【作者】 李玉龙；

【导师】 段芳龄；

【作者基本信息】 郑州大学 ， 消化内科， 2004， 硕士

【摘要】 目前公认肝细胞癌(hepatocellular carcinoma，HCC)的发病是多因素协同作用的结果，其中乙肝病毒(HBV)感染是HCC的主要病因之一。流行病学调查表明HBV感染者HCC的发病率较对照人群高出200倍以上。虽然HBV的确切致癌机制尚未完全阐明，但随着分子病毒学的研究进展，人们对HBVX基因(HBx)及其产物X蛋白(pX)与HCC之间的关系已有了多方面，多层次的了解，积累了大量研究资料。 HBV基因组是一不全双链环状DNA分子，其长链含有4个开放读码框(open reading frame，ORF)，分别编码前S1，前S2及S蛋白的S基因，编码前C蛋白与C蛋白的C基因，编码DNA聚合酶蛋白的P基因，以及编码pX的X基因。X基因是HBV DNA中最小的一个ORF，编码基因区位于1374～1838核苷酸之间，其编码的pX(或称为乙肝病毒X抗原，HBxAg)由154个氨基酸组成。Kwee等研究表明X基因至少编码Mr 17 000，8 000和6 600三种蛋白，基因分析显示靠近5′的AUG起始密码开始的序列表达完整长度的Mr 17 000蛋白，即pX。Mr8 000和6 600两种蛋白是由框架内起始密码子编码的两种缺失氨基端部分序列的pX。X基因编码的三种蛋白在功能上互相联系，具有多种转录调节活性。 实验证明，在胞核内pX可以和多种反式因子结合，发挥转录调控作用，如：TBP，TFⅡA，TFⅡB，TFⅡD，bzip等，而在胞浆中，pX主要通过激活细胞内信号传导通路如Ras-Raf-MAP激酶级联反应激活AP-1、NF-κB等与转录有关的因子。信号传递途径往往与细胞有丝分裂、细胞运动和分化、细胞周期有关，因此pX激活信号传递途径与其导致肝细胞癌变有密切关系。本试验通过稳定表达HBx的肝癌细胞株HepG2对顺铂敏感性的研究，探讨了丝裂原活化蛋白激酶2004年郑州大学硕士研究生毕业论文表达HBx的HePGZ细胞对顺铂敏感性的变化及其机制研究(mitogen activated protein侧比ase，MApK)家族主要成员之一细胞外信号调节激酶(e xtraCenual卜signal regulated Protein ki川”e，ERK)及其信号传导通路在HBx对细胞的保护作用中所起到的作用。方法 1 HBx一ORF的克隆从pCPlo质粒中通过PCR扩增得到HBx一oRF，与T-A载体连接后，转化感受态大肠杆菌JM109，通过蓝白筛选得到可疑阳性克隆，酶切鉴定，得到阳性克隆。 2测序及分析对阳性重组质粒中的HBx-ORF进行测序，并与pCP10中的HBx一ORF序列比较。 3重组表达载体的构建和鉴定将测序鉴定后的HBx-ORF片段从T-A克隆中纯化，回收，与表达载体pcDNA3连接，并转化感受态大肠杆菌JMloo，蓝白筛选得到可疑阳性克隆，然后进行酶切鉴定，确定阳性克隆。 4转染以及阳性细胞克隆的筛选和鉴定用脂质体法将阳性重组质粒转染肝癌细胞株HePGZ，G418筛选获得抗性克隆，Rl’- pCR鉴定稳定表达HBx-oRF的阳性转染细胞株。 5 westemblot比较阳性及阴性细胞克隆中p一ERKI/2水平的变化。 6M仃法比较阳性及阴性细胞对顺铂敏感性的差异分别将HBx+细胞，转染空载体细胞和转染空脂质体细胞接种于%孔板，含2“创ml CDDP的培养基孵育24h，MTr检测对各组细胞的抑制率。 7M竹法检测阻断ERKI/2的激活对细胞的影响HBx+细胞接种%孔板后，先以loopmo比PD98059孵育Zh，再作用以Zpg/mlConP，24h后检测抑制率。 8用SPSS10.O软件对各组细胞在CDDP作用下的抑制率进行统计分析。多组间均数的比较采用单因素方差分析，以a=0 .05为检验水准。结果 1 HBx-ORF的克隆PCR扩增，电泳得到482bP片段，凝胶回收后与T-A载体连接，转化JM109。从蓝白筛选得到的可疑阳性克隆中抽提质粒，Hindm，Ec。犯双酶切得到471坤的目的条带和3015如的质粒条带，与预期相同。 2测序及分析HBx一ORF片段测序结果显示，克隆片段与质粒中HBx序2004年郑州大学硕士研究生毕业论文表达HBx的HePGZ细胞对顺铂敏感性的变化及其机制研究歹d 100%相同。 3重组表达载体的构建和鉴定对构建好的表达载体pCDNA3一HBx进行双酶切，得到47lbp和5 446bp两个条带。与预期完全符合。 4转染后细胞的筛选和鉴定G418筛选4~5周后，HBx+组和空载体组均得到抗性克隆3一5个，经Rr-PCR鉴定后，HBx+组中有两个克隆扩增出482bP片段，为阳性克隆。 5各组细胞中p一ERKI/2水平的比较在44扔和42kD处有目的条带存在，HBx+细胞组条带明显强于空载体组和脂质体组。 6 HBx阳性和阴性细胞对CnnP敏感性的差异Zpg/m ICDDp作用24h后，对HBx+HePGZ的抑制率为19.56%士2.23，明显低于空载体组和脂质体组。 7阻断ERK的激活后，HBx+细胞对CDDP敏感性的变化先以100 p mol/LPngso59孵育HBx+细胞Zh以阻断E双的激活，再用2 09/m 1 eDnP孵育24h，CDDP对其抑制率上升至32.28%士4.22，与未加 PD98059时相比，尸仗0.05。结论 1成功构建包含HBx的表达载体pCDNA3一HBx。 2筛选得到2个稳定表达HBx的HePGZ细胞克隆。 3 HBx+细胞中p一ERKI/2的表达明显强于阴性细胞，证明px可以激活E双 通路。 4 HBx+细胞对CDDP的抗性明显强于阴性细胞。 5阻断ERK的激活后，HBx+细胞对CDDP的抗性消失。证明ERK通路在 保护细胞免于CDDP的杀伤中发更多还原

【Abstract】 Hepatocellular carcinoma(HCC) is now generally accepted as a consequence of multiple factors, and HBV infection is the major one of them. Epidemiological study showed that the incidence rate of HCC in patients with HBV was 200 times higher than those who were not infected. Although it is not exactly understood how HBV induces HCC, with the development of molecular virology, the relationship between HCC and HBV X gene (HBx), and its protein pX was more and more revealed.HBV has a genome of incomplete double strain DNA circle. The longer strain includes four open reading frames(ORF). S gene encodes pre-S 1, pre-S2 and S protein, c gene encodes pre-C and C protein, p gene encodes the polymerase and the x gene encodes pX. HBx is the smallest ORF of HBV genome and distributes between 1374-1836nt, encodes a protein of 154aa which can regulate the transcription of many genes.It has been proved that in nulear, pX binds to many trans-acting factors such as TBP, TFII A, TFIIB, TFII D,bzip etc, functioning as a transcription regulator. While in the plasma, pX can activate several signal pathways such as Ras-Raf-MAP kinase cascades to interact with the transcription factors. Signal transduction pathways can often induce the alteration of cell cycle, mitosis and differentiation and are generally believed to contribute to the occurrence and development of HCC. In this experiment, HepG2 cells stably expressing pX were employed to demonstrate in which way can pX influence the cells’ sensitivity to CDDP and protect the infected cells from death. METHODS1 Cloning of HBx-ORF The fragment of HBx-ORF aquired from the plasmaid pCPIO by PCR was ligated into a T-A vector. Transform the competent E.coli JM 109 with the reconstructed plasmid. The positive clones were identified bythe blue-white trial and restriction enzyme digestion.2 Sequencing The plasmids in the positive clones were sequenced and compared with the HBV genome sequence in pCP10.3 Construction of expression vector The fragment of HBx-ORF was cut from the sequenced T-A vector and ligated into the expression vector pCDNAS. The reconstructed plasmid was transformed into competent E.coli JM109 and positive clones were identified by blue-white trial, and restriction enzyme digestion.4 Transfection and positive cell clone selection Transfect the human hepatoma cell line HepG2 with the identified vector pCDNA3-HBx by liposome. Add G418 into the medium to select the cell clones that were resistant to it. Identify the clones containing HBx-ORF by RT-PCR.5 Comparing the expression of p-ERKl/2 in the positive and negative clones by Western blot.6 Comparing the sensitivity of HBx positive and negative clones to CDDP by MTT Plant the HBx+ HepG2, mock vector transfected HepG2 and liposome transfected HepG2 into 96-well plate and incubate with 2 u g/ml CDDP for 24h. Test the inhibitory rate of differtent cell groups with the method of MTT.7 After blocking the activation of ERK1/2, detect the sensitivity of HBx+ HepG2 to CDDP Incubate the HBx+ HepG2 first with 100 u mol/L PD98059 for 2h to block the activation of ERK1/2. Then incubate the cells with 2 u g/ml CDDP for 24h, Test the inhibitory rate with the method of MTT.RESULTS1 Cloning of HBx-ORF By PCR, a fragment of 482bp was got from pCP10. Digeste the reconstructed T-A vector with two restriction enzymes HindIII and EcoRI , two bands of 471bp and 3015bp were got as expected.2 Sequencing The fragment in the reconstructed T-A vector was sequenced and showed that it had a 100% homology with the HBx-ORF sequence in pCPlO.3 The identification of expression vector Digestion of pCDNA3-HBx with the same restriction enzymes got 471bp and 5.4kb bands, just as expected.4 Selection and Identification of the transfected cell After 4~5 weeks of selection in G418, 3~5 cell clones resistant to G418 were got both in pCDNA3-HBx group and mock vector group. RT-PCR showed 482bp bands in 2 clones in pCDNA3-HBx group which proved that it contained the HBx-ORF.5 The le更多还原