›› 2014, Vol. 32 ›› Issue (5): 462-466.

• Original Article • Previous Articles     Next Articles

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

 LI Xiaoling1, LIU Chunlei2, SUN Lirong3    

  1. (1. Department of the Third Pediatrics, the Linyi People's Hospital, Linyi 276000, Shandong, China; 2. Department of the Network and Data Center, the Linyi People's Hospital, Linyi 276000, Shandong, China; 3. Department of Pediatric Hematology, the Affiliated Hospital of Medical College, Qingdao University; Qingdao 266003, Shandong, China)
  • Received:2013-11-01 Online:2014-05-15 Published:2014-05-15

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.