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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

The specific expression mediated by promoters of hepatitis B virus in hepatocarcinoma cells

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The Chinese-German Journal of Clinical Oncology 2006年第5期5卷 328-331页

作者： Na Xie Xiaoyan Wang Qiong Zhang Yuanyuan lin Kuohuan Liang jusheng lin Institute of Liver Diseases Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China

Objective: To investigate four expression vectors carrying enhanced green fluorescent protein (EGFP) genes, which under the control of different HBV promoters, were detected and compared as to their expressions in hepatocytes and non- hepatic cell lines. Methods: Four HBV promoters (including enhancers) were amplified by PCR, subcloned into the expression vector pEGFP-1. The four recombinants controlled by HBV promoters were analyzed and identified by restriction enzymes and sequencing. After transfection into human hepatoma cell lines and non-liver cells, transfection efficiency was measured by EGFP expression through fluorescence microscopy and analyzed with fluorescence activated cell sorter (FACS). Results: All target fragments were separately obtained and successfully cloned into the expression vector. The transfection results showed that in hepatoma cells, the expression of EGFP was more obvious than it was in non-hepatic cells. Among the four promoters, S gene promoter presented the strongest transfection efficiency. Conclusion: Our findings indicate that HBV promoters could lead specific expression in hepatocytes, different promoters had different outcomes, which might be a potential for the gene therapy of liver diseases.

关键词： HBV promoter EGFP hepatocellular carcinoma (HCC) gene therapy

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肝肺综合征

临床内科杂志 2001年第2期18卷 85-86页

作者： lin jusheng WU Jinming 林菊生吴金明 ##Institute of liver diseases tongji hospitaltongji medical college Huazhong Sci. techn. University武汉430030 ##华中科技大学同济医学院附属同济医院肝病研究所

肝肺综合征(heptaopulmonary syndrome，HPS)指慢性肝功能不全患者因肺内血管扩张而出现的严重低氧血症，是各种慢性肝脏疾病终末期的一种严重并发症，发病率为5％～29％，预后差，呈三联征的特点，即基础肝脏疾病-肺血管扩张-严重低氧... 详细信息

肝肺综合征(heptaopulmonary syndrome，HPS)指慢性肝功能不全患者因肺内血管扩张而出现的严重低氧血症，是各种慢性肝脏疾病终末期的一种严重并发症，发病率为5％～29％，预后差，呈三联征的特点，即基础肝脏疾病-肺血管扩张-严重低氧血症，其中基础肝脏疾病主要包括各种原因的肝硬化和慢性肝功能不全以及非肝硬化性门脉高压和肝外门静脉阻塞等。Rrowka[l]指出，发生HPS的决定因素是门静脉高压而不是广泛性肝细胞坏死。严重低氧血症是由于肺内血管扩张、通气血流比例(V/Q)失调所致动脉血氧合功能障碍所引起。此时，在呼吸室内空气时肺泡-动脉血氧分压差(A-aDO2)增大。

关键词：肝肺综合征诊断治疗发病机制

靶向瘦素基因小干扰RNA抑制肝星状细胞增殖和细胞内钙离子浓度

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华中科技大学学报（医学版） 2014年第6期43卷 631-635页

作者：薛秀兰林菊生厦门大学附属第一医院感染科厦门361000 华中科技大学同济医学院附属同济医院肝病研究所武汉430030

目的探讨靶向瘦素(leptin)基因的小干扰RNA(small interfering RNA,siRNA)对leptin表达,肝星状细胞(hepatic stellate cell,HSC)的增殖、凋亡及细胞内钙离子浓度(intracellular Ca2+concentration,[Ca2+]i)的影响,为肝纤维化基因治疗提供可行的方案。方法 HSC分为4组:正常对照组、空载体组、siRNA对照组、leptin-siRNA组。依分组要求分别转染HSC后,采用MTT法和流式细胞技术检测各组细胞的凋亡和增殖;应用免疫细胞化学SP法、Western blot法检测leptin蛋白的表达;Fura-2/AM负载检测[Ca2+]i变化。结果与HSC的正常对照组、空载体组及siRNA对照组相比,leptin-siRNA组可以有效抑制leptin基因的表达和细胞增殖(P<0.05),且不同时段(24、48、72h)细胞抑制率之间有显著差异(P<0.05)。leptin-siRNA组细胞凋亡比例明显增加(P<0.01);[Ca2+]i显著降低(P<0.05)。结论leptin-siRNA可以通过沉默leptin基因表达而抑制HSC的增殖、促进凋亡和减少[Ca2+]i。leptin在肝纤维化发展中起着重要作用,有望成为肝纤维化一个新的预测和预后的标志。

关键词：瘦素小干扰RNA 细胞凋亡肝星状细胞细胞内钙离子浓度

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AFP启动子调控PNP载体的构建及分析

肿瘤 2005年第3期25卷 208-210页

作者：蔡晓坤林菊生刘址忠沈霞梁扩寰华中科技大学附属同济医院消化内科肝病所武汉430030

目的分别构建三种启动子调控的大肠杆菌嘌呤核苷磷酸化酶(PNP)基因表达载体,检测、分析三者的表达差异。方法构建三种启动子调控的PNP基因表达载体。将三个PNP基因载体转染不同细胞株,检测PNP基因在各细胞株中表达,分析三者表达的特点... 详细信息

目的分别构建三种启动子调控的大肠杆菌嘌呤核苷磷酸化酶(PNP)基因表达载体,检测、分析三者的表达差异。方法构建三种启动子调控的PNP基因表达载体。将三个PNP基因载体转染不同细胞株,检测PNP基因在各细胞株中表达,分析三者表达的特点。结果AF0.3和[HRE]AF调控的PNP基因在AFP阳性肝癌细胞中实现了靶向性表达,而且后者在AFP阴性肝癌细胞中也实现了靶向性表达。结论三种PNP基因表达载体的成功构建为利用PNP/MeP-dR系统进行肝癌的靶向性基因治疗打下坚实的基础。

关键词：肝肿瘤嘌呤核苷磷酸化酶遗传载体基因疗法细胞系,肿瘤

HLA-DQA1基因rs9272346位点多态性与湖北汉族人群乙型肝炎不同临床转归的关联研究

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中华医学遗传学杂志 2014年第1期31卷 93-96页

作者：余金玲姚津剑里进安会敏陈曼宋起龙林菊生江苏省扬州市第一人民医院消化科 225000 华中科技大学同济医院肝病研究所

目的探讨中国湖北地区人类白细胞抗原（human leukocyte antigen，HLA—DQA1）基因rs9272346单核苷酸多态性与慢性乙型肝炎不同转归的关联。方法采用病例一对照研究，招募湖北地区汉族35岁以上住院乙型重型肝炎（acute liver failure，... 详细信息

目的探讨中国湖北地区人类白细胞抗原（human leukocyte antigen，HLA—DQA1）基因rs9272346单核苷酸多态性与慢性乙型肝炎不同转归的关联。方法采用病例一对照研究，招募湖北地区汉族35岁以上住院乙型重型肝炎（acute liver failure，ALF）173例、乙肝相关性肝硬化（1iver cirrhosis，LC）292例、乙肝相关性肝癌（hepatocellular carcinoma，HCC）325例、无症状乙肝携带者（asymptomatic HBV carrier，AHC）238例。应用TaqManMGB探针实时PCR技术检测rs9272346位点基因型，应用T检验、卡方检验和非条件Logistic回归进行统计分析。结果G等位基因型在ALF、LC、HCC组与AHC组分布差异无统计学意义（P=0．312、0．314、0．264）。在显性模型下，校正性别年龄因素后，GG＋GA与AA基因型相比，与ALF（OR=1．08，95％CI：0．70～1．68）、LC（OR=1．11，95％CI：0．87～1．26）、HCC（OR=0．93，95％CI：0．65～1．33）患病风险无相关性。性别分层后显性模型下显示的女性患者中，GG＋GA基因型与AA基因型相比为进展为重型肝炎的保护因素（OR=0．30，95％CI：0．1～0．87）。结论HLA—DQA1基因rs9272346位点单核苷酸多态性与乙型肝炎不同转归无相关性，女性患者中G等位基因携带可能是进展为重型肝炎的保护因素。

关键词：乙型重型肝炎肝硬化肝癌转归单核苷酸多态性 HLA—DQA1基因

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

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原发性肝癌基因治疗的研究进展

胃肠病学和肝病学杂志 2008年第12期17卷 959-961页

作者：高新生林菊生黄石市第五医院消化内科湖北黄石435005 华中科技大学同济医学院附属同济医院肝病研究所

针对原发性肝癌的传统治疗方法疗效欠佳、预后较差,基因治疗成为新的研究方向和热点。新的基因治疗策略不断涌现,载体系统不断更新,导入方法不断优化,动物模型不断改进,本文就以上方面进行综述。

关键词：肝细胞癌基因治疗