【作者】 张伯；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 普通外科学， 2014， 博士

【摘要】 第一部分eNOS重组腺病毒载体的制备目的：构建含有eNOS基因的重组腺病毒载体。方法：（1）将重组腺病毒载体Ad-eNOS行基因测序。（2）将重组腺病毒载体Ad-eNOS用脂质体介导转染293细胞包装成病毒颗粒，应用合成的eNOS基因引物行Real-time PCR鉴定。（3）通过测定吸光值计算病毒滴度并分装。结果：（1）经测序，Ad-eNOS基因序列与GeneBank中的eNOS序列一致，比对符合率100%。（2）Real-time PCR鉴定重组腺病毒Ad-eNOS包装成功。（3）经病毒滴度测定，Ad-eNOS滴度为9.35×109PFU/ml。结论：成功构建了重组腺病毒载体Ad-eNOS质粒，采用HEK293细胞进行腺病毒包装，最终获得重组腺病毒Ad-eNOS。第二部分eNOS基因转染对L02肝细胞缺复氧损伤的保护作用目的：探讨eNOS基因转染对L02肝细胞缺复氧损伤的保护作用。方法：采用L02肝细胞，放入低氧环境（含95%N2+5%CO2、PO2≤4Kpa）及复氧环境（85%O2+15%C O2，PO2≥13Kpa）孵箱中培养，建立缺复氧模型，实验分三组：实验组加入Ad-eNOS处理；对照组和正常组加RPMI-1640培养液。实验组和对照组放入缺复氧模型中培养，正常组在5%CO2、饱和湿度的孵箱内培养。检测培养液中ALT及NO的含量，Real-time PCR检测eNOS的基因表达，Western-blot检测eNOS的蛋白表达，流式细胞检测细胞凋亡。结果：实验组细胞培养液中ALT含量(26.26±3.78u/l)明显低于对照组(48.42±5.31u/l)（P﹤0.05）,但高于正常组(17.20±2.64u/l)，差别有显著意义（P﹤0.05）；而NO的含量实验组（18.89±3.30umol／L）明显高于对照组（6.44±2.11umol／L）和正常组（8.85±2.40umol／L）（P﹤0.05）；Real-time PCR及Western-blot结果显示实验组eNOS在基因及蛋白水平的表达均明显高于对照组及正常组（P﹤0.05）；流式细胞结果显示实验组L02肝细胞凋亡明显低于对照组（P﹤0.05），而正常组L02肝细胞凋亡很少。结论：（1）腺病毒载体成功将eNOS基因转染到L02肝细胞上，使其高表达eNOS基因及蛋白；（2）eNOS基因转染可减轻L02肝细胞缺复氧损伤的细胞凋亡；（3）eNOS基因转染可能通过升高NO途径对L02肝细胞缺复氧损伤有保护作用。第三部分大鼠小体积肝脏移植模型的建立目的：用改良二袖套法建立稳定的大鼠小体积肝脏移植模型。方法：在Kamada二袖套法的的基础上，对大鼠原位肝移植术在取肝、灌注、修植肝等手术操作以及围手术期的处理进行改良，建立稳定的大鼠原位肝移植模；在此基础上再建立稳定的大鼠小体积肝脏移植模型。结果：通过三阶段比较，正式实验组在手术时间、无肝期、手术成功率、并发症等方面均有明显提高。结论：（1）改良的二袖套法具有无肝期短、手术成功率高、大鼠术后存活时间长的优点，是大鼠原位肝移植的理想术式；（2）在建立稳定的大鼠原位肝移植模型的基础上开展大鼠小体积肝移植模型有利于提高模型的稳定性。第四部分eNOS基因转染对大鼠小体积肝移植缺血再灌注损伤的保护作用目的：探讨eNOS基因转染对大鼠小体积肝移植缺血再灌注损伤的保护作用。方法：SD大鼠12对，随机分两组（n=6）行小体积肝移植。对照组供体用空载体进行腹腔注射，实验组供体用Ad-eNOS进行腹腔注射。36小时后取肝行小体积肝移植。门静脉复流后6小时后处死模型，采血送检肝功能；硝酸还原法检测NO浓度；免疫组化检测TNF-α、巨噬细胞；TUNEL法检测肝组织的细胞凋亡；流式细胞检测肝细胞凋亡率；Real-time PCR检测肝脏组织的eNOS基因表达；Western-blot检测肝组织中的eNOS蛋白表达。结果：（1）实验组的ALT、AST、LDH显著低于对照组；细胞凋亡率显著低于对照组；（2）肝组织NO含量实验组明显高于对照组；（3）Real-time PCR及Western-blot结果显示实验组eNOS在基因及蛋白水平的表达均明显高于对照组；（4）免疫组化发现TNF-α及巨噬细胞在对照组中高表达，实验组中低表达。结论：eNOS在大鼠小体积肝移植缺血再灌注损害中对肝细胞有保护作用，其作用机制可能与下调TNF-α，抑制巨噬细胞浸润有关。更多还原

【Abstract】 Part One Construction of Recombinant Adenovirus of eNOSObjective: To construct the recombinant adenovirus vector containing eNOS gene.Methods:(1) The recombinant adenovirus vector Ad-eNOS using gene sequencing.(2) The recombinant adenovirus vector Ad-eNOS by liposome mediated transfection293cells packaged into viral particles, using eNOS gene primers for identification of PCRsynthesis.(3)By measuring the absorbance of the virus titer was calculated and packed.Results:(1) By DNA sequencing, the Ad-eNOS gene sequence is consistent withGeneBank.(2) Identification of recombinant adenovirus Ad-eNOS packing is successfulby Real-time PCR.(3) The virus titer was9.35×109PFU/ml by the determination of virustiter.Conclusion: To construct the recombinant adenovirus vector Ad-eNOS by usingHEK293cells.To obtain recombinant adenovirus Ad-eNOS.Part Two Protective effects of eNOS gene transfer onhypoxia-reoxygenation injury in hepatocellular L02Objective: To investigate the protective effect of eNOS gene transfer on hypoxia-reoxygenation injury in hepatocellular L02.Methods:Using the L02liver cells, in the hypoxia model (including95%N2+5%CO2、PO2≤4Kpa) and reoxygenation model (85%O2+15%CO2，PO2≥13Kpa), constructinghypoxia-reoxygenation model, The experiments were divided into three groups:inexperimental group treated with Ad-eNOS; in control group and in normal group with RPMI-1640medium. The experimental group and the control group in hypoxia-reoxygenation model, in normal group were cultured in5%CO2, saturated humidityincubation box. The contents of ALT and NO was detected in the medium, the expressionof eNOS gene was detected by Real-time PCR, the expression of eNOS protein wasdetected by Western-blot, apoptosis cell was detected by FCM.Results: In experimental group, the content of ALT (26.26±3.78u/l) lower than thatof in control group (48.42±5.31u/l)(P <0.05), but higher than that in normal group(17.20±2.64u/l)(P <0.05); and the content of NO in experimental group (18.89±3.30umol/L) was significantly higher than in control group (6.44±2.11umol/L) and innormal group (8.85±2.40umol/L)(P <0.05); Real-time PCR and Western-blot resultsshowed that the expression of eNOS in experimental group in the gene and protein levelswere significantly higher than in control group and in normal group (P <0.05); FCMshowed that in experimental group of L02liver cell apoptosis was significantly lower thanin control group (P <0.05), and in normal group L02apoptosis of hepatic cells rarely.Conclusion:(1) Ad-eNOS gene was transfected into the L02liver cells, the highexpression of eNOS gene and protein;(2) eNOS gene transfer can reduce cell apoptosis onhypoxia-reoxygenation injury in hepatocellular L02;(3) eNOS gene transfer could haveprotective effect on hypoxia-reoxygenation injury in hepatocellular L02by rising the NOpathway.Part Three To establish the model of small-for-size liver transplantationin ratsObjective: to establish the rat model of small-for-size liver transplantation bymodified two cuff method.Methods: Based on the Kamada two cuff technique, improvement of the ratorthotopic liver transplantation in perfusion, repair, treatment with liver operation and perioperation period, to establish a stable rat orthotopic liver transplantation model; on thisbasis to establish the small-for-size liver transplantation in rat model. Results: By comparison of the three phase, the formal experimental group in theoperation time, anhepatic phase, the success rate of operation, complication were obviouslyincreased.Conclusion:(1)The modified two cuffed technique has the advantages of longsurvival time, short anhepatic phase operation, high success rate, which is an idealoperative method of orthotopic liver transplantation in rats;(2) Based on the stable modelof orthotopic liver transplantation in rats establishing of small-for-size liver transplantationin rats model is helpful to improve the stability of the model.Part Four The protective effect of eNOS gene transfer on IRI of ratsmall-for-size liver transplantationObjective: To investigate the protective effect of eNOS gene transfer on IRIsmall-for-size liver transplantation in rats.Methods:12SD rats were randomly divided into two groups (n=6) for small-for-sizeliver transplantation.The control group were intraperitoneal injection of donor with emptyvector, the experimental group with Ad-eNOS donor. After36hours small-for-size livertransplantation is operated. Six hours after reperfusion, the recipients were sacrificed, NOwere measured, TNF-α、macrophage levels were detected by immunohistochemistry.Apoptosis of the hepatocytes was analyzed by TUNEL and FCM; the expression of eNOSgene was detected by Real-time PCR, the expression of eNOS protein was detected byWestern-blot.Results:(1) In experimental groups ALT, AST, LDH were significantly lower thanin the control group; apoptosis rate was significantly lower than in control group;(2) thecontent of NO in liver tissue in experimental group was significantly higher than in controlgroup;(3) Real-time PCR and Western-blot results showed that in experimental groupeNOS at the level of gene and protein were significantly higher than in control group;(4)Immunohistochemistry showed that TNF-α and macrophages high expression in controlgroup and low expression in experimental group. Conclusion: eNOS has a protective effect on IRI of small-for-size livertransplantation in rats, and its mechanism may be related to the down-regulation of TNF-α,inhibiting the macrophage infiltration.更多还原