PCV2对体外培养仔猪淋巴细胞凋亡的影响

Apoptosis of lymphocytes in piglets after porcine circovirus 2 inoculation in vitro

选用4头猪圆环病毒2型(porcine circovirus 2,PCV2)血清抗体阴性的普通断奶仔猪,无菌取脾脏,分离淋巴细胞,分为对照组和试验组进行体外培养。试验组在培养体系中加入一定量的PCV2。分别在培养后的0、2、4、6、12、24、36和48 h收获淋巴细胞。用电子显微镜、DNA琼脂糖凝胶电泳对细胞凋亡进行定性测定,用流式细胞术对细胞周期和细胞凋亡率进行定量测定,同时测定了凋亡相关蛋白半胱天冬酶3(Caspase-3)的表达情况。结果显示,PCV2作用后不同时间,DNA电泳均呈现出具细胞凋亡特征的梯形条带;电镜下出现细胞凋亡各期的形态学变化。从培养后4 h开始,试验组淋巴细胞凋亡率均显著高于对照组(P<0.05);而表示细胞增殖活性的增殖指数(PI),试验组在2、4、6、12和24 h均比对照组显著下降(P<0.05);表达Caspase-3的细胞百分率,除0 h外,试验组均显著高于对照组(P<0.05)。上述结果表明,PCV2能引起体外培养的仔猪淋巴细胞凋亡率增加,细胞增殖活性下降,且Caspase-3是PCV2诱导淋巴细胞凋亡的重要调节物。

Four conventional piglets with free of porcine circovirus 2 （PCV2） antibody were used. Lymphocytes isolated from spleen with pinhead of injectors were distributed into two groups： control and PCV2 inoculation. Cell apoptosis was qualitatively analyzed by agarose gel electrophoresis DNA and electronic microscope at 0, 2, 4, 6, 12, 24, 36 and 48 hours, and cell cycle, apoptotic rate and expression of Caspase-3 were measured with flow cytometry （ FCM ） at same time. In PCV2 group, the special DNA ladder of apoptosis was detected by agarose gel electrophoresis DNA, and the morphological changes of apoptosis at different phase were observed under electronic microscope. The rate of apoptosis in PCV2 group was significantly higher than that of the control group after 4 hours cell culture （ P 〈 0. 05 ）. The proliferation index （PI） was greatly decreased in the PCV2 group at 2, 4, 6, 12 and 24 hours, compared with the control group （P 〈0. 05）, indicating that PCV2 may restrain lymphocytes proliferation in vitro at the beginning of infection. The percentage of expression of Caspase-3, which plays a key role in the progress of apoptosis, prominently increased in PCV2 group during the experiment, but at 0 hour, compared with the control group （P 〈 0. 05 ）. The results demonstrate that PCV2 infection could induce lymphocyte apoptosis and inhibit proliferation of lymphocytes in vitro, and the Caspase-3 is an important conditioner in lymphocyte apoptosis induced by PCV2 infection.