靶向c-Met的shRNA重组质粒的构建及生物活性鉴定

目的设计以人c-Met基因为靶向的短发夹RNA（short hairpin RNA,shRNA）,构建携带此shRNA的重组质粒,检测其抑制c-Met mRNA及蛋白表达的效应,筛选RNAi作用最强的shRNA片段。方法设计3对针对c-Met基因不同位点的shRNA片段,构建携带此shRNA的重组质粒pSilencer 2.0/c-Met-shRNA,通过脂质体介导的方法将其转染到喉癌Hep-2细胞株中。采用RT-PCR及Western-Blot法检测c-Met mRNA及蛋白表达情况,比较转染前后其表达的变化,判断各shRNA的干扰效应。结果经测序,模板序列与设计序列完全正确,经过RT-PCR及Western-Blot法检测,转染干扰质粒后,c-Met基因的mRNA水平及蛋白表达水平均下调,以重组质粒pSilencer 2.0/c-Met-sh RNA2效应最显著。结论成功构建了针对c-Met基因的重组RNAi质粒,为下一步探讨c-Met基因对喉癌Hep-2细胞的生物学行为的作用奠定了基础。     

Expression of hepatocyte growth factor, keratinocyte growth factor and their receptors in experimental chronic pancreatitis

BACKGROUND: Hepatocyte (HGF) and Keratinocyte growth factors (KGF) are key factors of tissue organization and regeneration. These peptide growth factors and their receptors c-met and keratinocyte growth factor receptor (KGFR) are overexpressed in pancreatic cancer. AIM: Expression and localization of ligands and receptors were investigated during the development of experimental chronic pancreatitis. METHODS: Chronic pancreatitis was induced in rats by intravenous injection of dibutyltin dichloride. One to 60 days after treatment, the expression of growth factors and receptors was analysed by competitive polymerase chain reaction, Western blot analysis and immunohistochemistry. RESULTS: HGF mRNA expression increased (10-fold) until days 7-14 followed by a decrease to control level. Expression of c-met mRNA constantly increased (15-fold). KGF and KGFR mRNA expression were increased after 14-28 days (5-fold) and then returned to control levels. mRNA expression patterns correlated with changes in the protein expression, whereas protein levels of KGF remained unchanged. Ligands were localized in mesenchymal cells and their receptors on epithelial cells. CONCLUSIONS: The significant increase of HGF and c-met expression suggests an essential role of this growth factor in the morphological changes during the development of chronic pancreatitis. Changes in the expression of KGF and KGFR are less pronounced.     

肝细胞生长因子及其受体c-Met在卵巢癌细胞侵袭转移中的作用

肝细胞生长因子(HGF)是一种多肽生长因子,具有促进包括肝细胞、上皮细胞、内皮细胞、造血细胞等多种类型细胞的生长、迁移和形态发生的作用. 它还参与多种细胞的增殖、迁移,对各类肿瘤的侵袭转移有着重要的诱导作用. 本文通过对近年来国外有关文献的回顾,希望能够为进一步探讨HGF及其受体在卵巢癌治疗中的具体意义提供新的思路.     

HGF/c-met在小鼠毛囊生长周期中的表达

目的HGF及其受体c-met是很多系统中间充质-上皮互相作用的主要的介导者,毛囊周期性生长是典型的间充质-上皮互作模型,为证实HGF/c-met信号在小鼠毛囊周期生长过程中是否发挥作用。方法 本实验运用免疫组织化学的方法对HGF/c-met在ICR小鼠毛囊生长期、退化期和休止期进行组织定位。结果 HGF主要位于真毛乳头和皮脂腺,c-met主要位于毛基质、根鞘部和表皮。HGF及其受体在小鼠毛囊生长期表达量达到最高值,在退化期基本不表达,在休止期-生长期过渡时HGF及其受体表达量均上升。结论 HGF/c-met信号在ICR小鼠毛囊生长过程中起调控作用。     

原癌基因c—met和半乳糖凝素-3在甲状腺肿瘤中的表达

目的探讨原癌基因c—met和半乳糖凝素-3（Gal-3）在甲状腺肿瘤中的表达特点及在鉴别诊断中的价值。方法采用PV-6000通用型免疫组织化学二步法检测正常甲状腺组织10例、结节性甲状腺肿15例、甲状腺腺瘤25例、甲状腺滤泡癌20例、甲状腺乳头状癌31例、甲状腺未分化癌2例中c—met和Gal-3的表达。结果c—met和Gal-3在滤泡癌、乳头状癌及未分化癌中的阳性表达率高于甲状腺腺瘤、结节性甲状腺肿。c—met表达的强阳性率与淋巴结转移有关,Gal-3表达的强阳性率与甲状腺癌组织分化程度、临床分期、淋巴结转移有关。癌旁正常甲状腺组织中c—met和Gal-3表达均为阴性。甲状腺癌c—met的阳性表达与Gal-3的阳性表达呈正相关关系。结论c．met和Gal-3过表达与甲状腺肿瘤的发生、发展密切相关,对甲状腺肿瘤的鉴别以及预后的评估有-定的价值。     

补髓生血颗粒对CAA患者骨髓单个核细胞c-met受体mRNA表达水平的影响

目的探讨补髓生血颗粒对慢性再生障碍性贫血（CAA）患者骨髓单个核细胞C-metmRNA表达的影响。方法采用逆转录-聚合酶链反应（RT-PCR）检测CAA患者治疗前后骨髓单个核细胞c-metmRNA表达水平的变化。结果CAA患者骨髓c-metmRNA表达水平高于正常组,治疗后二者表达水平均有不同程度下降,并且补髓生血颗粒对二者表达水平的下调程度优于再障生血片（P〈0．05）。结论CAA患者骨髓c-metmRNA的表达存在异常,补髓生血颗粒剂可能通过调节骨髓c-metmRNA的表达来改善CAA患者的骨髓造血功能,促进骨髓造血功能恢复。     

细胞外调节蛋白激酶信号通路在乙型肝炎病毒X基因调控c-met基因启动子中的作用

目的 明确在乙型肝炎病毒X基因(HBx)对原癌蛋白质(c-met)基因启动子区的调控中所涉及的信号传导通路及其在肝癌侵袭和转移中的作用.方法 用Western blot比较可能涉及的信号通路特异性阻断剂对HBx调控c-met表达的影响,分析在该调控过程中所涉及的信号通路.体外侵袭试验验证信号传导通路在HBx调控c-met表达中的作用.对数据进行析因设计的方差分析.结果 HBx引起c-met表达水平的增加,细胞外调节蛋白激酶信号通路抑制剂U0126能够降低该作用,而p38MAPK信号通路抑制剂SB203580、PI-3K信号通路抑制剂wortmanin则未显示出该作用.体外细胞侵袭试验也证实了U0126能够降低HBx引起的HepG2细胞的强侵袭性[(74.3±6.2)个与(34.6±5.2)个,F=113.45,P＜0.01].结论 HBx引起肝癌细胞的侵袭转移可能与其通过细胞外调节蛋白激酶信号通路对c-met基因启动子区(-183 bp～-100 bp)的调控有关.     

肝细胞生长因子及其受体与肾脏疾病

1984年,Russell等在大鼠血浆中发现一种可刺激大鼠肝脏DNA合成的阳离子交换剂多肽,并称之为肝细胞生长因子(HGF).在随后的20余年中,人们对HGF的研究不断深入,目前认为HGF是一种多效性细胞因子,在促有丝分裂、细胞移动、血管生成、造血、抗凋亡、抗纤维化、修复组织损伤和抑制肿瘤细胞生长等多方面发挥苇要生物学功能.     

Expression of c-met/HGF Receptor in Human Non-small Cell Lung Carcinomas in vitro and in vivo and Its Prognostic Significance

The expression of c- met /HGF receptor was evaluated in non-small cell lung cancers (NSCLC) by western blot analysis of 11 established cell lines and 104 surgically resected tissues. All cancer cell lines (eight adenocarcinomas, two squamous cell carcinomas and a large cell carcinoma) showed strong c-met protein bands of 145 kDa and 170 kDa. Moreover, c-met protein was demonstrated in 34 (72.3%) of 47 surgically resected adenocarcinomas, 20 (38.5%) of 52 squamous cell carcinomas and 3 of 5 others, and the results were mostly confirmed immunohistochemically in formalin-fixed and paraffin-embedded tumors of the same case. Although squamous cell carcinomas showed relatively high c-met protein expression in established cell lines, more adenocarcinomas than squamous cell carcinomas showed c-met protein expression in the original cancers. Furthermore, two cell lines used in this study originated from primary cancers negative for c-met protein expression, suggesting that c-met protein expression might be influenced by cultivation. Furthermore, clinicopathological study revealed that NSCLC with c-met protein expression tended to be in a higher pathological tumor stage and to have a worse outcome than those without such expression. In conclusion, c-met protein is expressed in cell lines and primary tumors of NSCLC, and this phenomenon is probably closely related to the aggressive behavior or progression of NSCLC, especially of adenocarcinomas.     

经phoenix细胞包装含人全长HGF基因的逆转录表达载体及体外感染骨髓间充质干细胞的研究

目的：构建含人全长肝细胞生长因子（HGF）基因与绿色荧光蛋白（GFP）基因融合的逆转录表达载体plEGFP-HGF，经phoenix细胞包装，病毒液感染骨髓间充质干细胞（BM—MSCs），研究phoenix细胞的包装能力与病毒感染效率。方法：将人全长HGFcDNA亚克隆到逆转录病毒载体plEGFP-N1中，获得HGF定向插入藿组于plEGFP-HGF，采用酶切法和测序法分别鉴定。用磷酸钙法经phoenix细胞包装，收集病毒上清，感染MSCs，经G418筛选获得阳性克隆，命名为HGF／MSCs细胞。ELISA法检测细胞培养上清中HGF，RT-PCR检测HGF受体c-met在MSCs中的表达。结果：重组逆转录病毒载体体系plEGFP-HGF经限制性内切酶切电泳后显示有2．3kb目的基因片段和6．9kb的线性化plEGFP-N1载体片段：荧光显微镜观察到GFP基因获得了表达。重组于测序结果与Genebank中HGF-cDNA序列相符。RT-PCR检测转染细胞中c-met基因mRNA表达水平升高。ELISA法检测细胞培养上清中HGF升高。结论：重组真核表达载体构建正确并在MSCs中获得高效、稳定表达，为其在进一步的体内外研究奠定基础。