【作者】 黄建康；

【导师】 卢榜裕；

【作者基本信息】 广西医科大学 ， 微创外科， 2010， 博士

【摘要】 第一章BRL大鼠肝细胞HSP70的表达及意义目的：通过热休克预处理提高BRL大鼠肝脏细胞HSP70的表达水平,建立体外实验研究的热休克模型。方法：选择生长良好的且未铺满培养瓶底的BRL大鼠肝细胞做热休克反应。从37℃C02培养箱中拿出培养瓶,放入预设温度(40～50℃)的水浴箱中,孵育30′后,再放入培养箱复温培养。分别培养2h后测细胞成活率,选择成活率在95%以上的温度再进一步进行热处理30′后37℃培养48小时,再次测定细胞成活率,发现40～42℃水浴30′,37℃培养48h后细胞成活率在95%以上。BRL大鼠肝脏细胞40～42℃热处理30′后分别培养2h、8h、12h、24h、48h,用Western-blot检测HSP70蛋白表达情况。正常BRL大鼠肝细胞用不同浓度的谷氨酰胺、槲皮素分别进行培养2h后,42℃热浴30′,于培养2h、8h、12h、24h、48h后测细胞成活率及用Western-blot检测HSP70蛋白、GR蛋白表达情况。分组：37℃培养BRL大鼠肝细胞为正常对照组,42℃热处理BRL大鼠肝细胞30′为热休克组,槲皮素、谷氨酰胺培养肝细胞2h再热处理分别为槲皮素干预组、谷氨酰胺干预组。结果：40～42℃热处理BRL大鼠肝细胞30′后,37℃培养48h细胞的成活率大于95%。正常BRL大鼠肝细胞HSP70含量较低,热处理后2hHSP70表达迅速增加,8h明显升高,12h达到顶峰,24小时仍处在较高水平,48h稍高于正常。40℃、41℃、42℃热处理后BRL大鼠肝细胞HSP70的表达在各时相点均高于正常对照组(P＜0.05),发现在42℃2h、8h、12h、24hHSP70的表达明显高于40℃、41℃(P＜0.05),48小时稍高于40℃、41℃(P＞0.05)。41℃各时相点HSP70表达均高于40℃,但无显著性差异(P＞0.05)。因此选用42℃热处理30′做为BRL大鼠肝细胞的热休克反应温度及时间。用不同浓度的谷氨酰胺培养肝细胞2h、42℃热处理30′,37℃培养2h后检测HSP70表达,发现浓度为8mmol／L谷氨酰胺诱导HSP70表达最高；用不同浓度的槲皮素培养肝细胞2h,然后42℃热处理30′,37℃培养2h,发现150umol／L浓度槲皮素干预后HSP70表达最低。分别用浓度8mmol／L谷氨酰胺、150umol／L浓度槲皮素培养肝细胞2h后进行热休克反应并检测2h、8h、12h、24h、48hHSP70、GR的表达。谷氨酰胺干预组HSP70的表达在各时相点均显著高于正常对照组(P＜0.05)；与热休克组相比,在8h、12h、24h有显著性差异(P＜0.05)。槲皮素干预组HSP70的表达在2h、8h时相点显著低于正常对照组(P＜0.05),12h、24h稍低于正常组,48h接近正常(P＞0.05)。谷氨酰胺干预组HSP70的表达在各时相点均显著高于槲皮素干预组(P＜0.05)。谷氨酰胺干预组各时相点细胞成活率均高于95%,槲皮素干预组细胞成活率2h、8h明显降低,12小时后逐渐升高,48h仍低于85%。BRL大鼠肝细胞42℃热休克30′后37℃培养,在各时相点GR表达均较正常对照组下降,2h开始下降,8h下降明显,12h降至最低,24小时仍较低,48h接近正常。8mmol／L谷氨酰胺干预组在各时相点GR均较热休克组有所下降,8h、12h有显著性差异(P＜0.05)；150umol／L槲皮素干预组在各时相点GR均较正常组下降,2h、8h较热休克组有轻度升高,但无统计学意义；槲皮素干预组GR表达较谷氨酰胺干预组在2h、8h有所升高(P＜0.05)。结论：(1)正常BRL大鼠肝细胞HSP70表达较低,42℃热处理30′方法可以成功诱导BRL大鼠肝细胞高表达HSP70,此方法不会导致连续培养48h后细胞成活数量的明显变化,成功建立了BRL大鼠肝细胞热休克模型。(2)谷氨酰胺可以诱导HSP70的高表达,对细胞有保护作用,槲皮素可以明显抑制HSP70的表达,导致细胞成活数量的明显降低。8mmol／L谷氨酰胺、150umol／L槲皮素分别为诱导、抑制HSP70表达的最佳浓度,并为动物实验提供剂量参考。(3)热休克反应及谷氨酰胺可以诱导BRL大鼠肝细胞HSP70高表达,GR呈低表达。槲皮素干预后,GR也呈低表达,但较谷氨酰胺干预组有所升高。在热休克反应中HSP70与GR有一定的负相关,HSP70调节GR的活性,两者关系有待进一步研究。第二章不同切肝量大鼠HSP70的表达及意义目的：检测大鼠不同程度手术创伤后血清及肝脏组织中HSP70的表达,研究HSP70与手术创伤程度、创伤致内环境及肝功能改变、GC、GR之间的关系,探讨HSP70评价手术创伤程度及对内环境稳态、肝脏的保护作用及机制。方法：1.动物模型建立：雄性健康Sprague-Dawley(SD)大鼠随机分为(1)正常对照组：大鼠不作任何手术处理。(2)手术创伤组：①轻度创伤组：大鼠肝脏左外叶切除组；②中度创伤组：大鼠肝脏左外叶和右叶切除组；③重度创伤组：大鼠肝脏左外叶、右叶、乳头叶和尾状叶切除组。(3)谷氨酰胺干预组：在手术前2h尾静脉推注0.75g／kg谷氨酰胺,分为谷氨酰胺干预轻度创伤组、谷氨酰胺干预中度创伤组、谷氨酰胺干预重度创伤组。(4)槲皮素干预组：在手术前2h腹腔注入7mg／kg,分为槲皮素干预轻度创伤组、槲皮素干预中度创伤组、槲皮素干预重度创伤组。(5)米非司酮干预组：在手术前2h肌肉注射5mg／kg米非司酮,分为米非司酮干预轻度创伤组、米非司酮干预中度创伤组、米非司酮干预重度创伤组。每一程度创伤组有50只大鼠,在5个时间点(术后2h、8h、12h、24h、48h)进行分批处理大白鼠(每时间点均为10只),每组大鼠在同样的环境中饲养,不同组间及时间点间大鼠体重、年龄无差异。2.指标检测：分别采取正常对照组、手术创伤组及干预组各组术前、术后2h、8h、12h、24h、48h血液标本及肝脏组织,用ELISA法检测血清HSP70水平,用血气分析仪检测血PH,全自动生化仪检测血K+、Na+及Ca2+浓度；化学发光仪检测血清皮质醇含量；用western-blot、实时荧光定量PCR检测大鼠肝脏组织HSP70、HSP70mRNA及GR、GRmRNA表达变化情况。结果：(1)模型的成功建立：采用对SD大鼠行肝叶切除术成功建立不同程度手术创伤的模型,手术轻度创伤组、中度创伤组、重度创伤组大鼠存活率分别为98.0%、94.0%、92.0%,组间比较无显著性差异(P＞0.05),为研究不同程度手术创伤对机体的影响建立了动物模型。(2)血清HSP70的表达：①手术创伤组：手术创伤后2h血清HSP70表达开始增加,12h达到高峰,以后逐渐下降,48h仍高于正常水平。随着手术创伤程度的增加血清HSP70表达水平均升高；术后8、12、24h时相点轻度、中度、重度创伤组之间比较有显著差异性(P＜0.05)。血清皮质醇于术后2h迅速增加,8h达到高峰,24小时仍有较高浓度,48h略高于正常。随着手术创伤程度的增加,皮质醇浓度亦增加,术后2、8、12、24h时相点轻度、中度、重度之间比较有显著差异(P＜0.05)。②谷氨酰胺干预组：在每一时相点较手术创伤组血清HSP70的表达均增加,在术后8、12、24h时相点增加明显,差异有显著性(P＜0.05)。与手术创伤组比较,每一时相点皮质醇浓度均有所下降,在术后2、8、12h有显著性差异(P＜0.05)。③槲皮素干预组：较手术创伤组相应的各时相点HSP70表达均有所下降,在术后2、8、12、24h下降明显(P＜0.05),但组内之间无显著性差异；与手术创伤组相比各时相点的皮质醇浓度有所增加,在术后2、8、12h有显著性差异(P＜0.05)。与谷氨酰胺干预组相比在各时相点HSP70均显著下降(P＜0.05)；而皮质醇浓度有所升高,在术后2、8、12、24h差异有显著性(P＜0.05)。④米非司酮干预组：较手术创伤组相对应的各时相点血清HSP70表达和皮质醇浓度均明显升高(P＜0.05)。与槲皮素干预组相比,HSP70表达和皮质醇浓度在每一时相点都显著升高,差异有显著性(P＜0.05)。(3)肝功能的变化：①手术创伤组：不同程度的手术创伤术后随着时相延长ALT、TB有增高趋势,24h高峰,以后开始下降；随着创伤程度的增加,每一时相点ALT升高明显(P＜0.05)；在术后8、12、24h随着创伤程度的增加,TB升高明显(P＜0.05)；轻度创伤组术后8h、12h、24h ALT高出正常范围,TB均正常；中度、重度创伤术后每一时相点ALT均显著高出正常范围,中度创伤术后8h、12h、24h及重度创伤术后8h、12h、24h、48hTB高于正常。②谷氨酰胺干预组：手术创伤组异常ALT、TB指标经谷氨酰胺干预后均有明显改善,但中度创伤术后12h、24h及重度创伤术后8h、12h、24h的ALT虽然明显下降,仍高于正常。中度创伤术后24h、重度创伤术后12h、24hTB略高于正常外,其余各时相TB均恢复正常。③槲皮素干预组：与手术创伤组相比在相同创伤程度下ALT在(2、8、12、24h)、TB在中度(24h)及重度(12、24h)时相点差异有显著性(P＜0.05)。槲皮素干预组与谷氨酰胺干预组相比,ALT在轻度(2、8、12、24h)、中度及重度各时相点差异有显著性差异(p＜0.05)；TB在轻度(24h)、中度(8、12、24、48h)、重度每一时相点差异有显著性差异(p＜0.05)。④米非司酮干预组：手术创伤组各时相点的异常ALT经米非司酮干预后均有明显升高(P＜0.05),TB在术后8、12、24h升高明显(P＜0.05)。与谷氨酰胺干预组相比ALT在每一时相点、TB在轻度(12、24h)中度(8、12、24、48h)及重度各时相点有显著性差异(P＜0.05)；与槲皮素干预组相比ALT在(8、12、24h)、TB在重度(12、24h)时相点有显著性差异(P＜0.05)。(4)手术创伤组术后内环境的变化(一)K+的变化：①手术创伤组：手术创伤术后K+均迅速下降,至2h明显降低,后逐渐上升恢复至正常水平；在术后2h K+浓度随创伤程度增加而下降明显(P＜0.05)。②谷氨酰胺干预组：术后2h K+有所升高,中度创伤组至正常水平,重度创伤组接近正常(P＜0.05)。③槲皮素干预组：较手术创伤组在各时相点K+有所下降,术后2h K+与手术创伤组、谷氨酰胺干预组比较有显著性差异(p＜0.05)。④米非司酮干预组：术后血K+在各时相点较创伤组、谷氨酰胺组有所下降,在术后2h有显著性差异(p＜0.05)。(二)Na+的变化：①手术创伤组：术后Na+逐渐下降,至12h达最低水平,后逐渐上升至正常水平；在术后12h随创伤程度增加而下降明显(p＜0.05)。②谷氨酰胺干预组：术后各时相点均较手术创伤组有所上升,轻、中度创伤术后12h Na+恢复至正常,重度组仍稍低于正常。③槲皮素干预组：术后Na+较手术创伤组均有所下降,在术后12h有显著性差异(p＜0.05)。术后12h与谷氨酰胺干预组相比有显著性差异(P＜0.05)。④米非司酮干预组：术后各时相点Na+均较手术创伤组下降,在12h与手术创伤组比较有显著性差异(P＜0.05)。(三)PH：①手术创伤组：重度手术创伤组术后2h降低较明显,低于正常水平,与其余组间有差异(p＜0.05)。②谷氨酰胺干预组：重度创伤组术后2h PH值升为正常。③槲皮素干预组：术后PH在各时相点均有所下降,且中度创伤组术后2hPH低于正常(p＜0.05)。④米非司酮干预组：术后PH在各时相点均有所下降,且轻、中度创伤组术后2hPH也低于正常(p＜0.05)。(5)肝脏组织HSP70和HSP70mRNA的表达①手术创伤组：肝脏组织HSP70和HSP70mRNA的表达有一致性。正常大鼠肝脏HSP70和HSP70mRNA含量较低,术后2h表达增加,12h达到高峰,以后逐渐下降,24h仍维持在较高水平,48h稍高于正常。随着手术创伤程度的增加肝脏组织HSP70和HSP70mRNA表达水平均升高；HSP70在术后8、12、24h时相点轻度、中度、重度之间比较有显著性差异(P＜0.05)；HSP70mRNA在术后2、8、12h时相点轻度、中度、重度之间比较有显著性差异(P＜0.05)。②谷氨酰胺干预组：较手术创伤组在每一时相点肝脏组织HSP70、HSP70mRNA的表达水平均增加,HSP70在术后8、12、24h时相点增加明显(P＜0.05)；HSP70mRNA在术后2、8、12h时相点增加明显(P＜0.05)。与槲皮素干预组相比各时相点HSP70、HSP70mRNA表达均有显著性差异(p＜0.05)。③槲皮素干预组：较手术创伤组各时相点HSP70和HSP70mRNA表达均有所下降,在术后2、8、12、24h下降明显(P＜0.05)。④米非司酮干预组：HSP70表达在术后各时相点均高于手术创伤组(P＜0.05),HSP70mRNA表达在术后2、8、12、24h时相点高于手术创伤组(P＜0.05)；与槲皮素干预组相比HSP70和HSP70mRNA表达在术后各时相点有显著性差异(p＜0.05),肝脏组织的HSP70、HSP70mRNA的表达与血清HSP70的表达在创伤程度及时程上也具有一定的一致性。(6)肝脏组织GR和·GRmRNA的表达：①手术创伤组：肝脏组织GR和GRmRNA的表达有一致性。正常大鼠肝脏GR和GRmRNA有一定含量,术后表达开始下降,8h明显降低,12h达到最低,24h有所恢复仍较低,48h接近正常。在每一时相点GR和GRmRNA的表达均低于正常,随着手术创伤程度的增加肝脏组织中GR和GRmRNA表达水平逐渐下降；术后8、12、24h时相点轻度、中度、重度之间比较有显著差异性(P＜0.05)。②谷氨酰胺干预组：在各时相点GR和GRmRNA表达均较正常有所下降,随着手术创伤程度的增加肝脏组织中GR和GRmRNA表达水平逐渐下降；术后8、12h时相点轻度、中度、重度之间比较有显著性差异(P＜0.05)。与手术创伤组比较在术后8h、12h有显著性差异(P＜0.05)。③槲皮素干预组：在各时相点GR和GRmRNA表达均较正常组下降,较手术创伤组有轻度升高,但无统计学意义。槲皮素干预组较谷氨酰胺干预组在术后8h、12h、24h有所升高(P＜0.05)。手术创伤大鼠肝脏GRmRNA的表达和GR的表达有一致性。结论：(1)在不同程度手术创伤后,血清HSP70和皮质醇随创伤程度增加而升高,两者有正相关性,血清HSP70可作为判断手术创伤程度的指标。(2)手术创伤后大鼠内环境指标Na+、K+、PH值随着HSP70升高逐渐趋于至正常水平；术前谷氨酰胺诱导HSP70的高表达可使手术创伤后内环境指标和肝功能明显改善；HSP70对内环境及肝功能有保护作用。(3)血清HSP70、肝脏HSP70及HSP70mRNA的表达在创伤程度及时程上有一定的一致性,手术创伤后HSP70在转录和翻译水平同时增强。(4)在手术创伤中,随着HSP70的高表达,GR表达下降,两者具有一定的负相关性；诱导HSP70高表达后,GR仍有所下降,肝功能及稳态指标趋于正常或好转；阻断GR后,HSP70更为升高,但肝功能及稳态指标趋于恶化,在手术创伤中HSP70可能参与并启动了抗损伤,在一定程度上反映了肝脏损伤的程度。(5)HSP70与GC、GR均参与手术应激,HSP70影响GR的释放与代谢,GC与GR亦有负相关性；在手术创伤中HSP70与GR之间如何调控有待进一步研究。第三章临床肝癌病人手术后HSP70的变化及意义目的：探讨肝脏切除患者血清HSP70在评价手术创伤程度中的意义。方法：用酶联免疫吸附试验(ELISA)检测肝癌患者肝脏切除术前、术后2、12、24、48、72h血清HSP70表达并同时检测血清皮质醇的浓度及检测2、24、48、72、120h肝功能的变化,并进行比较。肝脏切除患者分为开腹左外叶切除、开腹左半肝切除组、开腹左三叶切除组、腹腔镜左外叶切除组。结果：肝脏切除术后2h血清HSP70明显升高,12h维持在高水平,24h达到高峰,48h仍高于正常水平,72h略高于正常；在术后2、12、24、48h随着切除肝量的增加HSP70的表达增加(p＜0.05)；开腹肝脏左外叶切除、左半肝切除组、左三叶切除组三组之间比较在术后2、12、24、48h时相点HSP70的表达有显著性差异(p＜0.05)。血清皮质醇于术后2h迅速增加,12h达到高峰,24小时仍有较高浓度,48h高于正常,72小时恢复正常；在术后12h、24h随着切除肝量的增加皮质醇的浓度明显增加；开腹肝脏左外叶切除组、左半肝切除组、左三叶切除组三组之间在术后12、24h时相点皮质醇浓度有显著性差异(p＜0.05)。腹腔镜左外叶切除组HSP70表达在术后12、24h低于开腹左外叶切除组(P＜0.05),腹腔镜左外叶切除组在术后12h皮质醇浓度低于开腹左外叶切除组(P＜0.05)。术后2hALT、TB开始升高,24h达到峰值,72h明显高于正常,120h接近正常；术后2、24、48、72h ALT、TB均明显高于术前(P＜0.05)。开腹左外叶切除组、开腹左半肝切除组、开腹左三叶切除组三组之间比较在术后24、48、72h ALT、TB浓度有显著性差异(P＜0.05)。腹腔镜左外叶切除组与开腹左半肝切除组、开腹左三叶切除组之间比较在术后24、48、72hALT、TB浓度有显著性差异(P＜0.05)。总蛋白、白蛋白术后2h开始下降,分别于术后24h、72h降至最低,术后120h未恢复到术前水平,术后与术前比较有显著性差异(P＜0.05)；总蛋白在术后24、48、72h开腹左外叶切除组、腹腔镜左外叶切除组分别与开腹左半肝切除组、开腹左三叶切除组之间比较有显著性差异(P＜0.05)。白蛋白在术后72h开腹左三叶切除组与开腹左外叶切除组、开腹左半肝切除组、腹腔镜左外叶切除组之间比较有显著性差异(P＜0.05)。结论：1.手术创伤可以引起血清HSP70和皮质醇浓度的明显升高,两者具有一定的正相关性,且随着手术创伤程度的增加而显著升高；HSP70的表达与肝功能的变化在手术创伤程度及时程上有一致性。2.血清中检测HSP70的表达可以作为判断手术创伤程度的指标。3.腔镜左外叶肝切组较相应开腹组血清HSP70表达及皮质醇浓度降低,提示：腔镜左外叶肝切术较相应开腹术创伤小。更多还原

【Abstract】 CHAPTER ONE VARYIATION AND SIGNIFICANCE OF HSP70 IN BRL RAT LIVER CELLObjective:To establish the cell model of heat shock in vitro by heat shock pretreatment for the BRL rat liver cell to promote the expression level of HSP70.Methods:Selecting the BRL rat liver cells that growing well and un-confluent bottom in culture bottle to make heat shock reaction.Take out the culture bottle from the CO2 incubator of 37℃, put into water bath case for 30 minutes that the temperature is preset at 40～50℃, then rewarm cultivate in incubator again, to measure cell survival rate after 2 hours cultivation。Making 30minutes heat treatment further at the temperature which the survival rate is over 95%,Then measure cell survival rate again after 48 hours cultivation at 37℃.It is found that the cell survival rate is above 95% after 40～42℃water bath for 30 minutes and then 48 hours cultivation at 37℃.The BRL Rat liver cell are heat treated at 40～42℃for 30 minutes and cultivated for 2h、8h、12h、24h、48h, the HSP70 protein expression are detected by Western-blot respectly。The BRL rats liver cells are cultivated for 2h by different concentrations of glutamine、quercetin and heat treated In the condition of 42℃for 30minutes, then cell survival rate and the expression of HSP70、GR are detected by Western-blot at 2h、8h、12h、24h、48h time point.Results:BRL Rats live cells were heat treated with 40～42℃for 30 minutes and cultivated at 37℃for 48s hours, that the cell survival rate is more than 95%。The HSP70 expression of normal BLR rats liver cell is low,however it increases rapidly at the 2h after being treated with heat shock, significantly higher at the 8h,and reach the top at the 12h; Though at the 24h,it remain at much more high level,and slightly higher than normal at 48h. In the condition of 40℃、41℃、42℃, the HSP70 expression of BLR rats liver cell is higher than normal control at every phase point(P<0.05).It is found that the HSP70 expression in the condition of 42℃is significantly higher than the condition of 40℃、41℃at 2h、8h、12h、24h points (P<0.05).At 41℃, every phase point is higher than at 40℃though without significant difference between them(P>0.05).So choosing 42℃for 30minutes as the heat shock temperature and time to treat with the BRL rats’liver cells。Using glutamine of different concentrations to cultivate liver cells for two hour,then heat treated for 30minutes at 42℃and cultivate 2h at 37℃before detecting the expression of HSP70,We can discover that the concentration of 8mmol/L of glutamine can induce the highest expression of HSP70. Using quercetin of different concentrations to cultivate liver cells for two hour,then heat treated for 30minutes at 42℃and cultivate 2h at 37℃before detecting the expression of HSP70.We discover that the lowest expression of HSP70 is induced when the concentration of quercetin is 150umol/L。Using glutamine of 8mmol/L and quercetin of 150umol/L to cultivate cells for two hour, then detectde the expression of HSP70 at the point of 2h、8h、12h、24h、48h after heat-shock reaction。The expression of HSP70 at every phase point in the glutaminate group is much higher than the expression of HSP70 in the normal control group (p<0.05).The expression of HSP70 at every phase point in the glutaminate group is higher than the expression of HSP70 in heat shock group,it has Significant difference at the piont of 8h、12h、24h between the two group(p<0.05).The quercetin group’s HSP70 expression of 2h、8h phase point is Significantly lower than normal control (P<0.05) and 12h、24h phase point slightly lower than normal control,however the 48h phase point gets close with normal(P>0.05).In every time point, the expression of HSP70 in the glutaminate group is higher than the quercetin group(P<0.05).Cell survival rate in each phase point of glutaminate group is higher than 95%; It cuts down at the piont of 2h、8h in quercetin group,step-up gradually after 12 hour and remain lower than 90% at 48h.The GR expression in heat shock group after being treated with 30minutes at 42℃and incubation at 37℃is lower than those in normal group at all time points; it decreases at the 2h,significantly decreases at the 8h,and reach minimum at the 12h; still low at the 24h,near normal at 48h.The GR expression of 8mmol/L glutamine intervention group is lower than those in heat shock group at all time points, There is significant difference between them at 8、12h (P<0.05).The GR expression of 150umol/L quercetin intervention group is lower than those in in normal group at all time points;slightly elevated than heat shock group at 8、12h,but no statistical significance between them (p<0.05)。GR expression in the intervention group is higher than those in glutamine intervention group at 2h,8h (P<0.05).Conclusion:(1) The pretreating method of 42℃for 30 minutes can successfully induced high expression of HSP70 in the BRL rat liver cells, that will not obviously cause the change of cell survive quantity after continuous cultivation 48 hours; The method can establish the heat shock cell model successfully. (2) The glutamines can induce high expression of HSP70 in the BRL rat liver cells, which has protective function for the cell; while the quercetin may inhibit the expression of HSP70 obviously, which result in the reduction of cell survive quantity obviously。8mmol/L glutamine、150umol/L quercetin are optimum concentration induced、inhibit the expression of HSP70 respectively. (3) Heat shock reaction and glutamine can induced high expression of HSP70 and low expression of GR in rat liver cells; expression of GR is also low after quercetin intervention.HSP70and GR have a certain relevance In the heat shock reaction.CHAPTER TWO VARYIATION AND SIGNIFICANCE OF HSP70 IN DIFFERENT HEPATECTOMY RATObjective:The HSP70 expressions were detected in the liver tissue and serum of surgical trauma rat,to study the relationship among HSP70、the degree of surgical trauma and trauma-induced changes of internal environment、in liver function, to discuss the significance of HSP70 in the evaluation of degree of surgical trauma and protective effect and mechanism of the Steady-state internal environment、the liver function.Methods:1.The establishment of animal model:Male healthy Sprague-Dawley rats were randomly divided into groups. (1) Normal control group:rats without any surgical treatment. (2) Surgical trauma groups.①mild traumatic Group:rat hepatic left lateral lobectomy group;②moderate trauma group:rat hepatic left lateral lobe and right lobe resection group;③severe trauma group:rat hepatic left lateral lobe, right lobe, nipple and the caudate lobe resection group. (3) Glutamine intervention group:tail intravenous injection 0.75g/kg glutamine at 2h before surgery:mild trauma of glutamine intervention group, moderate trauma of glutamine intervention group, severe trauma group of glutamine intervention group. (4) Quercetin intervention group:intraperitoneal injection 7mg/kg quercetin at 2h before surgery:mild trauma of quercetin intervention group, moderate trauma of quercetin intervention group, severe trauma group of quercetin intervention group. (5) Mifepristone intervention group.Intramuscular injection of 5mg/kg mifepristone at 2h before surgery: mild mifepristone of quercetin intervention group, moderate trauma of mifepristone intervention group, severe trauma group of mifepristone intervention group. Each degree of trauma included 50 rats, each point in time included 10 rats,5 time points were 2h,8h,12h, 24h,24h after surgery. Each rat was kept in the same environment, there are no difference between groups、the body weight, and age.2. Indicators detection:to take blood samples and liver tissue at 2h before surgery、2h、8h、12h、24h、48h of control group、surgical trauma group and intervention group respectively。To detect serum HSP70、cortisol、Na+、K+、Ca2+、PH, HSP70、HSP70mRNA、GR、GRmRNA by ELISA,arterial blood gas analyzer,automatic biochemical analyzer, Western-blot and real-time fluorescence quantitative PCR respectively.Results:(1)The success of model-building:model of varying degrees of surgical trauma in SD rats successfully established by different hepatic lobectomy, survival rates among mild surgical trauma group, moderate surgical trauma group, severe surgical trauma group were 98.0%,94.0%,92.0%, there are no significant difference among three groups (P>0.05), the animal models will be used to study the effects of surgical trauma on animal.(2)The HSP70 expression in serum.①Surgical trauma groups:the HSP70 expression in serum increased at 2h after the surgical trauma,12h peaked,48h at a high level.More degree of surgical trauma,more HSP70 expression(P<0.05);there are significant difference at 8,12,24h time points among three groups, serum corticosterone increased rapidly at 2h after operation,8h peak, still relatively hig at 24h,48h slightly higher than normal。More degree of surgical trauma,more serum cortisol(P<0.05);there are significant difference at 2,8,12,24h time points among three groups.②glutamine intervention groups:the HSP70 expression in serum is significantly higher than surgical trauma groups at 8,12,24h after surgery(p<0.05); the serum cortisol is significantly lower than surgical trauma groups at 2,8,12h after surgery(p<0.05).③Quercetin intervention group:the HSP70 expression in serum is significantly lower than surgical trauma groups at 2,8,12,24h after surgery(p<0.05);the serum cortisol is significantly higher than surgical trauma groups at2,8,12,24h After surgery (P<0.05).there are significant difference between quercetin intervention group and glutamine intervention groups about HSP70 expression(P<0.05), the serum cortisol in quercetin intervention group is significantly higher than glutamine intervention groups at 2,8,12,24h after surgery(P<0.05).④Mifepristone intervention group:the HSP70 expression in serum is significantly higher than surgical trauma groups at each piont time after surgery(p<0.05); the serum cortisol is significantly higher than surgical trauma groups at each piont time after surgery(p<0.05), the serum cortisol is still high than normal at 48h.(3) Changes in liver function:①Surgical trauma groups:the indicators of ALT、TB increased in different degree of Surgical trauma;with the increasing degree of trauma, ALT increased significantly (P<0.05). ALT in Mild trauma group were above the normal range at 8h,12h,24h;TB were normal. ALT in moderate、severe trauma were significantly higher than the normal range at each time point; TB in moderate trauma 8h,12h,24h and severe trauma 8h,12h,24h,48h were higher than normal.②glutamine intervention groups:the abnormal indicators of ALT、TB in Trauma group were significantly improved by L-glutamine intervention, while ALT in moderate trauma at 12h,24h and severe trauma at 8h,12h,24h of ALT weer still higher than normal,TB in Moderate trauma at24h and severe trauma at 12h, 24h were slightly higher than normal, the rest of the phase TB are back to normal.③Quercetin intervention group:ALT, TB has significantly increased compared with the surgical trauma in the same degree of trauma at 2,8,12,24 h postoperative time points (P <0.05),48h ALT, TB in moderate、severe trauma group was significantly higher than normal;ALT, TB in quercetin intervention group were higher than glutamine intervention group(p<0.05).④Mifepristone intervention group:the abnormal ALT, TB in trauma group at all time points were significantly increased by Mifepristone intervention; ALT, TB Of the mild trauma group at 48h was significantly higher than normal. There are significant difference compared with glutamine intervention group, quercetin intervention group(p<0.05).(4) changes of indictors within the environmental.1.K+:①surgical trauma group:the K +were rapidly declined to the lowest level at 2h after trauma in three groups, but then it gradually increased to normal levels. The K+was significantly decreased at 2h after surgery with the degree of trauma (P<0.05).②K+at 2h after the glutamine intervention has increased to normal levels in moderate trauma group; severe trauma group close to normal (P<0.05).③The K+ in quercetin intervention group decreased at each postopreativetime point compared with surgical trauma group, there were significantly different between quercetin intervention group and surgical trauma group or glutamine intervene-tion group at 2h (p<0.05).④The K+ in Mifepristone intervention group decreased at each postoperative time point compared with surgical trauma group, there were significantly different between Mifepristone intervention group and surgical trauma group at 2h(p<0.05). 2.Na+.①surgical trauma group:it was gradually decline to the lowest level at 12h, then increased to preoperative levels; The Na+ is significantly decreased at 12h after surgery with the degree of trauma (P<0.05). (2) Na+ at 12h after the glutamine intervention has increased to normal levels in mild、moderate trauma group; severe groups was still slightly lower than normal.③The Na+ in quercetin intervention group decreased at each postopreativetime point compared with surgical trauma group, Na+ decreased significantly at 12h(P<0.05);there were significantly different between quercetin intervention group and glutamine intervention group at 12h (p<0.05).④The Na+in Mifepristone intervention group decreased at each postoperative time point compared with surgical trauma group, there were significantly different between Mifepristone intervention group and surgical trauma group at 12h(p<0.05). 3.PH.①PH is reduced more obvious at 2h after surgery in severe trauma group, and it significant differences between other groups (P<0.05).②PH at 2h after surgery was promoted to the normal value in severe trauma groups after the glutamine intervention.③PH at various time points were decreased after the quercetin intervention, PH at 2h in the moderate trauma group was lower than normal (P<0.05).④PH at various time points were decreased after the Mifepristone intervention, PH at 2h in the mild、moderate trauma group was lower than normal (P<0.05)(5) The expression of HSP70 and HSP70mRNA in liver tissue①Surgical trauma groups:The expression of HSP70 and HSP70mRNA have consistency in Liver tissue. A low level of HSP70 and HSP70mRNA in normal rat liver,the HSP70 and HSP70mRNA expression in liver tissue increased at 2h after the surgical trauma,12h peaked, 24h remained at a high level,48h slightly higher than normal.HSP70 and HSP70mRNA expression were increased with the degree of surgical trauma. There were significant difference for the expression of HSP70 among mild, moderate and severe surgical trauma at 8,12,24 h time points after surgery(p<0.05). There were significant difference for the expression of HSP70mRNA among mild, moderate and severe surgical trauma at 2,8,12h time points after surgery(p<0.05).②glutamine intervention groups:HSP70 expression and HSP70mRNA were increased Compared with surgical trauma group at each time point, the HSP70 expression in Liver tissue is significantly higher than surgical trauma groups at 8,12,24h after surgery(p<0.05),while HSP70mRNA at 2,8,12h(p<0.05).HSP70 and HSP70mRNA expression in glutamine intervention groups were higher than quercetin intervention group at each time poin (p<0.05).③Quercetin intervention group:the HSP70 HSP70mRNA expression in Liver tissue is significantly lower than surgical trauma groups at 2,8,12,24h after surgery(p<0.05);④Mifepristone intervention group:the HSP70 expression in Liver tissue is significantly higher than surgical trauma groups at each piont time after surgery(p<0.05);while HSP70mRNA expression at 2、8、12、24h after surgery(p<0.05).The expression of HSP70 and HSP70mRNA in Liver tissue and serum HSP70 have consistency in the degree of surgical trauma and time points.(6) The expression of GR and GR70mRNA in liver tissue①Surgical trauma groups:The expression of GR and GRmRNA have consistency in Liver tissue..A high level of GR and GRmRNA in normal rat liver,the GR and GRmRNA expression in liver tissue decreased after the surgical trauma,8h significantly reduced,12h minimum,24h some recovery is still relatively low,48h near normal.GR and GRmRNA expression were decreased with the degree of surgical trauma. There were significant difference for the expression of GR and GRmRNA among mild, moderate and severe surgical trauma at 8,12,24 h time points after surgery(p<0.05).②glutamine intervention groups:GR and GRmRNA expression were decreased compared with surgical trauma group at each time point, GR and GRmRNA expression were decreased with the degree of surgical trauma. There were significant difference for the expression of GR and GRmRNA among mild, moderate and severe surgical trauma at 8,12h time points after surgery(p<0.05).GR and GRmRNA expression in Liver tissue is significantly lowerer than surgical trauma groups at 8,12h after surgery(p<0.05).③Quercetin intervention group:the GR、GRmRNA expression in Liver tissue is significantly lower than normal control group(P<0.05);but slightly high than surgical trauma groups at (p>0.05); the GR、GRmRNA expression in Quercetin intervention group were higher than glutamine intervention groups at 8h,12h,24h postoperative(p<0.05). The expression of GR and GRmRNA in Liver tissue have consistency in the degree of surgical trauma and time points.Conclusion:(1) Serum HSP70 and corticosterone concentration are markedly elevated with varing degree of trauma, Serum HSP70 expression can be used as indicator to diagnose the degree of surgical trauma. (2) The Na+, K+, PH of Internal environmental indicators can be gradually returned to normal level with HSP70 levels increased after surgical trauma; high expression of HSP70 induced by glutamine Preoperative can significantly improved liver function and Internal environmental indicators after surgical trauma; HSP70 has a protective effect on internal environment and liver function.(3)Serum HSP70, HSP70 and HSP70mRNA expression in liver have a certain consistency with trauma degree and time course in the trauma (4) GR expression decreased with the high HSP70 expression after surgical trauma; High expression of HSP70 induced by glutamine after surgical trauma, but GR is still decreased, liver function and indicators of internal environment tended to normal or improve. GR was blocked by mifeprostone,HSP70 increased, but indicators of internal environment and liver function worse after surgical trauma; HSP70 may be involved in and started anti-injury. (5) HSP70 and GC, GR are involved in the surgical stress, HSP70 affectthe GR release and metabolism, the relevance of HSP70 and GR to be further studied In thesurgical trauma.CHAPTER THREE THE VARIATION AND SIGNIFICANCE OF SERUM HSP70 IN LIVER CANCER PATIENTS AFTER HEPATECTOMYObjective:To discuss the significance of serum HSP70 in the evaluation of degree of surgical hepatectomy.Methods:To detect the expression of serum Hsp70 By enzyme-linked immuno-sorbent assay (ELISA) and the concentration Of serum cortisol before and after 2h、12h、24h、48h、72h for the patients of hepatocellular carcinoma with liver resection, compared with the healthy control group。The patients of liver resection were divided into open left lateral lobectomy、open left hepatic resection group、open left trisegmentectomy group and laparoscopic left lateral lobectomy.Results:The level of serum HSP70 was significantly higher at 2h after liver resection, maintained at a high level at 12h,reached a peak at 24h,still higher at 48h than normal levels, slightly higher at 72h than normal; The more volume of liver resection,the more expression of HSP70 in serum at 2,12,24,48h after surgery(p<0.05); The expression of Hsp70 was significantly difference among open left lateral lobectomy、open left hepatic resection group、open left trisegmentectomy group at 2,12,24,48h time point after liver resection(p<0.05). The level of serum cortisol increased rapidly at 2h after liver resection, reached a peak at 12h, still above the normal at 48h, returned to normal at 72h; The more volume of liver resection, the more concentration of cortisol in serum at 2,12,24h after surgery(p<0.05), The concentration of cortisol was significantly difference among open left lateral lobectomy、open left hepatic resection group、open left trisegmentectomy group at 2,12,24h time point after liver resection (p<0.05).The expression of HSP70 in Laparoscopic left lateral lobectomy group is lower than open left lateral lobectomy at 12,24h after surgery(p<0.05); The concentration of cortisol in Laparoscopic left lateral lobectomy group is lower than open left lateral lobectomy at 12h after surgery(p<0.05). The level of serum ALT、TB was significantly higher at 2h after liver resection, reached a peak at 24h, still higher at 72h than normal levels, slightly higher at 120h than normal; There was significantly difference among before and after operation at 2、24、48、72h (p<0.05);The level of serum ALT、TB was significantly difference among open left lateral lobectomy、open left hepatic resection group、open left trisegmentectomy group at 24,48、72h time point after liver resection(p<0.05). The expression of ALT、TB in Laparoscopic left lateral lobectomy group is lower than open left hepatic resection group、open left trisegmentectomy group at 24、48、72h time point after liver resection (p<0.05). Total protein, albumin began to decrease at 2h after liver resection, Total protein at 24h、albumin at 72h to a minimum respectively,5 days (120h) after surgery are not returned to preoperative levels; There was significantly difference among before and after operation(p<0.05). Total protein at 24、48、72h in Laparoscopic left lateral lobectomy group、open left lateral lobectomy group are higher than open left hepatic resection group、open left trisegmentectomy group resrectively (p<0.05).Albumin at 72h in open left trisegmentectomy group is lower than open left lateral lobectomy group are、open left hepatic resection group、Laparoscopic left lateral lobectomy group(p<0.05).Conclusion:Serum HSP70 and corticosterone concentration are markedly elevated after Surgical trauma; with increased degree of trauma, Serum HSP70 and corticosterone concentration are significantly increased.HSP70 expression have consistent with changes of liver function about degree of surgery trauma and timely way. Serum HSP70 expression can be used as indicator to diagnose the degree of surgical trauma.更多还原