Experiment Study Of Placental Immunoregulatory Factor Preparation And Its Activity On CIK

Objective:To prepare placental immunoregulatory factor(PF)and PF preliminary purified products;select the most effective active components from preliminary purified products.Method:①PF preparation:removed anadesma from fresh human placenta that had examined,then washouted it by sotonic Na chloride and homogenated it.Placed it in -20℃48h and freeze thawing three times,then dialyzed it by bag filter and collect dialysate.Sterile filtered it though a 0.2 micron filter,and subpackaged.Eventually,took of samples to Erythrocyte rosette forming cell test(ERFC).②PF preliminary purified products preparation:to purify PF by NS balanced Sephadex G-25 gel filtration column;to detect products activity by erythrocyte rosette forming cell test(ERFC).Result:The Ea%of experimental group(contain PF)was much higher than control group(NS group)(p＜0.05),especially when PF concentration was 1/10～1/100.By Sephadex G-25 gel column chromatography purification,the PF purified products were four components.The most notable activity component was the peak four component.Conclusion:During this experiments,we had prepared PF and preliminarily purified PF.By used of erythrocyte rosette forming cell test,we had found out the most notable activity PF preliminary purified product:peak four component. Objective:The purpose was to investigate the effect of placental immunoregulatory factor(PF)on proliferation and cytoxicity of cytokine-induced killer(CIK),and the effect on LO2 cells and K562 cells.Method:Peripheral blood mononuclear cells(PBMNCs)were from healthy human donors.After being induced to be cytokine induced-killer(CIK),they were divided into two groups:experimental group(add PF)and control group(add NS).Then observed the proliferation and cytotoxicity of the two groups.Cultured K562 cells and the cell line L02 in 96 orifices plates respectively,then added different concentration PF,observed the growth of K562 cells and LO2 after 24hours.Result:①The proliferation of cells in experimental group was obviously different from control group(p＜0.05).The cytotoxicity of experimental group was much higher than control group.When PF concentration was 1:16,CIK had most notablely proliferation.When PF concentration was 1:32,it up-regulated cytotoxicity of CIK most notablely.②PF had a function to inhibit the growth of K562;PF could regulate the growth of LO2 at the same time.Conclusion:CIK of experimental group was more notable proliferation and cytotoxicity than control group.It is proved that PF has an immunomodulation effect on CIK.PF has a function to inhibit the growth of K562;PF can regulate the growth of LO2 at the same time. Objective:In order to investigate the immunoregulatory activity of placental immunoregulatory factor(PF),we had observed the ultrastructure and immunophenotype of cytokine-induced killer cells which were cultured by the culture medium containing PF.Method:①Peripheral blood mononuclear cells(PBMNCs)from healthy donors were induced to be cytokine induced killer(CIK).Then divided them into two groups:one was control group cultured by traditional method,another was experimental group cultured by traditional method which medium contains PF.Finally observed ultrastructure and immunophenotype of CIK.②Prepared electron microscope samples and observed ultramicrostructure of CIK.Used flow cytometry to analyze percentages of CD4+,CD8~+,CD3~+CD4~+,CD3~+ CD8~+,CD25~+,HLA-DR,CD3~+CD56~+,CD80~+ of CIK.Result:Comparing Experimental group with control group,the ultrastructure had some changes,such as the density of mitochondrias increased,lysosomes proliferated,heterochromatin increased,and microvillus grew longer.Percentage of CD4~+CD8~+ and CD3~+CD4~+ was down-regulated,and percentage of CD3~+CD8~+ and CD25~+ was up-regulated(P＜0.05)Conclosion:PF have an effect on cytokine-induced killer cells that can change ultramicrostructure and immunophenotype of CIK.For this reason,we extrap that PF have certain undiscovered immunoregulatory activity on on development process of CIK. Objective:SELDI-TOF-MS technology was used to detect the changes of protein expression in cytokine-induced killer cells(CIK)that the culture medium contains placental immunoregulatory factor(PF).Metho:Peripheral blood mononuclear cells(PBMNCs)were from healthy human donors.After being induced to be cytokine induced-killer(CIK),they were divided into tow groups:the control group was cultured by using NS,and the experimental group was cultured by using a 10%FBS culture media that contains placental immunoregulatory factor(PF).The proteomic spectra of CIK were obtained by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS)on WCX-2 chips.Then compared the different proteins between control group and experimental group.Result:When compare experimental group with control group,SELDI showed that a total of 6 protein was significantly different(P＜0.05),specifically the molecular weight of 5636.077,6860.359,6072.814 and 5984.191.Conclosions:PF have a function that can change proteins expression of CIK. And SELDI technology can find out the different proteins in CIK effectively.