外源hTERT基因对原代培养肝细胞增殖作用的研究

目的探讨表达hTERT蛋白的重组腺病毒rAd-hTERT感染体外原代培养的肝细胞后对其增殖情况的影响。方法将表达hTERT蛋白的重组腺病毒rAd-hTERT感染体外原代培养的肝细胞。同时设空载体对照组及空白对照组。通过定量PCR及Western-blot检测基因在细胞中的表达情况；MTT法观察其对细胞增殖的影响；采用流式细胞术检测rAd-hTERT对细胞周期和凋亡的影响。结果（1）以50MOIrAd-hTERT感染肝细胞可明显促进其生长增殖，但不会引起细胞毒性作用。（2）定量PCR在转录水平检测rAd-hTERT感染后的细胞中hTERT表达量明显增加。（3）Western-blot在蛋白水平验证rAd-hTERT感染后的细胞中hTERT蛋白的表达及其蛋白活性。（4）流式细胞DNA含量分析显示，rAdhTERT可引起肝细胞周期S＋62／M期增加，并抑制细胞的凋亡。结论rAd-hTERT在体外能有效促进原代肝细胞的生长。与阴性对照比较，同期生长的肝细胞S＋62／M期细胞增加，凋亡细胞减少（P〈0．05），并未引起细胞的不典型增生表现。     

转染HGF基因对肝细胞的生物学效应

目的探讨肝细胞生长因子(hepatocyte growth factor，HGF)基因对肝细胞生长增殖能力的影响。方法通过脂质体介导法，将HGF基因导入肝细胞中。用荧光显微镜以及原位杂交观察到HGF基因表达。采用检测细胞生长曲线、Ki-67蛋白和嗜银蛋白免疫组化观察肝细胞生长增殖能力及DNA合成能力的变化。结果荧光显微镜观察可见到绿色荧光蛋白的表达，用原位杂交方法进一步证实了HGF蛋白在细胞中的表达。细胞生长曲线显示，转染HGF基因的肝细胞增殖速度明显增快，Ki-67蛋白和嗜银蛋白表达明显增多，提示转导HGF基因使肝细胞增殖活性增加。结论本实验显示转染的HGF可表达并有促细胞分裂活性。为进一步了解HGF分子生物学特性及治疗应用提供了理论基础。     

白细胞介素1β对大鼠肝细胞毒性作用的细胞内信号传导途径

目的研究白细胞介素-1β(IL-1β)对原代培养大鼠肝细胞细胞毒性作用的细胞内信号传导机制. 方法使用雄性Wistar大鼠,原位胶原酶灌注分离肝细胞.应用比色法测定乳酸脱氢酶(LDH)活性,Wes tern Blot方法分析c-Jun N末端激酶(JNK)、p38激酶的表达,凝胶电泳移动抑制实验检测激活物蛋白-1(AP-1)的结合活性. 结果 IL-1 β促进原代培养大鼠肝细胞LDH释放(IL-1β刺激组与对照组LDH活性分别为21.9%±3.6% 和11.0%±1.8%,P＜0.01);IL-1β通过激活JNK途径,激活转录因子AP-1,对原代培养大鼠肝细胞产生细胞毒性作用,而同时激活的p38激酶途径对这一过程起负性调节作用. 结论 IL-1β通过激活JNK途径,激活转录因子AP-1,对原代培养大鼠肝细胞产生细胞毒性作用.     

大鼠肝细胞、Kupffer和Ito细胞的分离与培养

我们采用Ｃｏｌｌａｇｅｎａｓｅ体内门静脉灌注结合体外Ｔｒｙｐｓｉｎ皿内消化制备游离肝细胞，进而采用ＰｒｏｎａｓｅＥ和Ｎｙｃｏｃｌｅｎｚ离心处理获单－Ｋｕｐｆｆｅｒ细胞和Ｉｔｏ细胞。三种细胞的产生率分别为１．２×１０８，１．１×１０７和２．３×１０７；其细胞纯度分别为９５％，８１％和８８％；存活率都在９０％以上。作者对三种细胞原代培养过程的形态特征和生物学活性进行了观察与讨论。     

PCNA反义cDNA基因转导抑制人膀胱癌细胞增殖的研究

目的 探讨增殖细胞核抗原（PCNA）基因反义cDNA对人膀胱癌细胞体外增殖活性的抑制作用。方法 构建PCNA反义cDNA真核表达载体并转染膀胱癌DJ细胞,通过细胞生长曲线、MTT比色分析、H^3-TdR掺入法检测癌细胞增殖活性,流式细胞仪（FCM）分析细胞周期时相变化,SABC免疫组化观察癌细胞PCNA蛋白表达水平。结果 PCNA反义cDNA导入后,膀胱癌DJ细胞生长速率显著减慢（P＜0．01）,增殖活性抑制率59．02％（P＜0．05）,DNA合成速率降低52．31％（P＜0．01）,S期细胞减少,细胞周期阻滞于G0／G1期,PCNA蛋白表达显著减弱（P＜0．05）,差别均有显著性意义。结论 PCNA反义cDNA基因转导能有效抑制膀胱癌细胞的PCNA蛋白表达及体外增殖活性,为肿瘤基因治疗提供了有效途径。     

重组腺相关病毒转导NIH3T3建立神经生长因子真核表达细胞株的研究

目的探讨重组腺相关病毒(recombinantadeno-associatedvirus,rAAV)转导美国国家健康研究中心建系的小鼠胚胎成纤维细胞(mouseembryofibroblast,NIH3T3)建立神经生长因子β(nervegrowthfactorsubunitβ,NGFβ)真核表达细胞株可行性,并检测其生物活性。方法使用表达NGFβ的重组腺相关病毒感染NIH3T3细胞,取感染重组病毒的3T3细胞培养基上清,经表达蛋白提取和浓缩,肝素-SepharoseCL-6B亲和层析柱的纯化;酶免疫斑点法检测表达蛋白的NGFβ抗原活性;Coomassic亮蓝G250法测定表达蛋白的浓度;十二烷基硫酸-聚丙烯酰胺凝胶电泳(sodiumdodecylsulfat-polyarylanidegelelectrophoresis,SDS-PAGE)分析检测表达蛋白的分子量,计算表达蛋白占总蛋白的比率;鸡胚背根神经节突起生长实验和小鼠嗜铬瘤细胞(mousea鄄drenalpheochromocytomacells,PC12)无血清培养生长刺激5溴-2-脱氧尿嘧啶(5-bromo-2-deoxyurididne,Brdu)掺入实验观察真核表达蛋白的生物学活性。结果转导的3T3细胞具有13.6kd的表达蛋白,每升细胞培养基中可以获得400~500μgNGFβ蛋白,表达蛋白纯度可达95.4%以上。表达蛋白具有很强的NGF抗原活性。鸡胚背根神经节突起生长实验显示真核表达的NGFβ在10ng/ml时有明显的促进突起生长作用,PC12细胞的Brdu掺入实验显     

大鼠肝细胞,krpffer和Ito细胞的分离与培养

我们采用Ｃｏｌｌａｇｅｎａｓｅ体内门静灌注结合体外Ｔｙｐｓｉｎ皿内消化制备游离肝细胞。进而采用ＰｒｏｎａｓｅＥ和Ｎｙｅｏｃｌｅｎａ离心处理获－Ｋｕｐｆｆｅｒ细胞和Ｉｔｏ细胞。三种细胞的产生率分别１．２×１０＾８，１．１×１０＾７和２．３×１０＾７；其细胞纯度分别为９５％，８１％和８８％；存活率都在９０％以上。作者对三种细胞原代培养过程的形态特征和生物学活性进行了观察与讨论。     

聚集蛋白基因在原代培养成肌细胞中的表达

目的研究神经源性聚集蛋白（agrin）基因在原代培养成肌细胞中的表达。方法原代培养成年大鼠的成肌细胞，免疫细胞化学方法进行鉴定，将agrin—Y428基因cDNA片段亚克隆入pCDNA3真核表达载体，重组子经脂质体介导转染成肌细胞，G418筛选，获得具有抗性的克隆，增殖后以逆转录一聚合酶链反应（RT—PCR）和免疫荧光的方法检测基因的转录和蛋白表达，并初步测定其生物学活性。结果原代培养8周后，90％以上仍为成肌细胞，转染神经源性agrin基因后，成肌细胞可以表达相应的mRNA和有功能活性的蛋白。结论原代培养的成肌细胞可以作为agrin基因转移的有效载体，可用于进一步肌肉功能减退的基因治疗中。     

逆转录病毒介导的p16基因治疗原发性肝癌的实验研究

目的 调查转入野生型p16cDNA对肝细胞性肝癌细胞生物学行为的影响。方法 构建p16基因的逆转录病毒表达载体pCLXSN-p16。完成病毒包装后，分别感染p16表达阴性与阳性的肝癌细胞株，并筛选出各自稳定表达株，观测转基因后外源P16蛋白的表达及肿瘤细胞的生长情况。并检测肿瘤细胞的凋亡与生长周期。结果 我们包装产生的pCLXSN-p16假病毒具有感染肿瘤细胞并表达外源P16蛋白的功能，p16表达阴性的细胞SNU-449转入野生型p16基因后，细胞生长速度明显减慢，G0-G1期细胞明显多于转基因前，而p16表达阳性的细胞HepG2.2.15，转入外源p16cDNA后，其生长状况及细胞周期则未见明显改变，就SNU-449与HepG2.2.15细胞株而言，转入p16cDNA后，其生长状况及细胞周期则未见明显改变，就SNU-449与HepG2.2.15细胞株而言。转入p16cDNA均未能诱导细胞凋亡。结论 转入外源p16基因可抑制P16蛋白表达阴性的肝癌细胞的生长。     

原代猪肝细胞及主动脉内皮细胞的提取和培养

目的建立有效高活力的原代猪肝细胞和主动脉内皮细胞提取和培养的方法。为异种移植排斥反应的靶细胞——血管内皮细胞和肝细胞的研究提供基础。方法从野生巴马猪分离肝脏和主动脉血管,通过蠕动泵灌注Ⅱ型胶原酶消化的方法提取猪肝细胞,低速离心和差速贴壁法纯化肝细胞,过碘酸雪夫(PAS)染色、白蛋白与肝细胞核因子4α免疫荧光染色对原代肝细胞进行鉴定;利用Ⅰ型胶原酶血管管腔灌注消化法提取猪主动脉内皮细胞,并通过检测因子Ⅷ相关抗原vWF及内皮细胞吞噬乙酰化低密度脂蛋白的方法进行鉴定。结果通过本文方法提取到了大量高活力的原代猪肝细胞和主动脉内皮细胞,猪肝细胞可以连续培养一周,内皮细胞可进行传代培养和冻存。两种细胞分别表达肝细胞和内皮细胞标志性蛋白。结论本研究提供的原代猪内皮细胞和肝细胞提取方法是制备大量高活力的原代内皮细胞及肝细胞的可靠方案。     

人血管内皮细胞生长因子cDNA克隆和在大肠杆菌的高效表达

目的 在大肠杆菌高效表达人血管内皮生长因子蛋白质。方法 用PCR从人胎儿脑cDNA文库扩增血管内皮细胞生长因子（VEGF）cDNA,得到517bp的DNA片段。扩增片段重组到M13mP18中,经测序证实为VEGF165cDNA。将该片段重组到PRL621温控表达质粒中,在大肠杆菌表达20kd的重组蛋白。结果 该表达产物占菌体总蛋白的35%,其N-端15个氨基酸序列与天然VEGF165蛋白相应序列一致。结论 工程菌TG1/PRL621/VEGF高效表达人VEGF165蛋白质。     

肝特异性非病毒基因治疗载体介导增强型绿色荧光蛋白基因在小鼠肝细胞及肝脏中的表达

目的观察肝细胞特异性基因治疗载体蛋白介导增强型绿色荧光蛋白(EGFP)在体外培养小鼠肝细胞和小鼠肝脏中表达。方法将基因工程制备的肝细胞特异载体蛋白与真核表达质粒以所带电荷比为1：1的比例混合，两者结合形成蛋白-DNA复合物，将该复合物加入体外培养的小鼠肝细胞培养基中或经门静脉注射小鼠体内，用激光共聚焦显微镜观察EGFP在体外培养小鼠肝细胞及活体小鼠肝脏组织中的表达。结果在体外培养的小鼠肝细胞和小鼠肝脏切片中均可以观察到明显的绿色荧光，而对照组未能观察到绿色荧光蛋白。结论构建的肝细胞特异性载体蛋白可以有效的将真核表达质粒载体pcDNA3-EGFP带入肝细胞和活体肝脏组织。可能为肝细胞基因治疗提供有效载体。     

Morphology,viability and functions of suckling pig hepatocytes cultured in serum-free medium at high density

BACKGROUND: In bioartificial liver preparation, serum-contained medium is ordinarily replaced by serum-free medium and hepatocytes are generally cultured at high density. This study was to undertaken to evaluated the dynamic changes in morphology, viability and functions of porcine hepatocytes in serum-free medium at high density. METHODS: Hepatocytes were isolated from suckling pigs by modified two-step in situ collagenase perfusion method and cultured in serum-free medium at high density. Morphology, viability, protein and glucose syntheses, G-6-Pase activity, diazepam transformation of hepatocytes and release of LDH in supernatant during 7 days of culture were evaluated. These measurements were also determined on both groups of hepatocytes cultured at low-density in serum-free medium and serum-contained medium, which served as control groups. RESULTS: Morphology and protein synthesis of hepatocytes cultured in serum-free medium at high density were stable over the course of 7 days. High viability (>90%) was obtained though it declined with time. Diazepam transformation of cells was higher on days 2 and 3. Glucose synthesis of cells declined from day 3 to day 7. G-6-Pase activity of the hepatocytes declined apparently after 1 day of culture and it was maintained at a low level from day 1 to day 7. Release of LDH in supernatant was higher on days 1, 2 and 3. There were no significant differences in viability and functions of hepatocytes except for G-6-Pase activity at low-density culture between the serum-free medium group and the serum-contained medium group. The functions of hepatocytes cultured at high density were lower than at low-density culture. CONCLUSIONS: The results showed that the morphology, viability, protein synthesis and diazepam transformation of hepatocytes cultured in serum-free medium at high density were maintained during 7 days of culture. The serum-free medium provided indices of cell viability and functions that were comparable to serum-contained medium. The functions of hepatocyte cultured at high density (1 x 10(7) cells/ml) were lower than at low density (5 x 10(5) cells/ml).     

Effects of anti-insulin receptor antibodies (AIRA) on downregulation and turnover of insulin receptors on cultured hepatocytes

We have studied the effects of two polyclonal anti-insulin receptor antibodies (AIRA) on insulin receptor downregulation and turnover in rat hepatocytes in primary culture. Downregulation was determined by measurement of insulin binding after acid washing of cells to remove AIRA. Insulin receptor turnover was estimated by measurement of insulin binding after inhibition of synthesis of functional receptors with tunicamycin (0.5 micrograms/ml). Exposure of hepatocytes to AIRA (both sera were of comparable effectiveness) resulted in progressive, time- and dose-dependent losses of insulin binding (maximal loss was about 55% after 24 h of incubation with AIRA diluted 1:25). Cycloheximide (100 microM) prevented AIRA-mediated downregulation. The t1/2 of disappearance of cell surface insulin binding capacity determined with tunicamycin was 8.0 h. Addition of insulin (1000 ng/ml) or AIRA to tunicamycin reduced the t1/2 to 2.6 h (insulin), 2.2 h (patient B10), and 2.0 h (patient 1). These data suggest that AIRA downregulated insulin receptors on cultured hepatocytes by accelerating their rate of disappearance, inhibition of protein synthesis prevented AIRA-mediated downregulation, and downregulation by AIRA of insulin binding may be partially responsible for the desensitization of target cells to some of the insulin-like actions of these autoantibodies.     

胎牛血清在体外培养肝细胞极性形成的作用

目的观察胎牛血清在体外培养肝细胞极性形成的作用。方法分别利用含胎牛血清和不含胎牛血清培养液培养成鼠肝细胞,并观察血清对肝细胞形态、特异性膜区域蛋白分布以及蛋白分泌的影响,以确定胎牛血清在体外肝细胞极性形成的作用。结果不含血清组,肝细胞形成肝板样结构,肝细胞膜区域蛋白特异性分布,肝细胞保持稳定的蛋白分泌并持续增长达2周之久。而含血清组肝细胞培养3d后,分化良好的小管样结构消失,肝细胞膜区域蛋白失去特异性分布,蛋白分泌逐渐减少。结论胎牛血清可阻止肝细胞极性的形成,对于肝细胞移植及生物人工肝支持具有重要意义。     

Modulation by a sulfonylurea of insulin-dependent glycogenesis, but not of insulin binding, in cultured rat hepatocytes. Evidence for a postreceptor mechanism of action

To detect potential direct effects of the sulfonylurea glyburide on hepatic carbohydrate metabolism, we tested whether the drug was capable of modulating insulin binding and glycogenesis in primary cultured hepatocytes. After 24-h culture under serum- and hormone-free conditions, cells were incubated with or without 10(-8) M insulin and/or glyburide (0.1-5.0 micrograms/ml) for another 24 h. Then, specific 125I-insulin binding and basal and insulin-stimulated glycogen synthesis were determined. Acute addition of glyburide to previously untreated cells did not modulate any of these parameters. Incubation for 24 h with 2 micrograms/ml of glyburide did not affect the DNA and protein content of the dishes. Cellular glycogen content and basal glycogenesis also remained unchanged by glyburide in hepatocytes incubated in the absence of insulin, but glycogen content was increased and basal glycogen synthesis decreased in insulin-pretreated cells. In contrast, glyburide increased insulin-stimulated glycogenesis in a dose-dependent fashion in both insulin-pretreated and control cells by enhancing responsiveness, but not sensitivity, toward insulin. Pretreating hepatocytes with 10(-8) M insulin caused a 40% reduction in specific insulin binding. Glyburide did not modulate insulin binding or degradation in control cells nor was insulin-induced regulation of insulin receptors affected. These results demonstrate a direct dose-dependent effect of a sulfonylurea on an insulin action toward hepatic carbohydrate metabolism, and suggest that this effect is mediated by a postreceptor mechanism.     

人降钙素基因相关肽重组腺相关病毒在原代培养大鼠阴茎海绵体平滑肌细胞中的表达及其作用

目的:观察腺相关病毒载体介导的人降钙素基因相关肽(hCGRP)基因在原代培养的大鼠阴茎海绵体平滑肌(CCSM)细胞的表达及其作用,探讨其应用于勃起功能障碍(ED)基因治疗的可行性。方法:原代培养的SD大鼠CCSM细胞随机分为4组:实验组、空病毒组、示踪剂组和对照组,分别以可分泌表达hCGRP重组腺相关病毒VssH-GCMV-hCGRP、空病毒VssHGCMV和表达绿色荧光蛋白(GFP)重组腺相关病毒VssCMV-GFP进行体外转染或不予任何处理。采用斑点印迹试验检测培养液中的hCGRP和放射免疫法检测转染细胞中的环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)水平,观察hCGRP和示踪剂GFP的表达及其对CCSM细胞的影响。结果:重组腺相关病毒载体能有效地将外源基因hCGRP导入大鼠CCSM细胞并稳定表达,与空病毒组和对照组相比,该病毒明显刺激大鼠CCSM细胞中cAMP水平升高[(48.7±1.1)nmol/L对(12.3±1.2)nmol/L和(7.8±1.4)nmol/L,P均<0.01],培养液中可检测到免疫反应性hCGRP。结论:可分泌表达生物活性肽重组腺相关病毒基因转移系统可有效进行多肽类在CCSM细胞过表达,并影响该细胞的cAMP水平,可被应用于ED的基因治疗。