重组人FHIT真核表达质粒的构建及其干预胆管癌细胞株QBC939增殖、凋亡及侵袭性的研究
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摘要
序言真核表达质粒的构建和转染是目前医学研究中常用的分子生物学手段。而肿瘤的发生与抑癌基因和癌基因密切相关。FHIT是一种重要的抑癌基因,在多种肿瘤中被发现表达缺失。Cyclin D1为常见的癌基因,它可以促使肿瘤细胞度过细胞周期的限制点,如G1/S限制点。它们的相互调控作用,尤其是FHIT干预胆管癌细胞的增殖、凋亡及侵袭性成为本次研究的重点。
目的通过构建重组人脆性组氨酸三联体(FHIT)基因真核表达质粒,探讨FHIT基因对胆管癌细胞株QBC939增殖、凋亡及侵袭性的影响,并进一步研究FHIT基因对细胞周期蛋白Cyclin D1的调控作用。
方法首先通过Trizol法提取新鲜标本组织中的RNA进行RT-PCR,将产物与载体pcDNA 3.1用BamH I/Xho I双酶切纯化后相连接,构建出重组真核表达质粒,并进行序列鉴定。然后通过脂质体瞬时转染法,将构建的FHIT-pcDNA 3.1转染入QBC939细胞,挑选出新霉素抗性克隆并进行荧光定量RT-PCR鉴定。将实验对象随机分为三组:自然对照组(NCG)、空白对照组(BCG)和实验组(EPG)。分别使用MTT法检测细胞增殖情况、流式细胞仪检测细胞凋亡情况、Transwell小室侵袭实验检测侵袭能力的变化、荧光定量RT-PCR检测Cyclin D1 mRNA表达情况及Western Blot法检测Cyclin D1蛋白质表达情况。所得数据进行单因素方差分析。
结果构建出了重组人FHIT真核表达质粒并经过序列鉴定。荧光定量RT-PCR检测转染后的QBC939细胞FHIT基因表达有限恢复。实验组与两个对照组比较,QBC939细胞增殖被抑制(P<0.05),促进了其凋亡,传过小室膜的细胞数量明显减少;并且Cyclin D1在基因和蛋白质水平的表达均被下调(P<0.05)。
结论成功构建了重组人FHIT真核表达质粒。FHIT基因可以干预QBC939细胞的增殖和凋亡,减弱其侵袭力,并能下调Cyclin D1的表达水平。为胆管癌进一步的基础与临床研究提供了分子生物学工具和方向。
Introduction The construction and transfection of eukaryotic expression plasmid are commonly molecular biology method used in medical research. And the occurrence of tumor is closely related to oncogenes and anti-oncogenes. FHIT is an important tumor suppressor gene, in a variety of tumors expression was found missing. Cyclin D1 gene is a common oncogene. It can promote tumor cell through the cell-cycle restriction point like G1/S. The coadjustment of them, especially the intervention of FHIT on the proliferation, apoptosis and invasive ability in human cholangiocarcinoma cell become the focus of this study.
Objective Through the construction of recombinant human fragile histidine triad (FHIT) gene eukaryotic expression plasmid, to discuss the effect of FHIT gene on the proliferation, apoptosis and invasive ability of cholangiocarcinoma cell line - QBC939. And the regulation functions of FHIT gene on Cyclin D1 was studied further.
Methods Genomic RNA of the fresh specimens’tissue was extracted by Trizol for RT-PCR. The product above and the pcDNA 3.1 vector were purified and connected by double digestion with BamH I/Xho I. All of above, the recombinant eukaryotic expression plasmid was constructed and sequence identification. Then FHIT-pcDNA 3.1 was transfected into QBC939 by lipofectamine instant method and selected neomycin-resistant clones to identified by real-time RT-PCR. Experimental subject was randomly divided into three groups: natural control group (NCG), blank control group (BCG) and experimental group (EPG). Their situations of proliferation were observed using the MTT method and the apoptosis were detected by Flow Cytometer (FCM). The invasive ability was determined by Transwell chamber test. The expression of Cyclin D1 mRNA was detected by real-time RT-PCR and the protein was detected by Western Blot. All of data were analyzed by one-way ANOVA, using Bonfferoni test or rates by chi-square test.
Results Human FHIT recombinant eukaryotic expression plasmid was constructed successfully and identified through sequencing. Real-time RT-PCR detection of transfected QBC939 showed that FHIT gene expression was restored limitedly. Experimental group compared with two control groups showed QBC939 cell proliferation was inhibited (P<0.05) and the apoptosis was promoted. The amount of cells which passed the filter was significantly less. At the same time, the expression of Cyclin D1 in gene and protein level was down-regulated (P<0.05).
Conlusion The human FHIT recombinant eukaryotic expression plasmid was constructed successfully. FHIT gene can interfere proliferation and apoptosis of QBC939, weaken the invasive ability and reduced the expression level of Cyclin D1. Molecular biology tool and direction were provided for further fundamental and clinical research of cholangiocarcinoma.
目的通过构建重组人脆性组氨酸三联体(FHIT)基因真核表达质粒,探讨FHIT基因对胆管癌细胞株QBC939增殖、凋亡及侵袭性的影响,并进一步研究FHIT基因对细胞周期蛋白Cyclin D1的调控作用。
方法首先通过Trizol法提取新鲜标本组织中的RNA进行RT-PCR,将产物与载体pcDNA 3.1用BamH I/Xho I双酶切纯化后相连接,构建出重组真核表达质粒,并进行序列鉴定。然后通过脂质体瞬时转染法,将构建的FHIT-pcDNA 3.1转染入QBC939细胞,挑选出新霉素抗性克隆并进行荧光定量RT-PCR鉴定。将实验对象随机分为三组:自然对照组(NCG)、空白对照组(BCG)和实验组(EPG)。分别使用MTT法检测细胞增殖情况、流式细胞仪检测细胞凋亡情况、Transwell小室侵袭实验检测侵袭能力的变化、荧光定量RT-PCR检测Cyclin D1 mRNA表达情况及Western Blot法检测Cyclin D1蛋白质表达情况。所得数据进行单因素方差分析。
结果构建出了重组人FHIT真核表达质粒并经过序列鉴定。荧光定量RT-PCR检测转染后的QBC939细胞FHIT基因表达有限恢复。实验组与两个对照组比较,QBC939细胞增殖被抑制(P<0.05),促进了其凋亡,传过小室膜的细胞数量明显减少;并且Cyclin D1在基因和蛋白质水平的表达均被下调(P<0.05)。
结论成功构建了重组人FHIT真核表达质粒。FHIT基因可以干预QBC939细胞的增殖和凋亡,减弱其侵袭力,并能下调Cyclin D1的表达水平。为胆管癌进一步的基础与临床研究提供了分子生物学工具和方向。
Introduction The construction and transfection of eukaryotic expression plasmid are commonly molecular biology method used in medical research. And the occurrence of tumor is closely related to oncogenes and anti-oncogenes. FHIT is an important tumor suppressor gene, in a variety of tumors expression was found missing. Cyclin D1 gene is a common oncogene. It can promote tumor cell through the cell-cycle restriction point like G1/S. The coadjustment of them, especially the intervention of FHIT on the proliferation, apoptosis and invasive ability in human cholangiocarcinoma cell become the focus of this study.
Objective Through the construction of recombinant human fragile histidine triad (FHIT) gene eukaryotic expression plasmid, to discuss the effect of FHIT gene on the proliferation, apoptosis and invasive ability of cholangiocarcinoma cell line - QBC939. And the regulation functions of FHIT gene on Cyclin D1 was studied further.
Methods Genomic RNA of the fresh specimens’tissue was extracted by Trizol for RT-PCR. The product above and the pcDNA 3.1 vector were purified and connected by double digestion with BamH I/Xho I. All of above, the recombinant eukaryotic expression plasmid was constructed and sequence identification. Then FHIT-pcDNA 3.1 was transfected into QBC939 by lipofectamine instant method and selected neomycin-resistant clones to identified by real-time RT-PCR. Experimental subject was randomly divided into three groups: natural control group (NCG), blank control group (BCG) and experimental group (EPG). Their situations of proliferation were observed using the MTT method and the apoptosis were detected by Flow Cytometer (FCM). The invasive ability was determined by Transwell chamber test. The expression of Cyclin D1 mRNA was detected by real-time RT-PCR and the protein was detected by Western Blot. All of data were analyzed by one-way ANOVA, using Bonfferoni test or rates by chi-square test.
Results Human FHIT recombinant eukaryotic expression plasmid was constructed successfully and identified through sequencing. Real-time RT-PCR detection of transfected QBC939 showed that FHIT gene expression was restored limitedly. Experimental group compared with two control groups showed QBC939 cell proliferation was inhibited (P<0.05) and the apoptosis was promoted. The amount of cells which passed the filter was significantly less. At the same time, the expression of Cyclin D1 in gene and protein level was down-regulated (P<0.05).
Conlusion The human FHIT recombinant eukaryotic expression plasmid was constructed successfully. FHIT gene can interfere proliferation and apoptosis of QBC939, weaken the invasive ability and reduced the expression level of Cyclin D1. Molecular biology tool and direction were provided for further fundamental and clinical research of cholangiocarcinoma.
引文
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[2] Khan S A, Thomas H C, Davidson B R, et al. Cholangiocarcinoma [J]. Lancet,2005,366 (9493):1303-1314.
[3] Patel T. Increasing incidence and mortality of primary intrahepatic cholangiocacinoma in the United States [J]. Hepatology,2001,33:1353-1357.
[4]邹声泉,刘小方,郭仁宣,等.乙型肝炎和丙型肝炎病毒感染与胆管癌相关因素的调查分析[J].中华外科杂志,2003,41:417.
[5] Zhang YJ, Jiang W, Chen C , et al. Amplification and overexpression of cyclinD1 in human hepatocellular carcinoma [J]. Biochem Biophys Res Commun,1993,196(5): 1010-1016.
[6] Ito Y, Takeda T, Sasaki Y, et al. Bcl-2 expression in cholangiocellular carcinoma is inversely correlated with biologically aggressive phenotypes [J]. Oncology,2000,59 (1):63-67.
[7] Aishima S I, Taguchi K I, Sugimachi K, et al. c-erbB-2 and c-Met expression relates to cholangiocarcinogenesis and progression of intrahepatic cholangiocarcinoma [J]. Histopathology,2002,40(3):269-278.
[8] Limpaiboon T, Sripa B,Wongkham S, et al. Anti-p53 antibodies and p53 protein expression in cholangiocarcinoma [J]. Hepatogastroenterology,2004,51(55):25-28.
[9]赵坡,吕亚莉,钟梅,等.胆管细胞癌FHIT蛋白丢失与Cyclin D1蛋白表达的研究[J].中华肿瘤防治杂志,2007,14(20):1552-1555.
[10] Fujii K, YasuiW, Shimamoto F, et al. Immunohistochemical analysis of nm23 gene product in human gallbladder carcinomas [J].VirchowsArch,1995,426(4):355-359.
[11]Gillet C , Smith D , Gregory W , et al . Cyclin D1 and prognosis in human breast cancer [J]. Int J Cancer,1996,69 (1): 92-99 .
[12] Nisha N , Fukuda Y , Komeda T , et al . Amplification and overexpression of the Cyclin D1 gene in agressive human hepacellular carcinoma [J]. Cancer Res,1994,54 (10):3107-3110.
[13] Sherr CY. G1 Phase progeression: cycling on cue [J]. Cell,1994,79(2):551-555.
[14] Sugimaehi K. The role of overexpression and amplification of cyclin D1 in intrahepatic cholabgiocarcinoma [J]. J Hepato l,2001,35:74-79.
[15] Ohta M, Inoue H, Cotticelli MG, et al. The FHIT gene, spanning the chromosome 3p14. 2 fragile site and renal carcinoma-associated t (3:8) breakpoint, is abnormal in digestive tract cancers [J]. Cell,1996,84(2):587-597.
[16] Kannan K, Munirajan AK, Bhuvarahamurthy V, et al. FHIT gene mutations and single nucleotide polymorphism in Indian oral and cervical squamous cell carcinomas [J]. Oral Oncology,2000,36(1):189-193.
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