The Effects And Preliminary Study Of PCV2 On Ca²⁺ Signal In Lymphocytes Of Piglets

Porcine circovirus disease (PCVD) was mainly caused by Porcine circovirus type 2 (PCV2),which brought enormous loss to pig industry in whole world. because of immunosuppression. In the 20th international pig's disease conference in 2008, PCVD was classified as the NO.1 disease to endanger pig industry's development at present. Typical pathohistological findings of PCVD were lymphocyte depletion in lymphoid tissues and peripheral blood together with mononuclear cells infiltration, which also the basic reason of immunosuppression induced by PCV2. Some researches showed that lymphocyte depletion is caused by apoptosis. Under previous studies in our lab, the function of Ca2+ signal in the process of lymphocytes apoptosis and the mechanism of increase of Ca2+ concentration induced by PC V2 in vitro were stuied in this paper.The influence of PCV2 on lymphocytes apoptosis and Calcium signal in vitro. The spleen lymphocytes were collected from five 35-old piglets which were detected no PCV2 infection by ELISA method. The spleem lymphocytes were divided into two groups (control and group treated with PCV2). The lymphocytes were collected at Oh, 6h,12h and 24h after treated with PCV2. Lymphocytes apoptosis was detected by Annexin V-FITC/PI double staining method, the changes of the intracellar free calcium concentration ([Ca2+]i) were detected by fluometry probed with fluorescent indicator fura-2/AM, and the expressions of CaM and CaMKII were detected by Western-blot Results:The apoptosis rate of lympjocytes in PCV2 group is higher than that of control group, and significant differenceafter treated with PCV2 for 12h (P<0.05). [Ca2+]i increased with the increase of incubation time, and [Ca2+]i of PCV2 group were obviously higher than that of control group after incubating with 12h and 24h,respectively (P<0.05). The expression of CaM increased with incubation time, and significant difference in 12h(P<0.05).The were no significant changes of the expression of CaMKII after incubation with different times. Conclusion:PCV2 can induce piglets lymphocyte apoptosis, and it may be associated with calcium signaling. Mechanism of Calcium signal induced by PCV2 in lymphocytes of piglets in vitro. The homeostasis of intracellar Calcium depends on endoplasmic reticulum. There are three receptors associated with uptake and release of Ca+, RyR receptor is responsible for the release of Ca2+ to kytoplasm,1,4,5-IP3R and Ca2+ -ATPase for transport of Ca2+ to endoplasmic reticulum. The study is to investigate the expression changes of IP3R, RyR, Ca2+ -ATPase2 and Ca2+ -ATPase4 mRNA after lymphocytes infected with PCV2. lymphocytes were separated into two groups, control and PCV2 group, and were collected at 0 h,6 h,12 h,24 h after incubated with PCV2. Then the expression of IP3R-2, RYR-1, Ca2+ -ATPase2 and Ca2+ -ATPase4 mRNA was detected by realtime-PCR. The results showed that the expression of IP3R-2 mRNA elevated at 12 h, and PCV2 group significant higher than the control group (P<0.05). There is no significant change in the transcription of RyRmRNA between control and PCV2 group (P>0.05). The transcription of Ca2+ -ATPase2 mRNA elevated at 6 h and 12 h, and PCV2 group significant higher than the control group (P<0.05). Ca2+ -ATPase4 mRNA elevated at 6 h and 12 h, and the control group significant higher than PCV2 group (P<0.05).Conclusion:PCV2 induced lymphocyte [Ca2+]i increase may be related with the up-regulation transcription of IP3R mRNA and down-regulatio transcription of Ca2+ -ATPase4.