The Preliminary Study Of Immunological Functions Of Schistosoma Japonicum Calreticulin Protein

Studies have shown that radiation attenuated schistosome vaccine(RAV) induces high protective immune effect, the mechanism may be that the molecules of radiation attenuated schistosome stimulate the host immune cells in the lungs forming a Th1 proinflammatory environment. Thus, the discovery and identification of these molecules,clarifying the protective immune biological functions, are of great importance to the research and development of efficient and safe vaccine against Schistosoma japonicum. This paper aims to study the role and function SjCRT protein plays in innate immune response and the adaptive immune response.1,The expression of Schistosoma japonicum calreticulin(SjCRT) in both normal and irradiated Schistosoma at different periods. The schistosomula-derived cells were acquired by digesting the RA and normal schistosomula with trypsin in different stage of schistosomula. The results revealed that, compared with the normal schistosomula-derived cells,the expression level of SjCRT was significouldtly increased in RA schistosomula-derived cells at 4d by flow cytometry(FCM)(P<0.05), but it significouldtly decreased at 7d, 10 dand 14d(P<0.05). The expression of SjCRT in different stages of RA and normal schistosomula was detected by RT-PCR, we found that the mRNA expression of SjCRT inRA schistosomula at 4 d was significouldtly decreased compared with normal schistosomula(P<0.01), while it significouldtly increased at 7 d, but at 10 d there was no significouldt difference between the two groups. So it preliminary suggests that SjCRT was higher expressed and released in the early RA schistosomula.2, Exploring the function of SjCRT to macrophages of host. After the RAW264.7 cells were stimulated with SjCRT for 72 h. Detectcell proliferation by MTT, detect the secretion of NO by Griess assay, the expression of macrophages cytokines and chemokines receptor in transcription level were deceted by RT-PCR, the extracellular cytokines were detected by ELISA and the expression of surface molecules on macrophage RAW264.7was detected by FCM. The results show that SjCRT could promote macrophages’ proliferation; SjCRT could also improve the NO secretion of macrophages significouldtly(P<0.05); SjCRT promotes macrophages to increase the mRNA expression of COX-2, cPLA2, CCR7, TNF-α and IL-1β; ELISA result shows that SjCRT could be extremely significouldt to increasethe secretion of TNF-α(P<0.01); the FAC result indicates that the stimulation of SjCRT could cause the increasing CCR7 expression of macrophages significouldtly(P<0.05). It suggests that SjCRT can activate mouse macrophagocyte.3, Exploring the mechanism of how SjCRT activates dendritic cells(DC). Confocal laser and FCM were used to detect the bingding of SjCRT to DC, the mRNA expression of TNF-α, IL- 1β, IL-6, MCP-1, CXCL-2, CCR7 and CXCR4 were detected by RT-PCR, the secretion of TNF-α, IFN-γ and IL-4 were detected by ELISA, FCM was used to detect the expression of CCR7 and CXCR4 on the surface of DC, the proliferation of CD4+T cellswas detected by CFSE and ELISA was used to detect the secretion of extracellulacytokines. The results show that SjCRT could bind to the surface of DC, and it could bind to CD91. What’s more, with the help of CD91, SjCRT could be endocytosed by DC; RT-PCR results show that SjCRT could promote DC to significouldtly up-regulate the mRNA expression of TNF-α, IL-1β, IL-6, MCP-1, CXCL-2, CCR7 and CXCR4(P<0.05); ELISA detection shows that the stimulation of SjCRT could promote DC significouldtly to express and secret TNF-α, IFN-γ and IL-4(P<0.01); FCM results show SjCRT could causethe high expression of the surface chemokine receptor CCR7 and CXCR4 significouldtly(P<0.01); CFSE results indicate that DC after stimulated by SjCRT could cause the prolifcation of CD4+T cells significouldtly(P<0.01), the percentage of the proliferated cells were 47.7% and 44.1%; ELISA results showed that DCstimulated by SjCRT for 48 h could promote T cells to secrete TNF-α, IFN-γ, IL-4 and IL-10, and compared with the control group the difference was significouldtly(P<0.01). So it preliminary suggests that SjCRTcould activate DC and improve the functional mature of DC.4, Mixed lymphocyte reaction of SjCRT immunized mice.The sensitized lymphocyte had been stimulated with SjCRT for 48 h. MTT method was used to assay the proliferation of sensitized lymphocyte. The subgroup of sensitized lymphocyte and the cytokines secreted by sensitized lymphocyte were detected by FCM and ELISA. The results showed that SjCRT could induce the sensitized lymphocyte’s proliferation(SI>2); The value of ratio of CD4+/CD8+T from group immunized with SjCRT was 2.26, significantly higher than that of control group(P<0.01) which was 1.76. The expression of IFN-γ, TNF-α and IL-4 were also increased significouldtly(P<0.01). These results suggest that SjCRT could elicit proliferation of sensitized lymphocyte and enhance polarization of CD4+ T cells to Th1 phenotype.In conclusion, in this study we found that SjCRT was highly expressed in early RA schistosomula and can be release to the extracellula.SjCRT can activate macrophages,by binding to CD91 SjCRT can be endocytosed by DC and improve the functional mature of DC. It can also activate CD4+T cell and induceTh1 cellular immune response. This study will provide a basis for the special polarization of CD4+T cell and further clarifying the immunobiology functions and mechanisms of SjCRT in radiation attenuated schistosome vaccine.