卡介苗HSP70基因转染HL-60细胞瘤苗制备及抗瘤机制的研究

摘要/Abstract

摘要：

　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，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。

Abstract: 　Objective　To prepare the HL-60 cell vaccine expressing heat shock protein 70 (Hsp70) of Bacille calmette-Guérin (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 recombinant 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 72h, then the proliferation indices of T cells were assayed by CFSE-staining method, and IFN-γ were tested by enzyme-linked immunosorbent assay (ELISA). The HL-60 cells of different groups were cultured with peripheral 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 surface. (3) Detection of the immunogenicity: ① The 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). ② The contents of IFN-γ of the HSP70-HL60 group was the highest. ③ The 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.

李晓玲, 刘春雷, 孙立荣. 卡介苗HSP70基因转染HL-60细胞瘤苗制备及抗瘤机制的研究[J]. 临床儿科杂志, 2014, 32(5): 462-466.

　LI Xiaoling,LIU Chunlei,SUN Lirong . Preparation of HL-60 cell vaccine expressing BCG-HSP70 and its anti-leukemia effect[J]. , 2014, 32(5): 462-466.

参考文献

[1] Pui CH，Robison LL，Look AT. Acute lymphoblastic leukemia [J]. Lancet，2008，371(9617)：1030-1043.
[2] Pui CH，Carroll WL，Meshinchi S，et al. Biology，risk stratification，and therapy of pediatric acute leukemia：an update [J]. J Clin Oncol，2011，29(5)：551-565.
[3] Stow P，Key L，Chen X，et al. Clinical significance of low levels of minimal residual disease at the end of remission induction therapy in childhood acute lymphoblastic leukemia [J]. Blood，2010，115(23)：4657-4663.
[4] Yang J，Sun LR，Pang XY，et al. Effect of Bacillus Calmette-Guerin on the expansion of dendritic cells from peripheral blood of pediatric patients with leukemia in vitro [J]. Zhongguo Shi Yan Xue Ye Xue Za Zhi，2010，18(5)：1240-1243.
[5] Peng ML，Chen M，Ling NX，et al. Novel vaccines for the treatment of chronic HBV infection based on mycobacterial heat shock protein 70 [J]. Vaccine，2006，24(7)：887-896.
[6] Wei MQ，Menoesha A，Good D，et al. Bacterial targeted tumor therapy-drawn of new era [J]. Cancer Lett，2008，259(1)：16-27.
[7] Ye J，Chen GS，Song HP，et al. Heat shock protein 70/MAGE-1 tumor vaccine can enhance the potency of MAGE-1-specific cellular immune responses in vivo [J]. Cancer Immunol Immunother，2004，53(9)：825-834.
[8] Zhang Y，Li ZJ，Ma ZH，et al. Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene [J]. Cancer Res，2000，60(4)：1035-1042.
[9] Onishi H，Kuroki H，Matsumoto K，et al. Monocyte-derived dendritic cells that capture dead tumor cells secret IL-12 and TNF-alpha through IL-12/TNF-alpha/NF-kappa B autocrine loop [J]. J Cancer Immunol Immunother，2004，53(12)：1093-1100.
[10] Cheng WF，Hung CF，et al. Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene [J]. J Immunol，2001，166(10)：6218-6226.
[11] Hsu KF，Hung CF，Cheng WF，et al. Enhancement of suicidal DNA vaccine potency by linking Mycobacterium tuberculosis heat shock protein 70 to an antigen [J]. Gene Them，2001，8(5)：376-383.
[12] June CH. Adoptive T cell therapy for cancer in the clinic [J]. J Clin Invest，2007，117(6)：1466-1476.
[13] Ahmad M，Rees RC，Ali SA. Escape from immunotherapy possible mechanisms that influence tumor regression/ progression [J]. Cancer Immunol Immunother，2004，53(10)：844-854.
[14] Rouas - Freis N，Moreau P，Menier C，et al. Expression of tolerogenic HLA-G molecules in cancer prevents antitumor response [J]. Semin Cancer Biol，2007，17(6)：413-421.
[15] Peng ML，Chen M，Ling N，et al. Novel vaccines for the treatment of chronic HBV infection based on mycobacterial heat shock protein [J]. Vaccine，2006，24(7)：887-896.
[16] Mizukami S，Kajiwara C，Ishikawa H，et al. Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors [J]. Cancer Sci，2008，99(5)：1008-1015.
[17] Murshid A，Gong J，Calderwook SK. Heat-shock proteins in cancer vaccines：agents of antigen cross-presentatio [J]. Expert Rev Vaccines，2008，7(7)：1019-1030.
[18] Yamaoka A，Guan X，Takemoto S. Development of a novel Hsp70-based DNA vaccine as a multifunction antigen delivery system [J]. J Control Release，2010，142(3)：411-415.
[19] Hockertz S. Present and future of cancer vaccines [J]. Toxicology，2005，214(1-2)：151-161.
[20] Venken K，Thewissen M，Hellings N，et al. A CFSE based assay for measuring CD4+ CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses [J]. J Immunol Methods，2007，322(1-2)：1-11.
[21] Tsuge I，Okumura A，Kondo Y，et al. Allergen-specific T-cell response in patients with phenytoin hypersensitivity simultaneous analysis of proliferation and cytokine production by carboxyfluorescein succinimidyl ester (CFSE) dilution assay [J]. Allergol Int，2007，56(2)：149-155.
[22] Placek K，Coffre M，Maiella S，et al. Genetic and epigenetic networks controlling T helper 1 cell differentiation [J]. Immunology，2009，127(2)：155-162.
[23] Weidnann E，Brieger J，Jahn B，et al. Lactate dehydrogenase release assay：a reliable nonradioactive technique for analysis of cytotoxic lymphocyte-mediate lytic activity against blasts from acute myelocytic leukemia [J]. Ann Hematol，1995，70(3)：153-158.
[24] Pirious L，Chilmonczyk S，Genetetet N，et al. Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species [J]. Cytometry，2000，41(4)：289-297.