【作者】 王晓艳；

【导师】 沈守荣；

【作者基本信息】 中南大学 ， 内科学， 2003， 博士

【摘要】 结肠癌是最常见的消化道肿瘤之一，其发病机制涉及到多个抑瘤基因的失活和瘤基因的激活，尤其是抑瘤基因的失活。Vogelstein建立的结肠癌“APC→K-ras→DCC→p53→nm23”的多种瘤基因及抑瘤基因的突变和缺失的发病机制模式不能解释全部结肠癌的发生，且结肠癌细胞株发病机制的研究结果表明结肠癌的发病机制存在多样性，因此寻找新的抑瘤基因已成为揭示结肠癌发病机制的关键。我校肿瘤研究所细胞遗传室采用定位候选克隆的方法在9p11.1-12区域克隆了一个新基因NGX6，Genbank登录号：AF188239，其高表达可明显抑制鼻咽癌细胞系的生长，可能为抑瘤基因侯选者。并有研究初步证明NGX6在结肠癌，尤其是在有远处转移的结肠癌中表达下调或缺失。本研究通过脂质体介导NGX6转染结肠癌细胞系，进一步阐明NGX6在结肠癌发生发展中的作用及其分子机制。 我们通过脂质体转染方法将真核表达载体pcDNA3.1(+)/NGX6导入结肠癌细胞株HT-29细胞中，并用G418筛选，PCR、RT-PCR和点杂交鉴定，建立了稳定表达NGX6基因的HT-29细胞系。通过生长曲线、MTT、软琼脂集落形成、流式细胞术、裸鼠成瘤等方法探讨NGX6对HT-29结肠癌细胞系生物学行为的影响。借助免疫组化、免疫荧光、蛋白印迹等方法研究NGX6在体内、体外影响结肠癌细胞系生长的作用机制。 研究结果表明：转染了NGX6基因的HT-29细胞系的生长速度明显减慢，倍增时间由转染前的23.0小时延长至34.5小时；MTT显示NGX6转染组细胞在490nm处的吸光度较未转染组明显下降(p＜0.05)；软琼脂集落形成率降低(p＜0.05)；NGX6转染后HT-29细胞在裸鼠体内成瘤明显延缓，博士学位论文 中文摘要－－w＊”移植瘤体积大小和重量较HT上9组和 PCDNA3．1（）空载体转染组有所减少，将三组肿瘤进行病理切片，均为低分化腺癌，无组间差异，显示NGX6基因对裸鼠移植瘤生长具有抑制作用。因此，推断NGX6基因重表达在体内及体外均可逆转HT亿9结肠癌细胞系的恶性表型。 细胞周期调控机制的紊乱导致细胞失控性生长与分裂，显示出其恶性特征，故肿瘤又是一种细胞周期病。研究中采用流式细胞仪分析了细胞周期及细胞周期素的改变，结果显示稳定表达NGX6的HT七9细胞系 G／G；期细胞分布较对照组明显增加，S期细胞数减少；细胞凋亡率在转染前后无明显变化；cyclin E、cyclin B、cyclin DI的表达量下降，以 cyclin E和cyclin DI的改变最为明显。Western blot验证 cyclin E、cyclin DI在NGX6转染前后的变化，与流式细胞仪检测结果一致，以上结果说明在结肠癌细胞系中NGX6可宜主要通过下调cyclin E、cyclin DI的表达，延缓细胞周期的G；－S的进程，从而抑制HT七9细胞的过度增殖。 生物信息学预测NGX6具有－个EGF－like domain。研究表明转染NGX6后的鼻咽癌细胞系的EGFR和酪氨酸激酶传导通路上的MAPK磷酸化程度均较转染前明显降低，提示NGX6可能为EGFR的负性调控因子。为进一步探讨其在结肠癌细胞系的作用机制，本研究采用免疫组化、免疫荧光、蛋白印迹技术检测酪氨酸激酶传导通路上主要蛋白分子及细胞周期调控蛋白cycl＊s、CKI在转染NGX6前后的变化。结果显示：转染NGX6后细胞信号传导通路上的EGFR、K1as、p－JNK、c寸un均有不同程度的下调。细胞周期蛋白依赖性激酶抑制物家族成员P27在转染NGX6后上调；P16在NGX6转染前后HT《9结肠癌细胞系中均无表达，免疫组化结果与Wes朽＊ bio亡结果一致。c寸un的免疫荧光结果提示cJun主要分布于胞核，部分位于胞浆，且NGX6转染后核内cJun的分布较转染前减少。以上结果表明NG涨可能抑制酪氨酸激酶传导通路的信号传导、下调细胞周期素eye＊n E、cycl in DI和上调似 k27）的表达而阻滞细胞周期的进行，抑制细胞的 2博士学位论文 中文摘要增殖。 为了明确ATGX6蛋白在细胞中发挥作用的部位；我们进一步将NGX6MyC融合蛋白的真核表达载体系统pCMV十yC通过瞬时转染将该重组体导入HT－29结肠癌细胞系。采用兔疫荧光的方法检测NGX6－MyC融合蛋白的定位，荧光显微镜观察结果，显示该融合蛋白位于细胞胞浆内，少部分存在于胞膜上。根据生物信息学预测NGX6全长编码的蛋白具有两个跨膜区，去跨膜区后NGX6定位于胞浆的结果，推测NGX6编码的蛋白可能在胞浆中合成后，定位于膜上，分泌到胞外发挥其抑瘤作用。 为进一步研究了NGX6在体内作用的分子机制，实验中还采用免疫组化的方法分析了 EGFR． K－ras、c－Jun和 p27在NGX6转染前后细胞株所致的移植瘤中的表达变化。结果表明NGX6转染在移植瘤内亦可降低EGFR、K－ras和cJ八n的表达，上调p27的表达，与体外NGX6的作用机制一致，结果表明NGX6在体内也通过影响EGFR介导的酪氨酸激酶信号传导通路的激活及CKI家族P27的表达抑制肿瘤的增殖。更为重要的是发现NGX6在移植瘤中抑制血管内瘤栓形成，提示NGX6可能与结肠癌细胞的转移有关。借助更多还原

【Abstract】 Colon carcinoma is one of the most common gastrointestinal tumor.Its pathogenesis include inactivation of tumor suppressor genes and activation of oncogenes, especially the inactivation of tumor suppressor genes is very important .The model of pathogenesis of colon carcinoma which was established by Vogelstein laboratory theory ,"APC - K-ras - DCC - p53 - nm23",can’t elucidate the development of colon carcinoma completely, furthermore the pathogenesis of colon carcinoma is diverse .Therefore,it has become a critical subject to reveal the pathogenesis of colon carcinoma by identifying novel tumor suppressor genes.Cancer research institute has isolated a novel gene on the region of chromosome 9p by position- candidate cloning strategy, designated human NGX6, GenBank accession number : AF188239.NGX6 can suppress the proliferation of the nasopharyngea cell linel and maybe the important candidate of tumor suppressor genes. Some study states that NGX6 is down-regulated in colon carcinoma ,especially in that with distal metastasis.Our subject is to transfect mammalian expression vector pcDNA3.1(+)/NGX6 recombinant into colon cancer line HT-29 by lipofectin ,in order to further investigate the effect of NGX6 on colon carcinoma and elucidate its molecular mechanism.NGX6 was transfected into colon carcinoma cell line (HT-29)by lipofectin and a stable cell line of pcDNA3.1(+)/NGX6/ HT-29 overexpressing NGX6 gene was established .The overexpression of NGX6 was identified by PCR, RT-PCR and Dot blot analysis. The effect of NGX6 on the malignant behavior of HT-29was assessed by growth curves of cells , MTT, Clone formation in soft agar, FCM, tumor formation into nude mice.Its Molecular mechanism in vivo and in vitro was analyzed by immunohistochemical, Western Blot and immunofluorescence.pcDNA3.1(+)/NGX6/HT-29 ceils had a significant inhibition in cell proliferation by the growth curves of cells and the ability of clonogenesis in soft agar compared with HT-29 and pcDNA3.1(+) /HT-29 cells. Its double proliferation time of HT-29 cells prolonged from 23.0 hours to 34.5 hours after transfected with NGX6. The result of MTT showed that NGX6 had a negative effect on the cell viability. There was a delay in tumorigenesis and a reduction in tumor size when pcDNA3.1(+)/NGX6/ HT-29 cells were transplanted into nude mice. HE stain showed that xenografts were poorly differentiated adenocarcinoma. Taken together, these results indicated that the overexpression of NGX6 can reverse the malignant phenotype of the HT-29 cell line.The disorder of the cell cycle regulation can lead to the proliferation arid division of the cells out of control,Then the cells show its malignant characterization. So tumor is a kind of cell cycle disease .To investigate the possible effect of NGX6 gene on the distribution of cell cycle and apoptosis, FCM analysis were used to evaluate the cell cycle distribution and cyclins expression of cells. Flow cytometry analyses indicated that the overexpression of NGX6 could delay the progression of G1-S in cell cycle, while it had no effect on the apoptosis of cells. Flow cytometry analysis demonstrated that the expression of cyclin E, cyclin B, cyclin D1 decreased in NGX6-expressed HT-29 cells , especially the decrease of cyclin E and cyclin D1. All these studies provided evidence that NGX6 gene can repress the colon cancer cell’s uncontrolled proliferation,which mechanism was that NGX6 mainly exerted at the G1/S boundary of the cell cycle by down-regulating the expression of cyclin Eand cyclin D1.It was predicted that NGX6 gene possessed several structure including a EGF-like domain and two transmembrane regions. Study stated the phosphorylation degree of EGFR and MAPK , an important protein kinase on the EGFR relevant signal pathway, decreased in pcDNA3.1(+)/NGX6/HT-29 cells. Our study was designed to further investigate the alteration of critical molecular which included on the tyrsine kinase signal pathway , related cyclins and CKIs after transfected with NGX6 by of immunohistoc更多还原