本文選題：CAR-T + 間皮素�。� 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：癌癥已經(jīng)嚴(yán)重威脅到了人類(lèi)的健康,對(duì)人類(lèi)的生存造成了極大的危害。在全球范圍內(nèi),卵巢癌占婦科最常見(jiàn)癌癥的第八位,且死亡人數(shù)占婦科常見(jiàn)腫瘤的第七位。每年全世界約有230000名婦女被診斷為卵巢癌,而且超過(guò)75%的患者確診時(shí)已經(jīng)是晚期,卵巢癌的5年生存率低于45%。目前臨床上卵巢癌常見(jiàn)的治療手段包括手術(shù)、放化療等,但這些治療不能有效的控制腫瘤的復(fù)發(fā)及進(jìn)展,也不能有效的延長(zhǎng)患者的生存期及改善患者的生存質(zhì)量。因此我們迫切需要尋求新的治療手段。近年來(lái)生物治療成為一種重要的抗腫瘤治療手段,給攻克惡性腫瘤帶來(lái)了希望。研究表明大多數(shù)的腫瘤細(xì)胞能夠躲避免疫系統(tǒng)的識(shí)別,從而限制免疫治療的抗癌效果。CAR-T(嵌合抗原受體T細(xì)胞)技術(shù)應(yīng)運(yùn)而生,其具有較好的靶向性、殺傷性、增殖性及持久性。大量的臨床試驗(yàn)研究也表明該技術(shù)在改善惡性血液腫瘤患者生存質(zhì)量及延長(zhǎng)患者生存期方面取得了巨大的成功,CAR-T技術(shù)成為了免疫治療的研究熱點(diǎn)。然而該技術(shù)在實(shí)體腫瘤的研究還不夠成熟,未能得到期待的效果,仍然面臨許多問(wèn)題,包括:實(shí)體瘤組織特殊的病理學(xué)特征、表面缺乏合適的腫瘤特異性抗原,腫瘤周?chē)庖咭种莆h(huán)境、共刺激分子的優(yōu)化組合、自殺基因的設(shè)計(jì)、回輸后的不良反應(yīng)等。本課題構(gòu)建并檢測(cè)了靶向間皮素抗原的嵌合抗原受體T細(xì)胞對(duì)幾種卵巢癌細(xì)胞株的體外殺傷作用:1、構(gòu)建包含兩個(gè)胞內(nèi)共刺激區(qū)(CD3ζ和4-1BB)的mesoCAR的重組質(zhì)粒,再通過(guò)piggyBac轉(zhuǎn)座子系統(tǒng)轉(zhuǎn)染T細(xì)胞,獲得第二代mesoCAR-T細(xì)胞。其轉(zhuǎn)染效率為30%左右。進(jìn)一步證明mesoCAR能夠在T細(xì)胞中高水平穩(wěn)定表達(dá)。流式檢測(cè)經(jīng)過(guò)間皮素刺激后mesoCAR-T的活化表型的表達(dá)情況,結(jié)果表明間皮素抗原能使mesoCAR-T細(xì)胞活化。2、通過(guò)流式及Western Blot檢測(cè)多種腫瘤細(xì)胞株間皮素的表達(dá)水平,篩選出五種間皮素不同表達(dá)水平的卵巢癌細(xì)胞株。通過(guò)不同的方式檢測(cè)mesoCAR-T細(xì)胞對(duì)間皮素高表達(dá)的細(xì)胞株及低表達(dá)的細(xì)胞株的殺傷作用。結(jié)果顯示mesoCAR-T細(xì)胞對(duì)間皮素高表達(dá)細(xì)胞株的殺傷作用明顯強(qiáng)于低表達(dá)的細(xì)胞株,并且能更有效的抑制間皮素高表達(dá)細(xì)胞株的生長(zhǎng)。表明構(gòu)建的mesoCAR-T細(xì)胞對(duì)間皮素高表達(dá)的卵巢癌細(xì)胞株具有更強(qiáng)的殺傷活性及靶向性。3、構(gòu)建靶向間皮素抗原的嵌合抗原受體T細(xì)胞(mesoCAR-T),通過(guò)流式檢測(cè)間皮素抗原刺激后mesoCAR-T細(xì)胞的細(xì)胞因子分泌的情況,結(jié)果發(fā)現(xiàn)IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ的分泌量明顯高于對(duì)照組T細(xì)胞。同時(shí)用流式檢測(cè)mesoCAR-T細(xì)胞與兩種卵巢癌細(xì)胞共培養(yǎng)后細(xì)胞因子的分泌,發(fā)現(xiàn)MesoCAR-T細(xì)胞與間皮素高表達(dá)的細(xì)胞株(HO8910)共培養(yǎng)后,IL-2、IL-4、IL-6、IL-10、TNF-α及IFN-γ的分泌量相比間皮素低表達(dá)的細(xì)胞株(SKOV3)升高。本實(shí)驗(yàn)成功的構(gòu)建了靶向間皮素抗原的嵌合抗原受體T細(xì)胞(mesoCAR-T),其結(jié)果表明本實(shí)驗(yàn)室構(gòu)建的piggyBac轉(zhuǎn)座酶系統(tǒng)能夠很好的將外源基因整合T細(xì)胞中,并且構(gòu)建的mesoCAR-T細(xì)胞能夠特異性的殺傷間皮素表達(dá)的卵巢癌細(xì)胞,同時(shí)mesoCAR-T和卵巢癌細(xì)胞株共培養(yǎng)后能夠上調(diào)IL-2、IL-4、IL-6、IL-10、TNF-α及IFN-γ的分泌。為進(jìn)一步體內(nèi)及臨床試驗(yàn)的開(kāi)展提供了實(shí)驗(yàn)基礎(chǔ),也為卵巢癌的治療提供了新的治療方法。
[Abstract]:Cancer has been a serious threat to human health and has caused great harm to human survival. On the global scale, ovarian cancer accounts for eighth of the most common cancers in gynecology, and the death toll accounts for seventh of the common gynecologic cancers. About 230000 women all over the world are diagnosed with ovarian cancer every year, and more than 75% of the patients have been diagnosed at the time of diagnosis. The 5 year survival rate of ovarian cancer is lower than that of 45%.. The common treatment methods of ovarian cancer are surgery, radiotherapy and chemotherapy, but these treatments can not effectively control the recurrence and progress of the tumor. It can not effectively prolong the survival time of the patients and improve the quality of life of the patients. Therefore, we urgently need to seek new therapeutic hands. Biologic therapy has become an important antitumor therapy in recent years, which has brought hope to the attack of malignant tumor. The research shows that most of the tumor cells can avoid the identification of the immune system and restrict the anti-cancer effect of.CAR-T (chimeric antigen receptor T cell). A large number of clinical trials have also shown great success in improving the quality of life and prolonging the life of patients with malignant hematological tumors. CAR-T technology has become a hot topic in the study of immunotherapy. However, the study of this technique in solid swelling is not mature enough to be expected. The effect is still faced with many problems, including the special pathological features of solid tumor tissue, the lack of proper tumor specific antigen on the surface, the immunosuppressive microenvironment around the tumor, the optimal combination of CO stimulatory molecules, the design of suicide gene, the adverse reaction after the retransmission, and so on. The in vitro killing effect of primary receptor T cells on several ovarian cancer cell lines: 1, a recombinant plasmid containing two intracellular co stimulatory regions (CD3 zeta and 4-1BB) was constructed, and T cells were transfected through the piggyBac transposon system to obtain second generation mesoCAR-T cells. The transfection efficiency was about 30%. Further demonstrated that mesoCAR could be in the high level of T cells. The expression of the activated phenotype of mesoCAR-T after mesothelin was detected by flow test. The results showed that the mesothelin antigen could activate the mesoCAR-T cells to activate.2. The expression level of mesothelin in a variety of tumor cell lines was detected by flow and Western Blot, and five kinds of ovarian cancer cell lines with different levels of mesothelin were screened. The cytotoxicity of mesoCAR-T cells to mesothelin high expression cell lines and low expression cell lines was detected in the same way. The results showed that the killing effect of mesoCAR-T cells to mesothelin high expression cell lines was stronger than that of low expressed cell lines, and could effectively inhibit the growth of mesothelin high expression cell lines. The results showed that the constructed mesoCAR- was constructed. T cells have a stronger killing activity and targeted.3 for the ovarian cancer cell lines with high expression of mesothelin, and construct a chimeric antigen receptor T cell (mesoCAR-T) targeting mesothelin antigen. By flow cytometry, the cytokine secretion of mesoCAR-T cells after the stimulation of mesothelin antigen was detected. The results showed that IL-2, IL-4, IL-6, IL-10, TNF- a, IFN- gamma were found. The secretion of T cells was significantly higher than that of the control group. At the same time, the secretion of cytokines in the co culture of mesoCAR-T cells and two kinds of ovarian cancer cells was detected by flow cytometry. The secretion of IL-2, IL-4, IL-6, IL-10, TNF- alpha and IFN- gamma in the co culture of MesoCAR-T cells with the high expression of mesothelin (HO8910) was found to be higher than that of the cells with the low expression of mesothelin (SKOV3). High. This experiment successfully constructed the chimeric antigen receptor T cell (mesoCAR-T) targeting mesothelin antigen. The results show that the piggyBac transposing enzyme system constructed in this laboratory can integrate the exogenous gene in T cells well, and the constructed mesoCAR-T cells can specifically kill the ovarian cancer cells with the expression of mesothelin, and mesoCAR The co culture of -T and ovarian cancer cells can increase the secretion of IL-2, IL-4, IL-6, IL-10, TNF- alpha and IFN- gamma. It provides an experimental basis for further in vivo and clinical trials and provides a new treatment for the treatment of ovarian cancer.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 鹿萍;郝莎;袁衛(wèi)平;程濤;;嵌合抗原受體——癌癥免疫治療的新希望[J];中華醫(yī)學(xué)雜志;2015年04期

2 蔡慧;趙蓮君;鄒征云;;嵌合抗原受體基因修飾T淋巴細(xì)胞在腫瘤免疫治療中的研究[J];現(xiàn)代腫瘤醫(yī)學(xué);2014年11期

3 張少華;畢經(jīng)旺;;嵌合抗原受體修飾T細(xì)胞在惡性腫瘤中的研究進(jìn)展[J];國(guó)際腫瘤學(xué)雜志;2014年07期

4 陳杰;王宇環(huán);羅成林;王慧琴;羅曉玲;;嵌合抗原受體T細(xì)胞介紹及抗腫瘤臨床應(yīng)用[J];中國(guó)細(xì)胞生物學(xué)學(xué)報(bào);2014年02期

5 徐云云;金潤(rùn)銘;;血液腫瘤治療中嵌合抗原受體基因修飾T淋巴細(xì)胞作用[J];中國(guó)實(shí)用兒科雜志;2013年08期

，

本文編號(hào)：2052938