卵巢癌特异性结合肽的筛选及其对卵巢癌生物学行为的影响

发布时间：2018-06-24 04:15

本文选题：噬菌体展示技术 + 卵巢癌　；参考：《广州医科大学》2014年硕士论文

【摘要】：目的与背景卵巢癌病死率居女性生殖器三大恶性肿瘤首位，在确诊时75%的患者已是FIGOIII期以上病变。发生远处转移、侵润和化疗耐药性是卵巢癌高病死率、预后差的关键因素。因此，临床上迫切需要探索卵巢癌治疗及其转移、复发干预治疗的新方法。近年来，靶向药物输送系统治疗卵巢癌备受关注，但始终没有找到高特异性、高亲和力的载体。噬菌体展示技术通过确定表达在不同肿瘤细胞或组织器官上特异性分子的结合肽，并以此结合肽为载体与药物靶向相联，可以有效地提高定向传递化疗药物的能力。目前国内尚未见卵巢癌特异性结合肽的研究报道。因此，本实验拟使用噬菌体展示文库技术首次筛选卵巢癌细胞特异性结合肽，并通过体内外实验研究其对卵巢癌侵袭和转移生物行为的影响，为卵巢癌的靶向治疗提供理想的载体。方法 1.利用改良的细胞培养方法分离培养正常卵巢上皮细胞，创新性地运用细胞刷刷取人卵巢表面上皮（human ovarian surface epithelium，HOSE），红细胞裂解法、差速贴壁法对细胞进行分离纯化，并向无血清DMEM-F12培养基中添加人表皮生长因子（epidermal growth factor，EGF）进行细胞培养。在倒置显微镜下观察细胞形态，HE染色和免疫细胞化学染色法对细胞进行鉴定，为后续的筛选试验提供理想的体外试验材料。 2.以人卵巢癌细胞HO8910为靶细胞，分离获取的人正常卵巢上皮细胞为吸附细胞，对噬菌体随机环七肽库进行四轮差减筛选；细胞酶联免疫吸附法（ELISA法）验证阳性噬菌体克隆；对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体阳性克隆（phage1）与HO8910细胞的特异性结合，为针对人卵巢癌不同位点的靶向药物设计提供实验依据。 3.鉴定获取的噬菌体阳性克隆序列后，合成该特异性短肽序列（命名为peptide1），设立卵巢癌HO8910-k1组（peptide1组）、HO8910-k15组（control组）及HO8910组（blank组），通过细胞生长曲线、Transwell体外迁移和重组基底膜侵袭实验、粘附实验研究特异性短肽对HO8910细胞株侵袭和转移能力，探讨特异性合成短肽（peptide1）对卵巢癌体外恶性生物学行为的影响。 4.建立特异性短肽结合HO8910细胞裸鼠腹腔移植瘤模型，计算抑瘤率，免疫组化计算血管内皮生长因子(vascularendothelial growth factor，VEGF)的表达和原位凋亡TUNEL法检测肿瘤细胞凋亡指数（apoptotic index，AI），进一步探讨特异性合成短肽K1（peptide1）对卵巢癌体内恶性生物学行为的影响。结果 1.卵巢表面上皮培养24小时开始贴壁生长，7~12天后基本达到融合，细胞呈多角形或扁平型，透光性及折光性强。细胞形态符合正常上皮细胞特性，所分离的细胞几乎完全表达上皮细胞表面标志物CK19。细胞生长良好，可以传6~8代，细胞生长曲线呈“S”形，纯度达95%以上。细胞刷取培养法操作简单，能够快速分离获得大量卵巢表面上皮，所获得的细胞经红细胞裂解法和差速贴壁法处理后纯的达到95%以上，且细胞生长稳定，为组织工程研究提供了充足的种子细胞。 2.经过四轮筛选后，噬菌体在靶细胞HO8910上出现了明显的富集现象，第4轮筛选后与第1轮相比,富集了244倍；ELISA对20个随即挑选的克隆进行鉴定，12个可与HO8910特异性结合；DNA测序后得到一段与卵巢癌细胞特异性结合的七肽SWQIGGN（命名为K1），免疫荧光检测显示表达SWQIGGN序列的噬菌体克隆（phage1）能够与HO8910细胞结合，细胞呈绿色荧光反应，并不与人正常卵巢上皮细胞结合，提示phage1能与HO8910细胞特异结合。 3.合成并纯化后的的K1，，细胞生长曲线显示K1组HO8910细胞自第三天开始生长速度明显慢于其它两组，P＜0.05，差异具有统计学意义。粘附实验和细胞体外迁移侵袭实验表明合成的特异性短肽K1（SWQIGGN）能明显抑制HO8910PM的侵袭和转移能力，与对照组相比P＜0.05，差异具有统计学意义。表明特异性短肽K1（SWQIGGN）能够在体外抑制卵巢癌HO8910细胞的生长、侵袭及转移。 4.肿瘤侵袭转移体内实验表明特异性短肽K1（SWQIGGN）组裸鼠生长速度明显受到抑制、肿瘤体积、质量及播散数目均减少，与对照组及空白组相比，差异具有统计学意义（P＜0.5），而对照组与空白组相比，差异无统计学意义（P＞0.5）。表明SWQIGGN肽能够抑制HO8910细胞的增长，并能够在体内抑制卵巢癌HO8910细胞的恶性生物学行为。结论 1.运用细胞刷取法成功培养出人卵巢表面上皮，该方法不仅简便，而且高效实用，为卵巢癌的基础和临床研究提供了理想的试验材料。 2.采用BRASIL噬菌体展示技术和全细胞差减筛选策略，筛选出卵巢癌表面分子特异性的结合短肽SWQIGGN，为卵巢癌的靶向治疗提供了可能的作用靶向结合位点。 3.SWQIGGN短肽能够促进卵巢癌细胞的体内外生长、侵袭及转移能力。 4.SWQIGGN肽促进卵巢癌细胞侵袭和转移的机制可能与下调肿瘤内VEGF的表达有关，为进一步选择转移复发预测指标、肿瘤靶向治疗提供了重大线索。
[Abstract]:Purpose and background
The death rate of ovarian cancer ranks first in the three major female genitals, and 75% of the patients are more than FIGOIII stage. Distant metastasis, invasion and chemotherapeutic resistance are the key factors for the high mortality of ovarian cancer and poor prognosis. Therefore, it is urgent to explore new methods for the treatment of ovarian cancer and its metastasis and relapse. In recent years, targeted drug delivery systems have attracted much attention in the treatment of ovarian cancer, but there has been no finding high specificity and high affinity carrier. Phage display technology can effectively improve the determination of the binding peptide expressed in different tumor cells or tissues and organs with the binding peptide as the carrier and drug targeting. At present, there is no research report on the specific binding peptide of ovarian cancer. Therefore, we should use phage display library technology to screen the specific binding peptide of ovarian cancer cell for the first time, and study the effect on the invasion and migration of ovarian cancer in vivo and in vitro and in vitro, and the target treatment for ovarian cancer. Therapy provides an ideal carrier.
Method
1. the normal ovarian epithelial cells were isolated and cultured by improved cell culture method, and the epithelial cells of human ovarian surface (human ovarian surface epithelium, HOSE) were scrubbed innovatively, the erythrocyte lysis method, differential adherence method were used to separate and purify the cells, and the human epidermal growth factor (epiderma) was added to the DMEM-F12 medium of blood free DMEM-F12 (epiderma). Cell culture was carried out by L growth factor, EGF). Cell morphology was observed under inverted microscope, HE staining and immunocytochemical staining were used to identify the cells, which provided an ideal test material for the subsequent screening test.
2. the human ovarian cancer cell HO8910 was used as the target cell, the isolated human normal ovarian epithelial cells were adsorbed, and the phage random ring seven peptide library was screened by four rounds of differential screening, and the cell enzyme linked immunosorbent assay (ELISA) was used to verify the positive phage clones, and the positive clon was sequenced by DNA sequencing and bioinformatics analysis. The specific combination of phage positive clones (phage1) and HO8910 cells was identified by immunofluorescence test, which provided an experimental basis for the target drug design for different loci of human ovarian cancer.
3. after identifying the sequence of phage positive clones obtained, the specific short peptide sequence was synthesized (named peptide1), group HO8910-k1 of ovarian cancer (group peptide1), group HO8910-k15 (control group) and HO8910 group (blank group), through cell growth curve, Transwell in vitro migration and recombinant basement membrane invasion experiment, adhesion experiment study specific short peptide. To investigate the invasion and metastasis of HO8910 cell line, the effect of specific synthetic short peptide (peptide1) on malignant biological behavior of ovarian cancer in vitro was explored.
4. the tumor suppressor rate, the expression of vascular endothelial growth factor (vascularendothelial growth factor, VEGF) and the apoptosis index of the tumor cells (apoptotic index, AI) were calculated by immunohistochemistry, and the specific synthesis of short peptide K1 (peptide1) was further explored. Effects of malignant biological behavior in ovarian cancer.
Result
1. the surface epithelium of the ovary was cultured for 24 hours and began to adhere to the wall. After 7~12, the cells were fused, the cells were polygonal or flat, and the light transmittance and refraction were strong. The cell morphology accorded with the normal epithelial cells. The isolated cells almost completely expressed the epithelial cell surface marker CK19. cells, which could transmit the 6~8 generation and cell growth. The purity of the line is "S", with the purity of more than 95%. The cell brush culture method is easy to operate and can quickly separate a large number of ovarian surface epithelium. The obtained cells are more than 95% after the treatment of red cell lysis and differential adherence, and the cell growth is stable, which provides sufficient seed cells for tissue engineering research.
2. after four rounds of screening, the phage showed obvious enrichment on the target cell HO8910. After fourth rounds of screening, the enrichment was 244 times compared with the first round. 20 selected clones were identified and 12 could be specifically combined with HO8910. DNA sequencing, after DNA sequencing, obtained a seven peptide SWQIGGN (named after the specific combination of ovarian cancer cells). For K1), the immunofluorescence detection showed that phage clones expressing SWQIGGN sequences (phage1) were able to bind to HO8910 cells, and the cells showed a green fluorescence reaction, which did not combine with normal human ovarian epithelial cells, suggesting that phage1 could be specifically associated with HO8910 cells.
3. K1 after synthesis and purification, the cell growth curve showed that the growth rate of HO8910 cells in group K1 was significantly slower than the other two groups from third days, P < 0.05, the difference was statistically significant. The adhesion and cell migration and invasion experiments showed that the specific short peptide K1 (SWQIGGN) could inhibit the invasion and metastasis of HO8910PM obviously. Compared with P < 0.05, the difference was statistically significant. It showed that the specific short peptide K1 (SWQIGGN) could inhibit the growth, invasion and metastasis of ovarian cancer HO8910 cells in vitro.
4. in vivo experiments of tumor invasion and metastasis showed that the growth speed of nude mice was significantly inhibited in the specific short peptide K1 (SWQIGGN) group, and the tumor volume, mass and the number of spread were decreased. Compared with the control group and the blank group, the difference was statistically significant (P < 0.5), while the control group had no statistical significance (P > 0.5) compared with the blank group (P > 0.5). It can inhibit the growth of HO8910 cells and inhibit the malignant biological behavior of ovarian cancer HO8910 cells in vivo.
conclusion
1. the surface epithelium of the human ovary is successfully cultured by the method of cell brush extraction. This method is not only simple, but also efficient and practical. It provides an ideal experimental material for the basic and clinical research of ovarian cancer.
2. using the BRASIL phage display technique and the whole cell differential screening strategy, the specific binding short peptide SWQIGGN of the ovarian cancer surface molecule is screened, which provides the possible target binding site for the targeting therapy of ovarian cancer.
3.SWQIGGN peptide can promote the growth, invasion and metastasis of ovarian cancer cells in vivo and in vitro.
The mechanism of 4.SWQIGGN peptide to promote the invasion and metastasis of ovarian cancer cells may be related to the downregulation of the expression of VEGF in the tumor. It provides a major clue to the target therapy for tumor targeting.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】