Studies On HPV Protein Vaccine Of Cervical Cancer And Tumor SP-cell

Protein vaccine of cervical cancer and tumor SP cells were investigated in this study.Cervical cancer(CC)is the second most common cancer in women worldwide.Human papillomavirus(HPV)is the primary etiologic agent of cervical cancer.Two HPV oncogenic proteins,E6 and E7,are critical to the induction and maintenance of cellular transformation and are coexpressed in the majority of HPV-containing carcinomas.Most therapeutic vaccines target one or both of these gene products,E6 and/or E7 are administered in recombinant vectors,as peptides or protein vaccines.As advantages of safety and no HLA restriction,protein vaccine has become the most popular forms of HPV therapeutic vaccines.In study of protein vaccine of cervical cancer,the tumor specific immunological effect and inhibition of TC-1 cells growth were investigated by TC-1 cervical cancer model mice immunized with E6/E7 related proteins.HPV16 E6,E7 and mutant E7(mE7)genes were cloned into pET-28a(+)from cervical cancer cells.Compared with E7,mE7 contains a point mutation of 70th base(T→G)and removal of 15 bases of E7 3’ end.These three recombinants expression plasmids were successfully introduced into E.Coli Rosetta and were induced by IPTG.Then,proteins were purified by Ni-NTA Agarose and detected by SDS-PAGE and western blot.In tumor prevention model,C57BL/6 mice were vaccinated with 1.5 nmol HPV 16 E6,E7 or mE7 protein.No tumor was detected in all mice vaccinated E7 or mE7 protein by challenge with 1×105 TC-1 cells.Only 17%of E6 protein vaccinated mice did not develop tumor.Although the survival percentage was not a significant difference,tumor growth was obviously slow.In tumor therapy model,C57BL/6 mice were planted with 1×105 TC-1 cells,10d later,tumor mice were vaccinated with 1.5 nmol HPV 16 E6,E7 or mE7 protein,but the therapy effect was not significant.An adoptive immune model was used to improve the therapeutic effect,results showed that the development of TC-1 cells was obviously reduced by transfusion of T-cells but not serum from mice immunized with E7 protein.T-cells from E7 protein immunized mice also induced lysis of TC-1 cells in the cytotoxic T lymphocyte(CTL)assay.These findings provided that immunization with HPV16 E6/E7 protein was capable to elicit specific protective immunity against TC-1 tumor growth,and the mE7 was also seen bright future.Heat shock proteins(HSP)were reported to act as antigen vector and adjuvant to enhance antigen-specific tumor immunity,and participated tumor immune.Adjuvant-free immunization of C57BL/6 mice with recombinant protein of E6E7 and mycobacterium tuberculosis(MTB)HSP70 protected the animals against challenge with TC-1 cells.But MTB-HSP70 may induce immunity reaction in mouse or human beings.Mouse self-HSP70 as antigen vector and adjuvant will promote the clinical application of this type of vaccine.HSP70 cDNA was cloned to pET-28a(+)from C57BL/6 mice liver cells and E7-HSP70-pET-28a(+)recombinant expression plasmids was constructed.Two plasmids were transformed into E.Coli Rosetta and were induced by IPTG.Then,inclusion body proteins of HSP70 and E7-HSP70 were renatured and detected by SDS-PAGE and western blot analysis.In tumor prevention model,C57BL/6 mice were vaccinated with two purified proteins,respectively.No tumor was developed in all mice vaccinated with E7-HSP70 protein,whereas all HSP70 protein vaccinated mice developed tumors.This was the first evidence that immunization with E7-HSP70 protein could elicit specific protective immunity against TC-1 tumor growth.Tumor stem cells(TSC)are rare cells with idefinite proliferative potential that drives the formation and growth of tumors.Side population(SP)cells are tumor stem-like cells in tumor tissues or tumor cells.In study of tumor SP cells,mouse BM cells were separated.The method of separation and analysis of SP cells was established.Several tumor cell lines(LLC(FO),PC-12,CaSki and SiHa)were analyzed,all of these four cell lines contained SP cells.LLC(F3),origin from LLC(FO),contains higher percentage of SP cells.Animal experiment also indicated that LLC(F3)had stronger metastasis capability,these results proved that high percentage SP cells can promote tumor metastasis.