發(fā)布時間：2018-03-12 13:54

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

【摘要】：多發(fā)性骨髓瘤是最常見的惡性漿細胞病,由于骨髓瘤細胞的高侵襲性、轉移性和耐藥性,故所有患者最終都將復發(fā)或耐藥。目前還沒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,對于該疾病,首選的治療手段仍為干細胞移植術,但該方法不僅容易復發(fā),還易發(fā)生移植物抗宿主反應,帶來極高的發(fā)病率和死亡率。骨髓瘤的免疫治療目前存在的主要問題是:1)骨髓瘤細胞表面缺乏特異性表達的靶標分子;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細胞免疫療法的出現(xiàn)給骨髓瘤的治療帶來了新的希望。CAR分子具有同時特異性定位到靶標分子和活化T細胞的功能,并且通過與信號域的結合實現(xiàn)T細胞持續(xù)的激活。靶向CD19的CART細胞臨床試驗表現(xiàn)出驚人和持久的結果,隨著CART療法在治療血液病的成功,人們開始將目光轉向其他領域,關于骨髓瘤目前有三個臨床試驗正在進行,其中有兩個靶向BCMA和一個靶向CD138分子。本研究選取在骨髓瘤細胞表面高表達的CD138,BCMA,CD38,CD40分子和B細胞惡性腫瘤表面特異性表達的CD19分子為研究的靶標,通過freepatent online網(wǎng)站查找各研究靶標單抗序列的scFv段,獲取其序列。電泳鑒定完后片段與用EcoRI和XbaI酶切好的工具質粒進行重組,電泳結果顯示scFv片段、工具質粒、重組質粒的條帶位置符合預期結果。重組質粒DNA測序的結果對該結果進一步確認。各靶標重組質粒轉染293T細胞24小時,感染293T細胞48小時后均得到了很好的結果,通過對各靶標慢病毒的濃縮與純化,得到了平均滴度達到10^9 IU/ml的慢病毒。K562穩(wěn)定細胞株的抗原表達量均高于96%,CAR分子轉導T細胞,BCMA-CAR的轉導效率達64%,LDH法的殺傷實驗結果表明,各CART細胞殺傷效果明顯,最高達65%;我們成功的將殺傷實驗結果與IFN-γ、IL-2和TNF細胞因子釋放量建立相關性,本研究可為CAR-T細胞在應用到臨床時方案的選擇、細胞劑量的確定和臨床治療效果的預測提供重要依據(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.
【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.3
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本文編號：1601858