本文選題：多發(fā)性骨髓瘤　切入點(diǎn)：CAR-T細(xì)胞療法　出處：《華東師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：多發(fā)性骨髓瘤是最常見的惡性漿細(xì)胞病,由于骨髓瘤細(xì)胞的高侵襲性、轉(zhuǎn)移性和耐藥性,故所有患者最終都將復(fù)發(fā)或耐藥。目前還沒(méi)有一種有效的方法將其治愈,攻克骨髓瘤是我們不可懈怠的使命。免疫治療的發(fā)展給骨髓瘤的治愈提供了新方向,對(duì)于該疾病,首選的治療手段仍為干細(xì)胞移植術(shù),但該方法不僅容易復(fù)發(fā),還易發(fā)生移植物抗宿主反應(yīng),帶來(lái)極高的發(fā)病率和死亡率。骨髓瘤的免疫治療目前存在的主要問(wèn)題是:1)骨髓瘤細(xì)胞表面缺乏特異性表達(dá)的靶標(biāo)分子;2)患者骨髓的局部微環(huán)境易產(chǎn)生免疫抑制和免疫逃脫并且容易產(chǎn)生免疫耐受。CAR-T細(xì)胞免疫療法的出現(xiàn)給骨髓瘤的治療帶來(lái)了新的希望。CAR分子具有同時(shí)特異性定位到靶標(biāo)分子和活化T細(xì)胞的功能,并且通過(guò)與信號(hào)域的結(jié)合實(shí)現(xiàn)T細(xì)胞持續(xù)的激活。靶向CD19的CART細(xì)胞臨床試驗(yàn)表現(xiàn)出驚人和持久的結(jié)果,隨著CART療法在治療血液病的成功,人們開始將目光轉(zhuǎn)向其他領(lǐng)域,關(guān)于骨髓瘤目前有三個(gè)臨床試驗(yàn)正在進(jìn)行,其中有兩個(gè)靶向BCMA和一個(gè)靶向CD138分子。本研究選取在骨髓瘤細(xì)胞表面高表達(dá)的CD138,BCMA,CD38,CD40分子和B細(xì)胞惡性腫瘤表面特異性表達(dá)的CD19分子為研究的靶標(biāo),通過(guò)freepatent online網(wǎng)站查找各研究靶標(biāo)單抗序列的scFv段,獲取其序列。電泳鑒定完后片段與用EcoRI和XbaI酶切好的工具質(zhì)粒進(jìn)行重組,電泳結(jié)果顯示scFv片段、工具質(zhì)粒、重組質(zhì)粒的條帶位置符合預(yù)期結(jié)果。重組質(zhì)粒DNA測(cè)序的結(jié)果對(duì)該結(jié)果進(jìn)一步確認(rèn)。各靶標(biāo)重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞24小時(shí),感染293T細(xì)胞48小時(shí)后均得到了很好的結(jié)果,通過(guò)對(duì)各靶標(biāo)慢病毒的濃縮與純化,得到了平均滴度達(dá)到10^9 IU/ml的慢病毒。K562穩(wěn)定細(xì)胞株的抗原表達(dá)量均高于96%,CAR分子轉(zhuǎn)導(dǎo)T細(xì)胞,BCMA-CAR的轉(zhuǎn)導(dǎo)效率達(dá)64%,LDH法的殺傷實(shí)驗(yàn)結(jié)果表明,各CART細(xì)胞殺傷效果明顯,最高達(dá)65%;我們成功的將殺傷實(shí)驗(yàn)結(jié)果與IFN-γ、IL-2和TNF細(xì)胞因子釋放量建立相關(guān)性,本研究可為CAR-T細(xì)胞在應(yīng)用到臨床時(shí)方案的選擇、細(xì)胞劑量的確定和臨床治療效果的預(yù)測(cè)提供重要依據(jù)。
[Abstract]:Multiple myeloma is the most common malignant plasmacytosis. Due to the high invasion, metastasis and drug resistance of myeloma cells, all patients will eventually recur or resistant to drugs. The development of immunotherapy provides a new direction for the cure of myeloma. For this disease, the preferred treatment is stem cell transplantation, but it is not only easy to recur. Also prone to graft-versus-host response, The main problem with immunotherapy for myeloma is that the bone marrow microenvironment of patients with myeloma is prone to immunosuppression, which is a target molecule with no specific expression on the surface of myeloma cells. Immune escape and easy to produce immune tolerance. CAR-T cell immunotherapy brings new hope for the treatment of myeloma. Car molecule has the function of targeting target molecule and activating T cell at the same time. The clinical trials of CART cells targeting CD19 showed surprising and lasting results. With the success of CART therapy in the treatment of hematological diseases, people began to turn their eyes to other fields. With regard to myeloma, there are currently three clinical trials under way. In this study, we selected CD138BCMA-CD38 CD40 molecule and CD19 molecule specifically expressed on the surface of B cell malignant tumor. The scFv fragment of each target McAb sequence was searched by freepatent online website, and its sequence was obtained. After electrophoresis, the fragment was recombined with the tool plasmid digested by EcoRI and XbaI. The result of electrophoresis showed that scFv fragment, tool plasmid, and so on. The band position of the recombinant plasmid was in accordance with the expected results. The results of DNA sequencing of the recombinant plasmids confirmed the results. After transfection of each target recombinant plasmid into 293T cells for 24 hours, good results were obtained after 48 hours of infection with 293T cells. Through the concentration and purification of various target lentivirus, the antigenic expression of lentivirus. K562 stable cell line with an average titer of 10 ^ 9 IU/ml was higher than that of 96 car molecular transduction T cell line BCMA-CAR. The results showed that the transduction efficiency of BCMA-CAR reached 64%. The killing effect of each CART cell was significant, up to 65%. We successfully established a correlation between the killing results and the release of IL-2 and TNF cytokines. This study can be used as a choice for the regimen of CAR-T cells in clinical application. The determination of cell dose and the prediction of clinical therapeutic effect provide important basis.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.3
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本文編號(hào)：1601858