Study Of The PBMC(Peripheral Blood Mononuclear Cells)Which Are Stimulated By CpG-ODN And Polypeptides Killing HepG2

Objective: Via CpG-ODN and polypeptides independently or aliiedly stimulate peripheral blood mononuclear cells, linked incebation of peripheral blood mononuclear cells activated and HepG2, peripheral blood mononuclear cells killing HepG2, evaluate PBMC stimulated by CpG-ODNs and polypeptides killing HepG2 ,study the activating function of CpG-ODNs and mechanism; investigate the mechanism of CpG-ODN and polypeptides independently or aliiedly activating peripheral blood mononuclear cells, study the apoptosis of HepG2 killed peripheral blood mononuclear cells activated and HepG2, estimate the effect of PBMC killing HepG2.Method: The plamids of CpG-ODN were transformed into E.coli DH5α, the LB plate was spraided with E.coli DH5αand cultivanted at 37℃. Pick several colony from the freshly plate to a little doze LB medium cultivate the colony at 37℃with vigorous shaking. The plamids were extracted from the bacterial culture by lye clearage method and identified with enzyme shearing and agarose electrophoresis. A large scale culture was preperated when the plamids were identified correctly. The plamids of CpG-ODN were extracted from the bacterial culture by Endo-Free Plamid Maxi Kit. And the content and purity of plamids were identified with ultraviolet spectrophotometry. Design and synthesize the polypeptides which are corresponding to the epitope of alphafetoprotein, the purity of polypeptides is fit to the experiments of cells. PBMC were stimulated by polypeptides, CpG-ODN, HBsAg and plamid pUC18, phytohemagglutinin as control. Detectct the proliferation of PBMC with MTT, analyses and picture in excel. Synthesize the primers of MyD88,TLR9 and Hsp70 in polymerase chain reaction,β-actin as control. Stimulate the PBMC with CpG-ODN in different levels, extract the total RNA of PBMC, have first strand cDNA synthesis and polymerase chain reaction, detect in agarose electrophoresis. CpG-ODN and polypeptides independently or aliiedly stimulate peripheral blood mononuclear cells, PBMC killed HepG2, Detectct the survival of HepG2 with MTT, analyses and picture in excel. extract the total RNA of PBMC, have first strand cDNA synthesis and polymerase chain reaction with the primers of Bcl-XL/S,ich3,Bid and Bcl-2,β-actin as control, detect in agarose electrophoresis.Results: PBMC were proliferating with the stimulation of CpG-ODN. The proliferation was stable, while CpG-ODN as adjuvant, polypeptide stimulated the PBMC more strongly, HBsAg was the most. CpG-ODN stimulated the PBMC in different levels, at the concentration of 1μg/mL, mRNA of system of TLR9 was more activated than at the concentration of 0.1μg/mL and 10μg/mL. There was Bcl-2 at the concentration of 1μg/mL, not Hsp70 at any concentration. At 48 hours, PBMC stimulated by CpG-ODN killed HepG2 strongly, CpG was the most. It was more at 48 hours than at 72 hours. The survival of HepG were higher at at 72 hours than at 48 hours in CpG,polypeptides,CpG uniting polypeptide and control. Bcl-X and Bid were detected at CpG uniting polypeptide,Bid at CpG independent in PCR.Conclusion: PBMC stimulated by CpG-ODN and polypeptide have a proliferation effectly. CpG-ODN stimulated subpopulation of PBMC generally, polypeptide stimulated subpopulation of PBMC specificly. The proliferation of CpG-ODN stimulating PBMC was stbale, it was synergism in proliferation with protein or polypeptides. At the pertinent concentration, CpG-ODN was antiapoptosis of PBMC. After CpG-ODN and polypeptides stimulating PBMC, PBMC killed HepG2 more strongly,the polypeptides bind the epi-position of leukomonocytes correlated with tumours, activated T cells, it was synergism PBMC stimulated by CpG-ODN and polypeptides killing HepG2.