| Objective: Human Papillomaviruses (HPV) lead to positive or malignant pathologies of infected areas. They can be divided into two groups named low-risk or high-risk type according to their capability of leading to tumors. Infection with high-risk type of HPV is highly associated with the development of cervical cancer, half of which is related with the infection of HPV16. Now operation is the most common method in clinical therapy of cervical cancer. Gene therapy has becom one of the effective assistant means to control relapse of cervical cancer after operation through immunological principle. Now most of the targeting antigens for HPV 16 DNA vaccines are the early proteins of E6/E7. Although they can enhance specific cytotoxic T lymphocytes (CTLs) effect in some extent, they can not induce specific CD4+ T-help cells, which limits DNA vaccines'application. Therefore, improving the immunological potency of antigen has been a hot spot of rescent study. Dendritic cells (DCs) are the most important professional Antigen-pressing cells (APCs) and play a central role in inducing immune responses. The quantity of DCs in antigen producing site is not enough, which limites antigen's presentation by APCs and degrades immunogenicity of antigen. To deliver synthesis signals of both recruit and expanding DCs'population can enhance the quantity of DCs in antigen producing site, which provides a new strategy for DNA vaccine's study on HPV16E7.Macrophage inflammatory protein-1α(MIP-1α) is a kind of chemokine first isolated from macrophage supernatant which is induced by lippolysaccharide. It has a molecular weight of 8000~14000. When binding to CC chemokine receptor 1(CCR1), chemokine receptor 4(CCR4) or chemokine receptor 5(CCR5), it shows chemotactic activity to many kinds of immune responsive cells such as monocytes/macrophages, T lymphocytes, dendritic cells, eosinophile and granulocyte cells. Moreover, it is also an immune regulating factor of monocyte, T lymphocyte cells and B lymphocyte cells. Studies show that MIP-1αcan stimulate the production of IFN-γ, which is an effective adjuvant of Th1-related immune response. Fms-like tyrosine kinase 3 ligand (Flt3l) identified and successfully cloned in 1993, is an early growth factor of hematopoietic cells. It has part affinity with stem cell factor (SCF) and macrophage colony-stimulating factor (M-CSF).Marrow stroma and T lymphocyte express functional Flt3l protein. When combinding with Fms-like tyrosine kinase 3 receptor (Flt3R), tyrosine kinsae is activated to produce biological effect. Matural DCs do not express Flt3R while immatural ones can do so. Thus Flt3l can expand pro-dendritic cells selectively and then mature them. By this means DCs induced by Flt3l are typical dendritic pattern and can effectively initiate immune responses against invading pathogens. So Flt3l is one of the strongest and most important chemokine for expansion of DCs both in vivo and in vitro.In this study we construct eukaryotic expressing plasmids pcDNA3/Flt3l, pcDNA3/MIP-1α. Coinject them with pcDNA3/ HPV16E7 in C57BL/6 mice, to observe the CTL effect of spleen and the capability of mice against the challenge of TC-1 tumor cells.Methods: (1) The total RNA of spleen tissue was extracted from scald mice. The DNA fragment of MIP-1αwas amplified by RT-PCR from total RNA of spleen tissue. The DNA fragments of HPV16E7 were amplified by PCR from the plasmid contains HPV16E7; (2) the products of DNA fragments were linked to plasmids pGEM-T. E.Coli DH5αwas transformed and cultured in 2YT medium containing Ampicillin (Amp). When plasmid extracted, recombinant clones were selected and identified by endonucleases cutting and sequencing; (3) when confirmed by endonucleases cutting and sequencing, the constructed plasmids were cut by proper endonucleases and the target gene fragments of MIP-1αand HPV16E7 were linked to pcDNA3 which was cut by the same endonucleases to construct the eukaryotic expressing plasmids of pcDNA3/ MIP-1αand pcDNA3/HPV16E7; (4) the plasmids containing Flt3l were cut by proper endonucleases to acquire the DNA fragments of Flt3l. Then it was linked to pcDNA3 which was cut by the same endonucleases to construct the eukaryotic expressing plasmid of pcDNA3/Flt3l. E.ColiDH5αwas transformed and cultured in 2YT medium containing Ampicillin. When plasmids extracted, recombinant clones were selected and identified by endonuclease cutting and sequencing; (5) large preparation was performed to obtain the plasmids of pcDNA3/HPV16E7, pcDNA3/MIP-1α, pcDNA3/Flt3l and pcDNA3 from transformed E.Coli DH5α. Then they were purified by PEG; (6) Animal Experiment: female C57BL/6 mice aged 6~8 weeks were divided into five groups at random including pcDNA3 group, pcDNA3/HPV16E7group, pcDNA3/ MIP-1αand pcDNA3/HPV16E7 mixed group, pcDNA3/Flt3l and pcDNA3/HPV16E7 mixed group, pcDNA3/Flt3l, pcDNA3/ MIP-1αand pcDNA3/HPV16E7 mixed group. Each group contains fourteen mice. The plasmids were diluted by NS and inoculated to every mouse at a total dose of 150μg at one time. Among them each plasmid was 50μg and the rest is supplanted by pcDNA3 plasmid. The mice were injected intramuscularly (i.m.) in quadriceps muscle at 3 weeks intervals for 3 times. Two weeks after the last immunization six mice from each group were selected randomly and the spleens were harvested in free bacterium condition. Specific CTLs targeting to TC-1 cell were tested by CCK-8 kit. The rest mice were challenged with 5×104 TC-1 tumor cells/mouse via subcaneous injection in fold ingnen. Observation of tumor growth started one week after the challenge. At first the observation time was the next day, then once a week after a month. The growth of tumor and the survival of mice were noted. Neoplasms were harvested randomly when they grew up, dehydrated with grading ethand, embedded in paraflin. Sections were stained with hemotoxylin. Pathological changes were observed with microscope.Results: (1) The gene fragments of MIP-1αwere amplified by RT-PCR. The length of the fragment was the same as expected; (2) the prokaryotic clone vectors pGEM-T/MIP-1α, pGEM-T/HPV16E7 were successfully constructed. DNA sequencing showed that the sequences were identical to the sequence recorded in Genebank; (3) after these gene fragments were inserted to the pcDNA3, the recombinant plasmids were identified by endonucleases cutting and named the eukaryotic expressing plasmids of pcDNA3/MIP-1α, pcDNA3/HPV16E7 and pcDNA3/Flt3l; (4) the specific killing precentage of every immunized group targeting to TC-1 cells was as follows: pcDNA3/Flt3l, pcDNA3/MIP-1αand pcDNA3/HPV16E7 mixed group 88.83%, pcDNA3/Flt3l and pcDNA3/HPV16E7 mixed group 76.00%, pcDNA3/MIP-1αand pcDNA3/HPV16E7 mixed group 76.33%, pcDNA3/HPV16E7 group 79.00%, pcDNA3 group 65.33%. The One-Way ANOVA analysis showed that the differences among the five groups were significant (P<0.01). The difference between two groups showed that the specific killing precentage of pcDNA3 group was lower than other groups remarkably, besides the difference has statistic significance (P<0.05); the specific killing precentage of pcDNA3/Flt3l, pcDNA3/MIP-1αand pcDNA3/ HPV16E7 group was higher than other groups notablely and the difference has statistic significance (P<0.05); there were no statistic difference among pcDNA3/Flt3l and pcDNA3/ HPV16E7 mixed group, pcDNA3/MIP-1αand pcDNA3/ HPV16E7 mixed group and the group of pcDNA3/HPV16E7 (P>0.05); (5) 16 days after challenged with TC-1 tumor cells, all the mice of pcDNA3 group suffer tumors, while the tumor-forming rate of pcDNA3/Flt3l, pcDNA3/MIP-1αand pcDNA3/HPV16E7 mixed group was 12.5%, which lower than other groups. Statistics analysis showed that there were differences among five groups (P=0.005). By the multiple comparisons the difference between pcDNA3/Flt3l, pcDNA3/ MIP-1αand pcDNA3/HPV16E7 mixed group and the group of pcDNA3 was significant (P=0.001). The difference between pcDNA3/Flt3l, pcDNA3/MIP-1αand pcDNA3/HPV16E7 mixed group and the group of pcDNA3/HPV16E7 was significant (P=0.041). The difference between"pcDNA3/Flt3l, pcDNA3/MIP-1αand pcDNA3/HPV16E7 mixed group"and"pcDNA3/MIP-1αand pcDNA3/HPV16E7 mixed group"was significant (P=0.041). But the difference was not significant between other two groups (P>0.05). The statistics analysis showed that the differences of tumor-forming rate among any groups were not significant 60 days after challenge (P=0.229); (6) the pathological observation of neoplasm on mice was typical tumour cells pattern.Conclusions: (1) The gene fragments of MIP-1αwas successfully cloned from scalded mice; (2) the prokaryotic clone vectors pGEM-T/MIP-1αand PGEM-T/HPV16E7 were successfully constructed; (3) the eukaryotic expressing plasmids pcDNA3/Flt3l, pcDNA3/HPV16E7 and pcDNA3/MIP-1αwere constructed successfully; (4) the combinated vaccine of pcDNA3/Flt3l, pcDNA3/MIP-1αand pcDNA3/HPV16E7 was superior to DNA vaccine of HPV16E7 and delayed the tumor's growth in some extent; (5) the combinated vaccine of pcDNA3/Flt3l and pcDNA3/HPV16E7 did not enhance the immunological effect of HPV16E7; (6) the combinated vaccine of pcDNA3/MIP-1αand pcDNA3/HPV16E7 did not enhance the immunological effect of HPV16E7.
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