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Activity identification of ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in cell-free system and in hepatic Stellate cells

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Science China(Life Sciences) 2006年第1期49卷 73-81页

作者： song yuhu LIU Fang TIAN Dean XUE Xiulan LIU Nanzhi WU Xiaoli LIN Jusheng JIN Youxin Institute of Liver Diseases Tongji HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhan 430030China Institute of Hematology Union Hospital Tongji medical CollegeHuazhong University of Science and TechnologyWuhan 430022China State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell BiologyShanghai Institutes for Biological SciencesChinese Academy of SciencesShanghai 200031China

Transforming growth factorβ1 (TGFβ1) is known to be intimately involved inmany cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimerichammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulateTGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme againstTGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions ofboth ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activityin vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozymeinhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. Soit indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the researchon the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidatefor TGFβ1-related diseases.

关键词： RNA catalytic U1 snRNA TGFβ1 HSC

Construction and expression of hepatitis B virus vector encoding TC-tagged core protein

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Frontiers of Medicine 2009年第4期3卷 396-402页

作者： Yuanyuan LIN Xiaoming CHENG yuhu song Li ZHOU Peiyuan LI Ying CHANG Leiming XU Jinjian YAO Jusheng LIN Institute of Liver Diseases Tongji HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhan 430030China Department of Neurobiology Tongji Medical CollegeHuazhong University of Science and TechnologyWuhan 430030China Department of Pathophysiology Tongji Medical CollegeHuazhong University of Science and TechnologyWuhan 430030China

Virus tagged with greenﬂuorescent protein(GFP)contributes to the visualization and study of the virus in living ***,the hepatitis B virus(HBV)particle,which is a compact virion with limited internal space,cannot be incorporated with GFP tag as a large *** was recently reported that protein genetically inserted with a smaller size tetracysteine(TC)tag could be specially labeled by a biarsenicalﬂuorescent dye in living *** this study,we constructed a recombinant HBV vector encoding TC-tagged core protein for biarsenical labeling of HBV *** tag was genetically inserted near the immunodominant c/e1 site of HBV core protein by *** blot and enzyme-linked immu-nosorbent assay(ELISA)analysis showed that the TC-tagged core protein,hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)could be expressed in cells transfected with the recombinant HBV vector,which is similar to the cells transfected with wild-type HBV *** transcription-polymerase chain reaction(RT-PCR)and Southern blot analysis showed that HBV virion formation was affected by the genetic insertion of TC tag into core protein in some degree,but cells transfected with the HBV vector could still produce HBV virions incorporated with TC-tagged core *** together,the recombinant HBV vector can serve as a useful tool to produce HBV virions incorporated with TC-tagged core proteins to beﬂuorescently labeled by biarsencial dye for visualizing and studying HBV in living cells.

关键词： hepatitis B virus vector tetracysteine tag core protein

Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines

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Frontiers of Medicine 2009年第2期3卷 148-152页

作者： Qiong CHEN Qing YU yuhu song Peiyuan Li Ying CHANG Zhijun WANG Lifeng LIU Wei WU Jusheng LIN Institute of Liver Diseases Tongji HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhan 430030China

To investigate the regulation of tumor sup-pressor XAF1 gene expression in digestive system cancers,we studied XAF1 gene promoter transcription activity and mRNA level in digestive system cancer cell lines(human hepatoma cell line HepG2,human colon cancer cell line LoVo,and human gastric cancer cell line AGS)and nontumor cell lines(human embryonic liver cell line L02(L02 cells)and human embryonic kidney 293 cells[HEK293 cells])as controls.1395-bp-promoter fragment of XAF1 gene was ampliﬁed by polymerase chain reaction(PCR)and cloned into pGL3-basic vector and pEGFP-1 vector to assay its promoter transcription *** plasmids were transfected into a variety of cell lines by lipofectamine *** promoter transcription activity was determined by dual-luciferase report assay,and enhanced greenﬂuorescent protein(EGFP)-positive cells were detected byﬂuorescence *** expression of XAF1 mRNA in HEK293 and L02 were signiﬁcantly higher than that in any of the three digestive system cancer cell *** dual-luciferase reporter assay showed that the promoter transcription activity in digestive system tumor cell lines transfected with pGL3-XAF1p promoter was apparently lower than that of both HEK293 and L02 *** of greenﬂuorescent protein(GFP)under the control of XAF1 promoter in the three digestive system cancer cell lines was lower than that of both HEK293 and L02 *** activities of pGL3-XAF1p in the three digestive system cancer cell lines after treatment with heat stress were signiﬁcantly lower than those in the unstressed *** results suggested that remarkably down-regulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.

关键词： gene X-linked inhibitor of apoptosis protein associated factor-1(XAF1) promoter transcription regulation

Retrorsine Cooperates with Gut Microbiota to Promote Hepatic Sinusoidal Obstruction Syndrome by Disrupting the Gut Barrier

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Journal of Clinical and Translational Hepatology 2022年第6期10卷 1086-1098页

作者： Li Xiao Lilin Hu Huikuan Chu Liuying Chen Jingjing Yan Weijun Wang Xiaoqian Yang Qingjing Zhu Fan Du yuhu song Peng Chen Xiaohua Hou Ling Yang Division of Gastroenterology Union HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanHubeiChina Department of Gastroenterology Henan Provincial People’s HospitalPeople’s Hospital of Zhengzhou UniversitySchool of Clinical MedicineHenan UniversityZhengzhouHenanChina Wuhan Jinyintan Hospital WuhanHubeiChina Department of Pathophysiology School of Basic Medical ScienceSouthern Medical UniversityGuangzhouGuangdongChina

Background and Aims:Hepatic sinusoidal obstruction syndrome(HSOS)is a life-threatening syndrome,and a cause is exposure to pyrrolizidine alkaloid(PA)-containing *** is well-established that retrorsine(RTS),a rep-resentative Pas,insults hepatic sinusoidal endothelial cells and ensues congestion of hepatic ***,little known about the impact of Pas on gut microbiota and intesti-nal barrier and inflammation in ***:Mice were gavaged with or without nonabsorbable antibiotics(ABX),followed by a single dose of *** gut microbiota was examined by 16S rDNA ***:ABX pretreat-ment significantly reversed RTS-induced liver *** altered gut microbiota composition,increasing Gram-nega-tive bacteria and resulting in a sharp elevation of circulating lipopolysaccharides(LPS)in HSOS *** decontamina-tion with ABX alleviated RTS-induced intestine inflamma-tion,protected against disruption of the intestinal epithelial barrier and gut vascular barrier(GVB),and suppressed he-patic LPS-NF-κB pathway activation in RTS-induced ***,the LPS level was positively correlated with MELD score in patients with *** LPS in patients with HSOS confirmed that Gram-negative bacteria were in-volved in the pathogenesis of ***:RTS,a PA,cooperated with gut dysbiosis to cause intestinal inflam-mation and gut barrier compromise that increased transport of gut-derived LPS into the liver through the portal vein,which contributed to the pathology of *** the gut microbiota,protecting the intestinal barrier,and sup-pressing intestinal inflammation with prebiotics or antibiot-ics might be a useful pharmacologic intervention in HSOS.

关键词： Retrorsine Pyrrolizidine alkaloids Hepatic sinusoidal obstruction syndrome Gut microbiota Drug-induced liver injury

继发于造血干细胞移植后的肝窦阻塞综合征1例

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中华肝脏病杂志 2020年第6期28卷 521-522,531页

作者：杨小倩叶进宋宇虎华中科技大学同济医学院附属协和医院消化内科武汉430022

报道1例造血干细胞移植后的肝窦阻塞综合征,国内此类病例报道较少,在诊断和治疗上具有参考意义。

关键词：造血干细胞移植肝窦阻塞综合征急性白血病

Fenofibrate in a patient with primary biliary cholangitis with an inadequate response to bezafibrate

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Portal Hypertension & Cirrhosis 2023年第4期2卷 192-193页

作者： Yangyang Zhou Haonan Gao yuhu song Department of Gastroenterology Union HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanHubeiChina

To the Editor,A 48-year-old male patient presented to our hospital with abnormal liver *** results of his liver function tests were as follows:alanine transaminase,118 U/L(normal range,5-35 U/L);aspartate transaminase,86 U/L(8-40 U/L);alkaline phosphatase(ALP),246 U/L(40-150 U/L);andγglutamyl transferase(γ-GT),792 U/L(17-53 U/L).His immunoglobulin(Ig)G and IgM levels were 13.0 g/L(7.51-15.60 g/L)and 2.44 g/L(0.460-3.040 g/L),respectively,and his autoimmune antibody tests were positive for antinuclear antibodies and M2 subtype antimitochondrial antibodies.

关键词： patient function liver

自身免疫性肝炎与原发性胆汁性胆管炎临床特征比较

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临床肝胆病杂志 2019年第2期35卷 354-358页

作者：舒艳芸潘晓莉宋宇虎叶进华中科技大学同济医学院附属协和医院消化内科武汉430022

目的比较自身免疫性肝炎(AIH)、原发性胆汁性胆管炎(PBC)临床特征,以利于两者的鉴别及早期诊断。方法收集2010年1月-2018年2月华中科技大学同济医学院附属协和医院诊治的AIH患者(n=83)和PBC患者(n=108)临床资料,包括年龄、性别、血清生... 详细信息

目的比较自身免疫性肝炎(AIH)、原发性胆汁性胆管炎(PBC)临床特征,以利于两者的鉴别及早期诊断。方法收集2010年1月-2018年2月华中科技大学同济医学院附属协和医院诊治的AIH患者(n=83)和PBC患者(n=108)临床资料,包括年龄、性别、血清生化、免疫学指标及肝脏病理学检查结果,比较AIH与PBC的不同特征。正态分布计量资料两组间比较采用独立样本t检验;偏态分布计量资料两组间比较采用Mann-Whitney U检验。计数资料两组间比较采用X^2检验或Fisher确切概率法。结果纳入AIH及PBC患者均以女性为主,发病年龄高峰为40～60岁。二者临床表现无明显特异性,主要症状体征均为:乏力、腹胀、黄疸、纳差,其中AIH组纳差患者比例明显高于PBC组(25%vs 14%,X^2=6. 52,P=0. 011)。此外,AIH组肝硬化及合并风湿性疾病的比例亦明显高于PBC组(59. 0%vs 40. 7%,X^2=6. 23,P=0. 012; 23. 5%vs 15. 7%,X^2=7. 46,P=0. 006)。AIH组PLT、ALP、GGT、IgM、红细胞沉降率(ESR)及补体C3水平均低于PBC组,PT、国际标准化比值(INR)及IgG水平均高于PBC组,差异均有统计学意义(P值均<0. 05)。PBC患者抗线粒体M2型抗体(78. 8%vs 14. 8%)、抗核膜糖蛋白210抗体(46. 4%vs 5. 3%)、抗三联体抗体(抗3E抗体)(75. 0%vs 31. 6%)阳性率均高于AIH患者,差异均有统计学意义(X^2值分别为74. 70、7. 31、8. 73,P值分别为<0. 001、0. 007、0. 003)。肝组织学特点方面PBC主要以胆管改变(包括小胆管炎、胆管肉芽肿、胆管缺失、细胆管反应性增生)为主,AIH则以界面性肝炎为主(X^2值分别为31. 00、5. 88,P值分别为<0. 001、0. 015)。结论 AIH与PBC均好发于女性,临床表现无特异性,但入院时PLT、ALP、GGT、PT、INR、ESR、补体、免疫球蛋白水平,抗体的类型及肝脏病理学的差异均可用于二者鉴别,且抗3E抗体可辅助诊断PBC。

关键词：肝炎,自身免疫性原发性胆汁性胆管炎疾病特征

肝硬化食管胃底静脉曲张2型与孤立性胃静脉曲张1型的临床和影像学差异

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中华消化内镜杂志 2022年第9期39卷 725-730页

作者：司可可向红雨王泽慧宋宇虎李欣华中科技大学同济医学院附属协和医院放射科湖北省分子影像重点实验室武汉430030 华中科技大学同济医学院附属协和医院消化内科武汉430022

目的探讨肝硬化合并食管胃底静脉曲张2型(gastroesophageal varices type 2,GOV2)与孤立性胃静脉曲张1型(isolated gastric varices type 1,IGV1)患者在临床特征、影像学表现的差异。方法回顾性纳入2013年10月—2021年3月华中科技大学... 详细信息

目的探讨肝硬化合并食管胃底静脉曲张2型(gastroesophageal varices type 2,GOV2)与孤立性胃静脉曲张1型(isolated gastric varices type 1,IGV1)患者在临床特征、影像学表现的差异。方法回顾性纳入2013年10月—2021年3月华中科技大学同济医学院附属协和医院收治的肝硬化合并胃底静脉曲张患者,收集并分析其临床及影像学资料。结果共纳入患者210例,其中139例GOV2型,71例IGV1型。血常规结果显示患者中位血红蛋白降低,其中GOV2组较IGV1组更显著(91.00 g/L比112.00 g/L,P<0.05),GOV2组较IGV1组肝硬化门静脉高压性胃病发生率高[20.14%(28/139)比5.63%(4/71),P<0.05];而消化性溃疡发生率低[12.23%(17/139)比38.03%(27/71),P<0.05]。GOV2组门静脉主干中位直径大于IGV1组(15.09 mm比12.85 mm,P<0.05),胃底曲张静脉中位体积显著小于IGV1组(2.14 mL比10.00 mL,P<0.05)。GOV2组流入血管胃左静脉的构成比例高于IGV1组[98.43%(125/127)比77.78%(42/54),P<0.05]且胃左静脉中位直径较IGV1组更大(5.58 mm比4.53 mm,P<0.05);流出血管主要包括胃肾分流、脾肾分流,GOV2组与IGV1组相比,胃肾分流发生率[27.56%(35/127)比66.67%(36/54),P<0.05]与脾肾分流发生率[12.60%(16/127)比25.93%(14/54),P<0.05]均较低,而附脐静脉开放[38.58%(49/127)比12.96%(7/54),P<0.05]与腹膜后侧枝分流[30.71%(39/127)比11.11%(6/54),P<0.05]相对多见。结论肝硬化合并GOV2型与IGV1型患者在临床特征和影像学表现方面存在显著异质性。充分认识和理解两型患者间差异,可为临床采取适宜的治疗措施提供依据,有益于改善患者预后。

关键词：肝硬化胃底静脉曲张胃肾分流门静脉高压性胃病

Anti-viral Activity of Hairpin Ribozyme Directed against HBV Core Region In Vitro

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2001年第3期21卷 219-221页

作者：林菊生宋宇虎孔心娟谢娜吴小力刘南植王南下操二虎金由辛 Institute of Liver Diseases Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430030

To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core region in vitro, HRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5′-cis-Rz and 3′-cis-Rz. 32 P-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme. 32 P -labeled pKC transcript containing HBV core region as targets-RNA was transcribed by using T 7 RNA polymerase and purified by PAGE. Cold HpRz transcript was incubated with 32 P-labeled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis. The results showed that HpRz had the ability of cleavage at 37 ℃ and 12 mmol/L MgCl 2 and the design of ribozyme was correct. It is concluded that HpRz prepared in vitro possesses specific catalytic activity, indicating that it is possible for HpRz to intracellularly inhibit the replication of HBV. It may be developed into a nucleic acid drug in the treatment of hepatitis B in the future.

关键词： hairpin ribozyme hepatitis B virus plasmid-independent transcription RNA cleavage