卡介苗热休克蛋白70基因转染人原代白血病细胞瘤苗的制备及其抗瘤机制  

Preparation of leukemia cell vaccine expressing Bacille Calmette - Guerin heat shock protein 70 and its anti - leukemia therapeutic effect

作　　者：李晓玲[1] 刘春雷[2] 孙立荣[3] 

机构地区：[1]临沂市人民医院儿内三科,山东临沂276000 [2]临沂市人民医院网络与数据中心,山东临沂276000 [3]青岛大学医学院附属医院小儿血液科

出　　处：《中华实用儿科临床杂志》2015年第3期216-220,共5页Chinese Journal of Applied Clinical Pediatrics

摘　　要：目的通过体外培养白血病细胞，制备膜表面表达卡介苗（BCG）热休克蛋白70（HSP70）瘤苗，研究其抗瘤作用和机制。方法含重组人干细胞因子（SCF）、重组人flt-3配体（FL）、白细胞介素（IL）-3和IL-6的无血清培养液Stemspan体外培养急性髓系白血病（AML）细胞，含SCF、FL、IL-3和IL-7的无血清的改良Iscove培养基（IMDM）体外培养B系急性淋巴细胞白血病（B—ALL）细胞，通过观察细胞生长形态和免疫表型测定验证培养的白血病细胞。利用脂质体2000将重组载体pDisplay—HSP70转染到已被鉴定的白血病细胞，免疫荧光单克隆抗体进行细胞表面修饰鉴定。对转染后的白血病细胞免疫原性进行检测：实验组分为未进行转染的白血病细胞组（wt-LC组），转染空载体pDisplay的白血病细胞组（pDisplay-LC组）及转染重组载体pDisplay-HSP70的白血病细胞组（HSP70-LC组）。收获各组细胞，与自体外周血T淋巴细胞混合培养72h，羟基荧光素双乙酸盐琥珀酰亚胺酯（CFSE）标记，流式细胞术测定淋巴细胞增殖指数，酶联免疫吸附试验（ELISA）检测干扰素（IFN）-γ水平；各组白血病细胞与T淋巴细胞混合培养6d后，加入新鲜白血病细胞（效：靶分别为10：1、20：1、40：1、80：1）共培育12h，乳酸脱氢酶（LDH）释放改良法检测细胞毒性T淋巴细胞（CTL）的杀伤活性。结果短期培养的白血病细胞呈集落样悬浮生长，并保持增殖特性；1-10d细胞增殖较快，以后逐渐变慢。对培养10d的细胞进行免疫表型测定，AML细胞的CD13、CD33表达与培养前比较差异无统计学意义（P〉0．05）；B-ALL细胞的CD19、CD10、CD22表达与培养前比较差异无统计学意义（P〉0．05）。共聚焦显微镜观察证实BCG HSP70表达在转染后的白血病细胞表面。转染后的白血病细胞免疫原性检测结果：（1）自体淋巴细胞增殖：HSP70-LC组CFSE阳性�Objective To culture the acute leukemia cells in vitro, and to prepare cancer vaccine expressing heat shock protein 70 （HSP70） of Bacille Calemette- Gerrin（BCG） onto the cell surface,so as to study its anti - tumor effect and mechanism. Methods Acute myeloid leukemia （AML） cells were cultured in a serum - free Stemspan culture supplemented with eytokines [ stem cell factor（SCF）, fit- 3 ligand （FL）, interleukin （IL） -3 and IL - 6 ] in vitro. And B - lineage acute lymphoblastic leukemia （ B - ALL） cells were cultured in a Iscove modified medium（IMDM） culture supplemented with eytokines （SCF, FL, IL -3 and IL- 7 ） in vitro. Cellular morphology was observed by the microscopy and immunophenotype determination was used to verify the biological characteristics of acute leukemia cells after culture. Lipofeetamine 2000 was used to transfect the pDisplay - HSP70 plasmid into acute leukemia cells. The expression of HSP70 on the cell surface was detected by fluoreseene microscope. Then the immunogenicity of the leukemia cells expressing HSP70 were detected. The experimental groups were divided into 3 subgroups ： the wide - type acute leukemia cells （ wt - LC group）, the pDisplay - leukemia cells （ pDisplay - LC group）, and the pDisplay -HSP70 -leukemia cells （HSP70 -LC group） , respectively. The leukemia cells in different groups were cultured with autologous peripheral blood T cells for 72 hours. The proliferation indices of T cells were assayed by earboxyfluorescein diacetate succinimidyl ester （CFSE） - staining method, and the contents of interferon - 7 （ IFN - γ,） were tested by enzyme -linked immunosorbent assay （ELISA）. The leukemia cells in different groups were cultured with autologous peripheral blood T cells, and after 6 days, the fresh acute leukemia cells were added [ in the different ratios of cytotoxicity T lymphocyte （CTL） ：leukemia ceils were 10 ： 1,20 ：1,40 ：1 and 80 ：1 ] and continued to be cultured for another 12 hours. Cytotoxici

关 键 词：卡介苗 热休克蛋白70 白血病细胞 肿瘤疫苗 

分 类 号：R733.7[医药卫生—肿瘤] R392[医药卫生—临床医学]