中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
Chinese Journal of Anesthesiology
2015年
6期
762-765
,共4页
陈满丽%陈立建%万俐娟%张雷%都建%顾尔伟
陳滿麗%陳立建%萬俐娟%張雷%都建%顧爾偉
진만려%진립건%만리연%장뢰%도건%고이위
热休克蛋白质类%糖尿病%哌啶类%心肌再灌注损伤%缺血后处理
熱休剋蛋白質類%糖尿病%哌啶類%心肌再灌註損傷%缺血後處理
열휴극단백질류%당뇨병%고정류%심기재관주손상%결혈후처리
Heat-shock proteins%Diabetes mellitus%Piperidines%Myocardial reperfusion injury%Ischemic postconditioning
目的 评价葡萄糖调节蛋白78(GRP78)与糖尿病因素影响瑞芬太尼后处理心肌保护作用的关系.方法 H9c2细胞在含10％胎牛血清DMEM/F12培养基中培养、传代,接种于96孔板(100μl/孔)或6孔板(2 ml/孔),接种密度10 5个/ml,接种12 h后细胞贴壁.采用随机数字表法,将细胞分为6组(n=24):正常糖对照组(NC组)、正常糖缺氧复氧组(NHR组)、正常糖瑞芬太尼后处理组(NRP组)、高糖对照组(HC组)、高糖缺氧复氧组(HHR组)和高糖瑞芬太尼后处理组(HRP组).NC组、NHR组和NRP组细胞换用正常糖DMEM培养基(5.5 mmol/L)培养48 h;HC组、HHR组和HRP组细胞换用高糖DMEM培养基(25.0 mmol/L)孵育48 h.NHR组、NRP组、HHR组和HRP组弃去培养液,加入Tyrode液,将培养板置于95％N2-5％CO2培养罐中孵育5h,NHR组和HHR组更换为含10％胎牛血清的相应糖DMEM培养基孵育1h,NRP组和HRP组更换为含终浓度为1μmol/L瑞芬太尼的DMEM培养基孵育1h.于复氧1h时,每组取9孔细胞,采用CCK8法检测细胞活力;每组取12孔细胞,采用化学比色法测定培养液乳酸脱氢酶(LDH)活性;每组取3孔细胞,采用Western blot法测定GRP78表达.结果 与NC组比较,NHR组细胞活力降低,LDH活性升高,GRP78表达上调(P＜0.05);与NHR组比较,NRP组细胞活力升高,LDH活性降低,GRP78表达下调,HRP组细胞活力降低,LDH活性升高,GRP78表达上调(P＜0.05);与HC组比较,HHR组细胞活力降低,LDH活性升高,GRP78表达上调(P＜0.05);与HHR组比较,HRP组细胞活力、LDH活性和GRP78表达差异无统计学意义(P＞0.05);与NRP组比较,HRP组细胞活力降低,LDH活性升高,GRP78表达上调(P＜0.05).结论 糖尿病因素取消瑞芬太尼后处理心肌保护作用的机制可能与其上调GRP78的表达有关.
目的 評價葡萄糖調節蛋白78(GRP78)與糖尿病因素影響瑞芬太尼後處理心肌保護作用的關繫.方法 H9c2細胞在含10％胎牛血清DMEM/F12培養基中培養、傳代,接種于96孔闆(100μl/孔)或6孔闆(2 ml/孔),接種密度10 5箇/ml,接種12 h後細胞貼壁.採用隨機數字錶法,將細胞分為6組(n=24):正常糖對照組(NC組)、正常糖缺氧複氧組(NHR組)、正常糖瑞芬太尼後處理組(NRP組)、高糖對照組(HC組)、高糖缺氧複氧組(HHR組)和高糖瑞芬太尼後處理組(HRP組).NC組、NHR組和NRP組細胞換用正常糖DMEM培養基(5.5 mmol/L)培養48 h;HC組、HHR組和HRP組細胞換用高糖DMEM培養基(25.0 mmol/L)孵育48 h.NHR組、NRP組、HHR組和HRP組棄去培養液,加入Tyrode液,將培養闆置于95％N2-5％CO2培養罐中孵育5h,NHR組和HHR組更換為含10％胎牛血清的相應糖DMEM培養基孵育1h,NRP組和HRP組更換為含終濃度為1μmol/L瑞芬太尼的DMEM培養基孵育1h.于複氧1h時,每組取9孔細胞,採用CCK8法檢測細胞活力;每組取12孔細胞,採用化學比色法測定培養液乳痠脫氫酶(LDH)活性;每組取3孔細胞,採用Western blot法測定GRP78錶達.結果 與NC組比較,NHR組細胞活力降低,LDH活性升高,GRP78錶達上調(P＜0.05);與NHR組比較,NRP組細胞活力升高,LDH活性降低,GRP78錶達下調,HRP組細胞活力降低,LDH活性升高,GRP78錶達上調(P＜0.05);與HC組比較,HHR組細胞活力降低,LDH活性升高,GRP78錶達上調(P＜0.05);與HHR組比較,HRP組細胞活力、LDH活性和GRP78錶達差異無統計學意義(P＞0.05);與NRP組比較,HRP組細胞活力降低,LDH活性升高,GRP78錶達上調(P＜0.05).結論 糖尿病因素取消瑞芬太尼後處理心肌保護作用的機製可能與其上調GRP78的錶達有關.
목적 평개포도당조절단백78(GRP78)여당뇨병인소영향서분태니후처리심기보호작용적관계.방법 H9c2세포재함10％태우혈청DMEM/F12배양기중배양、전대,접충우96공판(100μl/공)혹6공판(2 ml/공),접충밀도10 5개/ml,접충12 h후세포첩벽.채용수궤수자표법,장세포분위6조(n=24):정상당대조조(NC조)、정상당결양복양조(NHR조)、정상당서분태니후처리조(NRP조)、고당대조조(HC조)、고당결양복양조(HHR조)화고당서분태니후처리조(HRP조).NC조、NHR조화NRP조세포환용정상당DMEM배양기(5.5 mmol/L)배양48 h;HC조、HHR조화HRP조세포환용고당DMEM배양기(25.0 mmol/L)부육48 h.NHR조、NRP조、HHR조화HRP조기거배양액,가입Tyrode액,장배양판치우95％N2-5％CO2배양관중부육5h,NHR조화HHR조경환위함10％태우혈청적상응당DMEM배양기부육1h,NRP조화HRP조경환위함종농도위1μmol/L서분태니적DMEM배양기부육1h.우복양1h시,매조취9공세포,채용CCK8법검측세포활력;매조취12공세포,채용화학비색법측정배양액유산탈경매(LDH)활성;매조취3공세포,채용Western blot법측정GRP78표체.결과 여NC조비교,NHR조세포활력강저,LDH활성승고,GRP78표체상조(P＜0.05);여NHR조비교,NRP조세포활력승고,LDH활성강저,GRP78표체하조,HRP조세포활력강저,LDH활성승고,GRP78표체상조(P＜0.05);여HC조비교,HHR조세포활력강저,LDH활성승고,GRP78표체상조(P＜0.05);여HHR조비교,HRP조세포활력、LDH활성화GRP78표체차이무통계학의의(P＞0.05);여NRP조비교,HRP조세포활력강저,LDH활성승고,GRP78표체상조(P＜0.05).결론 당뇨병인소취소서분태니후처리심기보호작용적궤제가능여기상조GRP78적표체유관.
Objective To evaluate the relationship between glucose-regulated protein 78 (GRP78) and diabetes mellitus-induced influence on myocardial protection provided by remifentanil postconditioning in vitro.Methods H9c2 cells were cultured in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum.The cells were seeded in 96-well (100 μl/well) or 6-well (2 ml/well) plates at the density of l05 cells/ml.The cells were then randomly divided into 6 groups (n =24 each) using a random number table:normoglycemic control group (group NC),normoglycemic hypoxia/reoxygenation (H/R) group (group NHR),normoglycemic remifentanil postconditioning group (group NRP),hyperglycemic control group (group HC),hyperglycemic H/R group (group HHR),and hyperglycemic remifentanil postconditioning group (group HRP).In NC,NHR and NRP groups,the cells were cultured in normoglyccmic culture medium (5.5 mmol/L) for 48 h.In HC,HHR and HRP groups,the cells were incubated in hyperglycemic culture medium (25.0 mmol/L) for 48 h.In NHR,NRP,HHR and HRP groups,after changing the culture medium for Tyrode solution,the cells were exposed to 95％ N2-5％ CO2 in an incubator at 37 ℃ for 5 h.Subsequently,in NHR and HHR groups,the culture medium was changed to DMEM/F12 culture medium supplemented with 10％ fetal bovine serum and glucose at the corresponding concentration,and the cells were incubated for 1 h;in NRP and HRP groups,the cells were incubated for 1 h in the DMEM culture medium containing remifentanil at the final concentration of 1 μmol/L.At 1 h of reoxygenation,the cells of 9 wells in each group were selected to measure the cell viability by CCK8 assay,the cells of 12 wells in each group were selected to determine the activity of lactic dehydrogenase (LDH) released in the supernatant using colorimetric method,and the cells of 3 wells in each group were selected to detect the expression of GRP78 by Western blot.Results Compared with group NC,the cell viability was significantly decreased,the LDH activity was increased,and the expression of GRP78 was up-regulated in group NHR.Compared with group NHR,the cell viability was significantly increased,the LDH activity was decreased,and the expression of GRP78 was down-regulated in group NRP,and the cell viability was significantly decreased,the LDH activity was increased,and the expression of GRP78 was up-regulated in group HRP.Compared with group HC,the cell viability was significantly decreased,the LDH activity was increased,and the expression of GRP78 was up-regulated in group HHR.There was no significant change in the parameters mentioned above between group HRP and group HHR.Compared with group NRP,the cell viability was significantly decreased,the LDH activity was increased,and the expression of GRP78 was up-regulated in group HRP.Conclusion Up-regulation of GRP78 expression may be involved in the mechanism by which diabetes mellitus negates myocardial protection induced by remifentanil postconditioning in vitro.
