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Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

World Journal of Stem Cells 2015年第1期7卷 223-234页

作者： Juan E Santiago-Torres Rebecca Lovasz Alicia L Bertone Comparative Orthopedic Research Laboratory Department of Veterinary Clinical Sciences College of Veterinary Medicine The Ohio State University Columbus OH 43210 United States College of Medicine The Ohio State University Columbus OH 43210 United States

AIM: To investigate adenoviral transduction in mesenchymal stem cells(MSCs) and effects on stemness in vitro and function as a cell therapy in ***: Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine(PEI)-mediated transfection of pc DNA3-e GFP or adenoviral transduction of green fluorescent protein(GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness(i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2(BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and ***: PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs(81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs(78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs(7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression(0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs(1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteo

关键词： adenovirus Bone morphogenic protein 2 Fetal Green fluorescent protein Mesenchymal stemcell Gene transfer technique

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The effect of Human LMP-1 gene on the expression of osteogenic gene in dog bone marrow mesenchymal stem cells

The effect of Human LMP-1 gene on the expression of osteogen...

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中华医学会第10届全国显微外科学术会议暨世界首例断肢再...

作者： Pu chao Li wei Ni wei-dong Qu yu Department of Orthopedics NanChong central Hospital

Objective To study the effect of Human LMP-1 gene on the expression of osteogenic gene in dog bone marrow mesenchymal stem *** Dog MSCs were infected by the recombinant adenovirus vector of human *** expression of LMP-1,BMP-7,ALP and osteocalcin gene was detected by RT-PCR. Results The best efficiency can get at the most appropriate MOI 100 after Ad-LMP-1 infected *** BMSCs can express mRNA and of LMP-1,expression of ALP increased,but not caused expression of BMP-7 and osteocalcin *** The recombinant adenoviral vector carring hLMP-1 gene can promote expression of osteogenic gene.

关键词： LIM mineralization protein-1 adenovirus bone marrow mesenchymal stem cells recombinant human bone morphogenetic protein -7 alkaline phosphatase

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Identification of the insulin gene as a novel RORαtarget gene

Identification of the insulin gene as a novel RORαtarget ge...

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第十二次全国医学遗传学学术会议

作者： Kuang Jiangying Hou Xiaoming Jiang Haiou Cao Guiqun Wu Sisi Su Zhiguang Molecular Medicine Research Center West China HospitalSichuan University

Objective Retinoic acid receptor related receptor a(RORa) is a nuclear receptor modulating physiopathological processes such as dyslipidemia,atherosclerosis,obesity and *** deficient mice display metabolic abnormalities and alterations in insulin sensitivity and glucose *** aim of this study is to investigate if RORa regulates the expression and secretion of *** Amplified rat Ins2 gene promoter was cloned into pGL3-Basic reporter *** cDNA encoding full-length mouse RORa was amplified by RT-PCR and then subcloned into the pAdTrack-CMV ***-1 cells were infected with RORα-adenovrius or RORa shRNA *** expressions of RORa and Beta2 were detected by both real-time quantitative PCR and *** amount of insulin was measured by *** binding of RORa to insulin promoter was examined by electrophoretic mobility shift assay(EMSA).Results Endogenous Ins2 mRNA expression was up-regulated by overexpression of RORa in rat INS-1 ***-down of RORa by shRNA produced a decrease in Ins2 mRNA. Furthermore,the altered expression of RORa also led to the alteration of secreted insulin and cellular content insulin *** addition,we identified a functional RORa response element(RORE) within the Ins2 *** of RORa levels through stable over-expression or shRNA knock-down also affected the level of transcription factor BETA2 in beta ***2 is a crucial factor for the assembly and function of the insulin gene transcription *** RORa modulates the insulin expression and *** regulates insulin expression directly by binding to RORE in insulin promoter and indirectly by affecting the expression of insulin transactivator Beta2.

关键词： RORα Insulin adenovirus shRNA EMSA

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Tumor Cell Apoptosis Induced After Treatment With Adenovirally Expressed TRAIL in Vivo

Tumor Cell Apoptosis Induced After Treatment With Adenoviral...

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贵州省2008年血液学年会

作者：王季石方琴吴颖亚胡晓燕沈璐杨远 Abraham N.G. 贵阳医学院附属医院血液科 Departments of Medicine and Pharmacology New York Medical CollegeValhallaNew YorkUnited States

Tumor necrosis factor related apoptosisinducing ligand(TRAIL) is a naturally occurring death inducing ligand that induces apoptosis preferentially in malignant cells as normal cells are protected by expression of deco... 详细信息

关键词： Tumor necrosis factor(TNF) adenovirus Gene trasnfer

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RIP140 overexpression impairs cardiac mitochondrial function and accelerates the transition to heart failure in chronically infarcted rats

RIP140 overexpression impairs cardiac mitochondrial function...

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2017年广东省药师周大会

作者：陈艳芳 ShaoRui Chen ZhongBao Yue YiQiang Zhang ChangHua Zhou WeiWei Cao Xi Chen LuanKun Zhang PeiQing Liu Department of Pharmacology and Toxicology School of Pharmaceutical SciencesSun Yat-sen University 广州医科大学附属第二医院药学部 Division of Cardiology and Institute of Stem Cell and Regenerative MedicineSchool of MedicineUniversity of Washington

Heart failure is associated with myocardial energy metabolic abnormality. Receptor-interacting protein 140(RIP140) is an important transcriptional cofactor for maintaining energy balance in high-oxygen consumption tissues. However, the role of RIP140 on pathological processes of heart failure remains to be elucidated. In this study, we investigated the role of RIP140 in mitochondrial and cardiac functions in rodent hearts under myocardial infarction(MI) stress. MI was created by permanent ligation of left anterior descending coronary artery(LAD) and exogenous expression of RIP140 by adenovirus(Ad) vector delivery. Four weeks after MI or Ad-RIP140 treatment, cardiac function was assessed by echocardiographic and hemodynamics analyses, and mitochondrial function was determined by mitochondrial genes expression, biogenesis and respiration rates. In Ad-RIP140 or MI group, a subset of metabolic genes changed, accompanied with slight reductions in mitochondrial biogenesis and respiration rates but no change in adenosine triphosphate(ATP) content. Cardiac malfunction was compensated. However, under MI stress, rats overexpressing RIP140 exhibited greater repressions in mitochondrial genes, state 3 respiration rates, respiration control ratio(RCR), and ATP content, and had further deteriorated cardiac malfunction. In conclusion, RIP140 overexpression leads to comparable cardiac function as resulted from MI, but RIP140 aggravates metabolic repression, mitochondrial malfunction and further accelerates the transition to heart failure in response to MI stress.

关键词： RIP140 heart failure energy metabolism mitochondria adenovirus

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Adenoviral -mediated transfer of hTGF-β2 induces chondrogenesis of human adipose derived stem cells

Adenoviral -mediated transfer of hTGF-β2 induces chondrogen...

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中华医学会第八届骨科学术会议暨第一届国际COA学术大会

作者： Xiao bing Jin Yong sheng Sun Ke Zhang Xiao dong Ju Si quan Lou Orthopaedics department Peking University Third Hospital.

＜正＞Objective:The chondrogenic potential of human adipose -derived stem cells （hADSCs） makes them a possible source of seeding cells for cartilage tissue engineering, however, little is known about the capacity of... 详细信息

关键词： adipose derived stem cells (ADSC) chondrogenic adenovirus human transforming beta 2 (hTGF-β2) pellet culture alginate tissue engineering

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在胶质瘤细胞中抑制FoxM1表达对其增殖和凋亡的影响初探

在胶质瘤细胞中抑制FoxM1表达对其增殖和凋亡的影响初探

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作者：王震东湖南大学

学位级别：硕士

Gliomas, which accounts for more than 50% as one of the primary tumors of the nervous system, is the most common primary central nervous system malignancies and also one of the most aggressive malignant *** are about 90,000 new patients per year in *** the in-depth understanding of the mechanism of glioma molecular biology pathogenesis, people gradually realized the occurrence and development of glioma is a multi-gene involvement, multi-link, multi-stage and multi-step complex process. The truth has now been found that inactivation of functional tumor suppressor genes such as p53,Rb, P16 *** over-expression of nuclear transcription factors such as FoxM1,MDM2,cdc2, VEGF,Survivin etc. can lead the tumor cells apoptosis suppressed and unlimited growth. So we need to find some of the key nuclear transcription factors, which can regulate multiple downstream tumor-associated genes or the expression of apoptosis-related genes.FoxMl is a proliferation-specific transcription factor. After FoxM1 protein activated, it can regulate many key factors with the G1/S phase, G2/M phase transition and mitotic cell cycle regulation. The research found that FoxM1 was continuously and highly expressed in many cancers including glioma. Over-expression FoxM1 will disorder downstream target genes, such as Survivin, Skp2, which can protect apoptosis, enhance cell proliferation, and induce the tumor cells out of control in cell growth. Studies have proved that FoxM1 makes high expression of angiogenic factors VEGF and promote glioma growth. In short, FoxM1 might be a potential target sites of the glioma's gene therapy.In this paper, we used RNA interference to block the FoxM1 signaling pathway, and studied the effect and mechanism of the inhibition of glioma proliferation. Therefore, we constructed Ad hFoxM1 siRNA, and got a large number of high-titer adenovirus by using the technolog of CsCl density gradient centrifugation. Make H4 and A172 cell infected by th

关键词： Glioma FoxMl adenovirus RNA interference Cell proliferation

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靶向性肿瘤增殖病毒的构建

靶向性肿瘤增殖病毒的构建

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作者：华汉巨浙江大学

学位级别：硕士

用病毒治疗肿瘤这个思路最早可以追溯到100年前，但是直到近10年才取得了飞速的发展。与以往的研究以病毒作为基因载体不同，最近的研究集中在靶向性肿瘤增殖病毒(Cancer-specific Replication-competent Virus)上，该病毒经转录调节修... 详细信息

用病毒治疗肿瘤这个思路最早可以追溯到100年前，但是直到近10年才取得了飞速的发展。与以往的研究以病毒作为基因载体不同，最近的研究集中在靶向性肿瘤增殖病毒(Cancer-specific Replication-competent Virus)上，该病毒经转录调节修饰后，可以以高度的特异性在目的肿瘤中复制并裂解肿瘤细胞，而对正常的体细胞无杀伤力。靶向性肿瘤增殖病毒的构建方法之一就是将肿瘤细胞内特异性表达的启动子／增强子插入到腺病毒某些必须基因的上游，替代腺病毒基因原有的启动子，使该基因只能在肿瘤细胞内表达，而在其它正常细胞内处于沉默状态，使经改建后的腺病毒只能以高度的特异性在肿瘤细胞内增殖并杀伤肿瘤细胞，而对正常体细胞无明显杀伤力，从而达到特异性杀灭肿瘤的目的。本课题拟以人端粒酶催化亚单位(human telomersse reverse transcriptase，hTERT)和人环氧合酶-2(Cyclo-oxygenase 2，Cox-2)不同长度的启动子作为腺病毒的增殖调控片断，使构建后的腺病毒只能在hTERT和Cox-2表达阳性的肿瘤细胞中特异性增殖，进而杀伤肿瘤细胞，而在正常体细胞内复制缺陷，具有高度的特异性和安全性。并通过比较筛选出特异性强、安全性高的转录调控片段作为腺病毒的调控序列。为下一步靶向性肿瘤增殖病毒治疗实体肿瘤奠定基础。浙江大学硕士学位论文材料和方法: 以腺病毒穿梭载体Pd306(俞德超博士友情赠送)作为质粒载体，核查Genbank，设计特异性引物，通过PCR方法将hTERT和Cox一的不同长度的启动子片段从人基因组中扩增出来。并将扩增的片断分别插入到腺病毒穿梭载体pd300所携带的腺病毒的Ela和Elb启动调控区，连接所得质粒通过PCR、酶切、测序等方法进行鉴定。为下一步进行腺病毒在293细胞的包装、纯化、以及病毒感染各种肿瘤细胞、病毒感染治疗动物实体瘤模型奠定基础。结果与讨论: 结果:通过不同的引物，从人基因组通过聚合酶联反应(PCR)亚克隆了hTERT的2个不同长度的启动子，大小分别为181bp、378bp。通过PCR亚克隆了Cox一2的2个不同长度的启动子，大小分别是400bp、8 14bp。将hTERT启动子片段通过Agel酶切，插入到经过Agel酶切、去磷酸化处理的po06上(Ela上游)，将Cox一2不同长度的启动子片段经过Eagl酶切，插入到经过Eagl酶切、去磷酸化处理的pd306上(Elb上游)。重组质粒经过转化到感受态细菌、质粒DNA抽提，重组质粒分别用对应的hTER’r和Cox一2启动子引物进行PCR扩增、Agel和Eagl酶切鉴定己含插入序列，最后经测序鉴定为正向克隆。将hTERT启动子准确插入腺病毒穿梭载体Pd306 Ela调控区，调控Ela的转录;将Cox一启动子插入到pd306 Elb的上游调控区域，调控Elb的转录。为下一步穿梭载体和腺病毒骨架质粒共转染293细胞进行病毒包装以及病毒感染肿瘤细胞和动物实验奠定基础。讨论:基因治疗肿瘤已经有悠久的历史，而腺病毒是基因治疗的一个常用载体。既往用腺病毒作为载体表达外源性基因，相对于质粒等非病毒类载体具有转染能力强、瞬时表达效率高等特点，但是由于经典的非复制型腺病毒载体不能稳定表达、需要重复感染、且具有较大的毒副作用等缺点使腺病毒的应用受到了严格限制。而本课题拟构建的复制型新型腺病毒具有转染率高、感染性强、且具有肿瘤细胞靶向攻击能力，对正常细胞杀伤力低下，安全性高等特点。本课题采用肿瘤组织特异性表达的启动子调控腺病毒必须基因的表达的方法来改建腺病毒。腺病毒穿梭载体Pd306包括了腺病毒早期基因Ela和Elb，Ela编码病毒复制必须的早期转录因子，其编码的转录因子与宿主细胞的转录因子协调作用激活其他早期基因，并使受染细胞进入s期，是腺病毒复制的必须基因。Elb编码的蛋白和p53的N端作用抑制p53介导的细胞凋亡作用，维持病毒复制的稳定环境。非复制型载体

关键词： hTERT Cox-2 Oncolytic virus adenovirus clone

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一起鸡心包积液病例的诊治

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家禽科学 2017年第3期 26-27页

作者：魏凤张文通苗立中沈志强山东省滨州畜牧兽医研究院山东滨州256600 山东绿都生物科技有限公司山东滨州256600

鸡包涵体肝炎（Inclusion Body Hepatitis,IBH）是由Ⅰ群禽腺病毒（Fowl adenovirusⅠ,FAV-Ⅰ）引起的一种急性传染病。该病最早发生在巴基斯坦卡拉奇市安卡拉地区,所以也称之为安卡拉病[1]。该病特征性病理变化为心包囊积液和肝炎。目... 详细信息

鸡包涵体肝炎（Inclusion Body Hepatitis,IBH）是由Ⅰ群禽腺病毒（Fowl adenovirusⅠ,FAV-Ⅰ）引起的一种急性传染病。该病最早发生在巴基斯坦卡拉奇市安卡拉地区,所以也称之为安卡拉病[1]。该病特征性病理变化为心包囊积液和肝炎。目前该病已在我国多地流行,可造成肉鸡、蛋鸡乃至水禽发病死亡,已严重影响养禽业健康发展[2-3]。

关键词：禽腺病毒病已包涵体淡黄色积液 adenovirus 急性传染病理变化剖检紧急接种高免卵黄抗体下游引物

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鸡包涵体肝炎的研究现状

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兽医导刊 2015年第21期 30-31页

作者：崔京腾李秀娟齐鲁动物保健品有限公司

鸡包涵体肝炎（Inclusion Body Hepatitis,IBH）,又称为鸡贫血综合症（Anemiasyndrome）,是由禽腺病毒（Fowl adenovirus,FAV）引起的鸡的一种急性传染病,主要侵害雏鸡。其特征为：病鸡死亡突然增多,严重贫血、黄疸、肝脏肿大、有出血... 详细信息

鸡包涵体肝炎（Inclusion Body Hepatitis,IBH）,又称为鸡贫血综合症（Anemiasyndrome）,是由禽腺病毒（Fowl adenovirus,FAV）引起的鸡的一种急性传染病,主要侵害雏鸡。其特征为：病鸡死亡突然增多,严重贫血、黄疸、肝脏肿大、有出血和坏死灶,肝细胞见有核内包涵体。临床表现为贫血、白色水样下痢,瘦小。

关键词：包涵体禽腺病毒下痢坏死灶肝细胞特征性病变纤突减蛋综合征 adenovirus 二十面体对称

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