SjGPR89蛋白对日本血吸虫生长发育的影响

The effect of SjGPR89 protein on the growth and development of Schistosoma japonicum

目的初步探索日本血吸虫G蛋白偶联受体89(SjGPR89)在日本血吸虫生长发育中的作用。方法利用SMART网站和TMHMM网站预测其SjGPR89蛋白的蛋白结构和跨膜结构。取40只SPF级6~8周龄雌性昆明小鼠经腹部贴片法感染日本血吸虫尾蚴(200±10)条/鼠,分别于感染后第14、16、18、20、22、24、26、28、30天解剖小鼠,收集虫体,提取虫体RNA并逆转录为cDNA,采用qRT-PCR检测SjGPR89 mRNA在血吸虫中的相对转录水平。取12只SPF级6~8周龄雌性昆明小鼠,经腹部贴片法感染日本血吸虫尾蚴(60±2)条/鼠,随机平均分为SjGPR89干扰组(干扰组)和对照组,干扰组于感染后第1、6、10、14、18、22、26天经尾静脉注射SjGPR89双链RNA(dsRNA)10μg/鼠,对照组注射等量绿荧光蛋白(GFP)dsRNA。小鼠感染后第30天收集虫体,取雌、雄虫各5条,提取虫体RNA,逆转录为cDNA,采用qRT-PCR检测虫体SjGPR89 mRNA相对转录水平;用多聚甲醛乙酸乙醇(AFA)固定分开的雌、雄虫后,采用Image J软件测量虫长,并统计虫荷;采用卡红染色对雌、雄虫的性腺进行染色,荧光显微镜下观察生殖腺发育状态的变化。另取12只SPF级6~8周龄雌性昆明小鼠,经腹部贴片法感染尾蚴(40±2)条/鼠,随机平均分为干扰组和对照组,干扰组于小鼠感染后第26、30、34、38天经尾静脉注射10μg dsRNA/鼠,于第42天解剖小鼠,取肝组织,qRT-PCR检测肝组织中胶原Ⅰ、胶原Ⅲ、α-平滑肌肌动蛋白(α-SMA)mRNA的相对转录水平;肝组织经5%NaOH消化后,计数每克肝组织虫卵数;制备肝切片,Masson染色后镜下观察肝纤维化程度。结果TMHMM网站预测结果显示,SjGPR89为一种较保守的9次跨膜的G蛋白偶联受体;SMART网站预测结果显示,SjGPR89蛋白由4个跨膜区域、1个高尔基pH调节受体(GPHR)结构域、1个低复杂度区域和一个脱落酸(ABA)G蛋白偶联受体(GPCR)结构域组成。qRT-PCR检测结果显示,小鼠体内不同发育时期(14~30 d)日本血吸虫的SjGPR89 m RNA相对转录水平较平稳,仅雌虫在感染小鼠后的第26~28天有大幅上升。感染后第30天,qRT-PCR检测结果显示,干扰组雌、雄血吸虫中SjGPR89的mRNA相对转录水平分别为307.70±58.21、97.88±11.38,较对照组的767.10±142.79、182.02±7.42(t=5.96、12.39,均P<0.01)敲低了59.89%和46.23%;干扰组雌、雄虫的虫长分别为(12.13±2.67)、(10.00±1.72)mm,较对照组的(13.67±1.74)、(11.48±1.94)mm(t=4.10、5.09,均P<0.01)减短了11.22%和12.93%;干扰组雌、雄虫的虫荷数分别为(23.00±1.83)、(23.75±2.99)条,较对照组的(26.75±0.96)、(31.00±3.56)条(t=3.64、3.12,均P<0.05)减少了14.02%和23.39%。卡红染色结果显示,干扰组雌虫的性腺发育出现延滞状态,卵巢和卵黄腺着色较浅,且卵巢的面积远小于对照组;雄虫的性腺发育不充分,形态大小较对照组小。干扰组的每克肝组织虫卵数为(2777.33±197.94)个,较对照组的(5871.32±875.25)个(t=5.97,P<0.01)减少了52.70%。感染后第42天,Masson染色观察结果显示,干扰组小鼠肝胶原纤维面积小于对照组;qRT-PCR检测结果显示,干扰组小鼠肝组织中的胶原Ⅰ、胶原Ⅲ、α-SMA mRNA相对转录水平分别为530.20±246.81、825.26±139.82、551.59±189.94,均低于对照组(t=3.81、2.50、4.72,均P<0.05)。结论干扰SjGPR89蛋白可抑制日本血吸虫的生长发育、生存能力和雌虫产卵,减轻对宿主肝脏的病理损伤。

Objective To preliminarily explore the role of G protein coupled receptors(Sj GPR89)in the growth and development of Schistosoma japonicum.Methods The protein structure and transmembrane structure of Sj GPR89 were predicted using the SMART website and TMHMM website.Forty femal Kunming mice at 6-8 week old of specific pathogen free(SPF)type were infected with S.japonicum cercariae 200±10 per mouse through abdominal skin,and the schistosomes were collected on the 14,16,18,20,22,24,26,28,and 30 days post-infection(dpi),respectively.The worm RNA was extracted and reverse transcribed into cDNA,and the relative expression level of SjGPR89 mRNA was determined by qRT-PCR.Twelve SPF Kunming mice were infected with(60±2)cercariae each,and then randomly assigned into SjGPR89 double-stranded RNA(dsRNA)group(interference group)and a green fluorescent protein(GFP)control group at even.To each of the interference group mice,10μg SjGPR89 dsRNA was injected through the tail vein on 1,6,10,14,18,22,and 26 dpi.On day 30,the worm RNA was extracted from 5 male and 5 female worms and reverse transcribed into cDNA,and the relative expression level of SjGPR89 mRNA was determined by qRT-PCR.The males and females separately manually were fixed using acetate formalin alcohol(AFA)to measure the worm body length using Image J software and to count the worm burden;the male and femal were stained with carmine to observe the developmental changes of gonads with fluorescence microscope.Additional 12 femal SPF Kunming mice of 6-8 week old were infected with 40±2cercariae each mouse,and then randomly assigned evenly into the interference group and control group;to each of the interference group mice,10μg dsRNA was injected through the tail vein,on the 2630,34 and 38 dpi,and on day 42 for collection of liver tissue to determine the relative transcription levels of collagenⅠ,collagenⅢ,andα-smooth muscle actin(α-SMA)mRNA by qRT-PCR.The liver tissues were digested with 5%NaOH to count the eggs and calculate the egg number/g tissue;liver sections were prepared and stained with Masson to observe the status of fibrosis.Results The TMHMM website predicted that SjGPR89 is a conserved nine-times transmembrane G protein-coupled receptor,and the SMART website predicted that SjGPR89 protein has four transmembrane regions,one GPHR_structure domain,one low complexity region,and one abscisic acid GPCR domain.SjGPR89 transcription in female and male S.japonicum at different developmental stages(14-30 days)in mice were found at a relatively stable level detected by qRT-PCR;continuous interference with tail vein injection of SjGPR89-dsRNA showed that the relative m RNA expression of SjGPR89 in interferece females(307.70±58.21)and males and(97.88±11.38)were significantly lower than the GFP control group(767.10±142.79)and(182.02±7.42)(t=5.96,12.39;P<0.01),with knockdown levels of 59.89%and 46.23%,respectively.Image J measurement showed that the lengths of the female(12.13±2.67)and male(10.00±1.72)in the interference group were significantly shorter than that of the control group(13.67±1.74),(11.48±1.94)(t=4.10,5.09;P<0.01),the female and male parasites were 11.22%and 12.93%shorter,respectively.The number of parasite loads of the female and male parasites in the interference group(23.00±1.83),(23.75±2.99)was less than that of the control group(26.75±0.96),(31.00±3.56)(t=3.64,3.12;P<0.05)significantly,and the degree of reduction of male and female parasite loads were 14.02%and23.39%.The results of the carmine staining re vealed that the gonads of the females in the interference group were delayed in development,the coloring of the ovaries and vitelline glands was lighter,and the area of the ovaries was significantly less than that of the control group;the male gonads were immature and smaller than those of the control group.The number of eggs per gram liver tissue in the interference group(2777.33±197.94)was significantly less than that in the control group(5871.32±875.25)(t=5.97,P<0.01),with a reduction of52.70%,and the degree of liver fibrosis in the interference group was further observed to be significantly less than that in control group using Masson staining.qRT-PCR detected the relative expressions of CollagenⅠ,CollagenⅢ,andα-SMA in the liver tissues of the interference group(530.20±246.81),(825.26±139.82),(551.59±189.94)were significantly lower than those of the control group(t=3.81,2.50,4.72;P<0.05).Conclusion S.japonicum development,viability,and egg production were impacted by interference with the SjGPR89 protein.Additionally,the host’s liver is far less likely to sustain pathological damages.