【作者】 李淑杰；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 放射医学， 2011， 硕士

【摘要】 目的:利用噬菌体展示技术筛选针对硫氧还蛋白过氧化物酶Ⅰ(PeroxiredoxinⅠ, PrxⅠ)抗原的肺腺癌特异性抗体(抗PrxⅠ抗体)并检测其性能,放射性核素标记抗PrxⅠ抗体后行荷瘤裸鼠体内分布及抑制肺腺癌生长作用研究。方法与结果:第一部分噬菌体抗体库的固相消减性筛选1.负性筛选:以正常支气管上皮HBE16细胞对抗体库负性筛选,除去抗体库中非肺腺癌细胞特异性抗体。2.全细胞筛选:以全肺腺癌A549细胞对经正常支气管上皮HBE16细胞负性筛选后的抗体库进行3轮筛选富集,得到肺腺癌A549细胞特异性的抗体。分别在每轮筛选富集前后测抗体滴度,结果显示收获率逐渐升高。3.已知抗原筛选:以PrxⅠ抗原对经肺腺癌A549细胞筛选后的抗体库进行3轮筛选,以获得PrxⅠ特异性抗体(抗PrxⅠ抗体)。分别在每轮筛选富集前后测抗体滴度,结果显示收获率逐渐升高。4.可溶性抗体的表达:制备单克隆抗体,ELISA检测证实为阳性噬菌体克隆转染大肠杆菌E.coli HB2151,IPTG诱导培养后纯化,得到纯化的抗PrxⅠ抗体。5.抗PrxⅠ抗体的鉴定:抗PrxⅠ抗体行SDS–PAGE检测,电泳分离得到30ku蛋白条带,此蛋白即为经大肠杆菌E.coli HB2151可溶性表达得到的抗体蛋白;抗PrxⅠ抗体经细胞ELISA及免疫细胞化学法检测,显示抗体具有较高的特异性,能与肺腺癌A549细胞特异性结合。第二部分抗PrxⅠ抗体体外作用研究1.抗PrxⅠ抗体内摄量测定:放射性核素131I经氯胺T法标记抗PrxⅠ抗体并纯化,37℃条件下,标记后的抗体(131I- PrxⅠ抗体)作用于肺腺癌A549细胞。作用后洗涤细胞,再溶解细胞,分别收集洗脱液及溶胞液,测洗脱液及溶胞液的放射性计数。与4℃孵育条件的对照组相比,37℃条件下,抗PrxⅠ抗体能有效结合并进入肺腺癌A549细胞内。2.抗PrxⅠ抗体体外作用:抗PrxⅠ抗体与肺腺癌A549细胞作用72小时后分别检测细胞增殖及凋亡情况,与对照组相比,MTT检测发现抗PrxⅠ抗体组有抑制细胞增殖作用;流式细胞检测发现,抗PrxⅠ抗体组细胞凋亡率较高。证实抗PrxⅠ抗体有抑制肺腺癌A549细胞增殖,促进凋亡作用。3.PrxⅠ蛋白表达测定:抗PrxⅠ抗体作用A549细胞72小时后,提总蛋白,Western blot检测显示,与对照组相比,抗PrxⅠ抗体组PrxⅠ蛋白表达水平较低。证实抗PrxⅠ抗体作用细胞后PrxⅠ蛋白表达受到抑制。第三部分抗PrxⅠ抗体在荷肺腺癌裸鼠模型体内分布及抑瘤作用1.~131I- PrxⅠ抗体稳定性检测: 131I- PrxⅠ抗体经层析柱纯化,三氯乙酸沉淀法测抗体的放射性核素标记率、放射化学纯度及比活度,结果显示标记抗体达到放免显像治疗要求。37℃条件下,131I- PrxⅠ抗体与人血清孵育48小时,放射化学纯度与孵育1小时的比较无明显降低。证明131I- PrxⅠ抗体稳定性较好。2.抗PrxⅠ抗体体内分布研究:建立荷人肺腺癌裸鼠模型,肿瘤长至要求大小后进行实验。131I- PrxⅠ抗体经尾静脉注射,不同时间处死裸鼠,测肿瘤及组织每克组织百分注射剂量率(%ID/g),同样裸鼠模型不同时间行单光子发射计算机断层成像术(SPECT)显像。结果示:131I- PrxⅠ抗体注射后,测瘤/血、瘤/肌肉放射性计数比值逐渐升高,即肿瘤组织对抗体的摄取随时间增加而逐渐增多,在第48小时时达到峰值(瘤/血、瘤/肌肉比分别为4.06±0.13、5.17±0.97)。SPECT显像肿瘤显示最清晰出现在第48小时。3.抗PrxⅠ抗体体内抑瘤作用研究:荷肺腺癌裸鼠随机分成3组,实验组经尾静脉注射131I- PrxⅠ抗体,对照组注射生理盐水、131I-IgG,1次/2天,共14次,4周后处死经各组治疗后的荷瘤裸鼠,剥离肿瘤组织,计算抑瘤率。剥离肿瘤组织病理切片,显微镜下观察病理学改变。结果显示,131I-PrxⅠ抗体组肿瘤质量及体积较其他2组均有明显减低,抑瘤率56.8%。病理切片HE染色后镜下观察131I-PrxⅠ抗体组可见大片肿瘤细胞坏死,核碎裂、核溶解明显。结论:本研究利用噬菌体展示技术从已构建的噬菌体抗体库中筛选抗PrxⅠ抗体,通过抗体的可溶性表达,性能检测,放射性核素标记后,应用于体内外抑制肺腺癌A549细胞增殖的研究。研究结果表明成功筛选出抗PrxⅠ抗体,该抗体能与肺腺癌A549细胞特异性结合,有效抑制体外肺腺癌A549细胞增殖。131I-PrxⅠ抗体荷瘤裸鼠体内研究显示,~131I-PrxⅠ抗体特异性及靶向性较好,得到了满意的体内分布及抑制肿瘤生长的结果,为肿瘤的诊断及靶向治疗提供思路。更多还原

【Abstract】 Objective: To screening specific antibodies from phage display library, and detect its efficiency. Label the antibodies with radionuclide 131I and make radio-immunoimaging study on xenograft in mice. Observe distribution of antibodies in the mice. Study against effect to lung adenocarcinoma. This study will lay the foundation for lung cancer radio-immunotherapy, and provide useful treatment to lung cancer patients.Methods and results:Part 1 Phage antibody library experienced mute solid phase screening1. Negativity screening: Normal human bronchial epithelial cells (HBE16) were used to deplete the phage library of nonspecific binders.2. Cell line screening: The phage library were screened on lung adenocarcinoma cells A549 3 rounds. Obtain the specific antibody against lung adenocarcinoma. The number of eluted phages increased after 3 rounds panning.3. Foregone antigen screening: The phage library after 3 rounds screening on A549 cells were then selected on PrxⅠ3 rounds. After these 3 rounds anti-PrxⅠwere obtained.4. Expresed soluble antibody: E. coli HB215l was infected with colonies which giving high signals against PrxⅠfor soluble expression. With IPTG. Induce cultivation, then fragments were purified, obtain the soluble antibody.5. Assess soluble antibodies: The soluble antibodies were identified by SDS–PAGE. A clear 30 ku band was observed, confirming the scFv fragments had been soluble expressed in E. coli HB215l. The soluble antibodies were analyzed by ELISA and immunocytochemistry, The result showed that the anti-PrxⅠantibodies specific purified with high affinity to A549 cells.Part 2 Study effect of anti-PrxⅠantibodies in vitro1. Internalize experiments: The anti-PrxⅠantibodies were labeled with radionuclide 131I by the chloramine T method(131I-PrxⅠantibodies). The A549 cells were incubated with labeled antibodies at 37°C. The temperature control was at 4°C. The cells were washed, then the cells were lysed. The acid washes and cell lysate radioactivity was measured on aγ–counter. The result showed that, compared with the control group, anti-PrxⅠantibodies the can combine with A549 cells and be internalized effectly.2. Effect of anti-PrxⅠantibodies in vitro: After anti-PrxⅠantibodies incubation with A549 cells 72 hs. MTT analysis showed growth inhibition to A549 cells. The apoptosis of A549 cells were analyzed by flow cytometry. The anti-PrxⅠcaused a significant apoptosis contrast the control group.3. PrxⅠprotein analysis: After 72 hs incubation with anti-PrxⅠantibodies, expression of PrxⅠprotein in A549 cells was examined with Western blot. Contrast control group, anti-PrxⅠantibodies caused a decrease in A549 cells.Part 3 Effect of anti-PrxⅠantibodies in vivo1. Text the anti-PrxⅠantibodies: 131I-PrxⅠantibodies purified by Sephadex G200 column. The labeling yield, specific activity, radiochemical purity and the stability of 131I- PrxⅠantibodies in serum were tested. The results showed that the labeling yield was up to the standard. After incubation with fresh human serum 48 hs, the radiochemical purity of the 131I–PrxⅠantibodies showed no significant depression Contrast to 1h group.2. Biodistribute of anti-PrxⅠantibodies in vivo: The xenograft mice were injected via the tail vein with 131I-PrxⅠantibodies. At various times, the tumor and organs were removed, and were weighed and counted on aγ-counter. The injected xenograft mice were fixed on boards for radio-immunoimaging analysis at various times. An index of the evaluation is percent injection dose per gram (%ID/g). The results showed that at 48 h tumor/blood and tumor/muscle values reached the peak of 4.06±0.13 and 5.17±0.97. The radioactivity was thicknessed at tumor locations and the tumor imaging was clearly showed.3. Against tumors of anti-PrxⅠantibodies in vivo: The xenograft mice were injected via the tail vein with 131I-PrxⅠa ntibodies. NS, 131I-IgG as control. One time per 2 days, and 14 times in all. Four weeks later, execute the mice. The tumors were removed, and were weighed. Calculate inhibition neoplasms efficiency was 56.8%. Pathology analysis of tumors after treatment showed that more tumor cells died, karyorrhexis and caryolysis in treat of anti-PrxⅠa ntibodies.Conclusion: We screening the anti–PrxⅠantibodies from phage display technology. Expressed soluble antibody and the specificity and proliferation ability were analyzed. Labeled the anti–PrxⅠantibodies with radionuclide. The study of the anti–PrxⅠantibodies against lung adenocarcinoma A549 cell line in votro. The result showed that the lung adenocarcinoma specificity human scFv antibodies have been prepared successfully and inhibition growth of A549 cell line. Biodistribution study and SPECT imaging showed that the anti–PrxⅠantibodies showed strong anti-proliferative effects on lung adenocarcinoma cells. The satisfactory radio-immunoimaging and specific affinity to target tumor implicates its potential application in lung adenocarcinoma imaging diagnosis and targeted therapy.更多还原