【作者】 李树钧；

【导师】 戚好文； 药立波；

【作者基本信息】 第四军医大学 ， 内科学， 2002， 博士

【摘要】 目的意义：Ndr2基因是1999年从正常人全脑cDNA文库中克隆到的新基因[GenBank登录号AFl59092]染色体定位于14q12.1，基因组中由15个内含子，16个外显子构成。Ndr2基因的cDNA全长为2024bp，编码357个氨基酸的蛋白质，分子量约41kD。目前研究表明，Ndr2基因在多种肿瘤组织或细胞系如结肠癌、胶质瘤、白血病、淋巴瘤和腮腺癌中无表达或低表达，相反在上述肿瘤相应的正常组织细胞中有较高水平的表达。另外在中枢神经系统的大脑皮质、白质、神经核，唾液腺上皮细胞和骨骼肌细胞中高表达。同时基因转染实验表明，Ndr2可抑制胶质瘤BT325细胞由G1期向S期的过渡，因此，推测Ndr2可能是一种新的抑癌基因。本研究的目的意义是探讨Ndr2基因在肺癌发病机制中的作用，确定Ndr2基因的功能，为肺癌的防治提供理论依据。 方法：采用RT-PCR，Westem blot和免疫组织化学方法确定Ndr2在正常组织和肺癌组织的表达。利用蜕皮素诱导的哺乳动物真核表达载体，将Ndr2 第四车医人学博士lr2上 基因构建到I匕载体pmD中，同时将含NdlZ 基因的融合载体转染肺8＆癌GLC｛2 细胞，经过鉴定确定NdrZ基因的成功转染。MTT法观察转染肺腺癌GLC－82 细胞的生长情况。细胞集落实验观察细胞表型的改变。流式细胞仪测定转染 肺腺癌CLC－82细胞的细胞周期变化。通过V尼stem blot观察细胞周期素＊ 在NdrZ基固转染肺腺癌 GLC－82细胞和空载体转染肺腺癌 GLC－82细胞的表 达差异。通过腺病毒p53基因转染肺腺癌 AX9细胞，观察p】基因对Ndl·2 基因表达的影响c 结果：经过RT－PCR方法提取正常肺组织的总RNA，反转录为CDNA，PCR 结果显示NdrZ在所检测的不同正常肺组织都有高表达。在正常肺组织中NdrZ 基因高表达；而其相应的肺癌组织呈现低表达或无表达，26例鳞癌中，有9 例无表达，8例4th达，9例高表达。14例腺癌中，4仔IJ无表达；3例低表达， 7例高表达。11例小细J腑癌有 4例无表达；3例4th达，4例高表达。正常 肺组织和肺癌的NdrZ表冰盯匕差异显著；P＜001。Western blot和免疫组织化 学方法进一步证实了这一结果。免疫组织化学结果显示NdrZ蛋白在正常气道 上皮细胞、平滑肌细胞、软骨细胞、浆液腺细胞、肺泡细胞胞浆中有高表达。 结果显示＊山2的表达与肺癌的分化相关，4例低分化鳞癌和二例低分化腺癌 冲瘤组织＊山二均无表达。3例高分化鳞癌和2例高分化腺癌与其正常肺组织 相比，NdrZ均高表达、19例中分化鳞癌中，有 5 ｛IJ无表达，8例低表达，6 例高表达。10例中分l匕腺癌，2例无表达；3伶低表达，5例高表达。11例 JJ、 细Ktw癌有4例无表达，3例fib达；4例高表达。另外，N山二的表达与肺癌 分期相关，在 40例 NSCLC中，6例 1期肺癌有 l伸J4th达。12例 11期肺癌 中 4例 NdrZ无表达，3例4ha达，5例高表达。22如1 Ill期肺癌中有 9伊J NdrZ 无表达，7例fto达，6例高表达。在门 例 SCLC中，l例 11期肺癌中 NdrZ 高表达。8 ｛Fd Ill期肺癌中 2 ｛II NdrZ无表达；3 ｛FJ｛Mi达，3 ｛FJ高表达，2 ｛FJ IV 期肺癌 NdrZ Jb无表达。 5 第四军医大学博士论文 利用蜕皮素诱导的哺乳动物真核表达载体转染NdrZ基因的肺腺癌GLC－82 细胞经过鉴定，NdrZ基因成功转染到肺腺癌GLC－82细胞内，蜕皮素诱导后 NdIZ基固得到高表达。M”IJ”法测定和细胞集落实验显示转染NdrZ基因的肺 腺癌＊＊G82细胞在蜕皮素诱导后，细Bbe殖受到抑制。流式细胞仪结果显示 NdrZ转染细胞存在GO／GI期细胞明显增多，NdrZ转染肺腺癌 GLC－82细胞 GO心1期细胞百分率为“．4O％，GZ－M期为6．刀％，S期为27．89％c而空载体 pmD季染肺腺癌 GLC－82 i田胞GO／GI期细胞百分率为 52．67O，GZ－A4期为 17．84地S期为29叩％。提示NdrZ能够通过细胞周期阻滞抑制肿瘤细胞增殖。 V乍stem Mot结果显示NdrZ转染肺腺癌GLC－82细胞的细胞周期素DI表达比 空载体转染肺腺癌hCE2 i邮胞的表达明显减弱。将腺病毒载体p53基因转染 肺腺癌 A刃9细胞，发现p53的表达能够上调NdrZ的表达。 结论：NdrZ在正常肺组织中高表达，而在肺癌组织中出现｛明达或无表 达。明确了NdrZ蛋白在正常气道上皮细胞、平滑肌细胞、软骨细胞、浆液腺 细胞、肺泡细胞胞浆中有高表达。NdrZ能够抑制肺腺癌 GLC七2细胞的增殖； 并能产生＊期阻滞，抑制细胞周期素＊的表达。p53的表达能够上调NdrZ 的表达。本研究的结果进一步支持NdrZ基因是一种新的抑癌基因；在肺癌中更多还原

【Abstract】 INTRODUCTION: Ndr2 is a newly found member of Ndr gene family which consists of Ndrl/RTP/Drgl, Ndr2, Ndr3 and Ndr4. The precise molecular and cellular function of members of this family is still unknown. But they are known to be involved in cellular differentiation events. Ndrl was first identified, and present investigation suggests it might be related to differentiation and anti-oncogenesis. Human Ndr2 was found by subtractive hybridization of human glioma tissue with its normal counterpart. Our preliminary investigation showed that the mRNA expression patterns of Ndr2 is dominantly in salivary gland, brain, skeletal muscle and mammary gland, and low expression in bone marrow, testis, periperal blood, and placenta, but it did not expressed in leukocyte, colorectal and some tumor cell line such as, Hela S3, leukemia cell, Bukitt’s lymphoma, adenocarcinoma SW480. The distinct expression patterns of the four family members suggest that Ndr2 might have relationship withtumor.AIM: To explore whether Ndr2 is related to tumor development, the expression of Ndr2 in lung cancer tissue and its role in lung cancer cell were observed.METHODS: RT-PCR analysis of Ndr2 expression in lung cacinoma specimens was used. Lung carcinoma specimens were taken from liquid azote and homogenized soaked in Trizol, Total RNA was isolated and quantified. Total RNA was used for RT-PCR reaction to amplify Ndr2. Western blotting assay was used . The frozen tissue was homogenized soaked in lysis buffer, the total protein quantity was determined by quantitative kit, and then applied to SDS-PAGE. The separated protein on SDS-PAGE was electro-transfered onto NC membrane. Western blotting assay was carried out as routine method described as reference . The routine immunohistochemistry was carried out using anti-Ndr2 monoantibody. Stable transfection and inducible expression of Ndr2 were used in lung adenocarcinoma ceh1. Full length cDNA encoding human Ndr2 was inserted into ecdysone-inducible vector pIND, named as pIND-Ndr2. Recombinant vector containg Ndr2 and another helper vector pV were co-transfected into GLC-82 lung carcinoma cells. Single clone resistant to both zeocin and G418 was isolated and cultured for enlarging its number. Cells were induced by ponasterone A , and the expression of Ndr2 was assayed by RT-PCR and Western blotting as described above. Cell growth is assayed by MTT method. Row Cytometry was used to determine cell cycle. To observe the expression of Ndr2, recombinant vector adenovirus-p53 was transfected in A549 lung carcinoma cell.RESULTS AND CONCLUSIONS: To explore whether Ndr2 is related to tumor development, its expression was evaluated firstly in 51 cases lung-carcinoma and equivalent number of normal tissue collected from clinical operation. RT-PCRassay indicated that according to tumor-free lung tissue, displayed completely negative in 9 of 26 cases of squamous carcinoma , low expression in 8 of 26 cases of squamous carcinoma; displayed completely negative in 4 of 14 cases adenocarcinoma, low expression in 3 of 14 cases adenocarcinoma; and displayed completely negative in 4 of 11 cases small cell lung carcinoma, low expression in 4 of 1 leases small cell lung carcinoma. RT-PCR results were confirmed by Western blotting and imrnunohistochernistry by using monoclonal antibody to Ndr2. The data above demonstrated that Ndr2 displayed down-regulated expression in lung carcinoma tissue in both transcription and protein level. These data indicated that Ndr2 expression was down-regulated in lung carcinoma, and related to tumor grade and phase development. The lower the tumor grade is, the higher the negative rate of Ndr2 expression is. The later the tumor phase is, the higher the negative rate of Ndr2 expression is. Additionally, human lung carcinoma cell line GLC-82 stably transfected with Ndr2 was acquired and the induced over-expression of Ndr2 resulted into Gl phase inhibition, and the growth of the tumor cell was reduced, and the clone forming ability was also inhibited, the expression of cy更多还原