卡介苗HSP70基因转染HL-60细胞瘤苗制备及抗瘤机制的研究

Preparation of HL-60 cell vaccine expressing BCG-HSP70 and its anti-leukemia effect

目的构建卡介苗(BCG)热休克蛋白70(HSP70)的真核表达载体,通过基因转染表达HL-60细胞表面,研究其抗瘤作用和机制。方法利用多聚酶链反应(PCR)方法扩增HSP70基因,与真核表达载体pDisplay连接,构建重组载体pDisplay-HSP70,并利用脂质体2000将其转染HL-60细胞。HL-60细胞分为未转染的HL60-wt组、转染空载体pDisplay的HL60-pDisplay组、转染重组载体pDisplay-HSP70的HL60-HSP70组,分别与外周血T淋巴细胞培养72 h,羟基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记和流式细胞术测定淋巴细胞增殖指数,ELISA法检测IFN-γ水平;各组HL-60细胞与T淋巴细胞混合培养6 d后获细胞毒性T淋巴细胞(CTL)效应细胞,加入HL-60靶细胞以不同效靶比共培育12 h,LDH释放改良法检测CTL细胞杀伤活性。结果扩增获得的HSP70基因大小与理论值一致,重组载体pDisplay-HSP70构建正确。HSP70表达在转染后的HL-60细胞表面。转染后的HL-60细胞,即HL60-HSP70组能明显促进异体T细胞扩增,该组CFSE阳性率、IFN-γ含量、CTL杀伤率均高于HL60-wt和HL60-pDisplay组,差异均有统计学意义(P<0.05);后两组间差异则无统计学意义(P>0.05);随效靶比增大,HL60-HSP70组CTL细胞杀伤活性逐渐增强,差异有统计学意义(P<0.05)。结论成功构建真核表达载体pDisplay-HSP70,并制备膜表面表达BCG HSP70的HL-60细胞瘤苗,HSP70基因转染后能明显增强HL-60细胞的免疫原性。

Objectives To prepare the HL-60 cell vaccine expressing heat shock protein 70 （HSP70） of Bacille calmette-Guerin （BCG）, so as to study its anti-tumor effect and mechanism. Methods The whole BCG HSP70 gene was amplified from BCG genome by polymerase chain reaction （PCR） and sub-cloned into the polyclone endonuclease sites in pDisplay. The recom-binant vector of pDisplay-HSP70 was verified by sequencing. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection. To detect the immunogenicity of HL-60 cells expressing HSP70, the test groups were divided into three subgroups, HL60-wt, HL60-pDisplay, and HL60-HSP70 respectively. Each group was cultured with peripheral blood T cells for 72 h, then the proliferation indices of T cells were assayed by CFSE-staining method, and IFN-γwere tested by enzyme-linked immunosorbent assay （ELISA）. The HL-60 cells of different groups were cultured with peripher-al blood T cells for 6d. The wild-type HL-60 cells were added and co-cultured for another 12h. Cytotoxicity assay was measured by LDH release. Results （1） The fragment of BCG HSP70 was consistent with the theoretical value. DNA sequencing showed that the recombinant vector of pDisplay-HSP70 was correctly constructed. （2） BCG HSP70 expressed onto the HL-60 cells sur-face. （3） Detection of the immunogenicity： ①The most significant T cell proliferation was observed in the group of HSP70-transfected HL-60 cells （P〈0.05）. There was no difference between the HL60-wt group and HL60-pDisplay group （P〉0.05）.②The contents of IFN-γof the HSP70-HL60 group was the highest.③The inhibiting activity of CTLs on HL-60 cells in the group of HSP70-transfected HL-60 cells was more significant than that of wide-type and pDisplay--transfected HL-60 cells. And with the increase of the E：T ratio, the inhibiting activity of CTLs in the HSP70-HL60 group was rising. Conclusions The recombinant eukaryotic expression vector （pDisplay-HSP70） of BCG HSP70 was successfully constructed. And the HL-60 cell vaccine expressing BCG HSP70 onto its surface was successfully prepared. The results showed that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells.