼠肺泡巨噬细胞凋亡率;RT-PCR检测Toll样受体蛋白(TLRs)的mRNA或miR-19b水平;Western blot检测Cleaved caspase 3、Bcl-2及TLRs蛋白的表达,应用Gel-pro4.0软件分析Western blots条带;所有数据采用独立样本t检验。结果:大鼠肺泡巨噬细胞经H37Rv感染后,miR-19b抑制剂组细胞总凋亡率[12 h为(13.55±0.45)%,24 h为(17.16±0.42)%]显著高于对照组[12 h为(9.42±0.47)%,24 h为(12.29±0.37)%];转染24 h后,细胞Cleaved caspase 3相对表达(0.59±0.07)显著高于对照组(0.45±0.05),Bcl-2相对表达(0.34±0.03)显著低于对照组(0.54±0.05),且细胞Toll样受体2(TLR2)相对水平(mRNA为0.283±0.03,蛋白为0.25±0.02)显著高于对照组(mRNA为0.20±0.02,蛋白为0.18±0.01),各组间TLR4、TLR7、TLR9表达无差异;以上变化在miR-19b模拟物组中则相反。此外,高表达的TLR2经其抗体中和后,miR-19b沉默使H37Rv感染大鼠肺泡巨噬细胞的促凋亡作用受到抑制。结论:大鼠肺泡巨噬细胞中,miR-19b沉默能够通过上调TLR2水平促进强毒力结核分枝杆菌诱导的大鼠肺泡巨噬细胞凋亡。
AIM: To investigate the effect of microRNA-19b (miR-19b) on the apoptosis of rat alveolar macrophages induced by virulent Mycobacterium tuberculosis (H37Rv). Methods: Rat alveolar macrophages transfected with miR-19b mimics or its inhibitors were infected with virulent H37Rv. The apoptosis rate of alveolar macrophages was detected by flow cytometry. Toll-like receptors The expression of Cleaved caspase 3, Bcl-2 and TLRs were detected by Western blot. Western Blots were analyzed by Gel-pro 4.0 software. All data were analyzed by independent sample t-test. RESULTS: After H37Rv infection, the total apoptotic rate in the miR-19b inhibitor group was (13.55 ± 0.45)% at 12 h and (17.16 ± 0.42)% at 24 h), significantly higher than that in the control group (9.42 ± 0.47% at 12 h and (12.29 ± 0.37)% at 24 h). The relative expression of Cleaved caspase 3 (0.59 ± 0.07) after 24 h transfection was significantly higher than that of control group (0.45 ± 0.05) The relative expression of Bcl-2 (0.34 ± 0.03) was significantly lower than that of the control group (0.54 ± 0.05), and the relative level of Toll-like receptor 2 (TLR2) mRNA was 0.283 ± 0.03 and 0.25 ± 0.02 (MRNA 0.20 ± 0.02, protein 0.18 ± 0.01). There was no difference in expression of TLR4, TLR7 and TLR9 between the groups. The above changes were opposite in miR-19b mimics. In addition, the high expression of TLR2 by its antibody neutralization, miR-19b silencing H37Rv infected rat alveolar macrophages apoptosis was inhibited. Conclusion: miR-19b silencing in rat alveolar macrophages can promote the apoptosis of rat alveolar macrophages induced by virulent M. tuberculosis by up-regulating the level of TLR2.