摘要
目的探讨白纹伊蚊唾液特异性34k2重组蛋白(rAlb-34k2)刺激(小鼠腹腔巨噬细胞)RAW264.7及C57BL/6小鼠后补体相关成分mRNA的变化。方法分别用不同浓度rAlb-34k2蛋白刺激RAW264.7细胞和C57BL/6小鼠足垫,采集样本,Trizol提取RNA,逆转录为cDNA后经实时荧光定量PCR方法检测刺激后不同时间各组标本中补体C3、C1q、CfB、CRIg的mRNA变化。结果rAlb-34k2诱导CfB、C3、C1q的mRNA表达上调,且皮肤组织上调幅度高于RAW264.7细胞,其中以CfB mRNA的上调幅度及持续更为显著,在诱导24h时各组(5μg、20μg)2-△△CT值分别是23.90±8.38、8.50±1.64(P<0.05);但刺激12-24h时高剂量组与低剂量组比较上调幅度差异无统计学意义(P>0.05)。在RAW264.7细胞,以刺激6h各种补体固有分成CfB、C3、C1q表达上调,但随时间延长均恢复至正常水平,各剂量组间差异不明显。在局部皮肤组织,rAlb-34k2刺激24h后CRIg表达上调,且呈浓度和时间依赖趋势;48h时,各组rAlb-34k2(5μg、20μg)2-△△CT值达到高峰,分别为8.67±2.44和32.98±9.74,有统计学差异(P<0.05)。在Raw264.7细胞,刺激6h时CRIg有上调表达趋势,但随时间延长而下调,各剂量组间比较差异无统计学意义(P>0.05)。结论白纹伊蚊唾液rAlb-34k2蛋白参与调控巨噬细胞及皮肤补体重要成分的表达变化,在组织中尤为显著,且显著上调定居组织的巨噬细胞特异性膜受体CRIg。
Objective To investigate changes in mRNA in some main complement components and CRIg in RAW264.7 and C57 BL/6 mice stimulated with Aedes albopictus saliva-specific 34 k2 recombinant protein(rAlb-34 k2).MethodsRAW264.7 cells and C57 BL/6 mouse footpads were treated with rAlb-34 k2 protein at different concentrations.Total RNA was collected at different times using the Trizol method and transcribed into cDNA using a reverse transcription kit.The mRNA of the complements C3,C1 q,CfB,and CRIg of different groups was evaluated with quantitative real-timePCR at different times after stimulation with rAlb-34 k2 protein.Results The levels of expression of CfB,C3,and C1 q in C57 BL/6 mouse skin tissues in vivo were up-regulated more markedly than levels in Raw264.7 cells in vitro.The2-△△CTfor CfB mRNA in local mouse skin increased most significantly,up to 23.90±8.38 in the low-dose group and 8.50±1.64 in the high-dose group at 24 h(P<0.05).Up-regulation of these main complement components by rAlb-34 k2 protein did not differ significantly between group receiving different doses(P>0.05).After stimulation with different doses of rAlb-34 k2 protein,the mRNA of these complement intrinsic fractions in Raw264.7 cells immediately increased at6 hand then quickly returned to normal levels at 24 h.The CRIg expression in local skin tissues was up-regulated in a concentration-and time-dependent manner.The 2-△△CTvalues peaked at 8.67±2.44 and 32.98±9.74,respectively(P<0.05),at 48 h,while the mRNA of CRIg in Raw264.7 cells transiently increased only at 6 hbut did not differ significant-ly between the groups(P>0.05).Conclusion rAlb-34 k2 protein,which is encoded by agene derived from the female Ae.albopictus saliva,acts to regulate the expression of complement components in local skin tissue.rAlb-34 k2 protein might influence macrophage function via the specific membrane molecule CRIg.
作者
金芮
田炎珅
朱亚妮
代薇露
卢涛
徐昊
朱俐兰
朱家洪
吴家红
商正玲
JIN Rui;TIAN Yan-shen;ZHU Ya-ni;DAI Wei-lu;LU Tao;XU Hao;ZHU Li-lan;ZHUJia-hong;WU Jia-hong;SHANG Zheng-ling(Department of Immunology,Basic Medical College,Guizhou Medical University,Guiyang,China 550025;Department of Human Parasitology,Basic Medical College,Guizhou Medical University;Characteristic and Key Laboratory of Modern Pathogen Biology,Guizhou Medical University;Microbiology and Biochemical Pharmacy Engineering Center,Guizhou Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2020年第5期551-555,579,共6页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.81960295)。
