嵌合抗原受体修饰的NK细胞在多发性骨髓瘤中的应用研究

发布时间：2019-01-14 15:32

【摘要】：研究背景：多发性骨髓瘤(Multiple Myeloma, MM)是一种浆细胞系统恶性克隆性疾病,主要特征为浆细胞克隆性增值并导致血和尿中出现大量免疫球蛋白M蛋白。目前治疗主要根据患者的危险度分层进行个体化治疗,虽然新药如免疫调节剂及蛋白酶体抑制剂的使用延长了患者的生存期,但大部分患者最终疾病会复发,因而就需要更新的治疗药物及方法。嵌合抗原受体(chimeric antigen receptors,CARs)修饰的淋巴细胞治疗恶性肿瘤是一种新的免疫治疗方法。前期研究发现嵌合抗原受体修饰的NK细胞具有抗骨髓瘤效应,为临床使用该项技术提供了理论依据。研究目的：该研究主要探讨嵌合抗原受体设计的NK细胞的抗骨髓瘤效应研究方法：本研究首先提取Jurkat细胞的总RNA，逆转录合成cDNA,通过PCR的方法合成CD28片段和CD3ζ片段,紫外光下切胶回收片段,通过双酶切连接的方法使每个片段连接到pcDNA3.1质粒,抗CD138scFv片段由我院生物治疗科提供片段由基因公司合成并连接到pcDNA3.1质粒,DNA测序正确后继续双酶切连接的方法使三个片段首尾彼此相连成融合片段,测序正确后,双酶切片段回收连接到pHBLV-CMVIE-Luc-T2A-puro质粒，通过三质粒慢病毒包装系统(pSPAX2, pMD2G,pHBLV-CMVIE-Luc-T2A-puro)转染293T细胞,收集病毒并测定病毒滴度,接着使用病毒感染NK92MI细胞系,并通过嘌呤霉素筛选阳性细胞株,建立稳定转染细胞株后，，通过PCR及Western Blot验证CAR(抗CD138-CD28-CD3ζ)在NK92MI细胞系的表达,验证正确后进行细胞试验。在细胞试验中,通过流式细胞仪检测NK92MI细胞分泌细胞因子cd107a的能力,并进行统计学分析。从而在细胞水平验证CAR-NK细胞的抗骨髓瘤作用。研究结果①通过基因工程技术,体外构建CAR基因片段,通过基因测序验证基因序列正确。②经过病毒包装技术,获得表达该片段的病毒株。③经过病毒转染实验,获得表达CAR融合蛋白的稳转NK细胞株。④经PCR及Western Blot证实CAR融合蛋白在NK细胞表达。⑤细胞杀伤实验证实CAR NK细胞具有明显的杀伤多发性骨髓瘤的效应，CARNK细胞分泌细胞因子cd107a的能力明显优于对照组。研究结论：①CAR(抗CD138-CD28-CD3ζ)能在体外构建并在NK92细胞表面表达②CAR NK细胞能杀伤表达cd138抗原的多发性骨髓瘤细胞株,从而发挥抗骨髓瘤效应。
[Abstract]:Background: multiple myeloma (Multiple Myeloma, MM) is a malignant clonal disease of plasmacyte system, which is characterized by cytoplasmic clonality increment and a large number of immunoglobulin M proteins in blood and urine. Although the use of new drugs, such as immunomodulators and proteasome inhibitors, prolongs the patient's survival, most patients will eventually recur. Therefore, the need for newer treatment drugs and methods. Lymphocyte modified by chimeric antigen receptor (chimeric antigen receptors,CARs) is a new immunotherapy method for malignant tumor. Previous studies have found that NK cells modified by chimeric antigen receptors have anti-myeloma effect, which provides a theoretical basis for clinical use of this technique. Objective: to investigate the anti-myeloma effect of NK cells designed by chimeric antigen receptor. In this study, the total RNA, reverse transcription synthesis (cDNA,) of Jurkat cells was first extracted. CD28 fragments and CD3 味 fragments were synthesized by PCR. Each fragment was ligated to pcDNA3.1 plasmid by double enzyme ligation. The CD138scFv fragment was synthesized by gene company and ligated to pcDNA3.1 plasmid by the biotherapeutic department of our hospital. After correct sequencing, the DNA sequence was sequenced correctly and the three fragments were linked to each other to form fusion fragments. After correct sequencing, the fragments were recovered and ligated to the pHBLV-CMVIE-Luc-T2A-puro plasmid, and the three plasmid lentivirus packaging system (pSPAX2,) was used. PMD2G,pHBLV-CMVIE-Luc-T2A-puro) was transfected into 293T cells, virus titer was collected and virus titer was measured, then virus was used to infect NK92MI cell line, then positive cell line was screened by purine mycin, and stable transfection cell line was established. The expression of CAR (anti CD138-CD28-CD3 味) in NK92MI cell line was confirmed by PCR and Western Blot. Flow cytometry was used to detect the ability of NK92MI cells to secrete cytokine cd107a. In order to verify the anti-myeloma effect of CAR-NK cells at the cell level. Results 1 the CAR gene fragment was constructed in vitro by genetic engineering technique, and the gene sequence was verified by gene sequencing. 2 the virus strain expressing the fragment was obtained by virus packaging technology. A stable NK cell line expressing CAR fusion protein was obtained. 4 the expression of CAR fusion protein in NK cells was confirmed by PCR and Western Blot. 5 cell killing experiments showed that CAR NK cells had obvious killing effect on multiple myeloma. The ability of CARNK cells to secrete cytokine cd107a was significantly better than that of control group. Conclusion: 1CAR (anti CD138-CD28-CD3 味) can construct in vitro and express 2CAR NK cells on the surface of NK92 cells, which can kill multiple myeloma cell lines expressing cd138 antigen, thus exerting anti-myeloma effect.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.3

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