【作者】 谭兴琴；

【导师】 陈玉培；

【作者基本信息】 重庆医科大学 ， 麻醉学， 2008， 硕士

【摘要】 目的:观察乳化异氟醚(Emulsified isofurane,EI,8 %体积分数)对培养乳鼠心肌细胞模拟缺血再灌注损伤后心肌细胞的保护作用,并对其可能的机制进行探讨,为EI以后在临床的研究及应用提供实验依据。方法:利用原代培养的Wister乳鼠心肌细胞建立模拟心肌缺血再灌注模型,分对照组(不加药物处理,常氧条件下培养6h)、模型(H/R)组(不加药物处理,缺氧3h后复氧3h)、脂肪乳组(在培养基中加入脂肪乳干预2h,余同H/R组)和EI组(在培养基中加入EI干预2h,余同H/R组)。各组心肌细胞做相应分组处理后,利用倒置显微镜观察心肌细胞的生长状态和搏动频率,并用透射电镜观察细胞超微结构改变;XTT比色法测定细胞存活率;收集心肌细胞行超声粉碎,离心取上清,按照试剂盒说明,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性,硫代巴比妥酸显色法测定丙二醛(MDA)含量,并测定缺氧复氧后细胞培养液中乳酸脱氢酶(LDH)活性;流式细胞仪检测心肌细胞凋亡率;RT-PCR检测心肌细胞凋亡基因bcl-2 mRNA、bax mRNA的表达;Western blot定量检测Bcl-2、Bax蛋白的表达。结果:1心肌细胞形态学观察:心肌细胞培养24h出现自发性搏动,72h后聚集成簇,呈现频率为80-100次/min的同步节律性搏动。细胞形态呈梭形或不规则三角形,伪足明显,可发生相互融合,且其超微结构完整、清楚。H/R后心肌细胞皱缩变圆,伪足减少,细胞搏幅减弱、频率减慢甚至出现停搏,超微结构出现线粒体肿胀,内质网空泡等改变。EI和脂肪乳组对H/R心肌细胞的形态和超微结构都有明显改善。2各组心肌细胞存活率比较:与对照组比较,其余各组心肌细胞存活率均降低,差异有统计学意义(P<0.05);而与H/R组比较,EI组和脂肪乳组心肌细胞存活率明显升高,差异有统计学意义(P<0.05)。3各组心肌细胞SOD、LDH活性与MDA含量的比较:与对照组比较,H/R组细胞SOD下降,MDA和LDH升高(均P<0.05)。EI组与H/R组比较,心肌细胞SOD活性提高,MDA和LDH含量降低(均P<0.05)。4 EI和脂肪乳均可显著降低H/R损伤心肌细胞的凋亡率(均P<0.05),但EI组与脂肪乳组比较降低更明显(均P<0.05);EI可显著上调Bcl-2表达(P<0.05)、下调Bax表达(P<0.05);脂肪乳对Bcl-2和Bax表达无影响(P>0.05)。结论:1 H/R对体外培养乳鼠心肌细胞可以造成明显的损伤,表现为细胞存活率降低,心肌酶的漏出,氧自由基的产生增加,心肌细胞超微结构改变,心肌细胞凋亡率增加等。2乳化异氟醚对体外培养乳鼠心肌细胞H/R损伤具有保护作用,表现为能提高细胞存活率,减少心肌酶的漏出,抑制氧自由基产生,维持心肌细胞超微结构完整,抑制心肌细胞凋亡,促进复氧时心肌细胞功能的恢复等。3乳化异氟醚对体外培养乳鼠心肌细胞H/R损伤的保护作用机制可能与其抗自由基损伤、bcl-2基因表达上调和bax基因表达下调有关。更多还原

【Abstract】 Objective: To investigate the protective effects and possible mechanism of emulsified isoflurane on myocardial cells after hypoxia-/ reoxygenation(H/R) in cultured myocardial cells from neonatal rat in order to provide experiment foundation for its later clinical reseach or application.Methods: Myocardial hypoxia/reoxygenation model was established by culturing primary myocardial cell from neonatal rat in vitro. The myocardial cells were divided into four groups, the control group(cultured in normoxia 6h), H/R group(cultured in hypoxia 3h and reoxgenation 3h) , fat milk group(fat milk pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h), EI group(EI pretreatment for 2h and cultured in hypoxia 3h and reoxgenation 3h). Every group cellular morphologic changes and pulsatile frequence were observed under inverted microscope and the ultrastructure of myocardial cells was observed under transmission electron microscope(TEM);Myocardial cell’s survival rate were determined by XTT;Cardiomyocytes were collected and ultrasonic-ccation for the later analysis of SOD and the content of MDA,the activities of plasma superoxide dismutase (SOD) were measured by method of xanthine oxidase and the contents of serum Malonicaldehyde (MDA) by the method of thiobarbituric acid;The activities of lactate dehydrogenase (LDH) in cell-cultured solution after H-R injury were measured according to relevant kits; The apoptotic rate of myocardial cells was determined by flow cytometry; The expression of bcl-2 mRNA and bax mRNA were detected by using RT-PCR method; The expression of Bcl-2/Bax protein were observed by Western blot.Results:1 The morphologic changes of cardiomyocytes: After H/R procedure cardiomyocytes beated unregularily and powerlessly,sells contracted and tended to get round in shape ,cellular pseudopods decreased in H/R,EI ,fat milk groups compared with control group. The ultrastrctural changes of cardiomyocytes were improved in EI,fat milk groups compared with H/R.2 The survival rate of myocardial cell:Compared with control group,the rate decreased in H/R,EI,fat milk groups(p<0.05).but compared with H/R group,the rate increased in EI and fat milk groups.3 SOD activities and LDH,MDA concentration: Compared with control group,SOD activity decreased and MDA,LDH increased in H/R group (p<0.05). Compared with H/R group, SOD activity increased, MDA and LDH decreased (p<0.05)in EI and fat milk groups.4 The apoptotic rate and the expression of correlated apoptotic gene Both EI and fat milk groups could significantly decrease the apoptotic rate (P< 0.05),while EI shoud have stronger effect(P<0.05). EI group signify -cantly up-regulated Bcl-2 expression and down-regulated Bax expression (P<0.05) while fat milk group can’t(P< 0.05).Conclusion1 H/R injured cultured myocardial cells from neonatal rat markedly,including the decreasing of myocardial cell’s survival rate, the increasing of serum myocardial enzyme leakage and myocardial MDA,the elevation of apoptotic rate ,injury of myocardial ultrastructure.2 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R.,which included reduction of myocardial cell’s survival rate,the reduction of the leakage of myocardial enzyme,reduction of oxygen-derived free radials,protection of myocardial ultrastructure,inhibition of apoptosis, better recovery of cardiac function during reoxygenation.3 EI pretreatment had the protective effects on cultured myocardial cells from neonatal rat injured by H-R. The possible mechanism may be that: one is by reducing Oxygen Free Radical,the other is by inhibiting myocardial apoptosis through up-regulating Bcl-2 expression and down- regulating Bax expression .更多还原