上海交通大学学报（医学版）
上海交通大學學報（醫學版）
상해교통대학학보（의학판）
JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY(MEDICAL SCIENCE)
2009年
11期
1324-1327
,共4页
知母活性成分%CHOm2细胞%毒蕈碱样乙酰胆碱2型受体mRNA%Real-time PCR%蛋白质合成抑制剂
知母活性成分%CHOm2細胞%毒蕈堿樣乙酰膽堿2型受體mRNA%Real-time PCR%蛋白質閤成抑製劑
지모활성성분%CHOm2세포%독심감양을선담감2형수체mRNA%Real-time PCR%단백질합성억제제
ZMS%CHOm2 cells%muscarinic M_2 receptor mRNA%Real-time PCR%protein synthesis inhibitor
目的 探讨知母活性成分ZMS提高CHOm2细胞毒蕈碱样乙酰胆碱2型受体 (M_2受体) mRNA表达的机制.方法 体外培养至80% ～90%融合的CHOm2细胞分为ZMS 1组(单纯添加1×10~(-5) mol/L ZMS作用24 h)、ZMS 2组(添加1×10~(-5) mol/L ZMS作用24 h后加入1 μg/mL环己酰亚胺作用12 h)、ZMS 3组(加入1 μg/mL 环己酰亚胺预处理4 h后加入1×10~(-5) mol/L ZMS作用24 h),以上各组分别设立对照组(以等体积DMSO替代ZMS,其他处理与相应ZMS组相同).各组细胞培养基中均加入放线菌素D抑制mRNA合成,于不同时间点收集CHOm2细胞,Real-time PCR检测M_2受体mRNA相对表达量并计算半衰期.结果 与相应对照组比较,ZMS 1组和ZMS 2组CHOm2细胞M_2受体mRNA的半衰期明显延长,分别为(4.75±0.54)h vs(2.13±0.23)h和(5.43±1.13)h vs (2.46±0.09) h(均P<0.05);添加1 μg/mL 环己酰亚胺预处理的ZMS 3组CHOm2细胞M2受体mRNA半衰期的(3.06±0.23)h与其相应对照组的(3.00±0.20)h比较,差异无统计学意义(P>0.05).结论 ZMS提高M2受体mRNA的稳定性需要有新蛋白质合成的参与.
目的 探討知母活性成分ZMS提高CHOm2細胞毒蕈堿樣乙酰膽堿2型受體 (M_2受體) mRNA錶達的機製.方法 體外培養至80% ～90%融閤的CHOm2細胞分為ZMS 1組(單純添加1×10~(-5) mol/L ZMS作用24 h)、ZMS 2組(添加1×10~(-5) mol/L ZMS作用24 h後加入1 μg/mL環己酰亞胺作用12 h)、ZMS 3組(加入1 μg/mL 環己酰亞胺預處理4 h後加入1×10~(-5) mol/L ZMS作用24 h),以上各組分彆設立對照組(以等體積DMSO替代ZMS,其他處理與相應ZMS組相同).各組細胞培養基中均加入放線菌素D抑製mRNA閤成,于不同時間點收集CHOm2細胞,Real-time PCR檢測M_2受體mRNA相對錶達量併計算半衰期.結果 與相應對照組比較,ZMS 1組和ZMS 2組CHOm2細胞M_2受體mRNA的半衰期明顯延長,分彆為(4.75±0.54)h vs(2.13±0.23)h和(5.43±1.13)h vs (2.46±0.09) h(均P<0.05);添加1 μg/mL 環己酰亞胺預處理的ZMS 3組CHOm2細胞M2受體mRNA半衰期的(3.06±0.23)h與其相應對照組的(3.00±0.20)h比較,差異無統計學意義(P>0.05).結論 ZMS提高M2受體mRNA的穩定性需要有新蛋白質閤成的參與.
목적 탐토지모활성성분ZMS제고CHOm2세포독심감양을선담감2형수체 (M_2수체) mRNA표체적궤제.방법 체외배양지80% ～90%융합적CHOm2세포분위ZMS 1조(단순첨가1×10~(-5) mol/L ZMS작용24 h)、ZMS 2조(첨가1×10~(-5) mol/L ZMS작용24 h후가입1 μg/mL배기선아알작용12 h)、ZMS 3조(가입1 μg/mL 배기선아알예처리4 h후가입1×10~(-5) mol/L ZMS작용24 h),이상각조분별설립대조조(이등체적DMSO체대ZMS,기타처리여상응ZMS조상동).각조세포배양기중균가입방선균소D억제mRNA합성,우불동시간점수집CHOm2세포,Real-time PCR검측M_2수체mRNA상대표체량병계산반쇠기.결과 여상응대조조비교,ZMS 1조화ZMS 2조CHOm2세포M_2수체mRNA적반쇠기명현연장,분별위(4.75±0.54)h vs(2.13±0.23)h화(5.43±1.13)h vs (2.46±0.09) h(균P<0.05);첨가1 μg/mL 배기선아알예처리적ZMS 3조CHOm2세포M2수체mRNA반쇠기적(3.06±0.23)h여기상응대조조적(3.00±0.20)h비교,차이무통계학의의(P>0.05).결론 ZMS제고M2수체mRNA적은정성수요유신단백질합성적삼여.
Objective To explore the mechanism of ZMS regulation of M_2 muscarinic receptor mRNA expression. Methods In vitro cultured CHOm2 cells were divided into ZMS 1 group (treatment with 1 × 10~(-5) mol/L ZMS for 24 h), ZMS 2 group (treatment with 1 × 10~(-5) mol/L ZMS for 24 h and 1 μg/mL cycloheximide for 12 h) and ZMS 3 group (treatment with 1 μg/mL cycloheximide for 4 h and 1 × 10 ~(-5) mol/L ZMS for 24 h), and their corresponding control groups were also established (substitution of ZMS by DMSO). Actinomycin D was added to cultured CHOm2 cells of each group to inhibit the synthesis of mRNA. CHOm2 cell samples were taken at different time points, the relative expression of M_2 receptor mRNA was detected by Real-time PCR, and half life of M_2 receptor mRNA was calculated. Results Compared with corresponding control groups, the half life of M_2 receptor mRNA of CHOm2 cells in ZMS 1 group and ZMS 2 group was significantly prolonged [(4.75h± 0.54) h vs (2.13 ±0.23) h, P<0.05; (5.43 ±1.13) h vs (2.46 ±0.09) h, P<0.05]. There was no significant difference in half life of M_2 receptor mRNA of CHOm2 cells between ZMS 3 group and its corresponding control [ ( 3.06 ±0.23) h vs (3.00 ± 0.20) h, P > 0.05]. Conclusion De novo protein synthesis is required for the enhancement of M_2 receptor mRNA stability regulated by ZMS.
