PCV2感染通过胞内Ca^(2+)超载诱导仔猪腹股沟淋巴结及脾脏淋巴细胞凋亡

The apoptosis of lymphocytes in lymph nodes and spleens of piglets induced by PCV2 via up-regulated of cytosolic Ca^(2+)

为探讨猪圆环病毒Ⅱ型(PCV2)感染仔猪的淋巴结和脾脏淋巴细胞内钙信号变化在细胞凋亡中的作用,选取19头PCV2抗原和抗体均为阴性的5周龄健康断奶仔猪,随机分成对照组(4头)和试验组(15头),试验组仔猪通过滴鼻接种PCV2,并于接种后14、21和35 d分别扑杀5头,对照组仔猪滴鼻同量PBS后当天扑杀。所有仔猪扑杀时均取腹股沟淋巴结及脾脏,流式细胞术检测细胞凋亡率,荧光分光光度法检测细胞内Ca2+浓度,孔雀绿比色测定法检测Ca2+-ATP酶的活性,荧光定量RT-PCR检测钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)mRNA转录水平。结果表明:所有接种病毒仔猪腹股沟淋巴结及脾脏细胞凋亡率均极显著高于对照组(P<0.01),Ca2+浓度均极显著高于对照组(P<0.01);接种病毒仔猪腹股沟淋巴结Ca2+-ATP酶活性在21 d时显著下降(P<0.05),脾脏中Ca2+-ATP酶活性在21 d和35 d时显著下降(P<0.05);腹股沟淋巴结CaMKⅡmRNA转录在整个试验过程中无明显变化,但脾脏中CaMKⅡmRNA在PCV2接种后21 d和35 d比对照组显著上调(P<0.05)。结论:细胞内Ca2+超载是PCV2感染引起仔猪淋巴结及脾脏淋巴细胞凋亡的重要机制。

To investigate the relationship between cytosolic Ca2＋ and the apoptosis in lymphocytes at the condition of porcine circovirus type 2（PCV2）,nineteen piglets without antibody to PCV2 and PCV2 nucleic acid were allotted into a control group of four piglets,and three experimental groups of five piglets per group.The control group was sacrificed immediately after inoculation with PBS,and the experimental groups were sacrificed at 14,21 or 35 d with PCV2.Collected inguinal lymph nodes and spleens,the apoptotic rates of lymphocytes were detected with flow cytometry;the cytosolic Ca2＋ concentrations were evaluated with spectrofluorometry;activities of Ca2＋-ATPase were determined by malachite green colorimetric assay and the transcriptions of CaMK ⅡmRNA were tested by real-time RT-PCR.Results：1）the apoptotic rates of spleen and inguinal lymph nodes in three experimental groups were significantly higher than in the control group（P〈0.01）;2）the concentration of cytosolic Ca2＋ were significantly up-regulated in three experiment groups（P〈0.01）in spleen and inguinal lymph nodes;3）in inguinal lymph nodes,the activities of Ca2＋-ATPase at 21 d were lower than those in the control group（P〈0.05）and in spleens both 21 and 35 day post-infected（DPI） were lower than those in the control group（P〈0.05）;4）the relative levels of CaMK ⅡmRNA were significantly up-regulated at 35 DPI（P〈0.05）in spleen but no statistical significant difference observed in inguinal lymph nodes（P〉0.05）.Conclusion：the up-regulated of cytosolic Ca2＋ was a very important intermedium when PCV2 induced the apoptosis of lymphocytes in inguinal lymph nodes and spleens.