摘要
目的:研究加味四君子汤含药血清对胃癌细胞SGC-7901凋亡相关因子表达的影响,进一步探讨其具体抗肿瘤作用机制。方法:40只SD大鼠随机分为加味四君子汤低、中、高剂量组(0.213,0.426,0.853 g·kg^(-1)),空白组,每组10只,空白组给予等体积的生理盐水灌胃,各组连续灌胃10 d,末次给药1.5 h后,水合氯醛腹腔麻醉,心脏采血,分离血清,56℃灭菌,0.22μm过滤,制备加味四君子汤高、中、低剂量组含药血清,各剂量组含药血清孵育胃癌细胞SGC-7901,运用流式细胞仪检测细胞早期和晚期凋亡情况,运用实时荧光定量聚合酶链式反应(Real-time PCR)技术检测肿瘤抑制基因p53,c-核蛋白类基因(c-Myc),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),凋亡相关基因B细胞淋巴瘤-2(Bcl-2)mRNA的表达,运用细胞免疫荧光技术检测p53,c-Myc,Caspase-3,Bcl-2蛋白的相对表达量情况。结果:与空白组比较,加味四君子汤含药血清高剂量组细胞凋亡比率显著升高(P<0.01),且可使胃癌细胞SGC-7901早期+晚期凋亡率达到22.58%(P<0.01)。Real-time PCR结果显示加味四君子汤中剂量组能显著促进Caspase-3 mRNA表达(P<0.01),加味四君子汤高剂量组能显著上调p53及c-Myc mRNA表达量(P<0.01),加味四君子汤高剂量组显著抑制Bcl-2 mRNA表达(P<0.01)。免疫荧光结果显示加味加味四君子汤高剂量组能使c-Myc,Caspase-3,p53蛋白表达水平显著升高(P<0.01),而Bcl-2蛋白表达水平显著降低(P<0.05)。结论:加味四君子汤含药血清能抑制抗凋亡蛋白Bcl-2的表达,促进凋亡相关分子p53,c-Myc,Caspase-3的表达,发挥抗肿瘤作用。
Objective: To explore the effect of modified Si Junzitang( MSJZT) drug serum on the expression of apoptosis-related molecules of gastric cancer cell SGC-7901 and further its anti-tumor mechanism.Method: A total of 40 SD rats were randomly divided into four groups: low-dose,middle-dose,high-dose MSJZT( 0. 213,0. 426,0. 853 g·kg^-1) groups and normal group( n = 10). The treatment groups were administrated through gastric perfusion,and the normal group was given the equivalent volume of normal saline for 10 days.1. 5 h after the last treatment,chloral hydrate peritoneal anesthesia was performed,blood was collected from heart,and different doses of serum were separated to prepare drug-containing serum of low-dose,middle-dose,high-dose MSJZT groups,in order to incubate SGC-7901 gastric cancer cell. Early and late apoptosis rates were detected with flow cytometry. Afterwards,the tumor suppressor gene p53,c-nucleoprotein gene( c-Myc),cysteine-aspartic acid protease-3( Caspase-3), B-cell lymphoma-2( Bcl-2) mRNA expressions were confirmed by fluorescence quantitative polymerase chain reaction( Real-time PCR). The protein expressions of p53,c-Myc,Caspase-3,Bcl-2 were detected by immunofluorescence. Result: Compared with the normal group,the high-dose MSJZT group could obviously increase the apoptosis rate to 22. 58%( P <0. 01). The results of Real-time PCR showed that the middle-dose MSJZT group significantly promoted the mRNA expression of Caspase-3( P <0. 01),the medium-dose group significantly increased the mRNA expressions of p53 and c-Myc( P <0. 01),and the high-dose group significantly inhibited the mRNA expression of Bcl-2( P <0. 01). The immunofluorescence results showed that the high-dose MSJZT group could significantly increase the protein expressions of c-Myc, Caspase-3 and p53( P <0. 01),while the protein expression of Bcl-2 was significantly reduced( P< 0. 05). Conclusion: MSJZT drug serum could exert an anti-tumor effect by inhibiting the expression of the anti-apoptotic protein Bcl-2,and promoting the expressions of pro-apoptotic-related molecules p53,c-Myc,Caspase-3.
作者
聂闪闪
李洵
赵玉航
王东生
NIE Shan-shan;LI Xun;ZHAO Yu-hang;WANG Dong-sheng(Xiangya Hospital, Central South University,Changsha 410008,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2019年第9期25-30,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81373554)
湖南省科技计划项目(2016SK2018)
