癌胚抗原相关黏附分子1对CVB3感染后CAR表达及心肌损伤的影响
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摘要
目的探讨癌胚抗原相关黏附分子1(CEACAM1或CC1)对柯萨奇病毒(CVB3)感染后柯萨奇病毒-腺病毒受体(CAR)表达和继发心肌损伤的影响。方法构建过表达小鼠CC1重组病毒,包装重组慢病毒pLVX-CEACAM 1-ZsGreen-Puro(rLV-CEACAM 1)并测定慢病毒生物学滴度。分为CC1正常组、CC1过表达组、CC1正常+CVB3组和CC1过表达+CVB3组;各组使用AnnxinV-PE/7-AAD双染法检测心肌细胞凋亡率,CCK8检测细胞活性;qPCR检测CAR基因表达;Western blot检测CAR蛋白表达,ELISA检测肿瘤坏死因子α(TNF-α)及白细胞介素1β(IL-1β)水平。结果 (1)CEACAM1重组载体测序提示目的基因序列连接,证明小鼠CEACAM1重组病毒载体构建成功,测定重组慢病毒滴度为1.5×10~(11) TU/L。(2)CC1过表达+CVB3组较其他组心肌细胞凋亡率明显升高,心肌细胞增殖最低(P<0.05)。(3)CAR基因相对表达量在CC1过表达+CVB3组最高,而在CC1正常组最低,CAR蛋白表达也有类似结果;CC1过表达组较CC1正常组胞内CVB3相对表达量显著升高(P<0.05)。(4)CC1过表达组TNF-α、IL-1β水平较高,CVB3感染后较前明显升高。结论 CC1可能可以促进CVB3感染心肌细胞后心肌组织或细胞上CAR的表达,CAR可能是CC1调控CVB3感染心肌致心肌损伤过程的潜在作用靶点。
Aim To investigate the effect of carcinoembryonic antigen-related cell adhesion molecule 1(CC1) on the expression of coxsackie and adenovirus receptor(CAR) and secondary myocardial injury after coxsackievirus B3(CVB3) infection. Methods The overexpressed mouse CC1 recombinant virus was constructed, and the recombinant lentivirus pLVX-CEACAM 1-ZsGreen-Puro(rLV-CEACAM 1) was packaged and the biological titer of lentivirus was determined. It was divided into CC1 normal cell group, CC1 overexpression group, CC1 normal +CVB3 group and CC1 overexpression+CVB3 group. The apoptosis rate of cardiomyocytes was detected by AnnxinV-PE/7-AAD double staining in each group, and cell activity was detected by CCK8. The expression of CAR gene was detected by qPCR. The expression of CAR protein was detected by Western blot, tumor necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β) were measured by ELISA. Results(1)Recombinant vector sequencing CEACAM1 showed a gene sequence connection, which proved mice CEACAM1 recombinant virus vector was built, determination of recombinant lentivirus was 1.5×10~(11) TU/L.(2)Apoptosis rate of cardiac myocytes in CC1 overexpression+CVB3 group was significantly higher than that in other groups, and the proliferation rate of cardiac myocytes was the lowest(P<0.05).(3)Relative expression of CAR gene was the highest in CC1 overexpression+CVB3 group, and the lowest in CC1 normal group. Relative expression of CVB3 was significantly higher in CC1 overexpression group than in CC1 normal group(P<0.05).(4)Level of TNF-α, IL-1β were higher in CC1 overexpression group, which increased significantly after CVB3 infection. Conclusion CC1 may promote the expression of CAR in cardiac tissue or cell after CVB3 infected cardiac myocytes. CAR might be a potential target for CC1 to regulate the process of cardiac injury caused by CVB3 infection.
Aim To investigate the effect of carcinoembryonic antigen-related cell adhesion molecule 1(CC1) on the expression of coxsackie and adenovirus receptor(CAR) and secondary myocardial injury after coxsackievirus B3(CVB3) infection. Methods The overexpressed mouse CC1 recombinant virus was constructed, and the recombinant lentivirus pLVX-CEACAM 1-ZsGreen-Puro(rLV-CEACAM 1) was packaged and the biological titer of lentivirus was determined. It was divided into CC1 normal cell group, CC1 overexpression group, CC1 normal +CVB3 group and CC1 overexpression+CVB3 group. The apoptosis rate of cardiomyocytes was detected by AnnxinV-PE/7-AAD double staining in each group, and cell activity was detected by CCK8. The expression of CAR gene was detected by qPCR. The expression of CAR protein was detected by Western blot, tumor necrosis factor alpha(TNF-α), interleukin 1 beta(IL-1β) were measured by ELISA. Results(1)Recombinant vector sequencing CEACAM1 showed a gene sequence connection, which proved mice CEACAM1 recombinant virus vector was built, determination of recombinant lentivirus was 1.5×10~(11) TU/L.(2)Apoptosis rate of cardiac myocytes in CC1 overexpression+CVB3 group was significantly higher than that in other groups, and the proliferation rate of cardiac myocytes was the lowest(P<0.05).(3)Relative expression of CAR gene was the highest in CC1 overexpression+CVB3 group, and the lowest in CC1 normal group. Relative expression of CVB3 was significantly higher in CC1 overexpression group than in CC1 normal group(P<0.05).(4)Level of TNF-α, IL-1β were higher in CC1 overexpression group, which increased significantly after CVB3 infection. Conclusion CC1 may promote the expression of CAR in cardiac tissue or cell after CVB3 infected cardiac myocytes. CAR might be a potential target for CC1 to regulate the process of cardiac injury caused by CVB3 infection.
引文
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[15] 方珊娟,吴学思,王春梅,等.神经调节蛋白-1对大鼠心肌缺血/再灌注损伤保护作用的研究[J].心肺血管病杂志,2016,35(5):396-400.
[16] 阮妙华,王凯,王丹,等.病毒性心肌炎小鼠心肌中MMP-2和NF-κB的表达[J].中国病理生理杂志,2016,32(9):1704-1707,1712.
[17] Massilamany C,Gangaplara A,Reddy J.Intricacies of cardiac damage in coxsackievirus B3 infection:implications for therapy[J].Int J Cardiol,2014,177(2):330-339.
[18] He X,Gao B,Zhou L,et al.Green tea polyphenol epigallocatechin-3-gallate-alleviated coxsackievirus B3-induced myocarditis through inhibiting viral replication but not through inhibiting inflammatory responses[J].J Cardiovasc Pharmacol,2017,69(1):41-47.
[19] Weber DA,Sumagin R,McCall IC,et al.Neutrophil-derived JAML inhibits repair of intestinal epithelial injury during acute inflammation[J].Mucosal Immunol,2014,7(5):1221-1232.
[20] Klaile E,Klassert TE,Scheffrahn I,et al.Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae,Moraxella catarrhalis,TLR3,and type I and II interferons[J].Respir Res,2013,14:85.
[21] Mao CS,Yin H,Ning HB,et al.Levels of HBx,VEGF,and CEACAM1 in HBV-related hepatocellular carcinoma and their correlation with cancer prognosis[J].Eur Rev Med Pharmacol Sci,2017,21(17):3827-3833.
[22] Khairnar V,Duhan V,Maney SK,et al.CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production[J].Nat Commun,2015,6:6217.
[23] Hosomi S,Grootjans J,Huang YH,et al.New insights into the regulation of natural-killer group 2 member D (NKG2D) and NKG2D-ligands:endoplasmic reticulum stress and CEA-related cell adhesion molecule 1[J].Front Immunol,2018,9:1324.
[24] Yuen S,Smith J,Caruso L,et al.The coxsackie-adenovirus receptor induces an inflammatory cardiomyopathy independent of viral infection[J].J Mol Cell Cardiol,2011,50(5):826-840.
[2] 王丽丽,梁斌,边云飞,等.心肌纤维化和肥厚型心肌病研究进展[J].中国动脉硬化杂志,2014,22(6):624-628.
[3] Yu M,Long Q,Li HH,et al.IL-9 inhibits viral replication in coxsackievirus B3-induced myocarditis[J].Front Immunol,2016,7:409.
[4] Pinkert S,Roger C,Kurreck J,et al.The coxsackievirus and adenovirus receptor:glycosylation and the extracellular D2 domain are not required for coxsackievirus B3 infection[J].J Virol,2016,90(12):5601-5610.
[5] Khairnar V,Duhan V,Patil AM,et al.CEACAM1 promotes CD8(+) T cell responses and improves control of a chronic viral infection[J].Nat Commun,2018,9(1):2561.
[6] Wechalekar H,Setchell BP,Peirce EJ,et al.Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice[J].Asian J Androl,2010,12(4):591-598.
[7] Ma L,Liu H,Xie Z,et al.Ginsenoside Rb3 protects cardiomyocytes against ischemia-reperfusion injury via the inhibition of JNK-mediated NF-kappaB pathway:a mouse cardiomyocyte model[J].PloS One,2014,9(8):e103628.
[8] Migaud M,Roques BP,Durieux C.Evidence for a high-affinity uptake system for cholecystokinin octapeptide (CCK8) in rat cortical synaptosomes[J].Eur J Neurosci,1995,7(5):1074-1079.
[9] Sapan CV,Lundblad RL,Price NC.Colorimetric protein assay techniques[J].Biotechnol Appl Biochem,1999,29 (Pt 2):99-108.
[10] 史晓娟,王品品,谭鑫.小儿病毒性心肌炎诊断中血清高敏C反应蛋白、肌酸激酶同工酶以及心肌肌钙蛋白Ⅰ联合检测的价值[J].心肺血管病杂志,2018,37(3):218-220,224.
[11] 陈小龙,王立峰,王璐,等.大蒜素对病毒性心肌炎小鼠的治疗作用[J].中国病理生理杂志,2016,32(6):1027-1030.
[12] Zhang Y,Sun L,Sun H,et al.Overexpression of microRNA-133b reduces myocardial injuries in children with viral myocarditis by targeting Rab27B gene[J].Cell Mol Biol (Noisy-le-grand),2017,63(10):80-86.
[13] Flynn CT,Kimura T,Frimpong-Boateng K,et al.Immunological and pathological consequences of coxsackievirus RNA persistence in the heart[J].Virology,2017,512:104-112.
[14] 李忠,张培华.小檗碱通过Toll样受体4/核因子κB信号通路对小鼠病毒性心肌炎发挥保护作用[J].中国动脉硬化杂志,2017,25(3):250-253.
[15] 方珊娟,吴学思,王春梅,等.神经调节蛋白-1对大鼠心肌缺血/再灌注损伤保护作用的研究[J].心肺血管病杂志,2016,35(5):396-400.
[16] 阮妙华,王凯,王丹,等.病毒性心肌炎小鼠心肌中MMP-2和NF-κB的表达[J].中国病理生理杂志,2016,32(9):1704-1707,1712.
[17] Massilamany C,Gangaplara A,Reddy J.Intricacies of cardiac damage in coxsackievirus B3 infection:implications for therapy[J].Int J Cardiol,2014,177(2):330-339.
[18] He X,Gao B,Zhou L,et al.Green tea polyphenol epigallocatechin-3-gallate-alleviated coxsackievirus B3-induced myocarditis through inhibiting viral replication but not through inhibiting inflammatory responses[J].J Cardiovasc Pharmacol,2017,69(1):41-47.
[19] Weber DA,Sumagin R,McCall IC,et al.Neutrophil-derived JAML inhibits repair of intestinal epithelial injury during acute inflammation[J].Mucosal Immunol,2014,7(5):1221-1232.
[20] Klaile E,Klassert TE,Scheffrahn I,et al.Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae,Moraxella catarrhalis,TLR3,and type I and II interferons[J].Respir Res,2013,14:85.
[21] Mao CS,Yin H,Ning HB,et al.Levels of HBx,VEGF,and CEACAM1 in HBV-related hepatocellular carcinoma and their correlation with cancer prognosis[J].Eur Rev Med Pharmacol Sci,2017,21(17):3827-3833.
[22] Khairnar V,Duhan V,Maney SK,et al.CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production[J].Nat Commun,2015,6:6217.
[23] Hosomi S,Grootjans J,Huang YH,et al.New insights into the regulation of natural-killer group 2 member D (NKG2D) and NKG2D-ligands:endoplasmic reticulum stress and CEA-related cell adhesion molecule 1[J].Front Immunol,2018,9:1324.
[24] Yuen S,Smith J,Caruso L,et al.The coxsackie-adenovirus receptor induces an inflammatory cardiomyopathy independent of viral infection[J].J Mol Cell Cardiol,2011,50(5):826-840.