【作者】 唐瑞峰； 孙超； 白建国； 纪军委； 殷兆麟； 李国耀； 杨伟； 马广军；

【机构】 河北医科大学四院；

【摘要】 目的:原发性肝癌(简称肝癌)是我国常见的消化系统恶性肿瘤之一,手术切除是目前治疗肝癌首选和最有效的方法。但肝癌患者临床症状出现较晚,症状出现后大多数患者已处于中晚期,手术切除率低,预后差。术后肿瘤的转移和复发是影响患者长期生存的首要原因。肿瘤的生长及转移需要新生血管的生成,血管内皮生长因子家族(vascular endothelial growth factor family members,VEGFs)可以直接或间接作用于每一个环节促进血管生长。迄今为止发现的VEGF家族有7个成员,包括VEGF-A(即通常所说的VEGF)、VEGF-B、VEGF-C、VEGF-D、VEGF-E(又称viral VEGF)、VEGF-F(又称snake venom VEGF)以及PLGF(胎盘生长因子placenta growth factor)。除了VEGF-E、VEGF-F外,其它VEGF家族成员均由哺乳动物基因编码。VEGF家族在脊椎动物中存在广泛的表达,在不同的物种中其编码序列高度保守。VEGF家族成员及其受体在众多肿瘤组织尤其是实体肿瘤组织(如肝癌)中存在异常表达,并与肿瘤的侵袭、转移、复发等生物学特性密切相关。VEGF-B是VEGF家族成员之一,在各种不同的肿瘤组织中存在异常表达,在肿瘤的侵袭、转移方面起着重要的作用。我们的前期研究显示:VEGF-B在肝癌的生长、侵袭及转移中起重要作用,并与肝癌患者的预后密切相关。因此,抑制VEGF-B基因的表达可望抑制肝癌细胞的增殖、转移。RNA干扰(RNA interference,RNAi)是近些年发展起来的一项新技术,RNA干扰的原理是将与内源性mRNA同源的外源性双链RNA (double stranded RNA,dsRNA)经不同途径导入体内或体外的组织细胞,致使特定基因沉默。小干扰RNA(small interfering RNA,siRNA)是RNA干扰的主要效应因子。利用质粒或病毒为载体,将基因靶向siRNA导入细胞内,可对特定基因产生沉默效应。本研究以VEGF-B为靶点,体外构建靶向VEGF-B siRNA表达质粒载体,并转染肝癌细胞HepG-2,用逆转录聚合酶链式反应技术(reverse transcription polymerase chainreaction,RT-PCR)检测分析实验组和对照组VEGF-B mRNA的表达,研究靶向VEGF-B siRNA对肝癌细胞HepG-2 VEGF-B基因的抑制作用,为肝癌的基因治疗提供新的靶点和方法,为临床应用提供理论依据。方法:扫描分析人类VEGF-B编码序列,设计合成靶向VEGF-B siRNA,利用大肠杆菌进行质粒扩增和提取;人肝癌细胞株HepG-2置于DMEM培养基中(含有l0%胎牛血清、100U/ml青霉素和100U/ml链霉素),在37℃、5%CO2的无菌培养箱中,视细胞生长状态及培养基颜色换液培养。待细胞培育至呈对数生长,用2.5 g/L胰酶消化并将细胞接种于6孔培养板,待细胞铺满率达80%后,按转染试剂说明书用Lipofectamine 2000将实验组及对照组质粒分别转染HepG-2细胞,于24～48小时在荧光显微镜下观察转染效率并收取细胞。以Trizol一步法提取细胞总RNA,用RT-PCR技术分析实验组及对照组肝癌细胞VEGF-B mRNA的表达(以β-action作为内参照标准)。结果:1、将重组的靶向VEGF-B siRNA质粒转入大肠杆菌进行扩增,利用高纯度小量质粒提取试剂盒,可成功提取纯度较高的靶向VEGF-B siRNA质粒。2、以质粒(含荧光标记)为载体,可将靶向VEGF-B siRNA成功转染人肝癌细胞HepG-2,于荧光显微镜下观察,实验组及对照组转染效率均达80%以上。3、将人肝癌细胞株HepG-2转染靶向VEGF-B siRNA后,与对照组相比,各实验组VEGF-B的基因扩增产物电泳条带亮度呈不同程度的减低(以β-action作为内参照标准);对照组、实验组1、实验组2、实验组3的VEGF-B的基因扩增产物电泳条带亮度与内对照β-action的比值分别为:0.396±0.037、0.135±0.019、0.271±0.011、0.222±0.030。各实验组VEGF-B mRNA的表达量显著低于对照组VEGF-B mRNA(P<0.05)。靶向VEGF-B siRNA可抑制HepG-2细胞VEGF-B mRNA的表达。结论:靶向VEGF-B siRNA在体外抑制肝癌细胞HepG-2细胞VEGF-B mRNA的表达。更多还原

【Abstract】 Objective:Primary liver cancer is one of the most common malignant tumors in our country,surgical resection is the preferred choice and the most effective methods.But,the clinical symptoms of liver cancer appear late,many patients with liver cancer are middle or advanced stage when symptoms show up,rate of surgery is low,prognosis is poor.Metastasis and recurrence is the most important reason to reduce survival time of patients with liver cancer.Growth and metastasis of tumor depend on angiogenesis.Vascular endothelial growth factor family members(VEGFs) can encourage angiogenesis directly or indirectly.So far,there are seven members in VEGFs,include:VEGF-A,VEGF-B,VEGF-C,VEGF-D,viral VEGF(VEGF-E),snake venom VEGF(VEGF-F) and PLGF(placenta growth factor).Except VEGF-E and VEGF-F,other VEGFs members are all encoded by mammals gene.VEGFs are widely expressed in vertebrate animals,and the coding sequence is highly conservative in different species.VEGF members and its’ receptors are over expressed in many tumor tissue,especially in the tumor tissue with more blood vessels(such as liver cancer),there is a close relationship between VEGFs members and characteristics of tumor such as metastasis and recurrence.As one of VEGF members,VEGF-B express highly in various tumor tissue and play an important role in tumor’s invasion and transfer.Our previous study suggested that VEGF-B played an important role in liver cancer’s invasion and metastasis,and have related closely with the prognosis of patients with liver cancer.Therefore,It is expected to inhibit liver cancer cell’s proliferation and metastasis by restraining VEGF-B express.RNA interference(RNA interference,RNAi) is a new technology developed in recent years,silenced specific gene in vivo or in vitro.Small interfering RNA(of small interfering RNA,siRNA) is the main effectors of RNA interference.Certain gene can be silenced effectively by targeted siRNA.In this study,we reconstruct plasmid carrier of targeted VEGF-B siRNA in vitro,then transfect the vector containing the object gene into liver cancer cell line HepG-2 cell,use reverse transcription-polymerase chain reaction(RT-PCR) to detect VEGF-B mRNA expression between the experimental group and the control group to research the inhibition of targeted VEGF-B siRNA to VEGF-B mRNA exprssion of HepG-2 cell.Provide new sites and method for gene therapy of liver cancer and theory basis for further clinical application.Methods:Scaned human VEGF-B coding sequence,designed and composed the targeted VEGF-B siRNA,amplified and extracted the carrying plasmid by using E.coli.Humanliver cancer cell lines HepG-2 cell was cultured in 37℃,5%CO2 sterile incubator in DMEM culture media containing 10%FCS,100 U/m1 streptomycin and 100 U/m1 penicilin,changed culture media depend on cell growth state and color of culture media.Digested cells with 2.5 g/L of trypsin when the cells grew logarithmically,then inoculated them to six holes training board.We transfected the HepG-2 cells by plasmid following the instruction of Lipofectamine 2000 when the cells covered the training board to 80% rate,and observed efficiency of transfection with fluorescence microscope after 24～48 hours.Collected cells and extracted total RNA using Trizol Reagents.RT-PCR was used to detected VEGF-B and β-action(internal stand) espression of experimental group and the control group.Results:1.The targeted VEGF-B siRNA plasmid was amplified by e.coli and extracted successfully.2.Target VEGF-B siRNA transfected the human liver cancer cell line HepG-2 successfully using the plasmid as a carrier and the efficiency of transfection can reach 80%.3.The brightness of each experimental group’s electrophoretic bands showed different degrees of reduction compared with the control group after transfected.The ratio of brightness between VEGF-B and the internal control β-action as follows:0.396±0.037,0.135±0.019,0.271±0.011,0.222±0.030.VEGF-B mRNA expression of each experimental group is lower than the control group Significantly(P<0.05).The expression of VEGF-B mRNA was inhibited by targeted VEGF-B siRNA in human liver cancer cell lines HepG-2.Conclusions:Targeted VEGF-B siRNA can inhibit expression of VEGF-B mRNA in human liver cancer cell line HepG-2 cell.更多还原