本文選題：噬菌體展示技術(shù) + 卵巢癌��； 參考：《廣州醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的與背景 卵巢癌病死率居女性生殖器三大惡性腫瘤首位，在確診時(shí)75%的患者已是FIGOIII期以上病變。發(fā)生遠(yuǎn)處轉(zhuǎn)移、侵潤(rùn)和化療耐藥性是卵巢癌高病死率、預(yù)后差的關(guān)鍵因素。因此，臨床上迫切需要探索卵巢癌治療及其轉(zhuǎn)移、復(fù)發(fā)干預(yù)治療的新方法。近年來(lái)，靶向藥物輸送系統(tǒng)治療卵巢癌備受關(guān)注，但始終沒有找到高特異性、高親和力的載體。噬菌體展示技術(shù)通過(guò)確定表達(dá)在不同腫瘤細(xì)胞或組織器官上特異性分子的結(jié)合肽，并以此結(jié)合肽為載體與藥物靶向相聯(lián)，可以有效地提高定向傳遞化療藥物的能力。目前國(guó)內(nèi)尚未見卵巢癌特異性結(jié)合肽的研究報(bào)道。因此，本實(shí)驗(yàn)擬使用噬菌體展示文庫(kù)技術(shù)首次篩選卵巢癌細(xì)胞特異性結(jié)合肽，并通過(guò)體內(nèi)外實(shí)驗(yàn)研究其對(duì)卵巢癌侵襲和轉(zhuǎn)移生物行為的影響，為卵巢癌的靶向治療提供理想的載體。 方法 1.利用改良的細(xì)胞培養(yǎng)方法分離培養(yǎng)正常卵巢上皮細(xì)胞，創(chuàng)新性地運(yùn)用細(xì)胞刷刷取人卵巢表面上皮（human ovarian surface epithelium，HOSE），紅細(xì)胞裂解法、差速貼壁法對(duì)細(xì)胞進(jìn)行分離純化，并向無(wú)血清DMEM-F12培養(yǎng)基中添加人表皮生長(zhǎng)因子（epidermal growth factor，EGF）進(jìn)行細(xì)胞培養(yǎng)。在倒置顯微鏡下觀察細(xì)胞形態(tài)，HE染色和免疫細(xì)胞化學(xué)染色法對(duì)細(xì)胞進(jìn)行鑒定，為后續(xù)的篩選試驗(yàn)提供理想的體外試驗(yàn)材料。 2.以人卵巢癌細(xì)胞HO8910為靶細(xì)胞，分離獲取的人正常卵巢上皮細(xì)胞為吸附細(xì)胞，對(duì)噬菌體隨機(jī)環(huán)七肽庫(kù)進(jìn)行四輪差減篩選；細(xì)胞酶聯(lián)免疫吸附法（ELISA法）驗(yàn)證陽(yáng)性噬菌體克��；對(duì)獲得的陽(yáng)性克隆進(jìn)行DNA測(cè)序及生物信息學(xué)分析,并進(jìn)一步利用免疫熒光實(shí)驗(yàn),鑒定噬菌體陽(yáng)性克�。╬hage1）與HO8910細(xì)胞的特異性結(jié)合，為針對(duì)人卵巢癌不同位點(diǎn)的靶向藥物設(shè)計(jì)提供實(shí)驗(yàn)依據(jù)。 3.鑒定獲取的噬菌體陽(yáng)性克隆序列后，合成該特異性短肽序列（命名為peptide1），設(shè)立卵巢癌HO8910-k1組（peptide1組）、HO8910-k15組（control組）及HO8910組（blank組），通過(guò)細(xì)胞生長(zhǎng)曲線、Transwell體外遷移和重組基底膜侵襲實(shí)驗(yàn)、粘附實(shí)驗(yàn)研究特異性短肽對(duì)HO8910細(xì)胞株侵襲和轉(zhuǎn)移能力，探討特異性合成短肽（peptide1）對(duì)卵巢癌體外惡性生物學(xué)行為的影響。 4.建立特異性短肽結(jié)合HO8910細(xì)胞裸鼠腹腔移植瘤模型，計(jì)算抑瘤率，免疫組化計(jì)算血管內(nèi)皮生長(zhǎng)因子(vascularendothelial growth factor，VEGF)的表達(dá)和原位凋亡TUNEL法檢測(cè)腫瘤細(xì)胞凋亡指數(shù)（apoptotic index，AI），進(jìn)一步探討特異性合成短肽K1（peptide1）對(duì)卵巢癌體內(nèi)惡性生物學(xué)行為的影響。 結(jié)果 1.卵巢表面上皮培養(yǎng)24小時(shí)開始貼壁生長(zhǎng)，7~12天后基本達(dá)到融合，細(xì)胞呈多角形或扁平型，透光性及折光性強(qiáng)。細(xì)胞形態(tài)符合正常上皮細(xì)胞特性，所分離的細(xì)胞幾乎完全表達(dá)上皮細(xì)胞表面標(biāo)志物CK19。細(xì)胞生長(zhǎng)良好，可以傳6~8代，細(xì)胞生長(zhǎng)曲線呈“S”形，純度達(dá)95%以上。細(xì)胞刷取培養(yǎng)法操作簡(jiǎn)單，能夠快速分離獲得大量卵巢表面上皮，所獲得的細(xì)胞經(jīng)紅細(xì)胞裂解法和差速貼壁法處理后純的達(dá)到95%以上，且細(xì)胞生長(zhǎng)穩(wěn)定，為組織工程研究提供了充足的種子細(xì)胞。 2.經(jīng)過(guò)四輪篩選后，噬菌體在靶細(xì)胞HO8910上出現(xiàn)了明顯的富集現(xiàn)象，第4輪篩選后與第1輪相比,富集了244倍；ELISA對(duì)20個(gè)隨即挑選的克隆進(jìn)行鑒定，12個(gè)可與HO8910特異性結(jié)合；DNA測(cè)序后得到一段與卵巢癌細(xì)胞特異性結(jié)合的七肽SWQIGGN（命名為K1），免疫熒光檢測(cè)顯示表達(dá)SWQIGGN序列的噬菌體克隆（phage1）能夠與HO8910細(xì)胞結(jié)合，細(xì)胞呈綠色熒光反應(yīng)，并不與人正常卵巢上皮細(xì)胞結(jié)合，提示phage1能與HO8910細(xì)胞特異結(jié)合。 3.合成并純化后的的K1，，細(xì)胞生長(zhǎng)曲線顯示K1組HO8910細(xì)胞自第三天開始生長(zhǎng)速度明顯慢于其它兩組，P＜0.05，差異具有統(tǒng)計(jì)學(xué)意義。粘附實(shí)驗(yàn)和細(xì)胞體外遷移侵襲實(shí)驗(yàn)表明合成的特異性短肽K1（SWQIGGN）能明顯抑制HO8910PM的侵襲和轉(zhuǎn)移能力，與對(duì)照組相比P＜0.05，差異具有統(tǒng)計(jì)學(xué)意義。表明特異性短肽K1（SWQIGGN）能夠在體外抑制卵巢癌HO8910細(xì)胞的生長(zhǎng)、侵襲及轉(zhuǎn)移。 4.腫瘤侵襲轉(zhuǎn)移體內(nèi)實(shí)驗(yàn)表明特異性短肽K1（SWQIGGN）組裸鼠生長(zhǎng)速度明顯受到抑制、腫瘤體積、質(zhì)量及播散數(shù)目均減少，與對(duì)照組及空白組相比，差異具有統(tǒng)計(jì)學(xué)意義（P＜0.5），而對(duì)照組與空白組相比，差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.5）。表明SWQIGGN肽能夠抑制HO8910細(xì)胞的增長(zhǎng)，并能夠在體內(nèi)抑制卵巢癌HO8910細(xì)胞的惡性生物學(xué)行為。 結(jié)論 1.運(yùn)用細(xì)胞刷取法成功培養(yǎng)出人卵巢表面上皮，該方法不僅簡(jiǎn)便，而且高效實(shí)用，為卵巢癌的基礎(chǔ)和臨床研究提供了理想的試驗(yàn)材料。 2.采用BRASIL噬菌體展示技術(shù)和全細(xì)胞差減篩選策略，篩選出卵巢癌表面分子特異性的結(jié)合短肽SWQIGGN，為卵巢癌的靶向治療提供了可能的作用靶向結(jié)合位點(diǎn)。 3.SWQIGGN短肽能夠促進(jìn)卵巢癌細(xì)胞的體內(nèi)外生長(zhǎng)、侵襲及轉(zhuǎn)移能力。 4.SWQIGGN肽促進(jìn)卵巢癌細(xì)胞侵襲和轉(zhuǎn)移的機(jī)制可能與下調(diào)腫瘤內(nèi)VEGF的表達(dá)有關(guān)，為進(jìn)一步選擇轉(zhuǎn)移復(fù)發(fā)預(yù)測(cè)指標(biāo)、腫瘤靶向治療提供了重大線索。
[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.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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