重组日本血吸虫虫卵核糖核酸酶SjCP1412体外抑制肝星状细胞LX-2活化作用的研究  

Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro

作　　者：李其凤 宋丽君 杨莹莹[2] 董盼盼[2] 梅丛进 李溢鑫 张键锋 熊春蓉[2] 余传信[2] 杨坤[1,2] LI Qi-feng;SONG Li-jun;YANG Ying-ying;DONG Pan-pan;MEI Cong-jin;LI Yi-xin;ZHANG Jian-feng;XIONG Chun-rong;YU Chuan-xin;YANG Kun(School of Public Health,Nanjing Medical University,Nanjing,Jiangsu 211166,China;National Health Commission Key Laboratory of Parasitic Disease Control and Prevention,Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology,Jiangsu Institute of Parasitic Diseases,Wuxi,Jiangsu 214064,China)

机构地区：[1]南京医科大学公共卫生学院,江苏南京211166 [2]江苏省血吸虫病防治研究所、国家卫生健康委员会寄生虫病预防与控制技术重点实验室、江苏省寄生虫与媒介控制技术重点实验室,江苏无锡214064

出　　处：《中国血吸虫病防治杂志》2022年第6期566-579,共14页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(82173586,81802035);江苏省无锡市科技局太湖之光科技攻关(医疗卫生技术攻关)项目(Y20222028);江苏省大型科学仪器开放共享自主研究课题(TC2021A033);江苏省无锡市卫生健康委科研项目面上项目(M202202)。

摘　　要：目的观察重组日本血吸虫虫卵核糖核酸酶SjCP1412(r SjCP1412)对体外培养的人肝星状细胞LX-2增殖、细胞周期、凋亡及活化的作用,并探索其潜在机制。方法在大肠埃希菌BL21中采用原核表达方式表达rSjCP1412蛋白,并通过Ni-NTA亲和层析法及尿素梯度复性透析制备高纯度可溶性rSjCP1412蛋白。使用12.5、25.0、50.0μg rSjCP1412蛋白分别于37℃酶解酵母RNA 2、3、4 h,酶解产物采用1.5%琼脂糖凝胶电泳分析,观察rSjCP1412蛋白RNA酶活性。采用CCK-8法检测经不同浓度rSjCP1412蛋白刺激48 h后LX-2细胞增殖,采用Annexin V-FITC/PI细胞凋亡双染法检测经不同浓度rSjCP1412蛋白刺激48 h后LX-2细胞凋亡,采用DAPI染色法检测经不同浓度rSjCP1412蛋白刺激48 h后LX-2细胞周期中G0/G1、S、G2/M期细胞百分比,采用荧光实时定量PCR(qPCR)检测经不同浓度rSjCP1412蛋白刺激48 h后LX-2细胞中Ⅰ型胶原、Ⅲ型胶原和α-平滑肌肌动蛋白(α-SMA)mRNA表达水平;采用Western blotting法检测经rSjCP1412蛋白刺激LX-2细胞48 h后Ⅰ型胶原、α-SMA和Smad4/7蛋白表达水平,以明确LX-2细胞活化。以上实验均设可溶性虫卵抗原(SEA)阳性对照和正常组(不含rSjCP1412蛋白的PBS)。采用rSjCP1412蛋白单独刺激或与转化生长因子-β1(TGF-β1)共同刺激LX-2细胞48 h后,采用Western blotting检测LX-2细胞中Ⅰ型胶原、α-SMA和Smad4蛋白表达水平,分析r SjCP1412作用于LX-2细胞的信号通路。结果成功表达并制备了高纯度可溶性的rSjCP1412蛋白,且rSjCP1412蛋白具有RNA酶活性。与正常组相比,12.5、25.0、50.0μg/mL r SjCP1412和SEA处理48 h后,LX-2细胞存活率均显著下降(F=22.417、20.448,P均<0.05);12.5、25.0、50.0μg/mL r SjCP1412蛋白处理48 h后,LX-2细胞凋亡率显著增加(F=11.350,P<0.05);12.5、25.0、50.0μg/mL rSjCP1412蛋白处理48 h后,可导致LX-2细胞G0/G1期阻滞(F=20.710,P<0.05)。12.5、25.0、50.0μg/mL rSjCP1412蛋白处理可导致LX-2细胞中Ⅰ型胶�Objective To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412(rSjCP1412)on proliferation,cell cycle,apoptosis and activation of human hepatic stellate cells LX-2 in vitro,and explore the underlying mechanisms.Methods The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression,and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis.Yeast RNA was digested using 12.5,25.0,50.0μg rSjCP1412 proteins at 37℃ for 2,3,4 h,and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of r SjCP1412 protein.The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay,and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining,while the percentage of LX-2 cells at G0/G1,S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining.The type Ⅰ collagen,type Ⅲ collagen and α-smooth muscle actin(α-SMA)mRNA expression was quantified using quantitative florescent real-time PCR(qPCR)assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of r SjCP1412 protein for 48 h,while soluble egg antigen(SEA)served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments.The expression of collagen Ⅰ,α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein,transforming growth factor-β1(TGF-β1)alone or in combination,to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells.Results The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared,which had a RNase activity.Compared with the no

关 键 词：日本血吸虫 核糖核酸酶 SjCP1412 肝星状细胞 Smad4信号通路 

分 类 号：R383.24[医药卫生—医学寄生虫学]