Pim-3基因对暴发性肝衰竭的肝保护效应研究
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摘要
暴发性肝衰竭(FHF)是一死亡率极高,以大量肝细胞凋亡、严重肝损害为特征的临床疾病。丝/苏氨酸激酶Pim-3基因具有显著的肝细胞凋亡抑制和生长促进效应,预期能在FHF的疾病治疗中发挥积极作用。为此,本课题进行了Pim-3基因转染动物活体肝组织的实验,目的是探讨Pim-3基因对FHF的组织修复或肝保护效应及其作用机制。课题研究采取了多步骤分阶段的方式进行。首先,采用RT-PCR的方法,从大鼠心肌组织中分离并克隆了Pim-3基因。通过基因重组技术,完成了Pim-3基因质粒载体的构建,并利用基因转染技术顺利地实现了重组体质粒pEGFP-N2/Pim-3在体外真核细胞的表达和活性测定;其次,采用常规和流体力学方法进行GFP表达质粒溶液的鼠尾静脉注射,证明常规注射和流体力学注射是两种不同的活体基因转染方法,具有不同的器官靶向性和基因转染效率。其中流体力学方法是一个高效的肝靶向性动物活体基因转染方法;再次,采用腹腔注射LPS/D-GalN的方法建立FHF大鼠模型。找到了制备动物模型最佳的药物剂量,证明小剂量LPS可诱导D-GalN致敏大鼠产生显著但非致死性的急性肝衰竭。其中,肝细胞凋亡是LPS/D-GalN诱导的肝衰竭主要的病理形态学特征;第四,通过构建体pEGFP-N2/Pim-3质粒的大鼠活体肝靶向性转染,证实该质粒能有效地在体内肝细胞表达,并发挥其对细胞凋亡的抑制效应;最后,采用致死剂量LPS/D-GalN处理大鼠,结果提示外源性Pim-3基因的体内转移极有效地降低了动物的死亡率和血清转氨酶水平、减轻了肝组织出血坏死性病变和炎性浸润,并使大片肝细胞凋亡得到逆转。同时,外源性Pim-3基因在体内的高表达也显著地降低了肝组织和血清内炎性细胞因子TNF-α和IL-1β水平以及肝组织损伤基因iNOS和凋亡诱导基因p53的表达,但促进了抗凋亡蛋白Bcl-2的分泌。这些结果提示,外源性Pim-3基因的活体转染能有效地保护大鼠免于LPS/D-GalN诱导的暴发性肝衰竭的发生。其保护作用的产生主要在于肝细胞凋亡的抑制和组织炎性反应的改善。
第一部分Pim-3基因克隆和质粒载体构建及其在真核细胞内的表达和活性研究
目的克隆Pim-3基因和构建其GFP表达质粒,并研究其在真核细胞内的表达及对细胞凋亡与生存能力的影响。
方法采用RT-PCR的方法从Wistar大鼠心肌组织中提取并克隆Pim-3基因,通过酶切和连接反应构建GFP表达质粒,随后经酶切证实和测序鉴定;肝癌SMMC7721细胞采用常规的方法进行培养和传代,原代肝细胞采用二步胶原酶灌注法进行分离;以阳离子脂质体介导的方法进行体外细胞的基因转染;基因转染效率借助荧光显微镜观察GFP的表达进行判断;细胞的凋亡和存活情况通过流式细胞术和MTT分析检测。
结果成功克隆大鼠Pim-3基因,并构建其真核表达质粒pEGFP-N2/Pim-3;构建体质粒转染肝癌SMMC7721细胞24h后即见GFP表达,在48h表达显著增强;外源性Pim-3基因的转染显著抑制了肝癌细胞的凋亡。在转染后48h,对照(非转染)、脂质体、空质粒和重组质粒组肝癌细胞的凋亡百分率分别为(10.2±6.3)%、(11.0±5.9)%、(10.7±4.1)%和(3.5±1.3)%。与其它三组相比,重组质粒组肝癌细胞的凋亡百分率显著降低(P＜0.01);通过MTT分析,Pim-3基因的转染显著地增强了肝癌细胞的增殖能力,且在观察期间,其增殖能力有随时间增强的趋势;成功分离培养大鼠原代肝细胞,并借助阳离子脂质体介导成功完成质粒体外肝细胞转染。在转染48h可见明显GFP表达;通过流式细胞检测,转染后48h,对照、脂质体、空质粒和重组质粒组肝细胞的生存率分别为74.12±11.20、69.87±13.45、72.03±12.47和90.11±9.27。与其它三组肝细胞相比,转染重组质粒后的肝细胞生存率明显提高(P＜0.01)。
结论成功构建Pim-3基因GFP表达质粒pEGFP-N2/Pim-3;该构建体能有效地在真核细胞包括肝癌细胞和原代培养肝细胞内表达,并发挥抑制凋亡和促进生长的作用。
第二部分绿色荧光蛋白表达质粒经鼠尾静脉注射后的器官靶向性研究
目的研究不同方式的鼠尾静脉注射对绿色荧光蛋白(GFP)基因器官靶向性的影响。
方法雄性昆明小鼠40只,随机分为正常对照组、流体力学注射组和常规注射组。流体力学和常规注射组再分为转染组和对照组,每组各8只小鼠。注射结束后24h采集血清检测转氨酶,并采集肝、脾、心、肾、肺和脑组织进行冰冻切片,部分肝组织采用4%多聚甲醛固定后切片。荧光显微镜下观察。
结果不同方式的注射对24h后的肝功能并无不良影响;常规尾静脉注射引起了少数肾小球细胞表达GFP,而肝、脾、心、肺及脑等组织未见明显GFP表达;流体力学注射引起了肝内GFP高水平表达,肝细胞表达率接近45%,其它组织则无GFP表达。
结论流体力学方法是肝靶向性的活体基因转染方法;GFP可作为该方法进行目的基因研究的一个可靠和方便的示踪剂。
第三部分内毒素诱导D-半乳糖胺致敏大鼠急性肝损伤或肝衰竭实验模型的研究
目的研究非致死剂量LPS/D-GalN的肝损伤效应和细胞凋亡在急性肝损伤或肝衰竭中的作用。
方法48只Wistar大鼠进行随机对照分组实验,分为6h、24h和48h取材3大组(各16只),每个大组再分为处理组和对照组(各8只)。处理组大鼠以LPS50μg/kg+D-GalN 300mg/kg,用1ml无菌生理盐水溶解后腹腔内注射,对照组动物仅腹腔内注射1ml生理盐水。在相应时间点,门静脉采血查血生化包括ALT、AST和TBIL;肝组织切片分别行光镜、透射电镜检查和TUNEL分析;基因表达通过RT-PCR的方法检测;24h处理鼠肝组织匀浆后行Caspases活性检测。
结果所有处理组大鼠ALT、AST和TBIL在6h内即显著升高,24h达到峰值,48h仍维持于高水平;在24h和48h,肝外形明显肿胀并失去正常光泽,表面见出血点;肝损伤的病理组织学改变包括肝细胞死亡、炎性渗出和出血等表现从6h开始出现,在24h和48h显著加重;LPS/D-GalN腹腔注射显著地诱导了肝细胞的凋亡,通过TUNEL分析,在24h和48h大鼠肝组织的凋亡指数(AI)均接近70%;肝组织炎性细胞因子IL-1βmRNA水平在6h显著升高,且在24h和48h维持于高水平,而TNF-α水平在处理组各时间点内均在正常范围;损伤基因iNOS mRNA表达被早期诱导,在6h达峰值,随后逐渐下降。相反,在早期(6h),并未发现凋亡诱导基因p53 mRNA高表达,但在肝凋亡的高峰期(24h),p53显著高表达,且维持于高水平直至后期(48h);此外,LPS/D-GalN的应用显著地诱导了24h处理大鼠肝组织caspase-3、-8、-9和-12的活性。
结论小剂量LPS可诱导D-GalN致敏大鼠产生显著但非致死性的急性肝衰竭;肝细胞凋亡是LPS/D-GalN诱导的肝衰竭主要的病理形态学特征,其发生在早期可能与iNOS基因、而中期和晚期可能与p53基因的高表达有关,且至少三条凋亡通路参与了这种病理过程。
第四部分Pim-3质粒构建体在活体肝组织表达和活性的研究
目的研究Pim-3质粒构建体pEGFP-N2/Pim-3在活体肝组织的表达和活性。
方法动物采用随机分组对照实验,基因活体内肝靶向性转染通过质粒溶液的大鼠尾静脉流体力学注射法完成,肝细胞凋亡的诱导则采用腹腔内注射LPS和D-GalN来实现。四组动物(8只/组)分别为:A组为正常对照组,B、C和D组分别以林格氏液、空载质粒和重组质粒溶液行尾静脉流体力学注射,1d后,予LPS/D-GalN腹腔内注射,24h后处死动物;肝组织报告基因GFP的表达通过荧光显微镜、目的基因Pim-3的表达通过RT-PCR方法检测;肝细胞凋亡采用TUNEL分析,并进行Caspase-3活性检测。
结果重组质粒和空质粒DNA通过流体力学注射法被成功转染入大鼠活体肝组织内,C组和D组大鼠肝组织切片中GFP呈现高水平表达;目的基因Pim-3的相对表达水平A组为0.06±0.02、B和C组为0、D组为0.49±0.15。D组与A、B、C三组间差异有显著统计学意义(P＜0.01);通过TUNEL分析,各组大鼠肝细胞的凋亡指数(apoptotic index,AI)分别为:A组3.1±0.7%,B组72.5±6.1%、C组69.8±5.7%和D组4.9±1.2%,D组与A组间差异无明显统计学意义(P＞0.05),而D组与B、C组之间差异存在显著统计学意义(P＜0.01)。B和C组间差异无统计学意义(P＞0.05);各组大鼠肝组织Caspase-3活性分别为:A组60.37±15.23pmol/min.mg、B组146.5±55.2pmol/min.mg、C组141.6±49.7pmol/min.mg和D组75.57±26.91pmol/min.mg,D与A组间和B与C组间差异无明显统计学意义(P＞0.05),而D组与B、C两组间差异有明显统计学意义(P＜0.01)。
结论已构建的重组体质粒pEGFP-N2/Pim-3能在大鼠活体肝组织内有效表达,并发挥其对肝细胞凋亡的抑制效应。
第五部分Pim-3表达质粒活体肝转移对LPS/D-GalN诱导暴发性肝衰竭大鼠的保护效应及其机制
目的探讨Pim-3基因对暴发性肝衰竭大鼠的肝保护作用及其机制。
方法32只大鼠随机分成四组(8只/组)。A组为正常对照组,B、C和D组分别以林格氏液、空载质粒和重组质粒溶液预处理大鼠,1d后,予LPS/D-GalN腹腔内注射诱导FHF,8h后或濒死期处死大鼠,采集标本。另设32只大鼠分组观察24h内的死亡情况;血清转氨酶水平通过全自动血生化分析仪检测;Pim-3基因的活体肝组织转染效率通过荧光显微镜观察GFP的表达来评估;细胞凋亡采用TUNEL分析和Caspase-3活性来测定;肝组织基因表达通过RT-PCR和Western blot进行检测,血清内蛋白质的分泌采用ELISA方法测定。
结果LPS/D-GalN攻击24h后,B和C组大鼠死亡率均为87.5%,D组为12.5%,A组均存活;Pim-3基因的应用显著地降低了LPS/D-GalN注射诱导的血清转氨酶水平;荧光显微镜观察,C组和D组肝组织有大量GFP表达,而A组和B组无GFP表达;肝组织病理检查,可见Pim-3质粒肝转移有效地减轻了LPS/D-GalN诱导的肝内出血坏死和炎性细胞浸润;肝组织TUNEL分析和Caspase-3活性测定显示外源Pim-3基因的体内转染显著地减轻了LPS/D-GalN诱导的肝细胞凋亡,并抑制了Caspase-3的活性;正常大鼠肝组织组成性表达Pim-3基因,LPS/D-GalN攻击显著地抑制了Pim-3基因表达,外源Pim-3基因的活体转染使这种抑制得到逆转并出现Pim-3 mRNA和蛋白质超高表达;LPS/D-GalN攻击大鼠肝组织和血清TNF-α和IL-1β水平均升高,但外源Pim-3基因使其增高程度显著减轻;LPS/D-GalN攻击诱导了大鼠肝组织iNOS和p53mRNA的表达,Pim-3基因的应用明显地降低了其增高的程度,但并不影响LPS/D-GalN诱导Bax的表达水平;外源Pim-3基因的应用也显著地诱导了Bcl-2蛋白表达。
结论外源性Pim-3基因的活体内转染能有效地保护大鼠免于LPS/D-GalN诱导暴发性肝衰竭的发生。其保护效应的产生主要在于肝细胞凋亡的抑制和组织炎性反应的改善。
Fulminant hepatic failure(FHF)is a life-threatening clinical syndrome characterized by massive hepatic apoptosis and severe impairment of liver function. Serine/threonine kinase Pim-3 gene may play an important role in protecting liver tissue against FHF because of its inhibition of hepatocyte apoptosis and promotion of cell growth.To probe the hypothesis,we finished an in vivo liver-target transfer of Pim-3 gene.We aimed to investigate the effect and mechanism of Pim-3 gene on FHF.First of all,by reverse transcription polymerase chain reaction(RT-PCR), Pim-3 gene was successfully isolated and cloned from rat myocardium tissues. Pim-3-expressed plasmid was then constructed by genetic recombination technology. After smoothly transfected into eukaryotic cells,the construct was found to have an effect on cell survival and apoptosis in vitro.Secondly,by an injection of green fluorescent protein(GFP)-expressed plasmid DNA into mouse tail vein using hydrodynamics-based or regular procedure,we found that different from regular injection,hydrodynamics-based procedure induced overexpression of GFP in mouse liver,thus was a convenient,efficient method of liver-target transfer of exogenous gene in vivo.Thirdly,FHF was induced by intraperitoneal injections of lipopolysaccharide(LPS)and D-galactosamine(D-GalN)in rats.The optimal dose of drugs was found to establish an animal model.We demonstrated that a challenge with low dose LPS in conjunction with D-GalN could induce nonlethal but marked liver failure,the morphological feature of which is mainly hepatic apoptosis,which may be associated with high expression of iNOS(in the early)and p53 gene(in the mid and late stage).Fourthly,by an intravenous injection of the naked pEGFP-N2/Pim-3 plasmid using hydrodynamics-based procedure,the construct was found to achieve high expression in living cells and had an inhibitory effect on hepatic apoptosis in rats.Finally,through a challenge of lethal doses of LPS/D-GalN, one hundred percent lethality was induced in the model rats within 72h.Treatment with foreign Pim-3 gene by an in vivo transfer protected D-GalN-sensitized rats from LPS-induced liver damage and mortality.Hepatic enzymes showed dramatic increases in vector and Ringer's groups,whereas only a slight increases was observed in Pim-3-treated rats.Liver architecture was preserved,whereas few neutrophil infiltrates and hemorrhagic necrosis were observed in the treated rats. Liver apoptosis was also strikingly inhibited by Pim-3 treatment.Moreover,levels of inflammatory mediator TNF-αand IL-1βwere declined,and expression of iNOS and p53 were dramatically suppressed by exogenous Pim-3 gene.However, overexpression of Bcl-2 protein was observed in Pim-3-treated rats after administrating LPS/D-GalN challenge.These results suggest that exogenous Pim-3 gene can protect rats from LPS/D-GalN-induced FHF by inhibiting liver apoptosis and improving inflammatory response of liver tissues.
PartⅠ:Cloning,plasmid construct of Pim-3 gene and its effects on eukaryotic cells
Objective To clone Pim-3 gene and construct its GFP-expressed plasmid,and to further investigate expression and activity of the construct in eukaryotic cells.
Methods Pim-3 gene was cloned from myocardium tissues of juvenile Wistar rat by RT-PCR and subcloned into GFP-expressed plasmid vecter pEGFP-N2 by restriction enzyme.The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-ligase and then identified by enzyme digestion and sequencing;Hepatoma SMMC7721 cells were cultured and passaged by conventional method,and primary rat hepatocytes were isolated by collegenase perfusion;The plasmid pEGFP-N2/Pim-3 containing Pim-3 and GFP gene was transfected into SMMC7721 cells and primary hepatocytes with cationic liposome;The efficiency of gene transfection was appraised by GFP expression under fluorescence microscope;Cell apoptosis and viability were detected by flow cytometry and MTT assay.
Results pEGFP-N2/Pim-3 plasmid was successfully constructed;In SMMC7721 cells,GFP expression was observed at 24h and markedly enhanced at 48h after transfection of the construct;Apoptosis of hepatoma cells was significantly inhibited by exogenous Pim-3 gene.48h after transfection,apoptotic percentage of hepatoma cells in control,liposome,vector and construct groups were(10.2±6.3)%, (11.0±5.9)%,(10.7±4.1)%and(3.5±1.3)%,respectively.Compared with apoptotic percentage of other groups,that of the construct group was markedly lower(P＜0.01);MTT assay showed viability of hepatoma cells was significantly enhanced after transfection of exogenous Pim-3 gene;Primary hepatocytes were successfully obtained and showed GFP expression at 48h after plasmid transfection.By flow cytometry assay,survival rates of primary hepatocytes in control,liposome,vector and construct groups were respectively 74.12±11.20,69.87±13.45,72.03±12.47 and 90.11±9.27.Survival rates of the construct group were significantly higher than those of other groups(P＜0.01).
Conclusions The constructed pEGFP-N2/Pim-3 plasmid can efficiently express in eukaryotic cells and have substantial effects of apoptotic inhibition or growth promotion on hepatoma cells and primary hepatocytes.
PartⅡ:Organ-specific transfer of naked green fluorescent protein (GFP)-expressed plasmid by tail vein injection in mice
Objective To investigate the target organ for gene transfer by an intravenous injection of GFP-expressed plasmid DNA using hydrodynamics-based or regular procedure.
Methods Forty mice were randomly divided into three groups:normal control (n=8),hydrodynamics-based procedure(n=16)and regular procedure group(n=16). The mice in the latter two groups were further divided into transfer(n=8)and control group(n=8),respectively.24 h after tail vein injection,the tissue and blood samples were collected.The contents of serum alanine aminotransferase(ALT)and aspartate transaminase(AST)were detected by blood biochemistry meter.The expression levels of GFP in liver,spleen,heart,lung,kidney and brain were appraised under fluorescent microscope by frozen sections.
Results The levels of serum transaminase between tail vein injection and normal control mice have no difference statistically;The hydrodynamics-based injection of naked GFP plasmid DNA into mice induces high expression level of GFP gene in liver,and approximately 45%of expression rate is achieved.However, the regular injection results in low expression level only in kidney,and no trace in liver,brain,lung,heart and spleen.
Conclusions Hepatic delivery of foreign gene can be accomplished by hydrodynamics-based injection with simplicity and high efficiency,and GFP is a reliable and convenient tracer agent in the hydrodynamics-based procedure in mice.
PartⅢ:Rat model of acute liver injury or hepatic failure induced by lipopolysaccharide in D-galactosamine-sensitized rats
Objective LPS is implicated in the pathology of acute liver injury and can induce lethal liver failure when simultaneously administered with D-GalN.Up to now,nonlethal liver failure,the liver injury of clinical implication,is incompletely understood after challenged by low dose LPS/D-GalN.Therefore,the aim of this article is to investigate effects of liver injury of nonlethal dose LPS/D-GalN and a role of apoptosis in this disorder.
Methods Forty eight rats were randomly divided into three groups(16 for each).The rats in each group were further divided into treatment(n=8)and control (n=8).The rats in the treatment group received intraperitoneal injections of LPS (50μg/kg body wt)and D-GalN(300mg/kg body wt)in 1 ml stroke-physiological saline solution(SPSS),while the rats in control were treated with SPSS only.The blood and tissue samples were collected at 6,24 and 48h,respectively.The contents of serum ALT,AST and total bilirubin(TBIL)were detected by autoassay meter of blood biochemistry.The morphological changes were observed under light microscope and electromicroscope.The apoptosis of liver cells was dectected by TUNEL assay.The expression of TNF-α,IL-1β,iNOS and p53 gene were dectected by RT-PCR,and the activities of caspase-3,-8,-9 and-12 were measured.
Results Blood biochemistry indexes,including ALT,AST and TBIL rose at 6h,reached the peak at 24h and sustained high levels at 48h after LPS/D-GalN injection.Abnormal liver appearance was found at 24h and 48h.Histopathological changes of hepatic injuries accompanied by hepatocellular death,inflammatory infiltration and hemorrhage began to appear at 6h,markedly aggravated at 24h and 48h.Cell apoptosis was significantly induced by nonlethal dose LPS/D-GalN challenge,and the apoptotic indexes(AIs)in 24h-and 48h-treated rats were approximately 70%by TUNEL assay.The inflammatory cytokine IL-1βmRNA levels rose markedly at 6h,and maintained high degrees at 24h and 48h,however, TNF-αlevels were normal in the liver tissues of 6h-,24h-and 48h-treated rats. mRNA expressions of damage gene iNOS were also induced early by LPS/D-GalN challenge and reached the peak at 6h and then gradually stepped down;contrarily, high expression levels of apoptosis-inducing gene p53 mRNA were not found in the early(6h)but emerged in the crest-time of liver apoptosis(24h)and maintained the level till the late stage(48h).In addition,we found that,in 24h-treated rats, caspase-3,-8,-9 and-12 were markedly activated by LPS/D-GalN challenge.
Conclusion These results suggest that a challenge with low dose LPS in conjunction with D-GalN can induce nonlethal but marked liver failure,the morphological feature of which is mainly hepatic apoptosis,which may be associated with high expression of iNOS(in the early)and p53 gene(in the mid and late stage);and at least three apoptosis pathways participate in the pathogenesis.
PartⅣ:In vivo expression and inhibitory role of hepatic apoptosis of GFP-expressed plasmid of rat serine/threonine kinase Pim-3 gene
Objective To investigate the in vivo expression of the construct pEGFP-N2/Pim-3 and its effect on cell apoptosis in rat liver.
Methods In vivo gene transfer to rat liver cells was achieved by an intravenous injection of the naked construct using a hydrodynamics-based procedure; The hepatic apoptosis was induced by intraperitoneal injections of LPS and D-GalN and detected by TUNEL assay and caspase-3 activity;The expression levels of reporter gene GFP and target gene Pim-3 were respectively appraised under fluorescent microscope and by RT-PCR.
Results High expression of target gene Pim-3 and reporter gene GFP was achieved in rat liver after transfer of the recombinant plasmid;Acute liver apoptosis induced by LPS/D-GalN was reversed by the efficient in vivo expression of exogenous Pim-3 gene.
Conclusions The construct pEGFP-N2/Pim-3 plasmid can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis in rat.
PartⅤ:In vivo transfer of Pim-3-expressed plasmid protects against lipopolysaccharide/D-galactosamine-induced fulminant hepatic failure
Objective To investigate the effects and mechanisms of serine/threonine kinase Pim-3 gene on fulminant hepatic failure.
Methods Thirty-two rats were randomly divided into four groups(eight for each group).Group A was normal control.Group B,C and D were pretreated with Ringer's solution,vector plasmid and recombinant plasmid,respectively,and received intraperitoneal injections of LPS and D-GalN after 1 day.8h after injections of LPS/D-GalN,liver tissues and blood samples were collected;The contents of serum transaminase was tested by automatic blood biochemistry meter;The morphological changes were observed by light microscopy using hematoxylin and eosin(HE)staining;GFP expression levels were appraised under fluorescent microscope by frozen sections;Cell apoptosis of liver tissues was detected by the assay of TUNEL and Caspase-3 activity.Gene expression was detected by RT-PCR and Western blot,and protein secretion in serum was determined by enzyme linked immunosorbent assay(ELISA).
Results 24h after LPS/D-GalN challenge,87.5%lethality was induced in group B and C,but only 12.5%in group D;Hepatic enzymes showed dramatic increases in group B and C,whereas only a slight increases was observed in Pim-3-treated rats;Overexpression of reporter gene GFP was induced by single rapid injection of a large amount of naked plasmid solution via rat tail vein (hydrodynamics-based gene transfer technique)and found in group C and D under fluorescence microscope.But group A and B had no trace of GFP;Histopathologic analysis performed on liver sections taken from group B and C after LPS/D-GalN challenge showed widespread destruction of liver architecture,erythrocyte agglutination and neutrophil infiltration.Numerous apoptotic cells were evidenced by the TUNEL assay.Strikingly,rats with Pim-3 gene before LPS/D-GalN injection were resistant to the lethal effect of the drugs' challenge.By marked contrast,liver apoptosis in Pim-3-treated rats was markedly inhibited,evidenced in isolated foci and Caspase-3 activity.Liver architecture was completely preserved,whereas few nentrophil or lymphoid infiltrates were observed;Expression of Pim-3 is constructively found in rat normal liver and inhibited by LPS/D-GalN challenge,but enhanced by in vivo transfer of exogenous Pim-3 gene;Inflammatory cytokines TNF-αand IL-1βsignificantly rised in liver tissues and systemic levels after injections of LPS and D-GalN,but this response was suppressed by in vivo transfer of Pim-3 gene;In addition,expressions of iNOS and p53(but not Bax)induced by LPS/D-GalN challenge were dramatically suppressed by exogenous Pim-3 gene. However,overexpression of Bcl-2 protein was observed in Pim-3-treated rats after administrating LPS/D-GalN challenge.
Conclusions Exogenous Pim-3 gene can protect rats from LPS/D-GalN-induced fulminant hepatic failure by inhibiting liver apoptosis and improving inflammatory response of liver tissues,which can be associated with many mechanisms including inhibiting expression of inflammatory mediators such as TNF-αand IL-1β,injury gene iNOS and apoptosis-induced gene p53,and promoting expression of antiapoptosis gene Bcl-2.
第一部分Pim-3基因克隆和质粒载体构建及其在真核细胞内的表达和活性研究
目的克隆Pim-3基因和构建其GFP表达质粒,并研究其在真核细胞内的表达及对细胞凋亡与生存能力的影响。
方法采用RT-PCR的方法从Wistar大鼠心肌组织中提取并克隆Pim-3基因,通过酶切和连接反应构建GFP表达质粒,随后经酶切证实和测序鉴定;肝癌SMMC7721细胞采用常规的方法进行培养和传代,原代肝细胞采用二步胶原酶灌注法进行分离;以阳离子脂质体介导的方法进行体外细胞的基因转染;基因转染效率借助荧光显微镜观察GFP的表达进行判断;细胞的凋亡和存活情况通过流式细胞术和MTT分析检测。
结果成功克隆大鼠Pim-3基因,并构建其真核表达质粒pEGFP-N2/Pim-3;构建体质粒转染肝癌SMMC7721细胞24h后即见GFP表达,在48h表达显著增强;外源性Pim-3基因的转染显著抑制了肝癌细胞的凋亡。在转染后48h,对照(非转染)、脂质体、空质粒和重组质粒组肝癌细胞的凋亡百分率分别为(10.2±6.3)%、(11.0±5.9)%、(10.7±4.1)%和(3.5±1.3)%。与其它三组相比,重组质粒组肝癌细胞的凋亡百分率显著降低(P＜0.01);通过MTT分析,Pim-3基因的转染显著地增强了肝癌细胞的增殖能力,且在观察期间,其增殖能力有随时间增强的趋势;成功分离培养大鼠原代肝细胞,并借助阳离子脂质体介导成功完成质粒体外肝细胞转染。在转染48h可见明显GFP表达;通过流式细胞检测,转染后48h,对照、脂质体、空质粒和重组质粒组肝细胞的生存率分别为74.12±11.20、69.87±13.45、72.03±12.47和90.11±9.27。与其它三组肝细胞相比,转染重组质粒后的肝细胞生存率明显提高(P＜0.01)。
结论成功构建Pim-3基因GFP表达质粒pEGFP-N2/Pim-3;该构建体能有效地在真核细胞包括肝癌细胞和原代培养肝细胞内表达,并发挥抑制凋亡和促进生长的作用。
第二部分绿色荧光蛋白表达质粒经鼠尾静脉注射后的器官靶向性研究
目的研究不同方式的鼠尾静脉注射对绿色荧光蛋白(GFP)基因器官靶向性的影响。
方法雄性昆明小鼠40只,随机分为正常对照组、流体力学注射组和常规注射组。流体力学和常规注射组再分为转染组和对照组,每组各8只小鼠。注射结束后24h采集血清检测转氨酶,并采集肝、脾、心、肾、肺和脑组织进行冰冻切片,部分肝组织采用4%多聚甲醛固定后切片。荧光显微镜下观察。
结果不同方式的注射对24h后的肝功能并无不良影响;常规尾静脉注射引起了少数肾小球细胞表达GFP,而肝、脾、心、肺及脑等组织未见明显GFP表达;流体力学注射引起了肝内GFP高水平表达,肝细胞表达率接近45%,其它组织则无GFP表达。
结论流体力学方法是肝靶向性的活体基因转染方法;GFP可作为该方法进行目的基因研究的一个可靠和方便的示踪剂。
第三部分内毒素诱导D-半乳糖胺致敏大鼠急性肝损伤或肝衰竭实验模型的研究
目的研究非致死剂量LPS/D-GalN的肝损伤效应和细胞凋亡在急性肝损伤或肝衰竭中的作用。
方法48只Wistar大鼠进行随机对照分组实验,分为6h、24h和48h取材3大组(各16只),每个大组再分为处理组和对照组(各8只)。处理组大鼠以LPS50μg/kg+D-GalN 300mg/kg,用1ml无菌生理盐水溶解后腹腔内注射,对照组动物仅腹腔内注射1ml生理盐水。在相应时间点,门静脉采血查血生化包括ALT、AST和TBIL;肝组织切片分别行光镜、透射电镜检查和TUNEL分析;基因表达通过RT-PCR的方法检测;24h处理鼠肝组织匀浆后行Caspases活性检测。
结果所有处理组大鼠ALT、AST和TBIL在6h内即显著升高,24h达到峰值,48h仍维持于高水平;在24h和48h,肝外形明显肿胀并失去正常光泽,表面见出血点;肝损伤的病理组织学改变包括肝细胞死亡、炎性渗出和出血等表现从6h开始出现,在24h和48h显著加重;LPS/D-GalN腹腔注射显著地诱导了肝细胞的凋亡,通过TUNEL分析,在24h和48h大鼠肝组织的凋亡指数(AI)均接近70%;肝组织炎性细胞因子IL-1βmRNA水平在6h显著升高,且在24h和48h维持于高水平,而TNF-α水平在处理组各时间点内均在正常范围;损伤基因iNOS mRNA表达被早期诱导,在6h达峰值,随后逐渐下降。相反,在早期(6h),并未发现凋亡诱导基因p53 mRNA高表达,但在肝凋亡的高峰期(24h),p53显著高表达,且维持于高水平直至后期(48h);此外,LPS/D-GalN的应用显著地诱导了24h处理大鼠肝组织caspase-3、-8、-9和-12的活性。
结论小剂量LPS可诱导D-GalN致敏大鼠产生显著但非致死性的急性肝衰竭;肝细胞凋亡是LPS/D-GalN诱导的肝衰竭主要的病理形态学特征,其发生在早期可能与iNOS基因、而中期和晚期可能与p53基因的高表达有关,且至少三条凋亡通路参与了这种病理过程。
第四部分Pim-3质粒构建体在活体肝组织表达和活性的研究
目的研究Pim-3质粒构建体pEGFP-N2/Pim-3在活体肝组织的表达和活性。
方法动物采用随机分组对照实验,基因活体内肝靶向性转染通过质粒溶液的大鼠尾静脉流体力学注射法完成,肝细胞凋亡的诱导则采用腹腔内注射LPS和D-GalN来实现。四组动物(8只/组)分别为:A组为正常对照组,B、C和D组分别以林格氏液、空载质粒和重组质粒溶液行尾静脉流体力学注射,1d后,予LPS/D-GalN腹腔内注射,24h后处死动物;肝组织报告基因GFP的表达通过荧光显微镜、目的基因Pim-3的表达通过RT-PCR方法检测;肝细胞凋亡采用TUNEL分析,并进行Caspase-3活性检测。
结果重组质粒和空质粒DNA通过流体力学注射法被成功转染入大鼠活体肝组织内,C组和D组大鼠肝组织切片中GFP呈现高水平表达;目的基因Pim-3的相对表达水平A组为0.06±0.02、B和C组为0、D组为0.49±0.15。D组与A、B、C三组间差异有显著统计学意义(P＜0.01);通过TUNEL分析,各组大鼠肝细胞的凋亡指数(apoptotic index,AI)分别为:A组3.1±0.7%,B组72.5±6.1%、C组69.8±5.7%和D组4.9±1.2%,D组与A组间差异无明显统计学意义(P＞0.05),而D组与B、C组之间差异存在显著统计学意义(P＜0.01)。B和C组间差异无统计学意义(P＞0.05);各组大鼠肝组织Caspase-3活性分别为:A组60.37±15.23pmol/min.mg、B组146.5±55.2pmol/min.mg、C组141.6±49.7pmol/min.mg和D组75.57±26.91pmol/min.mg,D与A组间和B与C组间差异无明显统计学意义(P＞0.05),而D组与B、C两组间差异有明显统计学意义(P＜0.01)。
结论已构建的重组体质粒pEGFP-N2/Pim-3能在大鼠活体肝组织内有效表达,并发挥其对肝细胞凋亡的抑制效应。
第五部分Pim-3表达质粒活体肝转移对LPS/D-GalN诱导暴发性肝衰竭大鼠的保护效应及其机制
目的探讨Pim-3基因对暴发性肝衰竭大鼠的肝保护作用及其机制。
方法32只大鼠随机分成四组(8只/组)。A组为正常对照组,B、C和D组分别以林格氏液、空载质粒和重组质粒溶液预处理大鼠,1d后,予LPS/D-GalN腹腔内注射诱导FHF,8h后或濒死期处死大鼠,采集标本。另设32只大鼠分组观察24h内的死亡情况;血清转氨酶水平通过全自动血生化分析仪检测;Pim-3基因的活体肝组织转染效率通过荧光显微镜观察GFP的表达来评估;细胞凋亡采用TUNEL分析和Caspase-3活性来测定;肝组织基因表达通过RT-PCR和Western blot进行检测,血清内蛋白质的分泌采用ELISA方法测定。
结果LPS/D-GalN攻击24h后,B和C组大鼠死亡率均为87.5%,D组为12.5%,A组均存活;Pim-3基因的应用显著地降低了LPS/D-GalN注射诱导的血清转氨酶水平;荧光显微镜观察,C组和D组肝组织有大量GFP表达,而A组和B组无GFP表达;肝组织病理检查,可见Pim-3质粒肝转移有效地减轻了LPS/D-GalN诱导的肝内出血坏死和炎性细胞浸润;肝组织TUNEL分析和Caspase-3活性测定显示外源Pim-3基因的体内转染显著地减轻了LPS/D-GalN诱导的肝细胞凋亡,并抑制了Caspase-3的活性;正常大鼠肝组织组成性表达Pim-3基因,LPS/D-GalN攻击显著地抑制了Pim-3基因表达,外源Pim-3基因的活体转染使这种抑制得到逆转并出现Pim-3 mRNA和蛋白质超高表达;LPS/D-GalN攻击大鼠肝组织和血清TNF-α和IL-1β水平均升高,但外源Pim-3基因使其增高程度显著减轻;LPS/D-GalN攻击诱导了大鼠肝组织iNOS和p53mRNA的表达,Pim-3基因的应用明显地降低了其增高的程度,但并不影响LPS/D-GalN诱导Bax的表达水平;外源Pim-3基因的应用也显著地诱导了Bcl-2蛋白表达。
结论外源性Pim-3基因的活体内转染能有效地保护大鼠免于LPS/D-GalN诱导暴发性肝衰竭的发生。其保护效应的产生主要在于肝细胞凋亡的抑制和组织炎性反应的改善。
Fulminant hepatic failure(FHF)is a life-threatening clinical syndrome characterized by massive hepatic apoptosis and severe impairment of liver function. Serine/threonine kinase Pim-3 gene may play an important role in protecting liver tissue against FHF because of its inhibition of hepatocyte apoptosis and promotion of cell growth.To probe the hypothesis,we finished an in vivo liver-target transfer of Pim-3 gene.We aimed to investigate the effect and mechanism of Pim-3 gene on FHF.First of all,by reverse transcription polymerase chain reaction(RT-PCR), Pim-3 gene was successfully isolated and cloned from rat myocardium tissues. Pim-3-expressed plasmid was then constructed by genetic recombination technology. After smoothly transfected into eukaryotic cells,the construct was found to have an effect on cell survival and apoptosis in vitro.Secondly,by an injection of green fluorescent protein(GFP)-expressed plasmid DNA into mouse tail vein using hydrodynamics-based or regular procedure,we found that different from regular injection,hydrodynamics-based procedure induced overexpression of GFP in mouse liver,thus was a convenient,efficient method of liver-target transfer of exogenous gene in vivo.Thirdly,FHF was induced by intraperitoneal injections of lipopolysaccharide(LPS)and D-galactosamine(D-GalN)in rats.The optimal dose of drugs was found to establish an animal model.We demonstrated that a challenge with low dose LPS in conjunction with D-GalN could induce nonlethal but marked liver failure,the morphological feature of which is mainly hepatic apoptosis,which may be associated with high expression of iNOS(in the early)and p53 gene(in the mid and late stage).Fourthly,by an intravenous injection of the naked pEGFP-N2/Pim-3 plasmid using hydrodynamics-based procedure,the construct was found to achieve high expression in living cells and had an inhibitory effect on hepatic apoptosis in rats.Finally,through a challenge of lethal doses of LPS/D-GalN, one hundred percent lethality was induced in the model rats within 72h.Treatment with foreign Pim-3 gene by an in vivo transfer protected D-GalN-sensitized rats from LPS-induced liver damage and mortality.Hepatic enzymes showed dramatic increases in vector and Ringer's groups,whereas only a slight increases was observed in Pim-3-treated rats.Liver architecture was preserved,whereas few neutrophil infiltrates and hemorrhagic necrosis were observed in the treated rats. Liver apoptosis was also strikingly inhibited by Pim-3 treatment.Moreover,levels of inflammatory mediator TNF-αand IL-1βwere declined,and expression of iNOS and p53 were dramatically suppressed by exogenous Pim-3 gene.However, overexpression of Bcl-2 protein was observed in Pim-3-treated rats after administrating LPS/D-GalN challenge.These results suggest that exogenous Pim-3 gene can protect rats from LPS/D-GalN-induced FHF by inhibiting liver apoptosis and improving inflammatory response of liver tissues.
PartⅠ:Cloning,plasmid construct of Pim-3 gene and its effects on eukaryotic cells
Objective To clone Pim-3 gene and construct its GFP-expressed plasmid,and to further investigate expression and activity of the construct in eukaryotic cells.
Methods Pim-3 gene was cloned from myocardium tissues of juvenile Wistar rat by RT-PCR and subcloned into GFP-expressed plasmid vecter pEGFP-N2 by restriction enzyme.The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-ligase and then identified by enzyme digestion and sequencing;Hepatoma SMMC7721 cells were cultured and passaged by conventional method,and primary rat hepatocytes were isolated by collegenase perfusion;The plasmid pEGFP-N2/Pim-3 containing Pim-3 and GFP gene was transfected into SMMC7721 cells and primary hepatocytes with cationic liposome;The efficiency of gene transfection was appraised by GFP expression under fluorescence microscope;Cell apoptosis and viability were detected by flow cytometry and MTT assay.
Results pEGFP-N2/Pim-3 plasmid was successfully constructed;In SMMC7721 cells,GFP expression was observed at 24h and markedly enhanced at 48h after transfection of the construct;Apoptosis of hepatoma cells was significantly inhibited by exogenous Pim-3 gene.48h after transfection,apoptotic percentage of hepatoma cells in control,liposome,vector and construct groups were(10.2±6.3)%, (11.0±5.9)%,(10.7±4.1)%and(3.5±1.3)%,respectively.Compared with apoptotic percentage of other groups,that of the construct group was markedly lower(P＜0.01);MTT assay showed viability of hepatoma cells was significantly enhanced after transfection of exogenous Pim-3 gene;Primary hepatocytes were successfully obtained and showed GFP expression at 48h after plasmid transfection.By flow cytometry assay,survival rates of primary hepatocytes in control,liposome,vector and construct groups were respectively 74.12±11.20,69.87±13.45,72.03±12.47 and 90.11±9.27.Survival rates of the construct group were significantly higher than those of other groups(P＜0.01).
Conclusions The constructed pEGFP-N2/Pim-3 plasmid can efficiently express in eukaryotic cells and have substantial effects of apoptotic inhibition or growth promotion on hepatoma cells and primary hepatocytes.
PartⅡ:Organ-specific transfer of naked green fluorescent protein (GFP)-expressed plasmid by tail vein injection in mice
Objective To investigate the target organ for gene transfer by an intravenous injection of GFP-expressed plasmid DNA using hydrodynamics-based or regular procedure.
Methods Forty mice were randomly divided into three groups:normal control (n=8),hydrodynamics-based procedure(n=16)and regular procedure group(n=16). The mice in the latter two groups were further divided into transfer(n=8)and control group(n=8),respectively.24 h after tail vein injection,the tissue and blood samples were collected.The contents of serum alanine aminotransferase(ALT)and aspartate transaminase(AST)were detected by blood biochemistry meter.The expression levels of GFP in liver,spleen,heart,lung,kidney and brain were appraised under fluorescent microscope by frozen sections.
Results The levels of serum transaminase between tail vein injection and normal control mice have no difference statistically;The hydrodynamics-based injection of naked GFP plasmid DNA into mice induces high expression level of GFP gene in liver,and approximately 45%of expression rate is achieved.However, the regular injection results in low expression level only in kidney,and no trace in liver,brain,lung,heart and spleen.
Conclusions Hepatic delivery of foreign gene can be accomplished by hydrodynamics-based injection with simplicity and high efficiency,and GFP is a reliable and convenient tracer agent in the hydrodynamics-based procedure in mice.
PartⅢ:Rat model of acute liver injury or hepatic failure induced by lipopolysaccharide in D-galactosamine-sensitized rats
Objective LPS is implicated in the pathology of acute liver injury and can induce lethal liver failure when simultaneously administered with D-GalN.Up to now,nonlethal liver failure,the liver injury of clinical implication,is incompletely understood after challenged by low dose LPS/D-GalN.Therefore,the aim of this article is to investigate effects of liver injury of nonlethal dose LPS/D-GalN and a role of apoptosis in this disorder.
Methods Forty eight rats were randomly divided into three groups(16 for each).The rats in each group were further divided into treatment(n=8)and control (n=8).The rats in the treatment group received intraperitoneal injections of LPS (50μg/kg body wt)and D-GalN(300mg/kg body wt)in 1 ml stroke-physiological saline solution(SPSS),while the rats in control were treated with SPSS only.The blood and tissue samples were collected at 6,24 and 48h,respectively.The contents of serum ALT,AST and total bilirubin(TBIL)were detected by autoassay meter of blood biochemistry.The morphological changes were observed under light microscope and electromicroscope.The apoptosis of liver cells was dectected by TUNEL assay.The expression of TNF-α,IL-1β,iNOS and p53 gene were dectected by RT-PCR,and the activities of caspase-3,-8,-9 and-12 were measured.
Results Blood biochemistry indexes,including ALT,AST and TBIL rose at 6h,reached the peak at 24h and sustained high levels at 48h after LPS/D-GalN injection.Abnormal liver appearance was found at 24h and 48h.Histopathological changes of hepatic injuries accompanied by hepatocellular death,inflammatory infiltration and hemorrhage began to appear at 6h,markedly aggravated at 24h and 48h.Cell apoptosis was significantly induced by nonlethal dose LPS/D-GalN challenge,and the apoptotic indexes(AIs)in 24h-and 48h-treated rats were approximately 70%by TUNEL assay.The inflammatory cytokine IL-1βmRNA levels rose markedly at 6h,and maintained high degrees at 24h and 48h,however, TNF-αlevels were normal in the liver tissues of 6h-,24h-and 48h-treated rats. mRNA expressions of damage gene iNOS were also induced early by LPS/D-GalN challenge and reached the peak at 6h and then gradually stepped down;contrarily, high expression levels of apoptosis-inducing gene p53 mRNA were not found in the early(6h)but emerged in the crest-time of liver apoptosis(24h)and maintained the level till the late stage(48h).In addition,we found that,in 24h-treated rats, caspase-3,-8,-9 and-12 were markedly activated by LPS/D-GalN challenge.
Conclusion These results suggest that a challenge with low dose LPS in conjunction with D-GalN can induce nonlethal but marked liver failure,the morphological feature of which is mainly hepatic apoptosis,which may be associated with high expression of iNOS(in the early)and p53 gene(in the mid and late stage);and at least three apoptosis pathways participate in the pathogenesis.
PartⅣ:In vivo expression and inhibitory role of hepatic apoptosis of GFP-expressed plasmid of rat serine/threonine kinase Pim-3 gene
Objective To investigate the in vivo expression of the construct pEGFP-N2/Pim-3 and its effect on cell apoptosis in rat liver.
Methods In vivo gene transfer to rat liver cells was achieved by an intravenous injection of the naked construct using a hydrodynamics-based procedure; The hepatic apoptosis was induced by intraperitoneal injections of LPS and D-GalN and detected by TUNEL assay and caspase-3 activity;The expression levels of reporter gene GFP and target gene Pim-3 were respectively appraised under fluorescent microscope and by RT-PCR.
Results High expression of target gene Pim-3 and reporter gene GFP was achieved in rat liver after transfer of the recombinant plasmid;Acute liver apoptosis induced by LPS/D-GalN was reversed by the efficient in vivo expression of exogenous Pim-3 gene.
Conclusions The construct pEGFP-N2/Pim-3 plasmid can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis in rat.
PartⅤ:In vivo transfer of Pim-3-expressed plasmid protects against lipopolysaccharide/D-galactosamine-induced fulminant hepatic failure
Objective To investigate the effects and mechanisms of serine/threonine kinase Pim-3 gene on fulminant hepatic failure.
Methods Thirty-two rats were randomly divided into four groups(eight for each group).Group A was normal control.Group B,C and D were pretreated with Ringer's solution,vector plasmid and recombinant plasmid,respectively,and received intraperitoneal injections of LPS and D-GalN after 1 day.8h after injections of LPS/D-GalN,liver tissues and blood samples were collected;The contents of serum transaminase was tested by automatic blood biochemistry meter;The morphological changes were observed by light microscopy using hematoxylin and eosin(HE)staining;GFP expression levels were appraised under fluorescent microscope by frozen sections;Cell apoptosis of liver tissues was detected by the assay of TUNEL and Caspase-3 activity.Gene expression was detected by RT-PCR and Western blot,and protein secretion in serum was determined by enzyme linked immunosorbent assay(ELISA).
Results 24h after LPS/D-GalN challenge,87.5%lethality was induced in group B and C,but only 12.5%in group D;Hepatic enzymes showed dramatic increases in group B and C,whereas only a slight increases was observed in Pim-3-treated rats;Overexpression of reporter gene GFP was induced by single rapid injection of a large amount of naked plasmid solution via rat tail vein (hydrodynamics-based gene transfer technique)and found in group C and D under fluorescence microscope.But group A and B had no trace of GFP;Histopathologic analysis performed on liver sections taken from group B and C after LPS/D-GalN challenge showed widespread destruction of liver architecture,erythrocyte agglutination and neutrophil infiltration.Numerous apoptotic cells were evidenced by the TUNEL assay.Strikingly,rats with Pim-3 gene before LPS/D-GalN injection were resistant to the lethal effect of the drugs' challenge.By marked contrast,liver apoptosis in Pim-3-treated rats was markedly inhibited,evidenced in isolated foci and Caspase-3 activity.Liver architecture was completely preserved,whereas few nentrophil or lymphoid infiltrates were observed;Expression of Pim-3 is constructively found in rat normal liver and inhibited by LPS/D-GalN challenge,but enhanced by in vivo transfer of exogenous Pim-3 gene;Inflammatory cytokines TNF-αand IL-1βsignificantly rised in liver tissues and systemic levels after injections of LPS and D-GalN,but this response was suppressed by in vivo transfer of Pim-3 gene;In addition,expressions of iNOS and p53(but not Bax)induced by LPS/D-GalN challenge were dramatically suppressed by exogenous Pim-3 gene. However,overexpression of Bcl-2 protein was observed in Pim-3-treated rats after administrating LPS/D-GalN challenge.
Conclusions Exogenous Pim-3 gene can protect rats from LPS/D-GalN-induced fulminant hepatic failure by inhibiting liver apoptosis and improving inflammatory response of liver tissues,which can be associated with many mechanisms including inhibiting expression of inflammatory mediators such as TNF-αand IL-1β,injury gene iNOS and apoptosis-induced gene p53,and promoting expression of antiapoptosis gene Bcl-2.
引文
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