【作者】 崔景秋；

【导师】 方佩华；

【作者基本信息】 天津医科大学 ， 内分泌与代谢病， 2004， 博士

【摘要】 钠／碘转运体（NIS）是位于甲状腺滤泡细胞基底膜上的一种糖蛋白，在其催化下，甲状腺完成对碘的主动转运，使碘的含量达到血浆的20～40倍，这是甲状腺激素合成的主要限速步骤。近年hNIS基因的克隆成功，使人们对多种甲状腺疾病如自身免疫性甲状腺疾病和先天性甲减等发病的分子机制有了进一步的了解，而其转染肿瘤细胞后介导的转基因治疗，可在非甲状腺肿瘤与不摄碘的甲状腺癌实现放射性¹³¹I治疗，从而为众多难治性肿瘤开辟了全新的治疗途径，具有广阔的临床应用前景。 目的： 在克隆甲状腺钠／碘同向转运体（hNIS）和甲状腺过氧化物酶（hTPO）基因全长的基础上，构建其真核表达质粒，并分别转染人神经胶质瘤细胞系和人卵巢癌细胞系，得到稳定表达hNIS和二者瞬时共转染的细胞株，然后在细胞水平分别研究其摄¹²⁵I功能和¹³¹I对细胞增殖的抑制作用；最后建立裸鼠卵巢癌荷瘤模型，在整体水平研究hNIS稳定转染后介导的卵巢癌移植瘤放射性核素^99mTcO₄^-显像和¹³¹I的治疗作用，从而为最终实现放射性碘治疗非甲状腺肿瘤与不摄碘的甲状腺癌提供理论依据。 方法： 1．分子生物学实验部分：根据发表序列和表达载体的需要自行设计引物，采用TRIzol一步法，从手术切除的Graves’病患者甲状腺组织中提取总RNA，逆转录合成人甲状腺cDNA第一链，再运用RT-PCR技术扩增出hNIS和hTPO编码基因全长，然后采用TOPO克隆技术将目的基因克隆入载体pcDNA3.1／D-V₅-His，氯化钙法转化宿主菌E. coli TOP10，筛选扩增阳性菌落后提取重组表达质粒pcDNA3.1D／FLhNIS和pcDNA3.1D／FLhTPO，并进行酶切鉴定和序列分析。 2．细胞生物学实验部分：在人神经胶质瘤细胞系TJ-905和人卵巢癌细胞系天津医料大学俘士学位论文ES一tw。中应用脂质体转染技术转染重组表达质粒pcDNA3.ID/FLhNIS，获得二种瞬时表达细胞系，初步鉴定其生物学活性后G418筛选，2周后得到稳定表达hNIS的细胞株TJ一905转和ES一two转，再用重组表达质粒peDNA3.ID/FLhTPO转染TJ一905转和ES一two转，获得hNIS和hTPO瞬时共转染的细胞株TJ一905共和ES一tw。共，然后研究4种细胞株TJ一905转、ES一two转、TJ一905共和ES一two共的摄‘，51活性、‘，51内流一时间曲线、”51外流一时间曲线和’zsI有机化程度等生物学活性，并利用MTT和细胞克隆形成实验在细胞水平研究”‘I对上述各种细胞增殖的抑制作用。3.动物模型实验部分:用稳定表达hNIS的细胞株ES一tw。转以1 07/0 .lmL的浓度接种裸鼠肩脚部皮下，建立卵巢癌荷瘤裸鼠模型，接种后2周，裸鼠饮水中加入优甲乐smg/L施行甲状腺封闭;接种后4周裸鼠皮下移植瘤直径约达到10Inln，腹膜内注射，竹c04一进行放射性核素物c0犷显像;并用丫一计数器测定腹膜内注射’25190分钟后不同脏器的cPm计数，进行裸鼠体内’zsI脏器分布定量实验，设立大小剂量组观察不同剂量’牡作用下，转染组和对照组裸鼠卵巢癌移植瘤体积、重量、抑瘤率和病理形态学的变化情况，研究不同剂量的’3，I对hNIS转染的裸鼠卵巢癌移植瘤的治疗作用。结果:1.先用RT一PCR法扩增出hNIS和hTPO编码基因全长，然后采用TOPO克隆技术分别构建T二者的重组表达质粒penNA3.ID/FLhNIS和peDNA3.10/FLhTPO，经酶切鉴定和序列分析证实与SM.Jhiang和KIInuraS等报告的序列完全一致。2.利用脂质体转染技术结合G418筛选实验得到了稳定表达hNIS的人神经胶质瘤细胞株TJ一905转及hN工S和hTPO瞬时共转染的神经胶质瘤细胞株TJ一905共，并进行了一系列摄‘2‘I活性研究，结果显示TJ一905转组摄‘251活性增高45.99士0.19倍（只0.05），并且其摄碘活性可被30 pM的过氯酸盐完全抑制;‘zs1在TJ一905转中快速聚集，20一30分钟内就达到了稳定阶段:同时‘与I从TJ一905转中亦快速外流，其有效半衰期约为5分;我们还用TCA沉淀法检测 3天津医料大学俘士学位论文了TJ一905共和TJ一905转的”，I有机化程度，结果表明TJ一905共的‘，51有机化程度比TJ一905转增高了4倍左右，并且实现hNIS和hTPO瞬时共转染后，125工外流减慢，其有效半衰期延长至10分钟左右。同时我们利用MTT和细胞克隆形成实验在细胞水平研究‘3‘工对上述各种细胞增殖的抑制作用，结果显示与空载体转染组TJ一905转空及未转染的对照组TJ一905相比，TJ一905转的细胞增殖率进行性降低，接种后第7天降至40.28士0.37%（只0.05），其细胞克隆存活率亦显著降低，降至31.83士0.52%（只0.05），而未转染的对照组TJ一905的细胞克隆存活率与未用‘3，I治疗者类似。3.利用脂质体转染技术结合G418筛选实验得到了稳定表达hNIS的人卵巢癌细胞株ES一two转及hNIS和hTPO瞬时共转染的卵巢癌细胞系ES一two共，并进行了一系列摄’zs1活性研究，结果显示和对照组ES一two相比，ES一two转组摄‘251活性增高92.52士1.90倍（只0.05），并且其摄碘活性可被30pM的过氯酸盐完全抑制:并且‘zsI在TJ一905转中快速聚集，20一30分钟内就达到了稳定阶段;‘25工从ES沈w。转中快速外流，其有效半衰期?更多还原

【Abstract】 Objectives:Based on the cloning of human sodium/iodide (hNIS) and human thyroid peroxidase(hTPO), we constructed their recombinant expression plasmids, then transfected human glioma cell lines and human ovarian cancer cell lines with Iipofectamine2000-plasmid complexes respectively, we accordingly obtained stably expressing hNIS cell lines TJ-905-t and ES-two-t, transient co-transfection of hNIS and hTPO cell lines TJ-905-co and ES-two-co, subsequently we investigated their radioiodide uptake function and 1311I inhibitory effect on cell proliferation; at last we established xenografted ovarian cancer nude mice model and investigated radioactive isotope 99mTcO4- imaging and radioiodine 131I treatment effect on xenografted ovarian cancer in vivo, thereby to provide an objective evidence for radioiodine therapy in nonthyroid tumor. Methods:1. Molecular biology experiment: After design PCR primer personally, total RNA was isolated from the thyroid tissue sample of GD patient by TRIzol reagent, the full length of hNIS gene and hTPO gene were amplified by RT-PCR, the target gene were inserted into cloning and expressive vector pcDNA3.1/D-V5-His by TOPO clone methods, then transformed into B. coli TOP10 by calcium chloride methods, after positive bacterium clone’s screening and amplifying, the recombinant expressive plasmids pcDNA3. 1D/FLhNIS and pcDNA3.1D/FLhTPO were isolated , then restrictive enzyme digested and sequenced.2. Cellular biology experiment: The human glioma cell lines and humanovarian cancer cell lines were tranfected with recombinant expressive plasmid-pcDNA3.1D/FLhNIS by lipofectamine 2000 respectively, we obtained two kinds of transient expressive cell lines, after their biologic activities were identified primary, screened them with G418 for 2 weeks, we obtained two kinds of stably expressing hNIS cell clones TJ-905-t and ES-two-t, the TJ-905-t and ES-two-t were again tranfected with recombinant expressive plasmid pcDNA3. lD/FLhTPO by lipofectamine 2000 respectively, we also obtained transient co-transfection of hNIS and hTPO cell lines TJ-905-co and ES-two-co, subsequently we investigated their biologic function including radioiodide uptake assay, l25I influx-course, 125I efflux-course and l2SI organification degree assay, furthermore we also investigated l3lI inhibitory effect on kinds of cell proliferation by MTT and cell clonogenic assays in vitro.3. Animal model experiment: Xenografted ovarian cancer were established in nude mice by subcutaneous injection.of 10V0. lmL ES-two-t on the shoulder blade, and controls were injected 107/0. lmL wild type ES-two on the same position. Two weeks after injection, nude mice were received L-T4 (5mg/L) supplementation in their drinking water to maximize radioiodine uptake in the tumor and reduce uptake by the thyroid gland. By 4 weeks after injection, Xenografts had reached 10mm in diameter, intraperitoneal injection of 0. 15mCi 99BTc04-, 40 minutes later, radioactive isotope 99mTc04- imaging was performed using ECT imaging system; for quantitative analysis of the amount of 125I in the tumors or other tissues, their radioactivity were quantified using a well-gamma counter for 1 minute. For 131I therapy studies , mice were divided into large dose group and small dose group, each groupincluding transfection subgroup and control subgroup, intraperitoneal injection of 6raCi or 3mCi 131I respectively, then Xenografted tumor’s volume, weight and pathologic morphology change were observed in order to investigate l3lI treatment effect on xenografted ovarian cancer in vivo. Results:1. The full length of hNIS gene and hTPO gene were amplified by RT-PCR, then their recombinant expressive plasmids pcDNA3.1D/FLhNIS and pcDNA3. 1D/FLhTPO were successfully constructed by TOPO clone methods respectively, and the sequences were the same as the results of SM. Jhiang and Kimura S. by restrictive enzyme digested and sequencing analysis.2.Stably expressing hNIS human glioma cell clones TJ-905-t and transient co-transfection of hNIS and hTPO cell lines TJ-905-co were更多还原