刚地弓形虫ROP16蛋白对MH-S细胞极化和凋亡的影响及其相关机制

doi:10.12140/j.issn.1000-7423.2022.05.003

摘要/Abstract

摘要：

目的探究刚地弓形虫棒状体蛋白16（ROP16）在小鼠肺泡巨噬细胞（MH-S细胞）中的表达及其对细胞极化和凋亡的影响和相关机制。方法利用携带绿色荧光标签的ROP16过表达慢病毒转染MH-S细胞,构建稳定表达ROP16的ROP16-MH-S细胞株（过表达组）,同时设空载体慢病毒转染对照组（空载体组）和无转染对照组（对照组）。转染72 h后免疫荧光法检测ROP16-MH-S中ROP16的表达及其在细胞中的定位。以甘油醛-3-磷酸脱氢酶（GAPDH）基因的转录水平为内参照,RT-qPCR检测ROP16-MH-S细胞中促炎（M1）因子白细胞介素-1β（IL-1β）、肿瘤坏死因子（TNF-α）、IL-18、IL-6、IL-12和抗炎（M2）因子IL-10、转化生长因子-β（TGF-β）,以及抑凋亡基因B细胞淋巴瘤/白血病-2（Bcl-2）和促凋亡基因Bcl-2相关X蛋白（Bax）、半胱氨酸蛋白酶3（Caspase 3）、Caspase 9等mRNA的相对转录水平;Western blotting检测ROP16-MH-S细胞中极化蛋白精氨酸酶1（Arg-1）、信号转导和转录激活因子3（STAT3）、705位点磷酸化（pTyr705）-STAT3、STAT6、pTyr641-STAT6和凋亡蛋白Caspase 9、Caspase 3、Bcl-2、Bax蛋白的相对表达水平;流式细胞术检测ROP16-MH-S细胞的凋亡情况。组间比较采用单因素方差分析。结果转染后72 h,空载体组和过表达组90%以上的细胞可见较强的绿色荧光,过表达组细胞中可见明显红色荧光聚集在ROP16-MH-S细胞核及其周围。RT-qPCR结果显示,过表达组ROP16 mRNA的相对转录水平为8 023.459 ± 39.325,高于空载体组（5.540 ± 0.001）（F = 83 188,P < 0.01）;Western blotting检测结果显示,过表达组ROP16蛋白的相对表达水平为16.349 ± 0.746,高于空载体组（1.291 ± 0.333）（F = 831.7,P < 0.01）。RT-qPCR结果显示,过表达组促炎（M1）因子IL-1β、TNF-α、IL-18、IL-6、IL-12 mRNA的相对转录水平分别为0.495 ± 0.002、0.337 ± 0.007、0.378 ± 0.014、0.474 ± 0.035和0.730 ± 0.021,均低于空载体组（0.994 ± 0.043、1.165 ± 0.034、0.943 ± 0.005、1.153 ± 0.028、0.926 ± 0.031）（F = 261.7、536.5、1 682.0、225.0、78.5,P < 0.01）;抗炎（M2）因子IL-10、TGF-β mRNA的相对转录水平分别为7.013 ± 0.032和1.608 ± 0.024,均高于空载体组（0.790 ± 0.031、1.091 ± 0.027）（F = 23 835.0、200.1,P < 0.01）。Western blotting检测结果显示,过表达组Arg-1、pTyr705-STAT3、pTyr641-STAT6蛋白相对表达水平分别为2.337 ± 0.089、3.471 ± 0.046、3.905 ± 0.045,均高于空载体组（0.871 ± 0.014、1.482 ± 0.071、1.514 ± 0.050）（F = 640.8、1 608.0、3 528.0,P < 0.01）。流式细胞术检测结果显示,过表达组细胞凋亡率为（2.990 ± 0.042）%,低于对照组（6.480 ± 0.071）%和空载体组（5.655 ± 0.290）%（F = 219.7,P < 0.01）。Western blotting检测结果显示,过表达组促凋亡蛋白Bax、Caspase 3、Caspase 9的相对表达水平分别为0.558 ± 0.005、0.640 ± 0.011、0.593 ± 0.026,Bax/Bcl-2为0.453 ± 0.011,均低于空载体组（0.991 ± 0.010、0.926 ± 0.006、0.963 ± 0.012、0.834 ± 0.008）（F = 2 850.0、1 200.0、359.3、2 337.0,P < 0.01）。RT-qPCR检测结果显示,过表达组促凋亡基因Bax、Caspase 3、Caspase 9 mRNA的相对转录水平分别为0.588 ± 0.086、0.563 ± 0.025和0.403 ± 0.014,Bax/Bcl-2为0.158 ± 0.008,均低于空载体组（0.924 ± 0.016、0.937 ± 0.041、0.807 ± 0.032、0.779 ± 0.014）（F = 24.7、78.6、154.9、265.5,P < 0.01）;过表达组抑凋亡基因Bcl-2 mRNA的相对转录水平为3.702 ± 0.362,高于空载体组（1.186 ± 0.006）（F = 104.1,P < 0.01）。结论 ROP16蛋白在ROP16-MH-S细胞中（主要位于细胞核及其周围）稳定表达,可激活STAT3、STAT6,磷酸化Tyr705-STAT3、Tyr641-STAT6,进而调控细胞向M2极化,抑制细胞凋亡。

关键词: 刚地弓形虫, 棒状体蛋白16, 小鼠肺泡巨噬细胞, 凋亡, 信号转导和转录激活因子3/6

Abstract:

Objective To investigate the expression of Toxoplasma gondii rhoptry protein 16 (ROP16) protein in mouse alveolar macrophages (MH-S) and its affect on cell polarization and apoptosis and the mechanisms involved. Methods MH-S cells transfected with ROP16 overexpression lentivirus labeled of green fluorescent were used to construct ROP16-MH-S cell line capable of stable expression of ROP16 (overexpression group). A blank vector lentivirus transfection control group (blank vector group) and no transfection control group (control group) were assigned in parallel. The expression of ROP16 in ROP16-MH-S and its localization within the cells were detected by immunofluorescence 72 h post-transfection. With the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal reference, and the mRNA relative transcription level of pro-inflammatory (M1) factor interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-18, IL-6 and IL-12; apoptosis-suppressing gene B-cell lymphoma/leukemia-2 (Bcl-2); and anti-inflammatory (M2) factors IL-10, transforming growth factor (TGF)-β, pro-apoptotic genes Bcl-2-associated X protein (Bax), cysteine protease 3 (Caspase 3), Caspase 9 in ROP16-MH-S cells was detected by RT-qPCR. The relative expression level of polarized protein arginase 1 (Arg-1), signal transducer and activator of transcription 3 (STAT3), phosphorylation at site 705 (pTyr705)-STAT3, STAT6, pTyr641-STAT6 and apoptosis proteins Caspase 9, Caspase 3, Bcl-2 and Bax in ROP16-MH-S cells were detected by Western-blotting. The apoptosis of ROP16-MH-S cells was detected by flow cytometry. One-way analysis of variance was used for comparison between groups. Results Strong green fluorescence was seen in the blank vector group and overexpression group at 72 h after transfection. Significant red fluorescence was also observed in the nucleus of ROP16-MH-S cells and its surrounding in the overexpression group. RT-qPCR results showed that the relative transcription level of ROP16 mRNA in the overexpression group was 8 023.459 ± 39.325 with green fluorescence, which was higher than that in the blank vector group (5.540 ± 0.001) (F = 83 188, P < 0.01); Western-blotting showed that the relative expression level of ROP16 protein in the overexpression group was 16.349 ± 0.746, which was higher than that in the blank vector group (1.291 ± 0.333) (F = 831.7, P < 0.01). RT-qPCR results showed that the relative transcription levels of pro-inflammatory (M1) factors IL-1β, TNF-α, IL-18, IL-6 and IL-12 in the overexpression group were 0.495 ± 0.002, 0.337 ± 0.007, and 0.378 ± 0.014, 0.474 ± 0.035, and 0.730 ± 0.021, respectively, which were lower than those in the blank vector group (0.994 ± 0.043, 1.165 ± 0.034, 0.943 ± 0.005, 1.153 ± 0.028, 0.926 ± 0.031) (F = 261.7, 536.5, 1 682.0, 225.0, 78.5; P < 0.01); the relative transcription levels of anti-inflammatory (M2) factors IL-10 and TGF-β mRNA were 7.013 ± 0.032 and 1.608 ± 0.024, respectively, which were significantly higher than those in the blank vector group (0.790 ± 0.031, 1.091 ± 0.027) (F = 23 835.0, 200.1, P < 0.01). Western-blotting revealed that that the relative expression levels of Arg-1, pTyr705-STAT3, and pTyr641-STAT6 proteins in the overexpression group were 2.337 ± 0.089, 3.471 ± 0.046, 3.905 ± 0.045, respectively, which were higher than those in the blank vector group (0.871 ± 0.014, 1.482 ± 0.071, 1.514 ± 0.050) (F = 640.8, 1 608.0, 3 528.0, P < 0.01). The flow cytometry results showed that the apoptosis rate in the overexpression group was (2.990 ± 0.042)%, which was lower than that in the control group (6.480 ± 0.071)% and the blank vector group (5.655 ± 0.290)%, respectively (F = 219.7, P < 0.01). Western-blotting showed that the relative expression levels of the pro-apoptotic proteins Bax, Caspase 3, Caspase 9 and Bax/Bcl-2 in the cells of the overexpression group were 0.558 ± 0.005, 0.640 ± 0.011, 0.593 ± 0.026 and 0.453 ± 0.011, respectively, which were lower than those in the blank vector group (0.991 ± 0.010, 0.926 ± 0.006, 0.963 ± 0.012, 0.834 ± 0.008) (F = 2 850.0, 1 200.0, 359.3, 2 337.0, P < 0.01). RT-qPCR showed that the relative transcription levels of pro-apoptotic genes Bax, Caspase 3, Caspase 9 and Bax/Bcl-2 mRNA in the overexpression group were 0.588 ± 0.086, 0.563 ± 0.025, 0.403 ± 0.014 and 0.158 ± 0.008, respectively, which were lower than those in the blank vector group (0.924 ± 0.016, 0.937 ± 0.041, 0.807 ± 0.032, 0.779 ± 0.014) (F = 24.7, 78.6, 154.9, 265.5, P < 0.01); while the relative transcription level of restraining-apoptosis gene Bcl-2 was 3.702 ± 0.362, which was higher than that in the blank vector group (1.186 ± 0.006) (F = 104.1, P < 0.01). Conclusion T. gondii ROP16 protein is stably expressed in ROP16-MH-S cells, mainly located in and around the nucleus,activating STAT3 and STAT6, phosphorylate Tyr705-STAT3 and Tyr641-STAT6, regulating ROP16-MH-S cells towards M2 polarization and thereby suppressing cell apoptosis.

Key words: Toxoplasma gondii, Rhoptry protein 16, Mouse alveolar macrophages cells, Apoptosis, Signal transducer and activator of transcription 3/6

中图分类号:

R382.5

李佳铭, 王艺璇, 杨宁爱, 马慧慧, 兰敏, 刘春兰, 赵志军. 刚地弓形虫ROP16蛋白对MH-S细胞极化和凋亡的影响及其相关机制[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 579-586.

LI Jia-ming, WANG Yi-xuan, YANG Ning-ai, MA Hui-hui, LAN Min, LIU Chun-lan, ZHAO Zhi-jun. Effects of ROP16 protein of Toxoplasma gondii on polarization and apoptosis of MH-S cells and their related mechanisms[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(5): 579-586.

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基因名称 Gene name	引物序列（5′→3′） Primer sequence（5′→3′）
GAPDH	F： GGTTGTCTCCTGCGACTTCA R： TGGTCCAGGGTTTCTTACTCC
Bax	F： TTGCCCTCTTCTACTTTGCTAG R： CCATGATGGTTCTGATCAGCTC
Bcl-2	F： GATGACTTCTCTCGTCGCTAC R： GAACTCAAAGAAGGCCACAATC
Caspase 3	F： AACAAAATGATTCTGTGAGCCC R： TTGACAAATGCTTTTCCCTGAG
Caspase 9	F： TGTGAATATCTTCAACGGGAGC R： GAGTAGGACACAAGGATGTCAC
IL-1β	F： TGGACCTTCCAGGATGAGGACA R： GTTCATCTCGGAGCCTGTAGTG
IL-6	F： CTTGCAAGACTTCCATCCAC R： AGTGGTAGACAGGTCTGTTGG
IL-10	F： ACCTGGTAGAAGTGATGCCCCAGGCA R： CTATGCAGTTGATGAAGATGTCAAA
IL-12	F： TTGAACTGGCGTTGGAAGCACG R： CCACCTGTGAGTTCTTCAAAGGC
IL-18	F： AGACCTGGAATCAGACAACTTT R： TCAGTCATATCCTCGAACACAG
TNF-α	F： CCTGTAGCCCACGTCGTAC R： GGGAGTAGACAAGGTACAACCC
TGF-β	F： ACGTCACTGGAGTTGTACGG R： GGGGCTGATCCCGTTGATT