中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
4期
295-299
,共5页
胡艳宁%李庆淑%李智%胡丹%曲彦
鬍豔寧%李慶淑%李智%鬍丹%麯彥
호염저%리경숙%리지%호단%곡언
慢病毒%热休克蛋白70%缺血/缺氧%PC12细胞%钙稳态
慢病毒%熱休剋蛋白70%缺血/缺氧%PC12細胞%鈣穩態
만병독%열휴극단백70%결혈/결양%PC12세포%개은태
Lentivirus%Heat shock protein 70%Ischemia and hypoxia%PC12 cell%Calcium homeostasis
目的：探讨慢病毒介导的热休克蛋白70（HSP70）基因表达对缺血/缺氧嗜铬细胞瘤（PC12）细胞钙稳态的影响及机制。方法取对数生长期的PC12细胞，分为重组慢病毒感染组（感染含HSP70及荧光蛋白基因的慢病毒）、慢病毒对照组（感染含荧光蛋白而不含HSP70基因的慢病毒）及未感染组3组。用反转录-聚合酶链反应（RT-PCR）检测转染细胞HSP70 mRNA表达，蛋白质免疫印迹试验（Western Blot）检测HSP70蛋白表达。PC12细胞缺血/缺氧处理4 h后，用四甲基偶氮唑盐比色法（MTT）检测细胞活性，乳酸脱氢酶（LDH）检测试剂盒测定细胞上清液LDH水平，流式细胞仪检测细胞内游离钙离子（[Ca2+]i）浓度，ATP酶试剂盒测定Na+-K+-ATP酶、 Ca2+-Mg2+-ATP酶、总ATP酶活性。结果重组慢病毒感染组PC12细胞HSP70 mRNA和HSP70蛋白表达呈阳性。经缺血/缺氧处理后，与慢病毒对照组和未感染组比较，重组慢病毒感染组细胞活性明显增强（A值：0.575±0.020比0.395±0.014、0.363±0.045，t1＝17.996，t2＝10.600，均P＜0.001），细胞上清液中LDH水平明显降低（U/L：743.46±23.68比935.43±34.77、962.89±26.68，t1＝11.179，t2＝15.044，均P＜0.001），[Ca2+]i浓度显著降低〔相对荧光强度：（31.60±2.43）%比（41.48±3.33）%、（40.40±3.05）%， t1＝5.853，t2＝5.502，均P＜0.001〕，Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶、总ATP酶活性显著升高〔Na+-K+-ATP酶（μmol·mg-1·h-1）：8.608±0.307比6.728±0.173、6.450±0.091，t1＝9.237、P1＝0.001，t2＝11.675、P2＜0.001；Ca2+-Mg2+-ATP酶（μmol·mg-1·h-1）：10.523±0.036比7.910±0.209、8.064±0.195，t1＝9.718、P1＝0.001，t2＝11.535、P2＜0.001；总ATP酶（μmol·mg-1·h-1）：17.041±0.324比14.150±0.182、13.983±0.085，t1＝16.113，t2＝17.602，均P＜0.001〕。慢病毒对照组与未感染组各指标差异均无统计学意义。结论 HSP70能维持缺血/缺氧PC12细胞钙稳态，可能是其抗凋亡的重要机制之一。
目的：探討慢病毒介導的熱休剋蛋白70（HSP70）基因錶達對缺血/缺氧嗜鉻細胞瘤（PC12）細胞鈣穩態的影響及機製。方法取對數生長期的PC12細胞，分為重組慢病毒感染組（感染含HSP70及熒光蛋白基因的慢病毒）、慢病毒對照組（感染含熒光蛋白而不含HSP70基因的慢病毒）及未感染組3組。用反轉錄-聚閤酶鏈反應（RT-PCR）檢測轉染細胞HSP70 mRNA錶達，蛋白質免疫印跡試驗（Western Blot）檢測HSP70蛋白錶達。PC12細胞缺血/缺氧處理4 h後，用四甲基偶氮唑鹽比色法（MTT）檢測細胞活性，乳痠脫氫酶（LDH）檢測試劑盒測定細胞上清液LDH水平，流式細胞儀檢測細胞內遊離鈣離子（[Ca2+]i）濃度，ATP酶試劑盒測定Na+-K+-ATP酶、 Ca2+-Mg2+-ATP酶、總ATP酶活性。結果重組慢病毒感染組PC12細胞HSP70 mRNA和HSP70蛋白錶達呈暘性。經缺血/缺氧處理後，與慢病毒對照組和未感染組比較，重組慢病毒感染組細胞活性明顯增彊（A值：0.575±0.020比0.395±0.014、0.363±0.045，t1＝17.996，t2＝10.600，均P＜0.001），細胞上清液中LDH水平明顯降低（U/L：743.46±23.68比935.43±34.77、962.89±26.68，t1＝11.179，t2＝15.044，均P＜0.001），[Ca2+]i濃度顯著降低〔相對熒光彊度：（31.60±2.43）%比（41.48±3.33）%、（40.40±3.05）%， t1＝5.853，t2＝5.502，均P＜0.001〕，Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶、總ATP酶活性顯著升高〔Na+-K+-ATP酶（μmol·mg-1·h-1）：8.608±0.307比6.728±0.173、6.450±0.091，t1＝9.237、P1＝0.001，t2＝11.675、P2＜0.001；Ca2+-Mg2+-ATP酶（μmol·mg-1·h-1）：10.523±0.036比7.910±0.209、8.064±0.195，t1＝9.718、P1＝0.001，t2＝11.535、P2＜0.001；總ATP酶（μmol·mg-1·h-1）：17.041±0.324比14.150±0.182、13.983±0.085，t1＝16.113，t2＝17.602，均P＜0.001〕。慢病毒對照組與未感染組各指標差異均無統計學意義。結論 HSP70能維持缺血/缺氧PC12細胞鈣穩態，可能是其抗凋亡的重要機製之一。
목적：탐토만병독개도적열휴극단백70（HSP70）기인표체대결혈/결양기락세포류（PC12）세포개은태적영향급궤제。방법취대수생장기적PC12세포，분위중조만병독감염조（감염함HSP70급형광단백기인적만병독）、만병독대조조（감염함형광단백이불함HSP70기인적만병독）급미감염조3조。용반전록-취합매련반응（RT-PCR）검측전염세포HSP70 mRNA표체，단백질면역인적시험（Western Blot）검측HSP70단백표체。PC12세포결혈/결양처리4 h후，용사갑기우담서염비색법（MTT）검측세포활성，유산탈경매（LDH）검측시제합측정세포상청액LDH수평，류식세포의검측세포내유리개리자（[Ca2+]i）농도，ATP매시제합측정Na+-K+-ATP매、 Ca2+-Mg2+-ATP매、총ATP매활성。결과중조만병독감염조PC12세포HSP70 mRNA화HSP70단백표체정양성。경결혈/결양처리후，여만병독대조조화미감염조비교，중조만병독감염조세포활성명현증강（A치：0.575±0.020비0.395±0.014、0.363±0.045，t1＝17.996，t2＝10.600，균P＜0.001），세포상청액중LDH수평명현강저（U/L：743.46±23.68비935.43±34.77、962.89±26.68，t1＝11.179，t2＝15.044，균P＜0.001），[Ca2+]i농도현저강저〔상대형광강도：（31.60±2.43）%비（41.48±3.33）%、（40.40±3.05）%， t1＝5.853，t2＝5.502，균P＜0.001〕，Na+-K+-ATP매、Ca2+-Mg2+-ATP매、총ATP매활성현저승고〔Na+-K+-ATP매（μmol·mg-1·h-1）：8.608±0.307비6.728±0.173、6.450±0.091，t1＝9.237、P1＝0.001，t2＝11.675、P2＜0.001；Ca2+-Mg2+-ATP매（μmol·mg-1·h-1）：10.523±0.036비7.910±0.209、8.064±0.195，t1＝9.718、P1＝0.001，t2＝11.535、P2＜0.001；총ATP매（μmol·mg-1·h-1）：17.041±0.324비14.150±0.182、13.983±0.085，t1＝16.113，t2＝17.602，균P＜0.001〕。만병독대조조여미감염조각지표차이균무통계학의의。결론 HSP70능유지결혈/결양PC12세포개은태，가능시기항조망적중요궤제지일。
ObjectiveTo investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia, and the mechanism involved.Methods PC12 cells at logarithmic phase were collected, and were divided into recombination lentivirus infection group (infected by lentivirus containing HSP70 and fluorescent gene), lentivirus control group (infected by lentivirus containing fluorescin without HSP70 gene) and non-infection group. HSP70 gene and protein expressions in PC12 cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. After being challenged with ischemia and hypoxia for 4 hours, the viability of cells was detected by methyl thiazolyl tetrazolium (MTT), the levels of lactic acid dehydrogenase (LDH) in cell supernatant were determined by LDH measurement test kit. The concentration of intracelluer calcium ([Ca2+]i) was assayed by flow cytometer. The activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and total ATPase were measured by ATPase test kits.Results The expressions of exogenous HSP70 gene and protein were found by RT-PCR and Western Blot in the recombination lentivirus infection group. After being challenged with ischemia and hypoxia, the viability of cells in the recombination lentivirus infection group was increased significantly as compared with the lentivirus control group and non-infection group (A value: 0.575±0.020 vs. 0.395±0.014, 0.363±0.045,t1= 17.996,t2= 10.600, bothP< 0.001), the levels of LDH in culture medium and the concentration of [Ca2+]i were decreased significantly [LDH (U/L): 743.46±23.68 vs. 935.43±34.77, 962.89±26.68,t1= 11.179, t2= 15.044, bothP< 0.001; [Ca2+]i relative fluorescence: (31.60±2.43)% vs. (41.48±3.33)%, (40.40±3.05)%, t1= 5.853,t2= 5.502, bothP< 0.001], and the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase and total ATPase were increased significantly [Na+-K+-ATPase (μmol·mg-1·h-1): 8.608±0.307 vs. 6.728±0.173, 6.450±0.091,t1=9.237,P1= 0.001,t2= 11.675,P2< 0.001; Ca2+-Mg2+-ATPase (μmol·mg-1·h-1): 10.523±0.036 vs. 7.910±0.209, 8.064±0.195,t1= 9.718,P1= 0.001,t2= 11.535,P2<0.001; total ATPase (μmol·mg-1·h-1): 17.041±0.324 vs. 14.150±0.182, 13.983±0.085,t1= 16.113,t2= 17.602, bothP<0.001]. There was no statistical difference in above indexes between lentivirus control group and non-infection group.Conclusion HSP70 can maintain the PC12 cells calcium homeostasis, which may be one of the important mechanisms of anti-apoptosis.
