热休克蛋白70对缺血／缺氧嗜铬细胞瘤细胞细胞膜钙通道的调节机制

The modulatory effects of heat shock protein 70 on membrane calcium homeostasis of PC12 cells induced by ischemic/hypoxia

目的探讨慢病毒介导的热休克蛋白70（HSP70）表达对缺血／缺氧神经细胞细胞膜钙离子通道的影响及其机制。方法取对数生长期的嗜铬细胞瘤细胞（PC12），以缺氧6h、复氧12h刺激PC12细胞模拟体内神经细胞遭受缺血／再灌注时的病理过程，将细胞分为非感染组，感染含绿色荧光蛋白（GFP）基因但不含HSP70基因的慢病毒对照组（GFP慢病毒对照组），及感染含重组HSP70和GFP基因的慢病毒实验组（HSP重组慢病毒感染组）。采用实时荧光定量反转录-聚合酶链反应（RT—PCR）及蛋白质免疫印迹试验（Western Blot）检测L型钙通道cav1．2、cav1．3亚基，受体门控通道NR1、NR2亚基，及Na^＋／Ca^2＋交换体（NCX）的mRNA和蛋白表达。结果缺氧／复氧处理后，HSP70重组慢病毒感染组细胞cav1．2、NR1、NR2的mRNA及蛋白表达均明显低于GFP慢病毒对照组和非感染组[cav1.2 mRNA（2^-△△Ct）：3．13±0．46比5．12±0．52、5．13±0．66，NR1 mRNA（2^-△△Ct）：1．61±0．44比3．23±0．82、3-31±0．78，NR2 mRNA（2^-△△Ct）：2．09±0．41比3．91±0．64、3．88±0．62；car1.2蛋白（灰度值）：2．82±0．39比3．98±0．23、3．96±0．24，NR1蛋白（灰度值）：1．84±0．35比2．79±0．21、2．86±0．23，NR2蛋白（灰度值）：0．87±0．24比1．57±0．31、1．33±0．44；均P〈0.01]；而GFP慢病毒对照组与非感染组间细胞car1.2、NR1、NR2的mRNA及蛋白表达差异均无统计学意义（均P〉0．01）。非感染组、GFP慢病毒对照组和HSP70重组慢病毒感染组间细胞cav1.3、NCX的mRNA和蛋白表达差异均无统计学意义[cav1.3 mRNA（2^-△△Ct）：4．82±0．32、4．72±0．36、4．82±0．29，NCX mRNA（2^-△△Ct）：3．49±0．78、3．47±0．71、3．56±0．65；cav1.3蛋白（灰度值）：2．63±0．40、2．64±0．39、2．68±0．39，NCX蛋白（灰度值）：3．27±0．48、3．34±0．48、3．31±0．42；均P〉0．01]。结论外源性HSP70可能通过抑制PC12细胞膜L型钙通道cav1.2亚基及受体门控通道NR1、NR2亚基的表达，对缺血／缺氧引起的PC12细胞损伤具有保护作用。

Objective To investigate the effects of lentivirus-mediated heat shock protein 70 （HSP70） gene on voltage-gated calcium channels on the cell membrane of pheochromocytoma cell 12 （PC12 cells） induced by ischemic/ hypoxia and its mechanisms. Methods PC12 cells at logarithmic phase were collected, which were challenged with hypoxia for 6 hours and reoxygenation for 12 hours to stimulate the nerve cells suffered ischemia/reperfusion pathological process in vitro. PC12 cells were divided into non-infection group, infected by lentivirus containing green fluorescent protein （GFP） without HSP70 gene lentivirus control group （GFP lentivirns control group）, and infected by lentivirus containing HSP70 and GFP gene recombined lentiviral infection group （HSP70 recombined lentiviral infection group）. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western Blot were used to determine mRNA and protein expressions of L-type voltage-gated Ca^2± channel subunits cav1.2 and car1.3, receptor gated channel subunits NR1 and NR2, and Na^±/Ca^2± exchange （NCX） in PC12 cells. Results After being challenged with hypoxia/reoxygenation, the mRNA and protein expressions of cav1.2, NR1 and NR2 in the PC12 cells were significantly lower in HSP70 recombined lentiviral infection group than those of GFP lentivirus control group and non-infection group [cav1.2 mRNA （2^-△△Ct）： 3.13±0.46 vs. 5.12±0.52, 5.13±0.66; NR1 mRNA （2^-△△Ct）： 1.61 ±0.44 vs. 3.23 ±0.82, 3.31 ± 0.78; NR2 mRNA （2^-△△Ct）： 2.09-4-0.41 vs. 3.91 ±0.64, 3.88 ±0.62; cav1.2 protein （gray value）： 2.82 ± 0.39 vs. 3.98 ± 0.23, 3.96 ± 0.24; NR1 protein （gray value）： 1.84 ± 0.35 vs. 2.79 ± 0.21, 2.86± 0.23; NR2 protein （gray value）： 0.87±_0.24 vs. 1.57±0.31, 1.33±0.44; all P 〈 0.01]. But there were no statistical differences in the mRNA and protein expressions of car1.2, NR1 and NR2 between GFP lentivirus control group and non-infection group （all P 〉 0.01）. There were no statistical differences in the mRNA and protein expressions of cav1.3 and NCX among non-infection group, GFP lentivirus control group and HSP70 recombined lentiviral infection group [cav1.3 mRNA （2^-△△Ct）： 4.82 ± 0.32, 4.72 ± 0.36, 4.82 ± 0.29; NCX mRNA （2^-△△Ct）： 3.49 ± 0.78, 3.47 ± 0.71, 3.56 ±_ 0.65; cav1.3 protein （gray value）： 2.63 ± 0.40, 2.64 ± 0.39, 2.68 ± 0.39; NCX protein （gray value）： 3.27 ± 0.48, 3.34 ± 0.48, 3.31 ± 0.42; all P 〉 0.01]. Conclusion Exogenous HSP70 affects the expression of L-type voltage-gated Ca^2＋ channel subunit car1.2 and receptor gated channel subunits NR1 and NR2, which may protect PC12 cells from the injury caused by ischemic/hypoxia.