猪链球菌2型对红细胞的黏附及溶血作用

Adhension and hemolysis of Streptococcus suis type 2 to red blood cells

为探讨猪链球菌2型(Streptococcus suis type 2,SS2)感染过程中对血液红细胞(RBC)损害及其机理,本研究采取无特定病原微生物感染的猪血液制备RBC悬液,根据预试验选用SS2∶RBC感染比(MOI)为100的SS2,分别体外感染RBC 0.5、1、1.5、2 h,光镜下观察BRC的形态变化;用G iem sa染色、CF D A-SE荧光标记检测SS2对红细胞的黏附和损伤,流式细胞仪分析不同作用时间的黏附率,real-time PCR检测SS2相关黏附因子溶菌酶释放蛋白(MRP)、荚膜多糖(CPS)、甘油醛-3-磷酸脱氢酶(GAPDH)、纤连蛋白和纤维蛋白结合蛋白(FBPS)及溶血因子溶血素(Sly)的转录水平。结果显示,SS2感染后第0.5~1小时,其主要黏附RBC,细胞呈现棘突样病变,少量细胞肿胀,出现轻微溶血,黏附率分别为18.7%和46.3%;感染后第1.5小时,SS2不仅可以黏附RBC,且多数细胞溶血,RBC开始发生破裂,黏附率可达到91.7%;感染后第2小时,细胞几乎全部破裂。MRP和CPS2J转录水平在感染后第0.5~1小时增高,并与黏附率呈正相关,感染后第2小时表达水平下降;Sly转录水平在感染后第0.5~2小时持续增加,与溶血现象呈正相关;GAPDH和FBPS在感染过程中变化不显著(P>0.05)。结果表明,SS2感染早期可对RBC产生黏附和溶血作用,先为黏附作用,后为溶血作用,且作用过程较快;主要参与黏附因子为MRP和CPS2J,溶血因子为Sly;GAPDH和FBPS是否参与SS2对RBC的黏附与溶血仍需证实;本试验结果为研究SS2传播和致病机制提供了重要的理论依据。

In order to find out the mechanisms of Streptococcus suis type 2（SS2） to red blood cells（RBC）,RBCs isolated from blood of SPF pigs aged 6-week-old,were infected by SS2 in vitro at the MOI（ multiplicity of infection） of 1 ∶ 100 for 0.5, 1, 1.5 and 2 h, respectively.The morphocytological changes of RBC were observed under light-microscopy. The adhension and damage of SS2 to RBCs were detected by Giemsa and CFDA-SE staining.The adhesiveness rate at different time was calculated by flow cytometry（FCM）.Real-time PCR was used to detect the genes related to adhension and hemolysis,such as muramidase released protein（MRP）,capsular polysaccharide（CPS）,glyceraldehyde-phosphate dehydrogenase（GAPDH）,fibrinogen binding protein（FBPS）,and suilysin（Sly）.The results showed that SS2 could adhere to RBCs,0.5-1 h post-infection,and more SS2 caused changes of RBCs such as spina,swelling and,hemolysis.The adhesiveness rate was 18.7% and 46.3%,respectively,and SS2 adhered to most of RBCs at1.5 h.Hemolysis of RBCs was increased.A small number of cells were broken by SS2,and adhesiveness rate was 91.7%.Most of RBCs were broken at 2 h,and the rest of the cells were not enough for FCM detection.The levels of MRP and CPS2 J m RNA enhanced when SS2 infected RBCs for 0.5-1 h,indicating a positive correlation with adhesiveness rate,while the levels of MRP and CPS2 J m RNA reduced at 2 h.The levels of Sly m RNA increased after SS2 infected RBCs for 0.5-2 h,indicating a positive correlation with the levels of hemolysis.However,the changes of GAPDH and FBPS m RNA were not significance during SS2 infected RBCs（P〈0.05）.The results indicated that the damage of SS2 to RBCs included adhension and hemolysis.The major damage at early stage of SS2 infected RBCs was adhension,and MRP and CPS2 J played key roles in SS2 adhered RBCs;while hemolysis as major damage at the later stage of SS2 infected RBCs,and Sly played key roles in hemolysis of RBC caused by SS2.The roles of GAPDH and FBPS need further studies.These results provide an important reference information for studies the mechanisms of SS2 infections and transmission.