A novel function of connexin 32: marked enhancement of liver function in a hepatoma cell line

Connexin 32 (Cx32) is the main gap junction protein in hepatocytes and plays an important role in the regulation of signal transfer and growth control in the liver by constructing gap junction channels and gap junctional intercellular communication (GJIC). In this study, the human Cx32 gene was transfected into a hepatoma cell line (HepG2) that showed aberrant expression of Cx32 and was deficient in GJIC. Cx32-transfected HepG2 not only expressed a higher level of Cx32 mRNA, but also showed increased GJIC compared with HepG2 and vector-transfected HepG2. Furthermore, the liver functions of ammonia removal and albumin secretion of HepG2 were markedly enhanced with Cx32 gene transfection. It may be expected to improve the cellular functions of the hepatoma cell line by Cx32 gene transfection and serve to develop an efficacious bioartificial liver.     

Organelle dynamics and viral infections: at cross roads

Viruses are obligate intracellular parasites of the host cells. A commonly accepted view is the requirement of internal membranous structures for various aspects of viral life cycle. Organelles enable favourable intracellular environment for several viruses. However, studies reporting organelle dynamics upon viral infections are scant. In this review, we aim to summarize and highlight modulations caused to various organelles upon viral infection or expression of its proteins.     

磁性纳米粒子作用于肝癌细胞的生物学效应的研究进展

磁性纳米粒子,是一类智能型的纳米材料,因其特有的性质,被广泛应用于生物医学领域,在肝癌的治疗方面也有大量的实验性研究和成果。研究和探索磁性纳米粒子治疗肝癌的新方法和途径,有着很大的现实意义。本文就磁性纳米粒子作用于肝癌细胞的生物学效应的研究现状和进展进行总结整理,从三个方面进行了综述:磁性纳米粒子直接作用于肝癌细胞,探索磁性纳米粒子的生物相容性、在肝癌细胞的分布方式以及磁性纳米粒子本身对肝癌细胞的生物学效应的影响;磁性纳米粒子协同外加磁场(稳恒磁场、极低频交变磁场和高频交变磁场)作用于肝癌细胞;磁性纳米粒子外加修饰(磁性白蛋白纳米颗粒、纳米磁流体、磁性脂质体等),作为药物载体作用于肝癌细胞。     

EGFP-Luc-Hepa1-6细胞系的构建及其在小鼠肝癌模型中的应用

目的:构建带有增强型绿色荧光蛋白EGFP及荧光素酶luciferase的真核表达载体p CMVLuciferase-IRES2-EGFP,对肝癌细胞系Hepa1-6转染后进行稳定筛选,并将表达该载体的细胞系应用于小鼠原位肝癌模型构建,以对小鼠肝癌细胞进行稳定标记与活体示踪。方法:对p GL3-Basic质粒进行Xba I酶切、Klenow片段补平黏端、Xho I酶切获得luciferase小片段,与Xho I/Sma I酶切p IRES2-EGFP质粒获得的载体大片段连接,获得重组质粒p CMV-Luciferase-IRES2-EGFP。酶切鉴定、测序比对确认序列完全正确后,以重组载体转染Hepa1-6细胞进行稳定筛选,以荧光显微镜观察EGFP表达,报告基因实验与LuminaⅡ成像系统检测荧光素酶活性。确认该细胞系中的质粒得到表达后,以该细胞系进行C57BL/6小鼠肝脏原位接种构建肝癌模型,以LuminaⅡ成像系统连续活体监测肝癌的生长,取离体的肝癌组织制备石蜡切片(HE染色)和冰冻切片(免疫荧光染色)分别观察离体肿瘤组织病理学特征及其中肝癌细胞绿色荧光蛋白表达情况。结果:成功构建表达p CMV-Luciferase-IRES2-EGFP载体的Hepa1-6细胞系(EGFP-Luc-Hepa1-6),并以该细胞系成功构建C57BL/6小鼠原位肝癌模型,实现小鼠肝癌细胞的活体示踪与体外标记。结论:成功构建EGFP-Luc-Hepa1-6细胞系,且以此细胞系构建的小鼠原位肝癌模型可以同时实现小鼠肝癌生长的连续活体监测与离体组织检测。     

两种方式接种肝癌模型的比较

目的观察直接注入法和瘤块种植法制作Wistar大鼠肝癌模型的差异。方法采用大鼠含有Walker-256肿瘤细胞的腹水离心洗涤后接种于另一组大鼠的后腿后外侧皮下,待肿瘤长到直径约为1．0em,取下肿瘤并切成1．0-2．0mm^3大小。然后将瘤块接种于15只大鼠的肝叶上;另一组（15只）按上述方式将癌性腹水接种于正常大鼠的肝叶上;两组均在第7天后采用CT和开腹后游标卡尺分别测量种植性肝癌的直径。结果腹水直接注射法与瘤块种植法的肿瘤成瘤率分别为：86．7％和80％,两者并不具有统计学意义（P〉0．05）。第7天时,直接注射法所形成肿瘤的直径大于瘤块种植法的肿瘤（P〈0．05）。但瘤块法所引起的腹腔转移的可能性要少于腹水直接种法。结论直接注射法和瘤块种植法制作大鼠肝癌模型均可以满足临床实验研究的需要,但直接注射法的成瘤时间短,腹腔转移可能性也大;瘤块种植法成瘤时间略长,但腹腔转移可能性少于直接法。     

Increased ROS generation and p53 activation in α-lipoic acid-induced apoptosis of hepatoma cells

α-lipoic acid (α-LA) is an antioxidant used for the treatment of a variety of diseases, including liver cirrhosis, heavy metal poisoining, and diabetic polyneuropathy. In addition to its protective effect against oxidative stress, α-LA induces apoptosis in different cancer cells types. However, whether α-LA acid induces apoptosis of hepatoma cells is unknown. Herein, we investigated whether α-LA induces apoptosis in two different hepatoma cell lines FaO and HepG2. The results showed that α-LA inhibits the growth of both cell lines as indicated by the reduction in cell number, the reduced expression of cyclin A and the increased levels of the cyclin/CDKs inhibitors, p27^Kip1 and p21^Cip1. Cell cycle arrest was associated with cell loss, and DNA laddering indicative of apoptosis. Apoptosis was preceded by increased generation of reactive oxygen species, and associated with p53 activation, increased expression of Bax, release of cytochrome c from mitochondria, caspases activation, decreased levels of survivin, induction of pro-apoptotic signaling (i.e JNK) and inhibition of anti-apoptotic signaling (i.e. PKB/Akt) pathways. In conclusion, this study provides evidence that α-LA induces apoptosis in hepatoma cells, describes a possible sequence of molecular events underlying its lethal effect, and suggests that it may prove useful in liver cancer therapy.     

Cytokine-mediated induction of metallothionein in Hepa-1c1c7 cells by oleanolic acid

Oleanolic acid (OA), a pentacyclic triterpene acid, has been reported to possess inducing activity of hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of OA on the regulation of MT expression in an in vitro model. OA that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the OA-treated conditioned media from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with OA and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in a decrease in the effects of OA on the MT induction. In the OA-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. However, the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the OA-treated conditioned media from the RAW 264.7 cells. These results suggest that the up-regulation of MT expression by OA was mediated by the TNF-alpha and IL-6 released from UA-activated macrophages.     

乙型肝炎病毒与肿瘤抑制基因p53相互作用的生物学功能及意义

以前的研究发现：ＨＢＶ Ｘ蛋白能与肿瘤抑制基因ｐ５３蛋白结合,在原发性肝癌的发生中具有重要意义。为进一步探讨二者之间的关系,经过计算机比较分析发现,在ＨＢＶ基因组中存在与经典的ｐ５３ＤＮＡ－蛋白质结合位点类似的序列（ＴＧＣＣＴ）,用含有此序列的ＨＢＶ基因片段作探针,与肝癌细胞核蛋白作用,通过凝胶电泳迁移率移位实验、凝胶电泳迁移率超移位实验和原位紫外线交联实验,确定其ＨＢＶ ＤＮＡ与ｐ５３蛋白的特异     

天然甾体皂甙化合物的抗肿瘤活性

采用MTT法,以长春新碱(VCR)为阳性对照,研究了6种从菝葜属植物中分离提取的天然甾体皂甙化合物对肝癌SMMC-7721、人宫颈癌HeLa和胃腺癌MGc80-3细胞生长的抑制作用.结果显示;6种甾体皂甙抗肿瘤活性与其化学结构密切相关,对三种癌细胞的抑瘤作用强度相同,抑癌活性的顺序为:薯蓣皂甙>VCR>SQD_4>SQD_3>M_1>SQD_1,甲基原薯蓣皂甙.甾体骨架的差异性是决定这类化合物抗肿瘤活性的主要因素.     

蓖麻毒素对肝癌细胞有丝分裂原激活蛋白激酶的影响

为进一步探讨蓖麻毒素的毒作用机理 ,采用 Western印迹和免疫组化方法研究蓖麻毒素对人肝癌细胞内磷酸化状态和有丝分裂原激活蛋白激酶 (MAPK)的影响 .其结果表明 :蓖麻毒素能够影响细胞内的磷酸化状态 ,并能激活细胞内的有丝分裂原激活蛋白激酶 ,对 MAPK的激活有时间依赖性和剂量依赖性 . MAPK信号传导途径参与了蓖麻毒素对肝癌细胞的毒作用 .