基质交联分子-1调控前列腺癌细胞PC-3中钙离子通道作用研究

The research of STIM1 on store-operated calcium pathway in PC-3 prostate cancer cells

目的探讨前列腺癌细胞PC-3在其细胞内外钙离子浓度改变时,基质交联分子-1(stromal interaction molecule 1,STIMI)实时动态表达水平及其调节钙通道的作用机制。方法利用带有STIM1-YFP荧光探针的腺病毒载体(105病毒颗粒/细胞),转染前列腺癌细胞PC-3 24h后,采用荧光显微镜和细胞计数方法检测病毒探针对细胞形态和数量的毒理影响;利用全内角反射荧光显微镜,观察STIM1在以下几种情况中在细胞膜附近的实时动态变化:100μmol/L肌浆网钙ATP酶(SERCA)抑制剂环匹阿尼酸(CPA)、2mmol/L钙离子螯合剂(EGTA)、100μmol/L卡巴胆碱。结果 STIM1荧光探针转染前列腺癌细胞不影响细胞形态和细胞数量。前列腺癌细胞PC-3在SERCA抑制剂CPA处理后,荧光强度增加到(1.45±0.2)(F/F0,n细胞数=18);当细胞外钙离子浓度降为0并加入EGTA钙离子螯合剂后,细胞荧光强度增加到(1.38±0.2)(F/F0,n=7);加入卡巴胆碱处理后,细胞荧光增加(1.62±0.3)(F/F0,n=23),刺激后荧光强度明显增加,有显著性差异(P<0.01)。结论 STIM1在前列腺癌细胞PC-3中表达并有活性;在其细胞内外钙离子浓度改变时,STIM1在细胞膜上形成多聚体,通过钙库操纵Ca^(2+)通道调节前列腺癌细胞的功能。

Objective To investigate STIM1real-time expression level and its role in adjusting calcium pathway when calcium concentration change in PC-3prostate cancer cells.Methods Using adenovirus with STIM1-YFP fluorescent probe（105viral particles per cell）we infected PC-3prostate cancer cells and after 24 hours fluorescence microscope and cell count methods were used to detect the toxicology of virus probe on cell morphology and number.Real-time changes of STIM1-YFP were observed by using total internal reflection fluorescence microscope under three different conditions：addition of 100 micromoles sarcoplasmic reticulum calcium ATP enzyme inhibitor cyclopiazonic acid;calcium removal in extracellular buffer together with 2micromoles EGTA;addition of 100 micromoles carbachol.Results There was no significant difference in cell morphology and number after transfecting with STIM1 fluorescent probe.Fluorescence intensity increased to（1.45±0.2）（F/F0,n=18）after adding of 100 micromoles CPA.Fluorescence intensity increased to（1.38±0.2）（F/F0,n=7）after adding of 2millimoles EGTA.Fluorescence intensity increased to（1.62±0.3）（F/F0,n=23）after adding of100 micromoles carbachol.There are significant differences（P〈0.01）.Conclusion STIM1 is expressed and active in PC-3prostate cancer cells.STIM1 in polymer is formed on the membrane when the calcium concentration change in the cells inside and outside.The function of prostate cancer cells are adjusting by store-operated calcium pathway.