The Role Of Protoscolices And Hydatid Fluid In Immune Escape Of Mouse Spleen Cells In Vitro

Obejective：This study was designed Protoscolices/Hydatid fluid and mouse spleen cells wereco-cultured in vitro,Observation the changes of the system of TGF-β secretion, CD4~+CD25~+Foxp3~+T(Treg cell) content, CD4~+/CD8~+T cells ratio and the expression level of NKG2D (NK cellsactivated receptor, recognized of the MHC-I class molecules) on the NK cell,and the NK cell killingactivity.Investigate whether the protoscolices/hydatid fluid through the promotion of spleen cellssecrete inhibitory molecule TGF-β,so as to promote the differentiation of CD4~+CD25~+Foxp3~+Tcells,further down the activity of NK receptor NKG2D expression level, ultimately leading to NKcells to killing protoscolex weakened, and the formation of host immune escape.Methods:1. After72h co-culture of spleen cells from BALB/C mouse and Hydatid fluid/Protoscolices withdifferent concentrations；Level of TGF-β in the co-culture supernatant were measured by ELISA,Collection of co-cultured cells as effector cells, Yac-1as target cells co-cultured detected by LDHenzymatic activity of NK cells, and NK cell surface receptor NKG2D expression detected by flowcytometry.2. Spleen cells from BALB/c mouse,co-culture with different concentration Hydatid fluid as the testgroup,PBS as control group. Percentage of T-lymphocyte subsets and CD4~+CD25~+T detected byFCM after co-culture72h,total RNA was extracted from co-culture cells in each group, RT-PCR wasused to detected gene expression of Foxp33. Another experiment FCM detected the Percentage of T-lymphocyte subsets of spleen cells at0、12、24、36、48h.Results:1. Compared with the control group, the experimental group TGF-β (the Hydatid fluid group andProtoscolices group) secretion were increased the cyst fluid group increased with the increase in theconcentration of antigen, which in high concentrations when compared with the control group,thereare statistically significant (p <0.01); Protoscolices Group reaches a larger value at1000/mlconcentration, and there are statistically significant (p <0.01)compared with control group, resultsshow the role of the cyst fluid antigen is stronger than Protoscolices.2. Hydatid fluid and Protoscolices are able to down-regulate the NK cell activity, with increased theconcentration of antigen and activity decreased more obvious. NK cell surface receptor NKG2D,mainly in the cyst fluid group, at the time of the high-concentration group decreased little effect inlow concentrations, the Protoscolices group of NKG2D.3.The ratio of CD4~+/CD8~+T cells from cyst fluid and protoscoleces groups were smaller than controlgroup detected by flow cytometry. 4. Compared with the control group, the low concentration of cyst fluid (100ul,200ul) andprotoscolices (500cells/ml) have high level of CD4~+CD25~+T cell (p <0.05).But with increasedthe antigens, CD4~+CD25~+T cells ratio is gradually decreased;The amount of specific proteinexpression of Foxp3in cyst fluid group (100ul,200ul) and protoscolices group (500cells/ml,1000cells/ml) have a statistically significant comprared with control group (p <0.05).Conclusion：1. Hydatid fluid/Protoscoliecs can stimulate the spleen cells of mice increased secretion of TGF-β invitro, suggesting that the Protoscoliecs and cyst fluid contain some antigen substances can induce thehost to produce TGF-β, and thus play a certain role in echinococcosis immune evasion.2. Hydatid fluid/Protoscoliecs antigen can reduce immune cytotoxic activity of NK cells, suggestingthat participation in the host’s natural immune function in the earlyregulation, and the role of cystfluid antigen is stronger than the Protoscoliecs; cyst fluid can down-regulate the expression of theNK cell surface receptor NKG2D,can be down the activity of NK cells, and at the same time caninhibition of NK cell surface receptor NKG2D expression,thereby reducing the host’s naturalimmunity.3. Echinococcus granulosus Hydatid fluid/Protoscolices antigens can promote the decline of CD4~+T cells, and makes the increase in the proportion of CD8~+T cells, CD4~+/CD8~+ratio decreased;,theexperimental results suggest that there may be conducive hydatid evade the host immunedestruction.4. Hydatid fluid/Protoscoliecs antigens can promote with immunosuppressive effects of CD4~+CD25~+T cells of proportion and its protein Foxp3increased，indicated that the stimulation of T cells in thecyst fluid and the Protoscoliecs Treg cell differentiation trend increase of CD4~+CD25~+Tcells;through the secretion of TGF-β inhibit the immune response of the host to survive in the host,the experimental results suggest that may be beneficial to evade the host immune killer.