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

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阳性率明显高于wt-LC组和pDisplay-LC组（t=17．89、19．58，P均〈0．05），pDisplay—LC组与wt—LC组间比较差异无统计学意义（P〉0．05）。（2）细胞因子水平：HSP70-LC组IFN-1水平明显高于wt-LC组和pDisplay-LC组（t=24．72、24．81，P均〈0．05）。（3）CTL杀伤活性：固定效：靶比例，与实验wt-LC组、pDisplay-LC组比较，HSP70-LC组能明显提高CTL的杀伤活性（F=13．66，P〈0．05）；当效：靶从10：1至80：1，HSP70-LC组组内CTL细胞杀伤活性逐渐增强（F=19．69，P〈0．05）。结论体外成功培养人原代急性白血病细胞，短期培养可以明显增加细胞数量，且对细胞表面抗原表达影响不大，能维持其生物学特性。成功制备膜表面表达BCGHSP70的白血病细胞瘤苗，且BCGHSP70基因转染后能明显增强白血病细胞的免疫原性。

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. Cytotoxicity assay was measured by lactate dehydrogenase（LDH） release. Results After short - term culture in vitro,the leukemia cells were in colony -like suspension and maintain the proliferation characteristics were maintained. The cell proliferation was rapidly cultured for about 10 days and then was gradually slowed down. But there was no difference between the day 10 and day 0 in the expressions of CD13 and CD33 in fifteen cases of AML cells （P 〉 0.05 ）. Equally, there was no difference between the day 10 and day 0 in the expressions of CD19, CD10 and CD22 in fifteen cases of B - ALL cells （ P 〉 0.05 ）. After BCG HSP70 gene transfection, the yellow - green fluorescence on the leukemia cells surface was observed under the confocal microscope. Detection of the immunogenicity ： （ 1 ） Autologous T cell proliferation ： the most significant T cell proliferation was observed in the group of HSP70 - transfected leukemia ceils （t = 17.89,19. 58, all P 〈 0.05 ）. There was no difference between the wt - LC group and pDisplay - LC group （ P 〉 0.05 ）. （2） The contents of cytokines ： the IFN - γ level in the group of HSP70 - transfected leukemia cells was higher than those of wide - type acute leukemia cells and the pDisplay - transfected ones （ t = 24. 72,24. 81, all P 〈 0. 05 ）. （3） Cytotoicity of CTL： the killing rate in HSP70 - transfected leukemia cells was significantly higher than those of wide - type acute leukemia cells and pDisplay transfected ones （F = 13.66, P 〈 0. 05 ）. And with the increase of the ratio from 10 ： 1 to 80 ： 1 ,the inhibiting activity of CTL in the HSP70 - LC group was raising（ F = 19. 69,P 〈 0. 05 ）. Conclusions Fresh acute leukemia ceils can be successfully cultured in vitro. Short - term cuhure can significantly increase the number of leukemia cells,but has little effect on surface antigen expression. So,the biological characteristics of the leukemia cells can be maintained. The leukemia cells vaccine expressing BCG HSP70 onto its surface was successfully prepared, and gene transfection of BCG HSPTO can significantly enhance the immunogenicity of leukemia cells.