【作者】 谢娟；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 药理学， 2004， 博士

【摘要】 目的：本课题采用受休介导癌基因肿瘤反义显像诊断乳腺癌。拟以乳腺癌c-erbB2原癌基因的核糖核酸mRNA作为反义靶目标，用^99mTc标记反义寡核甘酸与靶基因互补结合，联合表皮生长因子（EGF），通过表皮生长因子受体（EGFR）介导吸收，增加寡核苷酸的细胞摄取率。建立以扩增癌基因为靶位的，将基因互补及配体与受体结合的，两对结合体和结合力的特异性相结合的，高灵敏度、高选择性和高靶向性的肿瘤基因显像新方法。 意义：癌基因是一类具有潜在诱导细胞恶性转化的基因，癌基因激活过表达使胞浆中mRNA增加，使与目的mRNA互补的反义寡脱氧核苷酸（ASODN）识别及结合增加。用放射性核素标记ASODN，在体外显像可以诊断体内mRNA水平变化，肿瘤的存在和大小。理论上推论反义癌基因显像可以探测出只有癌基因扩增和过度表达还没有形成实体的肿瘤组织，因此可实现对肿瘤的早期无创性诊断。 乳腺癌（breast cancer）是妇女发病率最高的主要恶性肿瘤，其5年生存率和远期生存率较低。鉴别出具有真正临床意义的早期乳腺癌亚群，是施行“量体裁衣”式治疗提高生存率的关键。临床前后的研究不断显示，c-erbB2的有条件激活和扩增是乳腺癌的早期事件，蛋白的高表达多出现在乳腺肿瘤的早期阶段。20％～30％侵袭性乳腺重庆医科人学博卜学位论文中文摘要癌c一erbBZ基因扩增和过表达，c一erbBZ高表达者瘤细胞增殃旺盛，侵袭力强，肿瘤的转移潜能很高，患者存活率较低。c一erbBZ血清水平与肿瘤组织分级、临床分期、肿瘤复发、淋巴结侵袭、转移和人小，以及无疾病z卜存率和总生存率密切相关。以C一erbBZ的表达来估不}一‘tl-期乳腺癌的预后、无疾病生存率、总生存率，以及对内分泌治疗、化疗和放步犷的反应性。 农皮生长因子受体（EGFR）基因与c一erbBZ具有高度1司源性，EGFR在乳腺癌中的被检出率为35%。EGFR与c一erbBZ具有组织分布一致性的特点，联合EGF的反义显象能增加敏感性，早期诊断c一erbBZ激活并高表达的乳腺肿瘤亚群。以达到提高疗效，降低死亡率的[:I的。 方法: 1.反义寡核汁酸靶点定位于c一erbBZ基因mRNA转录起始位（Location 1 60一1 74），反义寡脱氧核普酸（AsODN）序列:5’一NHZGGTGCTCAcTGCGGC一3’;对照正义寡脱氧核营酸（SODN）序列:5’一NHZGeeGeAoToAGeAee一3’;对照无义寡脱氧核昔酸（NODN）序列:5’一NHZGGTeG灯oeCGeGTC一3’。以上三者总称寡脱氧核茸酸（ODN）O 2.合成EC一ODN:将鳌合剂乙酞双半耽氨酸（EC）与oDN联接，通过C18SeP一Pak反相柱纯化，冷冻干燥，得到自制冻干试剂盒EC一ODN。对EC一ODN进行’H^NMR、IR、UV和琼脂糖电泳结构鉴定。重庆医科人学博十学位论文中文摘要 3 .EC一ODN的”gn1Tc一标一记和生物学特性:”gmTc标记Ec一ODN，经SePhadexG25柱纯化，ITLC法测定标记率、比活度、放化纯和稳定性，PC法测定血浆蛋白结合率。 4.合成”gmTc一ODN一EGF:将表皮厂卜长因子（EGF）与多聚赖氨酸（PL）联接，得到EGF一PL，用PAGE电泳鉴定。”gmTc一EC一ODN与EGF一PL联合，形成”（）mTc一oDN一EGF，用Agarose电泳进行分析。 5.9（）lnTc一ODN一EGF的细胞摄取率和返流比率:培养SK一Br一3乳腺癌细胞，该细胞c一erbBZ基因扩增是正常细胞的16倍，p 1 85蛋白的过表达约为正常哺乳上皮细胞的100倍。对”gmTc一EC一ODN进行细胞3Omin一300min的摄取率和12h返流比率研究。 6.sK一Br--3细胞的生物学特性:（1） MTT法测”gmTc一ODN一EGF对细胞活力的影响;（2）流式细胞仪（FCM）和（3）免疫组化（IHC）测99mTc一oDN一EGF对细胞c一erbBZ蛋白表达的影响;（4） Rr-PcR半定量分析”，)mTc一ODN一EGF对c一erbBZ mRNA的影响;（5）电子显微镜观察9”mTc一ODN一EGF干预后细胞显微结构的变化和形态学改变。 7.1巨常BALB/c小鼠体内分布:早小鼠54只，随机分为3组，尾iv给子”gm几一ASODN一EGF、”gm几一ASODN和”gm几一EC 10一15风i/150林1，于o.sh、lh、Zh、4h、sh、24h，眼眶静脉取血，颈椎脱臼处死动物，取肝、脾、’肾、心、肺、胃、肠、肌肉、骨和脑，测epm，并准确称重，计算每克组织百分注射剂量率（%ID/g）。 8.兔药代动力学:早大白兔6只，称体重，随机分为2组，经左侧耳缘静脉注射”gm几一AsoDN一EGF和”gm几一AsoDN 3ouCi/Zml/重庆医科人学博十学位论文中文摘要兔，于lmin一360min从右侧耳缘静脉采血0.2ml，测定血‘液cPm，并准确称重，计算%ID/g，用3P97软件拟合曲线方程，计算分布半衰期、清除半衰期和清除率。 9.构建荷乳腺癌裸鼠模型:将对数生长期SK一B卜3细胞制成1.0又lo7ee一lszo.Zml的悬液。5一6周龄，体重129一189，早BALB/c裸鼠30只，一于右后肢内侧腋窝皮卜接种细胞1.0义lo7cefl:/0 .2ml/只。20天左右，肿瘤长至约1 .0 cm X 0.gcm大小。 10.荷瘤裸鼠体内分布试验:24只荷瘤裸鼠，随机分为两组，)毯iv给升10一15此i八50林1的”gm几一ASOoN一EGF和”gmTC一ASODN，于o.sh、lh、Zh和4h眼眶静脉取血，颈椎脱臼处死动物，取出肝、脾、’}矛、心、肺、胃、肠、肌IkJ、·胃·、脑和肿?更多还原

【Abstract】 Background and Objective The goals of this study were to image oncogene c-erbB2 using 99mTc-radiolabelled antisense oligodeoxy-nucleotides(ASODN) to bind complementally target gene to diagnose breast cancer. The target of antisense oligonucleotide is amplification ribonucleic acid mRNA of mammary tumor protooncogene c-erbB2. 9 mTc-ladled ASODN was connected with epidermal growth factor(EGF) to enhance uptake percentage of cell. To establish tumor oncogene imaging new method which has combined two specific conjugation force of DNA-mRNA complementation and ligand-receptor binding. So the new imaging method has such characters as higher sensibility, higher selectivity and higher target-tropism.Oncogene active can induce cell malignant changes and result in cell mRNA overexpression. ASODN can identify mRNA and specially conjugated mRNA to form mRNA/ASODN. Radiolabelled ASODN was designed complementation to target mRNA for imaging changes in the level of gene expression in vivo. In theory antisense oncogene imagingcan diagnose the position and size of tumor, and detect this neoplasm which have only had oncogene amplification and overexpression, but did not form entity tumor. To realize the earlier period and no-wound diagnosis for neoplasm.Breast cancer is mostly female malignant tumor which 5 year survival and long-term survival were lower. To investigate how to diagnose earlier mammary neoplasm subgroups is very important for performing clinical therapy projects that are the most suitable for personal treatment to rise survival rate. Compelling clinical studies on breast cancer have revealed that amplification of c-erbB2 has a high degree of correlation with disease recurrence and poor survival. Breast cancer genesis may dependent on overexpression of c-erbB2. c-erbB2 active and amplification were found in earlier period of mammary neoplasm and thought to play an important role in the process of breast cancer invasive and metastasis. 20%~30% invasive breast cancer have c-erbB2 amplification. The breast cancer of c-erbB2 overexpression have such characteres as higher tumor cell proliferation, higher invasiveness and metastasis potency, lower survival rate, poor prognosis, and tolerance for hormone treatment,chemistry therapy and radiation therapy. The blood serum c-erbB2 level is also associated with neoplasm constitution grades, clinical staging, tumor recurrence, lymph node invasive and metastasis, lymph node size, disease-free survival rate(DFSR), and overall survivalrate(OSR). Therefore c-erbB2 overexpression could be used to estimated the prognosis, DFSR and OSR of earlier period mammary neoplasm. Epidermal growth factor receptor (EGFR) have high homogeneity with c-erbB2. EGFR was detected overexpression in 35% breast cancer and distributed to specificity constitution.This study employed an 15-mer phosphorothioate oligodeoxynu-cleotide complementary to the zone which contains the translation initiation codon of c-erbB2 gene.The radiopharmaceutical of 99mTc-labeled ASODN combine EGF may identify intracellular c-erbB2 amplification at the level of gene transcription. The diagnosis would be extremely valuable for mammary neoplasm subgroups which are found in earlier period, and prescripted best treatment project to rise therapy efficiency and reduce mortality rate.Methods1. An 15 mer antisense oligodeoxynucleotide target is c-erbB2 mRNA translation initiation codon of location 160-174, ASODN sequence is 5’-NH2GGTGCTCACTGCGGC-3’. The control sense oligodeoxynucleotide (SODN) sequence is 5’-NH2GCCGCAGTGAG CACC-3’; The control nonsense oligodeoxynucleotide (NODN) sequence is 5’-NH2 GGTCGATGCCGCGTC-3’. ASODN,SODN and NODN are named oligodeoxynucleotide (ODN).2. Synthesis EC-ODN: Chelator L,L-ethylene dicysteine (EC)were connected with ODN. EC-ODN was purified by C18Sep-Pak reversecolumn and lyophilized, then EC-ODN frezze-drying reagent was obtained. EC-ODN was identified by ’H-NMRs IR, UV and agarose electrophorsis.3. WmTc-labeled EC-ODN and biology proper更多还原