摘要
目的探讨肝细胞局部补体对疟原虫红外期发育的影响。方法分别提取Hepa1-6和HepG2-CD81细胞RNA,逆转PCR扩增C3、C3aR1、C5、C5aR1基因。Hepa1-6细胞和HepG2-CD81细胞悬液分别加入蛋白酶和磷酸酶裂解,2,2-联喹啉-4,4-二甲酸二钠(BCA)法测定蛋白浓度,用蛋白质免疫印迹(Western blotting)测定肝细胞内补体和受体表达情况。将表达红色荧光蛋白(RFP)的约氏疟原虫BY265-RFP株、伯氏疟原虫ANKA株的昆明小鼠供斯氏按蚊吸血,17~19 d后解剖分离斯氏按蚊唾液腺,收集子孢子,在24孔板中按50000个子孢子/孔加入HepG2-CD81细胞(105个/孔)孵育6、12、24 h;另设置共孵育3 h后加入C5aR拮抗剂(40 nnmol/L,每孔500μl)的组别。经4%多聚甲醛固定、封闭,非透膜组加入一抗C3a兔抗人IgG抗体(1∶500)或rC5a兔抗人IgG抗体(1∶500)4℃孵育过夜,加入绿色Dylight 488荧光标记的山羊抗兔IgG二抗(1∶400)孵育1 h,加入DAPI染色液孵育5 min;透膜组加入一抗UIS4山羊多克隆抗体(1∶500)4℃孵育过夜,加入绿色IFKineTM荧光标记的驴抗兔IgG二抗(1∶400)孵育1 h,加入CD88兔多克隆抗体(1∶400)4℃孵育过夜,加入红色Dylight 649荧光标记的山羊抗兔IgG二抗(1∶400)孵育1 h,加入DAPI染色液孵育5 min,激光共聚焦显微镜观察补体在纳虫空泡周围的富集情况。取眼镜蛇毒因子(CVF)腹腔注射至C57BL/6小鼠,为CVF组;并设置C3-/-组(C3全基因敲除C3-/-小鼠)和对照组(C57BL/6小鼠)。取10000个约氏疟原虫子孢子感染各组小鼠,取肝脏提取总RNA后逆转录合成cDNA,荧光定量PCR测定疟原虫18S rRNA含量,肝脏虫荷用18S rRNA的相对含量表示。以尾静脉注射方式,用200个约氏疟原虫子孢子感染对照组(C57BL/6小鼠)10只、C3aR^(-/-)小鼠10只;1000个约氏疟原虫子孢子感染对照组(C57BL/6小鼠)5只、C5aR全基因敲除C5aR^(-/-)小鼠6只和肝脏C5aR条件性敲除Alb-cre+/+C5aRflox/flox杂交小鼠5只;1000个伯氏疟原虫子孢子感染对照
Objective To investigate the effect of hepatocyte local complement on the development of the liverstage of Plasmodium parasites.Methods Hepa1-6 cells and HepG2-CD81 cells were collected,lysed,and RNA was extracted,respectively.Reverse transcription PCR amplified C3,C3aR1,C5,C5aR1 gene.Hepa1-6 cells and HepG2-CD81 cells suspension were lysed with protease and phosphatase,respectively.The protein concentration was measured using the bicinchoninic acid(BCA)assay and the expression of complement and receptor in hepatocytes was measured using Western blotting.Kunming mice fed with the BY265-RFP strain of P.yoelii expressing red fluorescence and the ANKA strain of P.berghei were fed with Anopheles stephensi for blood sucking.After 17-19 days,anatomically separated salivary glands from An.stephensi mosquitoes and added 50000 sporozites into 24-well plate containing 105 HepG2-CD81 cells per well for co-incubation,set an additional group that the cells were incubated with C5aR antagonist(40 nnmol/L,500μl/well)3 h after co-incubation.Cells were fixed with 4%paraformaldehyde and blocked,the non permeable group was incubated overnight at 4℃with primary C3a rabbit anti human IgG antibody(1∶500)or rC5a rabbit anti human IgG antibody(1∶500),followed by 1 hour of incubation with green Dylight 488 fluorescent labeled goat anti rabbit IgG secondary antibody(1∶400),and 5 minutes of incubation with DAPI staining solution;added a primary antibody UIS4 goat polyclonal antibody(1∶500)to the permeable group and incubated overnight at 4℃,then added green IFKineTM fluorescent labeled donkey anti rabbit IgG secondary antibody(1∶400)and incubated for 1 h,added CD88 rabbit polyclonal antibody(1∶400)to incubate overnight at 4℃,added the red Dylight 649 fluorescent labeled goat anti rabbit IgG secondary antibody(1∶400)to incubate for 1 h,added DAPI staining solution to incubate for 5 min,and observed the enrichment of complement around parasitophorous vacuoles under laser confocal microscopy.Take cobra venom factor(CVF)and
作者
谭涅
焦世铭
丁艳
朱成宇
徐文岳
TAN Nie;JIAO Shiming;DING Yan;ZHU Chengyu;XU Wenyue(Department of Pathogenic Biology,Army Medical University,Chongqing 400038,China;School of Medicine,Chongqing University,Chongqing 400044,China)
出处
《中国寄生虫学与寄生虫病杂志》
CSCD
北大核心
2024年第2期169-176,共8页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(81830067,81802033)。
关键词
疟原虫
红外期
肝细胞局部补体
Plasmodium
Liver stage
hepatocyte local complement
