Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM To establish a cell culture system with long-termreplication of hepatitis C virus in vitro.METHODS Human hepatoma cell line 7721 was tested forits susceptibility to HCV by incubating with a serum from apatient with chronic hepatitis C.Cells and supernatantwere harvested at various phases during the culturingperiods.The presence of HCV RNA,the expression ofHCV antigens in cells and/or supernatant were examinedby RT-PCR,in situ hybridization and immunohisto-chemistry respectively.RESULTS The intracellular HCV RNA was first detectedon d2 after infection and then could be intermittentlydetected in both cells and supernatant over a period of atleast three months.The expression of HCV NS_3,CP_(10)antigens could be observed in cells.The fresh cells couldbe infected by supernatant from cultured infected cellsand the transmission of viral genome from HCV-infected7721 cells to PBMCs was also observed.CONCLUSION The hepatoma line 7721 is not onlysusceptible to HCV but also supports its long-termreplication in vitro.     

Significance of tumor necrosis factor (TNF) and interleukin-1 (IL-1) in the pathogenesis of fulminant hepatitis: Possible involvement of serine protease in TNF-mediated liver injury

The effect of tumor necrosis factor (TNF)-α and interleukin-1 (IL-l)α, which are thought to be principal mediators inducing homeostatic abnormalities during endotoxemia, were investigated on cultured hepatocytes in an attempt to understand their role in the pathogenesis of fulminant hepatitis. Both TNF and IL-1 had no direct cytotoxicity on cultured adult rat hepatocytes as assessed by their effects on protein synthesis and also cytosolic enzyme activity released into the culture medium in the presence of 5 mM Dgalactosamine (Ga1N). However, IL-1 caused a dose-dependent inhibition of DNA synthesis in cultured adult rat hepatocytes. Moreover, the serum from TNF-treated rats, prepared after intravenous administration of TNF (5 × 10⁴ U per rat), caused a significant increase of enzyme release into culture medium in contrast to control rat serum. The cytotoxicity disappeared when the serum from TNF-treated rats was pretreated by heating at 56°C for 30 min, and was decreased by the addition of the protease inhibitor, aprotinin.In vivo, gabexate mesilate, a serine-type protease inhibitor, prevented Ga1N/TNF-induced fulminant hepatitis, whereas MX-1, an anti-complement agent, had no such effect. These results strongly suggest that IL-1 has a inhibitory effect on hepatocytes in terms of DNA synthesis and that TNF indirectly induces hepatocellular damage through the serine proteases which are possibly activated by the cytokine in vivo.     

氟对小鼠神经细胞内游离钙的影响

目的观察氟对小鼠神经细胞内游离钙(Ca~(2 ))的影响,为探讨氟中毒的发生机制提供实验依据。方法①采用细胞培养方法,体外培养小鼠神经细胞。根据加氟剂量(NaF,0、5.0、10.0、15.0、20.0、40.0 mg/L)不同分为6组,在培养第15天,收集各组细胞,激光扫描共聚焦显微镜记录细胞内Ca~(2 )荧光图像;②出生后7d小鼠,按腹腔注射不同剂量的氟溶液(0、0.7、2.8、11.2 mg/kg)分为4组,于注射后8h处死小鼠,收集脑细胞,制备细胞悬液,记录细胞内Ca~(2 )荧光图像。结果体外培养加氟(≥5.0 mg/L)和体内腹腔注射氟溶液(≥0.7 mg/kg)均可导致神经细胞内Ca~(2 )增加,组间比较差异有统计学意义(F=191.20、93.83,P<0.01);各加氟组细胞内Ca~(2 )水平与对照组比较,差异均有统计学意义(P<0.01)。结论氟诱导神经细胞内Ca~(2 )增加,且随着加氟剂量的增加,细胞内Ca~(2 )水平也增加,二者呈剂量效应关系。     

Androgen receptors in cultured rat adipose precursor cells during proliferation and differentiation: regional specificities and regulation by testosterone

Different studies suggest that sex hormones affect adipose tissue metabolism and deposition. To investigate the possibility that androgens may play a role in adipose tissue development, we have studied androgen receptors (AR) in rat adipose precursor cells from two different anatomical fat deposits, one deep intraabdominal (epididymal) and one subcutaneous (inguinal) during the proliferation and differentiation processes. AR were quantified by [³H]R1881 specific binding in whole cells and the nuclear fraction and were localized by immunocytofluorimetry in both the cytosol and the nucleus. During the proliferative phase, total AR level decreased from D3 to D6. At confluence (D5), AR were higher in epididymal (64±4 fmol/mg protein) than in subcutaneous (33±3 fmoles/mg proteins) preadipocytes and were up-regulated by testosterone but not by 5α-dihydrotestosterone or by 17β-estradiol. At differentiation (D10-11), nuclear AR decreased by 50% in both precursor fat cell populations when compared to the confluent state (D5) and AR were no more up-regulated but rather down-regulated by testosterone. Because AR are present in preadipocytes and are differently regulated by testosterone depending on the stage of proliferation and differentiation, this study suggests that testosterone may play a role in the control of the adipogenic process.     

Persistence of colchicine resistance in Ehrlich-Lettré ascites tumors and cell strains

Summary The effect of colchicine on mitoses of mutant HD33 Ehrlich-Lettré ascites cells growing in vivo and in vitro was studied.HD33 mouse ascites tumors are colchicine-resistant. The LD₅₀ of colchicine in mice bearing HD33 ascites tumors was 1.4 mg/kg body weight (b.w.), but a single dose of 3.33 mg colchicine/kg b.w. failed to suppress the anaphase of HD33 tumor mitoses for 24h. No change in the level of colchicine resistance was observed after 269 weekly transplantations of HD33 ascites tumors without colchicine.In suspension culture, growth of HD33 ascites cells ceased at 1.5×10^-6 M colchicine. 10^-5 M colchicine suppressed the anaphase of HD33 mitoses and produced typical C-mitoses within one hour. The same effects on mitoses of colchicine sensitive Ehrilich ascites cells in vitro were achieved with 10^-6 M colchicine.In HD33 ascites cell cultures grown without colchicine, only a slight increase in colchicine sensitivity was registered after 5 years. Parallel cultures were propagated for the same period in the presence of 10^-7 M colchicine (HD33C ascites cells) without detectable growth alterations; the resistance level increased slightly. The limit of 10^-6 M colchicine was tolerated by the ascites cells in permanent culture without growth reduction (HD33CS ascites cells). ³H-colchicine binding studies suggest a permeability barrier of the plasma membrane as a mechanism of genetically fixed resistance.Some of the results have been reported at the International Symposium on Microtubules and Microtubular Inhibitors, Beerse, Belgium, Sept. 2–5, 1975     

胰腺癌SW1990细胞热疗温度及机制初探

目的观察多种温热疗法对人胰腺癌细胞SW1990生长的影响,确定对其进行热疗的最佳温度和时间,并初步探讨其机制。方法MTT检测不同温度、不同作用时间对SW1990细胞生长的抑制作用,光镜、电镜观察不同温度热疗后细胞形态的改变,RT-PCR检测各组凋亡相关基因bcl-2、bax的表达情况。结果MTT示44C对细胞增殖的抑制作用最明显,且44 C的IC50值在1 h左右;光镜示44C 热疗1 h较其他温度作用1 h出现的细胞凋亡小体数多;电镜示44 C热疗1 h细胞的超微结构破坏最明显,呈现明显凋亡。RT-PCR显示随温度的增高,凋亡相关基因bax的表达呈上升趋势。结论44C热疗1 h为SW1990细胞最佳的热疗温度和时间,热疗可能通过上调促凋亡基因bax的表达而诱导SW1990 胰腺癌细胞凋亡。     

环维黄杨星D对PC12谷氨酸损伤的保护作用的实验研究

目的:探讨环维黄杨星D(CBV-D)对PC12细胞谷氨酸(Glu)损伤模型的影响.方法:用Glu复制PC12细胞兴奋性氨基酸损伤模型,通过MTT染色和乳酸脱氢酶(LDH)活性的测定来研究CBV-D对PC12细胞的保护作用.结果:CBV-D抑制Glu造成的损伤,降低LDH的漏出.结论:CBV-D对兴奋性氨基酸引起PC12细胞损伤具有保护作用.     

HCV CE2融合蛋白在家蚕培养细胞、蚕幼虫中的表达及其初步应用

目的:高效表达HCV CE2融合蛋白并进行初步应用. 方法:将2a型HCV全长CE2基因的cDNA重组于质粒pBacPAK8,构建重组转移载体pBacPAK-CE2,与经线性化修饰的家蚕核型多角体病毒(BmNPV)DNA共转染家蚕培养细胞株,构建重组病毒. 双酶切及PCR鉴定重组病毒基因组中目的片段的表达. 重组病毒感染BmN细胞株及五龄家蚕幼虫后,SDS-PAGE分析细胞培养上清、细胞抽提物及幼虫体液样品中,HCV CE2融合蛋白的特异性条带. 并用间接ELISA法初步检测表达产物的生物活性. 结果:双酶切及PCR鉴定重组病毒基因组含有约1.6 kb的目的片段,重组病毒感染后的BmN细胞培养上清、细胞抽提物及幼虫体液样品中,均可见一Mr约90×103的特异性条带;用间接ELISA检测证明表达产物具有较好的免疫原性. 结论:HCV CE2融合基因在家蚕培养细胞及蚕幼虫中获得了高效表达,并具有生物活性,为进一步进行疫苗的研究及临床诊断试剂的开发奠定基础.     

人肝癌耐药细胞系的建立及生物学特性的研究

目的:培养建立耐药肝癌细胞模型QGY7703/DDP,分析人肝癌耐药细胞系生物学特性的改变.方法:应用人肝癌细胞株QGY7703,采用顺铂(DDP)逐步提高加间歇诱导历时6个月在体外建立QGY7703/DDP细胞系模型.观察该细胞的生长规律,利用四甲基偶氮唑蓝(MTT)法分析暴露前后细胞的增殖和药物对细胞毒性的差异.结果:经过将QGY7703持续暴露,细胞对化疗药物5-氟尿嘧啶(5-FU)、DDP、阿霉素(ADM)、丝裂霉素(MMC)的敏感性发生了改变.QGY7703/DDP对5-FU的耐药性是亲代细胞的1.533倍,对DDP的耐药性是亲代细胞的2.181倍,对ADM的耐药性是亲代细胞的7.080倍,对MMC的耐药性是亲代细胞的9.461倍.结论:化疗药物能够诱导培养的肿瘤细胞产生耐药性.     

骨髓贴壁细胞向破骨细胞的分化

背景：一般认为采用贴壁分离法得到的小鼠骨髓中能够贴壁的细胞为间充质干细胞,并能分化成为成骨、成脂肪及成软骨细胞,其中贴壁的细胞是否有分化为破骨细胞的潜能尚不明确。 目的：检测贴壁分离法得到的小鼠骨髓中能贴壁的细胞是否能够分化为破骨细胞。 方法：采用贴壁法得到小鼠原代间充质干细胞,以及通过贴壁1-5d后得到不同时间贴壁的骨髓细胞。原代间充质干细胞及不同时间贴壁的骨髓细胞分别用普通培养基,及含m-csf和RANKL培养基,培养9 d后进行碱性磷酸酶和抗酒石酸酸性磷酸酶染色。原代间充质干细胞传代后,第2代间充质干细胞分为4组,分别用普通培养基、含m-csf培养基、含RANKL培养基及含m-csf和RANKL的培养基培养,9 d后进行碱性磷酸酶及抗酒石酸酸性磷酸酶染色。 结果与结论：贴壁法得到较均一的间充质干细胞,原代及传代贴壁细胞的联合诱导组抗酒石酸酸性磷酸酶染色均为阳性,说明原代和传代的贴壁骨髓细胞中均有可以分化为破骨细胞的细胞。不同时间贴壁的细胞诱导后碱性磷酸酶及抗酒石酸酸性磷酸酶染色有差异,说明不同时间贴壁的细胞存在分化差异。