Th1/Th2类细胞因子的变化对急性免疫性肝损伤的影响研究

目的探讨Th1/Th2类细胞因子的变化对ConA诱导的急性免疫性肝损伤的机制,以及脾脏对急性免疫性肝损伤的影响作用。方法将Balb/c小鼠随机分为两组：正常对照组,肝损伤组。正常对照组尾静脉注射等量生理盐水,肝损伤组尾静脉注射12.5mg/Kg ConA一次。各组分别于ConA注射后8h,24h,72h取材,进行下列研究：①HE染色观察各组小鼠肝脏病理学改变。②经眼球取血,收集血清测ALT和AST。③收集各组小鼠血清及新鲜肝、脾组织（各100mg）,获取肝、脾组织裂解液。用多参数细胞因子检测技术即FlowCytomix技术,通过流氏细胞仪对荧光素PE信号强度的检测,实现对各组小鼠血清、肝组织、脾组织内多种Th1/Th2类因子的定性定量分析。结果①HE染色：正常对照组肝组织结构正常。肝损伤组8h时表现为急性肝损伤表现,24h时可见大片坏死灶,72h时肝损伤缓解。②血清ALT和AST检测：正常对照组3个时间点内无明显升高,肝损伤组3个时间段内ALT和AST均高于正常对照组,有显著性差异。③Th1/Th2细胞因子检测结果：肝脏：肝损伤组8h时Th1和Th2类细胞因子均明显升高,与正常对照组比较有显著性差异,24h后开始下降,降至正常水平或正常水平以下,呈明显下降趋势。血清：肝损伤组Th1,Th2类细胞因子8h均升高,24h后逐步降低。脾脏：肝损伤组Th1,Th2类细胞因子8h时均升高,与正常对照组比较,有显著性差异,24h时明显降低。结论①ConA诱导的急性免疫性肝损伤主要是由Th1类细胞、巨噬细胞和Th2类细胞分泌的炎性因子所造成。②脾脏通过Th1/Th2类细胞因子的分泌对急性免疫性肝损伤起到免疫调控作用。     

白细胞介素22对T细胞介导的小鼠肝损伤的治疗作用及初步机制探讨

目的研究人白介素22（IL-22）对T细胞介导的肝损伤小鼠的治疗作用。方法利用刀豆蛋白A（ConA）建立T细胞介导的肝损伤小鼠模型,检测静脉注射IL-22对肝损伤小鼠血清丙氨酸转氨酶（ALT）、天门冬氨酸转氨酶（AST）活性的影响,同时取小鼠肝组织进行病理学检查。并采用半定量RT-PCR方法检测IL-22刺激HepG2和LO2细胞后,对c-myc及Bcl-2基因转录表达水平的影响。结果IL-22明显降低ConA致小鼠急性肝损伤血清ALT、AST值的升高,减轻ConA对肝组织的病理损伤。体外检测IL-22对HepG2和LO2细胞表达c-myc及Bcl-2基因转录水平有促进作用。结论IL-22对T细胞介导的肝损伤小鼠模型具有治疗作用,该作用可能是通过IL-22促进肝细胞的抗凋亡因子的表达实现的。     

c-Myc抑制剂10058-F4减轻脂多糖/右旋半乳糖胺诱导的小鼠急性肝损伤

本研究探讨c-Myc抑制剂10058-F4对脂多糖(lipopolysaccharide, LPS)和右旋半乳糖胺(D-galactosamine, D-Gal)诱导的急性肝损伤的影响及其可能机制。32只雄性BALB/c小鼠(Mus musculus)分为4组：正常对照组、 10058-F4组、 LPS/D-Gal组和10058-F4+LPS/D-Gal组。通过腹腔注射LPS/D-Gal诱导小鼠急性肝损伤,10058-F4在注射LPS/D-Gal前0.5 h经腹腔注入。注射LPS/D-Gal 1.5 h后处死小鼠并采集样本,分别采用实时荧光定量PCR法和ELISA法检测肝组织中肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)的mRNA水平和血清中TNF-α的水平。另取32只小鼠经上述相同处理后,注射LPS/D-Gal 6 h后处死并采集样本,检测小鼠血清中转氨酶活性及白介素-6(interleukin-6, IL-6)的水平、肝组织中IL-6的mRNA水平及肝组织中半胱氨酸天冬氨酸特异性蛋白酶(cysteinyl aspartate specific p...     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

LPS/D-GalN诱导小鼠急性肝损伤模型的建立

目的建立脂多糖(lipopolysaccharide,LPS)/D-氨基半乳糖(D-galactosamine,D-GalN)诱导小鼠急性肝损伤模型。方法 40只雌性C57BL/6小鼠用于观察8种不同LPS与D-GalN剂量配比联合刺激后小鼠存活时间,以确定模型建立的最佳剂量。使用腹腔注射最佳剂量染毒32只雌性C57BL/6小鼠,分别在0、1、4、8 h处死,每组8只,0 h注射相同剂量生理盐水作为对照。观察染毒后小鼠肝组织病理损伤,检测血清中ALT及炎症因子IL-6、MCP-1和TNF-α表达水平变化。结果通过观察小鼠存活时间,确定腹腔注射最佳染毒剂量为LPS(2.5 mg/kg)/D-GalN(0.3 g/kg);小鼠染毒后肝组织呈进程性病变,最终发展为肝脏弥漫性坏死,肝细胞核崩解。与对照组相比,血清ALT显著升高(P0.001),IL-6、MCP-1、TNF-α均在1 h后达到最高水平(P0.001),然后持续下降。结论成功建立LPS/D-GaIN诱导小鼠急性肝损伤模型,为探索急性肝损伤的致病机制以及药物干预治疗提供有效的动物模型。     

正丁酸钠通过抑制高迁移率族蛋白B1的表达与释放减轻刀豆蛋白A诱导的小鼠急性肝损伤

本文旨在研究正丁酸钠预处理对刀豆蛋白A(concanavalin A,Con A)诱导的小鼠急性肝损伤的保护作用及机制。给小鼠腹腔注射正丁酸钠(500 mg/kg)0.5 h后,尾静脉注射Con A(15 mg/kg)诱导小鼠急性肝损伤模型,测定小鼠血清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)的水平;HE染色观察病理学改变;ELISA法检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、干扰素-γ(interferon-γ,IFN-γ)和高迁移率族蛋白B1(high-mobility group box 1,HMGB1)水平;RT-PCR和免疫组化法检测小鼠肝组织HMBG1的表达与释放。结果显示,正丁酸钠预处理能明显降低Con A诱导的急性肝损伤小鼠血清中ALT和AST水平(P0.01);显著改善肝组织损伤程度;明显降低血清中TNF-α和IFN-γ的水平(P0.01);显著抑制肝组织HMGB1的表达与释放(P0.05或P0.01)。以上结果提示,正丁酸钠预处理对Con A所诱导的小鼠急性肝损伤具有保护作用,可能与其减少炎性细胞因子TNF-α和IFN-γ的产生及抑制HMGB1的表达与释放有关。     

荷叶总生物碱激活肝脏AMPK/Nrf2通路缓解对乙酰氨基酚诱导的小鼠急性肝损伤

为探讨荷叶总生物碱(TAL)对对乙酰氨基酚(APAP)所致小鼠急性肝损伤的保护作用及可能机制,小鼠被随机分为正常组,模型组和TAL低、高剂量组(30、100 mg/kg),连续灌胃给药七天后,除正常组外,其余各组腹腔注射300 mg/kg的APAP诱导急性肝损伤。12小时后,收集各组小鼠的血清与肝脏样本,进行后续实验。结果显示,与模型组相比,TAL可显著降低血清中的ALT和AST活性,下调肝组织中TNF-α、IL-1β、IL-6和MDA含量,上调肝组织中SOD、CAT、GSH-Px和GSH的水平。此外,TAL还明显改善了APAP诱导的小鼠肝组织病变。在TAL的作用下,肝内AMPK磷酸化水平提高,Nrf2蛋白入核,HO-1和GCLC基因表达上调。综上所述,TAL对APAP诱导的急性肝损伤具有保护作用,其机制可能与激活AMPK/Nrf2通路相关。     

骆驼蓬脂转移蛋白基因真核表达载体的构建及其体内外抗瘤效应的研究

目的:构建骆驼蓬脂转移蛋白(lipid transfer protein from Peganum harmala,PhLTP)基因真核表达质粒,并探讨其对黑色素瘤B16细胞在体内外的抗肿瘤作用。方法:将PhLTP基因亚克隆至pcDNA3.1上,获得重组质粒pcDNA3.1-PhLTP;用脂质体转染法将重组质粒及空载体外转染B16细胞,MTT检测其对B16细胞生长的影响。建立B16荷瘤小鼠模型,设重组质粒(pcDNA3.1-PhLTP)、空载(pcDNA3.1)、生理盐水和阳性药物(CTX)组,分别处理小鼠后测量各组肿瘤体积并称瘤重,计算抑瘤率。光镜观察鼠脾、肝等组织变化;免疫组织化学法检测各瘤体中PhLTP、血管内皮生长因子(VEGF)及碱性成纤维细胞生长因子(bFGF)的表达。结果:pcDNA3.1-PhLTP转染B16细胞72 h后,细胞增殖能力明显受到抑制(P0.01)。注射pcDNA3.1-PhLTP组的小鼠肿瘤生长速度明显减慢,肿瘤体积小于空载和生理盐水组(P0.05)。显微镜下可见重组质粒组肿瘤细胞有不同程度的点、片状坏死,而肝、肺等无明显病理损伤。重组质粒组肿瘤组织中有PhLTP蛋白的表达,且VEGF和bFGF的阳性表达指数都低于空载和生理盐水组(P0.01)。结论:成功构建了重组表达质粒pcDNA3.1-PhLTP,体内外实验结果显示其能有效地抑制B16细胞的生长,预示了该重组质粒在治疗黑色素瘤中的潜在应用价值。     

Con A、α-Galcer与LPS/D-Gal N诱导免疫性肝损伤小鼠NKT细胞功能改变的比较研究

目的：通过比较三种免疫性肝损伤模型小鼠NKT细胞功能改变情况,寻找一种在病理生理机制上更接近临床特点的免疫性肝损伤动物模型。方法：40只小鼠随机分为空白对照组、Con A模型组、α-Galcer模型组、LPS/D-Gal N模型组,每组10只。Con A模型组尾静脉注射ConA溶液（18 mg/kg）,α-Galcer模型组腹腔注射α-Galcer溶液（40 μg/kg）,LPS/D-Gal N模型组腹腔注射LPS溶液（10 μg/kg）和D-Gal N溶液（700 mg/kg）。造模完成后8 h,检测小鼠血清转氨酶ALT、AST水平,计算肝指数,采用HE染色法观察肝组织病理改变情况,流式细胞仪分析肝组织NKT细胞含量,Western blot法检测肝组织中TNF-α、IFN-γ、IL-6蛋白表达情况。结果：与空白对照组比较,各模型组小鼠血清ALT、AST水平均显著升高（P<0.01）,肝指数增加（P<0.01或P<0.05）,肝组织病理损伤明显,NKT细胞含量显著增加（P<0.01或P<0.05）,TNF-α、IFN-γ、IL-6表达均明显上调（P<0.01）;各模型组之间比较,α-Galcer模型组血清ALT、AST含量显著低于Con A组（P<0.05）,病理损伤也相对较轻,LPS/D-Gal N模型组NKT细胞含量明显低于其余两组（P<0.05）。结论：Con A、α-Galcer和LPS/D-Gal N诱导的免疫性肝损伤模型小鼠NKT细胞明显激活,介导炎症紊乱;其中,以Con A诱导的动物模型肝脏病理损伤及免疫紊乱最为明显,更符合疾病临床特点,可作为免疫性肝损伤的首选模型。     

口服sST2质粒DNA在炎症性肠病小鼠中的免疫水平

目的制备口服可溶性ST2(Soluble ST2,sST2)质粒,观察其对于小鼠炎症性结肠黏膜免疫应答水平。方法提取小鼠脾脏总RNA,扩增sST2基因,并克隆至pcDNA3.1/myc-His A载体,构建pcDNA3.1-sST2-myc-His A质粒,借此转染COS-7细胞,使之表达sST2-myc-His A融合蛋白。利用脂质体LipofectamineTM2000包裹,制备口服sST2质粒,经灌胃给予用葡聚糖硫酸钠(Dextran sulfate sodium,DSS)诱生溃疡性结肠炎的C57BL/6实验小鼠,并用组织病理学方法观察结肠黏膜组织形态变化。用Real-time PCR检测sST2基因表达,用免疫印迹检测sST2质粒融合蛋白的表达;用ELISA检测小鼠肠固有层淋巴细胞培养上清中细胞因子(IL-4、IL-5和IL-13)的分泌水平。结果Real-time PCR检测到sST2基因的正确表达,经双酶切及测序鉴定,证明质粒pcDNA3.1-sST2-myc-His A构建成功,免疫印迹试验在转染的COS-7细胞检测到sST2-myc-His A融合蛋白的表达,其相对分子质量与预期的相符;观察到结肠黏膜组织形态发生的变化;Real-time PCR结果显示,sST2质粒组在结肠黏膜组织内sST2的表达水平明显高于pcDNA3.1组和生理盐水组(P0.01);ELISA结果显示sST2质粒组结肠固有层淋巴细胞培养上清中IL-4、IL-5和IL-13的分泌水平高于pcDNA3.1组和生理盐水组(P0.05)。结论实验成功制备口服sST2质粒;该质粒可抑制小鼠炎症性结肠黏膜组织内Th2型免疫应答。     

Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery

BACKGROUND: The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. METHODS: Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. RESULTS: PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. CONCLUSIONS: These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability.     

人乳头瘤病毒16型E5与IL-12联合基因疫苗的免疫活性

为了研制人乳头瘤病毒16型(HPV16)防治性疫苗,分析了HPV16 E5与IL-12联合基因疫苗的免疫活性。将构建的pcDNA3.1(+)/E5与pcDNA3.1(+)/IL-12联合免疫BALB/c小鼠,以ELISA测定小鼠血清中抗HPV16 E5 IgG水平、小鼠脾细胞培养上清中IFN-γ和IL-4含量;MTT法检测脾淋巴细胞增殖反应。结果显示末次免疫后,联合基因疫苗组和单基因疫苗组血清IgG A450值分别明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);且联合基因疫苗组显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组的IFN-γ和IL-4含量分别均明显高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组IFN-γ和IL-4含量(P<0.01),且联合基因疫苗组含量显著高于单基因疫苗组(P<0.01)。联合基因疫苗组和单基因疫苗组脾淋巴细胞刺激指数(SI)分别显著高于pcDNA3.1(+)组、pcDNA3.1(+)/IL-12组和PBS组(P<0.01);联合基因疫苗组与单基因疫苗组比较,SI差异无统计学意义(P>0.05)。结果表明HPV16 E5单基因疫苗以及与IL-12联合基因疫苗均能刺激机体产生较强的免疫应答,且联合基因疫苗优于单基因疫苗。     

Safety Assessment of Liver-Targeted Hydrodynamic Gene Delivery in Dogs

Evidence in support of safety of a gene delivery procedure is essential toward gene therapy. Previous studies using the hydrodynamics-based procedure primarily focus on gene delivery efficiency or gene function analysis in mice. The current study focuses on an assessment of the safety of computer-controlled and liver-targeted hydrodynamic gene delivery in dogs as the first step toward hydrodynamic gene therapy in clinic. We demonstrate that the impacts of the hydrodynamic procedure were limited in the injected region and the influences were transient. Histological examination and the hepatic microcirculation measurement using reflectance spectrophotometry reveal that the liver-specific impact of the procedure involves a transient expansion of the liver sinusoids. No systemic damage or toxicity was observed. Physiological parameters, including electrocardiogram, heart rate, blood pressure, oxygen saturation, and body temperature, remained in normal ranges during and after hydrodynamic injection. Body weight was also examined to assess the long-term effects of the procedure in animals who underwent 3 hydrodynamic injections in 6 weeks with 2-week time interval in between. Serum biochemistry analysis showed a transient increase in liver enzymes and a few cytokines upon injection. These results demonstrate that image-guided, liver-specific hydrodynamic gene delivery is safe.     

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.     

Effects of leptin gene expression in mice in vivo by electroporation and hydrodynamics-based gene delivery

In vivo electroporation and hydrodynamics-based gene delivery were utilized to test the effect of leptin gene transfer on food intake, and body and fat weights of mice. Gene transfer of pVRmob by electroporation caused a significant reduction in body weight compared with the control counterpart (p<0.05), although a lesser effect was found in food intake, and the weights of interscapular brown and epididymal fat by electroporation. As might be expected, the hydrodynamics-based transfection method significantly reduced body weight over 1 week post-transfection (p<0.05). Furthermore, epididymal fat was decreased by 50% at 1 week after gene transfer (p<0.001). These results suggest that both electroporation and hydrodynamics-based gene delivery may be effective approaches for systemic delivery of recombinant leptin to the central nervous system, and that the efficiency of gene transfer in hydrodynamics-based gene delivery was markedly higher than that in electroporation at least within the first week after transfection.     

Transgene expression of human PON1 Q in mice protected the liver against CCl4-induced injury

BACKGROUND: Oxidative stress, often in association with decreased antioxidant defenses, plays a pathogenetic role in both initiation and progression of liver injuries, leading to almost all clinical and experimental conditions of chronic liver diseases. Human paraoxonase 1 (hPON1) is a liver-synthesized enzyme possessing antioxidant properties. Here, we investigate the effects of transgene-expressed hPON1 Q on alleviating lipid peroxidation and preventing liver injury in a mouse model. METHODS: The hPON1 Q gene was cloned into pcDNA3.0 plasmid and electro-transferred into mouse skeletal muscle. After CCl4 had been administrated to induce liver injury, mice were monitored for serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malonyldialdehyde (MDA). The extent of CCl4-induced liver injury was also analyzed through histopathological observations. RESULTS: After gene delivery, hPON1 mRNA expression was detected in mouse muscle and serum PON1 activity was 1.5 times higher than that of the control counterpart. In the PON1 Q gene transferred mice, protection against CCl4-induced liver injury was reflected by significantly decreased serum ALT, AST and MDA levels compared to those in control mice (P < 0.01). Histological observations also revealed that hepatocyte necrosis, hemorrhage, vacuolar change and hydropic degeneration were apparent in control mice after CCl4 administration. In contrast, the damage was significantly prevented (P < 0.01) in the hPON1 Q transferred mice. CONCLUSIONS: Intramuscular electro-transfer of the hPON1 Q gene led to efficient expression of hPON1 in mice. Elevated levels of PON1, by virtue of its potency to alleviate oxidative stress, could protect mice from suffering CCl4-induced liver damage.     

HCV 5A基因在Hela细胞中的稳定表达及对细胞生长的抑制现象

应用PCR技术从含有HCV(Hepatitis Cvirus)全长开放阅读框的质粒pBRTM／HCV 1-3011中获得NS5A全长基因片断,利用基因重组技术将其克隆至真核表达载体pcDNA3．1(-)中。通过酶切、PCR及测序鉴定NS5A基因已正确插入到pcDNA3．1(-)中,再利用脂质体介导转染Hela细胞,48h后传代并利用pcDNA3．1(-)质粒上的neo抗性基因加入G-418进行筛选。大约两周后,获得稳定表达的细胞株。经RT-PCR及western blot验证,证实HCV的NS5A基因在Hela细胞中已经获得了表达。在培养条件完全一致的条件下,表达NS5A基因的Hela细胞与pcDNA3．1(-)转染的细胞相比,生长速度明显变慢,其倍增时间约为35-36h,比对照组细胞增加了约50％,而转染pcDNA3．1(-)的细胞的倍增时间与正常Hela细胞则无明显差别,都为23-24h。从而证明HCV的NS5A蛋白具有抑制Hela细胞生长的作用。     

流体动力学法表达乙肝病毒X蛋白小鼠模型的建立

目的建立表达乙肝病毒X蛋白的小鼠模型。方法实验动物分成模型组和对照组,模型组利用已构建的含有HBX基因的真核表达质粒pcDNA3.1（＋）-HBX,对照组以等量生理盐水代替,采用流体动力学法将质粒经尾静脉高压注入小鼠体内,24h后取小鼠肝组织,行免疫荧光、RT-PCR和Western blot法从不同水平检测HBX在小鼠肝组织内的表达情况。结果模型组小鼠RT-PCR显示肝组织内有HBX mRNA的存在,免疫荧光和Western blot检测均有HBX蛋白的表达;对照组小鼠则无HBX表达。结论成功建立了表达乙肝病毒X蛋白小鼠模型,为进一步探讨HBX蛋白在动物体内的生物学作用提供实验基础。     

小鼠白细胞介素-33基因的克隆及序列分析

目的:克隆小鼠IL-33基因全长编码区cDNA,并对其进行序列分析。方法:从BALB/c小鼠的脊髓组织中提取总RNA,逆转录为cDNA,用热启动PCR技术,扩增小鼠IL-33基因全长编码区cDNA,经双酶切后,克隆入pcDNA3.1( )载体中,构建真核表达载体pcDNA3.1-mIL-33,然后进行酶切鉴定与序列分析。结果:小鼠IL-33基因的PCR产物和重组载体经凝胶电泳和酶切鉴定、测序分析证实,其序列与GenBank中数据一致。小鼠IL-33基因的全长编码序列为801 bp,编码266个氨基酸。结论:小鼠IL-33基因成功的克隆并构建了其真核表达载体,为进一步进行IL-33的表达与功能研究奠定了基础。