肌质网IP3R/RyR介导胞浆内钙离子浓度变化与子宫肌瘤细胞缺氧凋亡的研究

The Relationship between Cytosolic Calcium Concentration Changes Mediated by Sarcoplasmic Reticulum IP3R /RyR and Uterine Leiomyoma Cells Apoptosis Under Hypoxic Condition

目的:探讨肌质网质膜上钙通道蛋白三磷酸肌醇受体(IP3R)/兰尼碱受体(RyR)介导胞浆内钙离子浓度变化与子宫肌瘤细胞缺氧凋亡的关系。方法:选择上海市杨浦区中心医院收治的子宫肌瘤患者20例,均行腹腔镜下子宫动脉阻断术。术中取子宫肌瘤及邻近平滑肌组织标本各20例,运用RT-PCR、Western blot方法检测并比较两种组织中IP3R/RyR的表达情况;原代培养子宫肌瘤与平滑肌细胞,取缺氧培养6小时后细胞分别分成4组:加入IP3R阻滞剂肝素(A组)、加入RyR阻滞剂钌红(B组)、加入肝素及钌红(C组)和空白组(D组),检测两种组织4组细胞中IP3R、RyR及凋亡因子细胞色素c(Cytc)的表达水平,同时检测细胞内钙离子(Ca2+)浓度。结果:①子宫动脉阻断前平滑肌组织中IP3RmRNA及蛋白表达水平均显著高于肌瘤组织(P=0.000);RyR mRNA及蛋白表达在两种组织中无差异(P>0.05)。②缺氧模型下,在D组肌瘤细胞中IP3R、RyR及Cytc蛋白表达水平均显著高于平滑肌细胞(P=0.000;P=0.007;P=0.011);而在A组和C组两种细胞中IP3R、RyR及Cytc蛋白表达水平差异均无统计学意义(P>0.05)。③缺氧模型下,4组肌瘤细胞中Ca2+浓度普遍高于平滑肌细胞,但仅B组及D组比较,差异有统计学意义(P=0.037;P=0.043)。结论:缺氧刺激下,肌瘤细胞内Ca2+浓度较平滑肌细胞明显升高,Ca2+释放主要受胞内肌质网IP3R钙通道调控,Ca2+浓度升高激活了内源性(线粒体)细胞凋亡途径,诱导大量肌瘤细胞发生凋亡。

Objective： To investigate the relationship between cytosolic calcium concentration changes mediated by sarcoplasmic reticulum IP3R/RyR and uterine leiomyoma cells apoptosis under hypoxic condi- tion. Methods ：20 cases with uterine myoma were recruited, myometrial and myomal tissues specimens were obtained during operation by lapar0scopic uterine artery blockage. By RT-PCR and Western blot, IP3R/RyR mRNA and protein expressions in both tissues were detected and compared. Uterine leiomyoma cells and smooth muscle cells were culture primarily. After 6 h of hypoxia incubation, the cells were taken out and then divided into 4 groups：group A （adding the IP3R blockers heparin ）, group B （ adding RyR blockers rutheni- um red ）, group C （ adding heparin and ruthenium red ）, group D （the blank group）, RT-PCR and West- ern blot were used to assayed IP3R, RyR and apoptosis factor Cytc expression levels, simultaneously the concentration of intracellular calcium ion（Ca2＋ ） were detected. Results：①IP3R mRNA and protein expres- sion levels of smooth muscle tissues were significantly higher than those of leiomyoma （ P = 0. 000 ）, the difference between RyR mRNA and protein expression in two tissues is not statistically significant（ P 〉 0.05）. ② Under hypoxia model, in group D, compared with smooth muscle cells, IP3R/RyR and Cytc protein ex-pression levels in leiomyoma cells were significantly higher（ P =0. 000;P = 0. 007;P = 0.011 ）. The levels of IP3R/RyR and Cytc protein expressions in group A were not significantly different from those of group C（ P 〉 0.05）. ③Under hypoxia model, intracellular Ca2＋ concentrations of 4 groups leiomyoma cells were generally higher than those of the smooth muscle cells, but only between group B and group D, the difference was statistically significant （ P = 0. 037; P = 0. 043 ） .i Conclusions： Under hypoxia stress, the intracellular Ca2＋ concentrations of smooth muscle cells increase more significantly than those of leiomyoma cells, Ca2＋ re- lease is dominantly regulated by intracellular Ca2＋ channel, IP3R of sarcoplasmic reticulum. The increasing of Ca2＋ concentration activates endogenous （mitochondrial） apoptosis pathways, which may subsequently induce large leiomyoma cells to apoptosis.