半夏泻心汤对siRNA干扰GIST-882细胞Cx43基因沉默后细胞间通讯的影响

发布时间：2018-05-14 17:59

本文选题：半夏泻心汤 + Cx43-siRNA　；参考：《北京中医药大学》2017年硕士论文

【摘要】：[目的]为进一步探究半夏泻心汤对胃肠运动障碍的调节机制,以细胞间缝隙连接通讯为核心,从Cx43与胃肠动力障碍的关系角度,揭示半夏泻心汤通过Cx43调节胃肠运动的细胞分子机制,进而从细胞间通讯的角度对"心下痞"的内涵进行揭示。这不仅可以为半夏泻心汤的治疗作用提供科学依据,同时也有助于在机理探究的基础上提高半夏泻心汤调节胃肠运动障碍的疗效,促进半夏泻心汤治疗消化系统疾病的现代化发展。[方法]采用SD雄性大鼠灌胃方法,制备半夏泻心汤含药血清;同时去离子水灌胃,制备SD雄性大鼠血清作为对照。将稳定培养传代后的胃肠间质瘤细胞株(GIST-882),随机分为空白对照组、Cx43-siRNA干扰模型组、半夏泻心汤含药血清组(即全方组)。利用CCK-8试剂盒检测细胞活力与细胞存活率,流式细胞仪测定细胞周期、分析细胞凋亡,钙离子荧光探针(Fluo-3 AM)检测细胞内钙离子浓度,免疫印迹法检测GIST-882细胞中缝隙连接蛋白43及其磷酸化MAPK、PKA、PKC、PKG等的蛋白表达水平,RT-PCR法测定 GIST-882 细胞中 Cx43 mRNA、MAPK mRNA、PKAmRNA、PKC mRNA等的表达。[结果](1)细胞活力与细胞存活率:与空白对照组相比,模型组的细胞活力与细胞存活率显著下降(P0.05);与模型组对比,全方组细胞活力与细胞存活率有着显著升高(P0.05)。(2)细胞凋亡与周期:各组能够不同程度的促进细胞凋亡,但均无显著性差异(P0.05)。各组间细胞在G1期,模型组上升,全方组下降,但无显著性差异(P0.05)。各组细胞在G2期,与空白对照组相比,模型组细胞在此期显著降低,有统计学差异(P0.01);与模型组相比,全方组细胞在此期周期较高,但无统计学差异(P0.05)。各组细胞在S期,与空白组相比,模型组在S期降低,但无统计学差异(P0.05);与模型组相比,全方组的周期在此期高于模型组(P0.05),有作用趋势,但无统计学差异。(3)细胞内钙离子水平:与空白对照组对比,模型组中细胞内钙离子浓度显著升高(P0.05);与模型组相比,全方组的细胞内钙离子浓度显著下降(P0.05)。(4)相关蛋白表达水平:与空白组对比,模型组Cx43、MAPK的蛋白表达降低明显(P0.05),模型组P-Cx43、PKA、PKC、PKG等的蛋白表达与正常组相比无显著性差异(P0.05);与模型组相比,全方组Cx43、MAPK的蛋白表达明显高于模型组(P0.01),全方组P-Cx43、PKA、PKC、PKG等的蛋白表达与模型组相比无显著性差异(P0.05)。(5)相关蛋白mRNA表达:与空白组相比,模型组Cx43mRNA、MAPKmRNA、PKAmRNA的表达明显降低,具有统计学差异(P0.05);与模型组相比,全方组Cx43 mRNA、PKAmRNA、MAPK mRNA等基因的表达明显升高,具有统计学差异(P0.05);PKC mRNA的表达中,与空白组相比,模型组表达增加;与模型组相比,全方组表达降低,均没有统计学差异。[结论](1)半夏泻心汤能够促进转染Cx43-siRNA的GIST-882细胞的增殖,且是通过细胞存活周期的增长而非抑制细胞凋亡使细胞存活增长,对转染Cx43-siRNA的GIST-882细胞具有一定的调节和保护功能;(2)半夏泻心汤能够降低转染Cx43-siRNA的GIST-882细胞内的钙离子浓度,从而调节和保护第二信使Ca2+通道;(3)半夏泻心汤能够增加转染Cx43-siRNA的GIST-882细胞的Cx43、MAPK蛋白的表达,同时增强Cx43 mRNA、MAPK mRNA及PKA mRNA等相关基因的表达,从而调控细胞间隙通讯,促进胃肠功能障碍恢复。综合以上结论,从现代分子机制的实验研究角度说明,半夏泻心汤可以改善细胞间缝隙连接,增强细胞间缝隙连接通讯功能,调节细胞内第二信使Ca2+通道、MAPK信号通路及PKA等蛋白激酶,从而促进转染Cx43-siRNA后GIST-882细胞的修复,促进胃肠功能障碍的恢复。这为半夏泻心汤治疗"心下痞"提供了可能性的依据,为该方现代分子生物学机制的研究打下了基础。
[Abstract]:[Objective] to further explore the regulation mechanism of Banxia Xiexin Decoction on gastrointestinal motility disorder, with intercellular gap junction communication as the core, from the angle of the relationship between Cx43 and gastrointestinal motility disorder, the molecular mechanism of regulating gastrointestinal motility by Cx43 is revealed, and then the connotation of "lower heart ruffian" is uncovered from the angle of intercellular communication. It can not only provide scientific basis for the treatment of Banxia Xiexin Decoction, but also help to improve the effect of Banxia Xiexin Decoction on regulating gastrointestinal dyskinesia on the basis of mechanism exploration, promote the modern development of digestive system disease with Banxia Xiexin soup. [Methods] the method of gavage of SD male rats was used to prepare Banxia Xiexin Decoction Serum containing medicine and serum of SD male rats were prepared with deionized water as control. The gastrointestinal stromal tumor cell line (GIST-882) was steadily cultured and divided into blank control group, Cx43-siRNA interference model group, and Banxia Xiexin Decoction serum group (the whole group). The cell viability and cell survival rate were detected by CCK-8 kit. Cell cycle was measured by cytometer, apoptosis was analyzed, calcium ion fluorescence probe (Fluo-3 AM) was used to detect intracellular calcium concentration. The protein expression level of gap connexin 43 and its phosphorylated MAPK, PKA, PKC, PKG in GIST-882 cells was detected by Western blot. Cx43 mRNA in GIST-882 cells was determined by RT-PCR method. MAPK [results] (1) cell viability and cell survival rate: compared with the blank control group, the cell viability and cell survival rate decreased significantly (P0.05). Compared with the model group, the cell viability and cell viability were significantly increased (P0.05). (2) cell apoptosis and cycle: each group could promote apoptosis in different degrees. There was no significant difference (P0.05). The cells in each group were in the G1 phase, the model group rose and the whole group decreased, but there was no significant difference (P0.05). Compared with the blank control group, the cells in each group were significantly lower than those in the blank control group (P0.01). Compared with the model group, the whole group cell cycle was higher in this period, but not statistically significant. Difference (P0.05). Compared with the blank group, the model group decreased in the S phase compared with the blank group, but there was no statistical difference (P0.05). Compared with the model group, the cycle period of the whole group was higher than that of the model group (P0.05), and there was no statistical difference. (3) the intracellular calcium level: compared with the blank control group, the intracellular calcium concentration in the model group was strong. Compared with the model group, the intracellular calcium concentration of the whole group decreased significantly (P0.05). (4) the expression level of related proteins: compared with the blank group, the protein expression of Cx43 and MAPK in the model group decreased significantly (P0.05), and the protein expression of P-Cx43, PKA, PKC and PKG in the model group was not significantly different from that of the normal group (P0.05); and the model group was compared with the model group (P0.05). The protein expression of Cx43 and MAPK in the whole group was significantly higher than that in the model group (P0.01). The protein expression of P-Cx43, PKA, PKC and PKG in the whole group was not significantly different from that of the model group (P0.05). (5) the expression of the related protein mRNA: compared with the blank group, the expression of Cx43mRNA, MAPKmRNA, PKAmRNA was significantly lower than that in the blank group; and the model was statistically different. The expression of Cx43 mRNA, PKAmRNA, MAPK mRNA and other genes in the group was significantly higher than that in the group (P0.05). The expression of the model group increased in the expression of PKC mRNA, compared with the blank group. Compared with the model group, the expression of the whole group decreased, and there was no statistical difference. [Conclusion] (1) Banxia Xiexin soup can promote the GIST-882 transfection of Cx43-siRNA GIST-882. Cell proliferation, and the growth of cell survival cycle, is not inhibited by cell apoptosis to increase cell survival, and has some regulatory and protective functions for Cx43-siRNA transfected GIST-882 cells. (2) Banxia Xiexin soup can reduce the concentration of calcium ions in GIST-882 cells transfected with Cx43-siRNA, thereby regulating and protecting the second messenger Ca2+ (3) (3) Banxia Xiexin Decoction can increase the expression of Cx43 and MAPK protein in transfected GIST-882 cells, and enhance the expression of Cx43 mRNA, MAPK mRNA and PKA mRNA, so as to regulate the intercellular space communication and promote the recovery of gastrointestinal dysfunction. Xiexin Decoction can improve the gap junction of cells, enhance the communication function of gap junction between cells, regulate the second messenger Ca2+ channel, MAPK signaling pathway and PKA protein kinase in cell, thus promote the repair of GIST-882 cells after Cx43-siRNA transfection and promote the restoration of gastrointestinal dysfunction. This provides the Banxia Xiexin Decoction for the treatment of "the lower heart". The basis of this study laid a foundation for the study of modern molecular biology mechanism.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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