乳腺癌原位和骨转移靶向载体研究

乳腺癌原位和骨转移靶向载体研究 研究生：董  坚 导  师：李世和 中文摘要 目的：建立达GFP的MDA-MB-231乳腺癌细胞骨转移动物模型，应用pC89噬菌体肽库体技术，筛选能特异性进入乳腺癌MDA-MB-231原代细胞及骨转移细胞的小肽，探讨其作为肿瘤靶向治疗载体的可行性，为提高乳腺癌骨转移治疗率及探讨乳腺癌体化治疗提供实验依据。 方法：将带有GFP表达基因的质粒PEGFP-N2采用脂质体转染法转染入处于对数生长期的人乳腺癌细胞MDA-MB-231，获得稳定表达GFP的MDA-MB-231/GFP细胞。扩增培养MDA-MB-231/GFP细胞，左心室注射裸鼠每只1×107个/0.2ml。45天后，小鼠行X线/PET-CT检查，确定骨转移部位。并同时取部分转移灶进行组织培养并在荧光显微镜观察细胞表达GFP情况。扩增培养PC89噬菌体肽库，使其扩增后的滴度达1011TU。将原代乳腺癌细胞以及从转移灶取得的乳腺癌细胞的肿瘤与pC89噬菌体肽库体外共培养。Subtilisin被用于灭活未进入细胞的噬菌体，进入细胞的特异性小肽噬菌体经细胞裂解、转化E.coli XLI-Blue得到扩增。经过五轮的反复共培养和扩增，富集得到能特异性进入乳腺癌细胞的小肽噬菌体。选用RGD-integrin binding噬菌体作为对照，分别检测乳腺癌细胞特异性小肽噬菌体与MDA-MB-231、其他乳腺癌细胞株、其他组织来源实体瘤细胞株以及正常细胞株的亲和性。测序并合成无噬菌体外壳蛋白的特异性多肽并标记FITC，排除噬菌体外壳蛋白以及氨基酸序列在特异性转导中的作用。将编码乳腺癌细胞特异性多肽的DNA片段定向插入能够表达GST融合蛋白的质粒pGEX-2T，构建原核表达载体，小肽与GST融合蛋白表达用SDS-PAGE和Western-blot进行鉴定，抗GST单克隆抗体免疫荧光检测小肽介导GST蛋白进入人乳腺癌原代细胞或从转移灶取得的乳腺癌细胞的能力，探讨特异性小肽作为靶向载体的携带能力。 结果：用PEGPF-N2转染入人乳腺癌细胞MDA-MB-231后，获得稳定表达细胞进行裸鼠心腔注射，62.5%的裸鼠出现胸廓和肋骨等处的转移。将亲代MDA-MB-231细胞和转移灶MDA-MB-231细胞分别与PC89噬菌体库体外培养，筛选获得五个能特异性进入靶细胞的小肽噬菌体，经DNA测序确定它们分别是CASPSGALRSC（PⅠ）, CFPVPGHDLVC（PⅡ）, CFSVPGHDIVC（PⅢ）, CYTYPLGWHIC（PⅣ）和 CTPMSLSLSEC（PⅤ）。嵌合蛋白（RGD-integrin）作为阳性对照研究发现，筛选的小肽噬菌体与其他大多数肿瘤细胞及正常细胞无特殊亲和性；PⅠ显示与亲代细胞和转移灶细胞均能高特异性结合，而PⅡ主要与转移灶细胞高特异性结合，并发现它们在细胞中的分布主要以核周为主。进一步研究发现噬菌体外壳蛋白对小肽的特异亲和性无影响，合成的无噬菌体外壳蛋白的多肽序列保持与原代细胞及及转移细胞的亲和特异性。成功构建pGEX-2T-PⅠ和pGEX-2T-PⅡ原核表达载体,并在大肠杆菌中分别表达融合蛋白PⅠ-GST和融合蛋白PⅡ-GST，两者经GST亲和层析，纯度达85%。免疫荧光显示PⅠ-GST进入MDA-MB-231亲代细胞和骨转移细胞中。 结论：本实验建立了一种利用噬菌体肽库研究能结合或进入不同细胞的未知小肽的技术；得到了一条能与原代细胞及转移细胞MDA-MB-231高特异性结合的小肽，并具有携带外源性蛋白质分子靶向性进入目标细胞的能力。这种特异性呈现氨基酸小肽序列高度依赖性。该小肽具有作为乳腺癌基因治疗高效靶向性载体的潜在临床应用价值。同时，本研究为肿瘤细胞特异性抗原或抗体的研究提供理论和实验依据。 关键词：乳腺癌; 骨转移; 动物模型; 噬菌体肽库; 小肽; 载体; 蛋白转导; 靶向治疗； The protein transduction peptide selected from phage peptide library to target the original and bone metastasis cells of breast cancer cell line-MDA-MB-231 Post Doctor student: Dong Jian Advisor: Professor Li Shi-he Biotherapy Center，The 1st affiliated hospital of kunming medical college Abstract Object: To estabilish a animal model of bone metastasis of breast carcinoma and select tumor cell specific peptide vector that be internalized into MDA -MB-231 cells/its bone metastasis cell and investigate the cell-type specificity and delivery efficiency in vitro. Method: To develop a targeting vector for target therapy of breast cancer. We establish animal model of breast cancer bone metastasis using human breast cancer cell lines MDA-MB-231 transfected with GFP via left ventricle of heart. After 45 days of injection, all mice were detected by X-ray /PET-CT to determine whether bone metastasis happened in mouse. The cells from bone metastasis were observed the GFP expression under inverted fluorescence microscope, and then cultured for further study. PC 89(11aa),a phage display library of random peptides ,was applifed until its tite reaching 1011TU.  MDA-MB-231 cells and bone metastasis cells were cocultured with pC89 (11aa) phage display library of random peptides. In multiple independent peptide-presenting phage screening trials, subtilisin was used as a proteinese inhibitor to inactivate extra-cellular phages. The internalized phages were collected by cell lysising and amplified in E.Coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. The internalization capacity of peptide-presenting phages isolated from MDA-MB-231 cells was following compared with RGD-integrin binding phage by coculturing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. Peptide without phage coat protein was synthesized labeled with FITC, and then their targeting capacity to MDA-MB-231 cells was detected by coculturing them with different cell lines. DNA encoding specific peptide to MDA-MB-231 were ligated into plasmid pGEX-2T for fusion protein expression. The expression of fusion protein was identified by SDS-PAGE and western-blotting analysis. The delivery efficiency of the peptide was tested by anti-GST antibody in different breast cancer cell lines. Result：A animal model of bone metastasis of breast cancer was established using human breast cancer cell lines MDA-MB-231 transfected with GFP via left ventricle of heart .62.5% mice were found the metastasis in bone ,lung and other tissues. The specific peptide presenting phages were isolated from phage libraries, which showed higher affinity to MDA-MB-231 cells and/or bone metastasis cells than other cell lines. Five peptide, including CASPSGALRSC（PⅠ）, CFPVPGHDLVC（PⅡ）, CFSVPGHDIVC（PⅢ）, CYTYPLGWHIC（PⅣ）and CTPMSLSLSEC（PⅤ）were identified by DNA sequencing. Compared with controlled RGD-integrin Phage，The phage ,encoding PⅠsequence, show high affility in both MDA-MB-231 cell and bone metastasis cell, but PⅡphage (encoding PⅡsequence) can be higher internalized in bone metastasis cells than MDA-MB-231 cells .Peptides(PⅠ, PⅡ)without phage coat protein keep internalization character and showed juxtanuclear localization. No affility from five peptides were detected in all other tumor cells and normal cells that we used in our study. The recombinant plasmid vector(PGEX-2T-PⅠ,PGEX-2T- PⅡ) encoding MDA-MB-231 specific peptide(PⅠ,PⅡ) were successfully constructed , both fusion proteins(PⅠ-GST,PⅡ-GST) were applified in E coli BL21 and purified with GST affinity chromatographthe. The ratio of purification reached to 85%. Immunoflenscense microscopy showed that the peptide mediated the intracellular transduction of fusion protein targetingly in MDA-MB-231 cells. Conclusion：In conclusion, we established a method to seek novel tumor specific peptide, which may also be a practical way to promote the research in the area of new cell receptor or antigen. Capacity of  PⅠ to deliver molecules into target cells confirms its therapeutic potent in individualization targeting therapy. Keyword: Breast cancer; bone metastasis; Phage peptide display library; MDA-MB-231 cell; Targeting therapy;     Ⅰ, PⅡ)without phage coat protein keep internalization character and showed juxtanuclear localization. No affility from five peptides were detected in all other tumor cells and normal cells that we used in our study. The recombinant plasmid vector(PGEX-2T-PⅠ,PGEX-2T- PⅡ) encoding MDA-MB-231 specific peptide(PⅠ,PⅡ) were successfully constructed , both fusion proteins(PⅠ-GST,PⅡ-GST) were applified in E coli BL21 and purified with GST affinity chromatographthe. The ratio of purification reached to 85%. Immunoflenscense microscopy showed that the peptide mediated the intracellular transduction of fusion protein targetingly in MDA-MB-231 cells. Conclusion：In conclusion, we established a method to seek novel tumor specific peptide, which may also be a practical way to promote the research in the area of new cell receptor or antigen. Capacity of  PⅠ to deliver molecules into target cells confirms its therapeutic potent in individualization targeting therapy. Keyword: Breast cancer; bone metastasis; Phage peptide display library; MDA-MB-231 cell; Targeting therapy;