半夏泻心汤调控Shh信号通路对BMSCs外泌体诱导的人胃癌细胞BGC-823增殖的影响

Effects of Regulation of Banxia Xiexin Decoction for Shh Signaling Pathway on BMSCs Exosomes-induced Proliferation of Human Gastric Cancer Cells BGC-823

目的观察半夏泻心汤对骨髓间充质干细胞(BMSCs)外泌体诱导的人胃癌细胞BGC-823增殖的影响,探讨其相关作用机制。方法利用Transwell小室将BMSCs外泌体与BGC-823细胞进行非接触共培养,实验分为空白组、模型组、阳性对照组和半夏泻心汤高、中、低剂量组,荧光染料Dil标记BMSCs外泌体,激光共聚焦显微镜观察BGC-823细胞对BMSCs外泌体的摄取,计算平均光密度(MOD);实时无标记细胞分析技术检测细胞增殖;流式细胞术检测细胞周期;RT-qPCR检测Shh、Ptch1、Smo、Gli1及C-myc mRNA的表达。结果与空白组比较,模型组细胞内可见大量红色荧光标记,MOD值显著增加(P<0.05);模型组20~50 h细胞指数(CI)显著增加(P<0.05),G1、G2期细胞比例降低,S期细胞比例升高(P<0.05),Shh、Smo、Ptch1、Gli1及C-myc mRNA表达显著升高(P<0.05);与模型组比较,半夏泻心汤各剂量组红色荧光标记减弱,其中半夏泻心汤高、中剂量组MOD值明显减少(P<0.05),半夏泻心汤各剂量组20~50 h CI显著减少(P<0.05),G1、G2期细胞比例升高,S期细胞比例降低(P<0.05),Shh、Smo、Ptch1、Gli1及C-myc mRNA表达显著降低(P<0.05)。结论半夏泻心汤可抑制BMSCs外泌体诱导的BGC-823细胞增殖,其机制可能与下调Shh、Smo、Ptch1、Gli1及C-myc mRNA表达,抑制Shh信号通路有关。

Objective To observe the effects of Banxia Xiexin Decoction on the proliferation of human gastric cancer cells BGC-823 induced by exosomes of bone marrow mesenchymal stem cells(BMSCs);To discuss its mechanism of action.Methods BMSCs exosomes were co-cultured with BGC-823 cells in a Transwell chamber.The experiment was divided into blank group,model group,positive control group,Banxia Xiexin Decoction high-,medium-and low-dosage groups.BMSCs exosomes were labeled with fluorescent dye Dil;the uptake of BMSCs exosomes by BGC-823 cells was observed with a laser confocal microscope,and the mean optical density(MOD)was calculated;Real-time label-free cell analysis technology was used to detect cell proliferation;flow cytometry was used to detect cell growth cycle;RT-qPCR was used to detect the expressions of Shh,Smo,Ptch1,Gli1 and C-myc mRNA.Results Compared with the blank group,a large number of red fluorescent markers were seen in the cells of the model group,and the MOD value significantly increased(P<0.05);the cell index(CI)of the model group increased significantly between 20 and 50 hours(P<0.05),the proportion of cells in G1 and G2 phases decreased,the proportion of cells in S phase increased(P<0.05),the expressions of Shh,Smo,Ptch1,Gli1 and C-myc mRNA significantly increased(P<0.05).Compared with the model group,the red fluorescent markers in Banxia Xiexin Decoction groups were weakened,and the MOD value of Banxia Xiexin Decoction high-and medium-dosage groups was significantly reduced(P<0.05);the CI of Banxia Xiexin Decoction groups decreased significantly between 20 and 50 hours(P<0.05),the proportion of cells in G1 and G2 phases increased,and the proportion of cells in S phase decreased(P<0.05),the expressions of Shh,Smo,Ptch1,Gli1 and C-myc mRNA were significantly reduced(P<0.05).Conclusion Banxia Xiexin Decoction can inhibit the proliferation of BGC-823 cells induced by BMSCs exosomes.The mechanism may be related to down-regulating the expressions of Shh,Smo,Ptch1,Gli1 and C-myc mRNA and inhibiting the Shh signaling pathway.