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Neural Regeneration Research 2012年第8期7卷 595-600页

作者： Ruizhi Wang Dongning Hao Wei Shi Jingnan Pu Zizhang Wang Department of Neurosurgery Second Affiliated Hospital Medical School ofXi＇an Jiaotong University Xi＇an 710004 Shaanxi Province China Department of Neurosurgery Yulin First People＇s Hospital Yulin 718000 Shaanxi Province China Department of Head and Neck Surgery Tumor Hospital of Shaanxi Province Xi＇an 710061 Shaanxi Province China

In the present study, a rat model of non-traumatic intracerebral hemorrhage was established by type IV collagenase injection into the right globus pallidus. Bax and Bcl-2 expression in tissues surrounding hematomas was significantly increased within 14 days after injury, and it then gradually decreased. Vascular endothelial growth factor, flk-1 and Flt-1 mRNA expression gradually increased over time. After intraperitoneal injection with minocycline, Bax expression was decreased 1 day after intracerebral hemorrhage. flk-1 and Flt-1 mRNA expression was decreased after minocycline injection, but Bcl-2 expression was increased, and vascular endothelial growth factor mRNA expression was decreased between 4-14 days. These results indicated that protective effects of minocycline on nerve tissues were associated with increased Bcl-2 expression and decreased Bax expression in the early stage after intracerebral hemorrhage. In the late stage, minocycline downregulated vascular endothelial growth factor and its receptor expression to inhibit brain tissue self-repair.

关键词： intracerebral hemorrhage minocycline vascular endothelial growth factor apoptosis inflammation Bax Bcl-2 flk-1 Flt-1 neural regeneration

Anti-angiogenesis Effect on Glioma of Attenuated Salmonella Typhimurium Vaccine Strain with flk-1 Gene

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2004年第4期24卷 389-391页

作者：冯珂珂赵洪洋陈剑姚东晓姜小兵周伟 Department of Neurosurgery Union Hospital Tongji Medical College Huazhong University of Science and technology

To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial Gl261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.

关键词： attenuated Salmonella typhimurium flk-1 glioma

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

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Journal of Genetics and Genomics 2020年第5期47卷 249-261页

作者： Xiaomin Wang Hui Shi Juanjuan Zhou Qingjian Zou Quanjun Zhang Shixue Gou Pengfei Chen Lisha Mou Nana Fan Yangyang Suo Zhen Ouyang Chengdan Lai Quanmei Yan Liangxue Lai CAS Key Laboratory of Regenerative Biology Guangdong Provincial Key Laboratory of Stem Cell and Regenerative MedicineSouth China Institute for Stem Cell Biology and Regenerative MedicineGuangzhou Institutes of Biomedicine and HealthChinese Academy of SciencesGuangzhou510530China University of Chinese Academy of Sciences Beijing100049China Guangzhou Regenerative Medicine and Health Guangdong Laboratory(GRMH-GDL) Guangzhou510005China Institute for Stem Cell and Regeneration Chinese Academy of SciencesBeijing100101China Research Unit of Generation of Large Animal Disease Models Chinese Academy of Medical Sciences(2019RU015)Guangzhou510530China School of Biotechnology and Health Sciences Wuyi UniversityJiangmen529020China Shenzhen Xenotransplantation Medical Engineering Research and Development Center Institute of Translational MedicineShenzhen University Health Science CenterShenzhen University School of MedicineFirst Affiliated Hospital of Shenzhen UniversityShenzhen Second People's HospitalShenzhen518035China Department of Central Laboratory Shenzhen Longhua District Central HospitalShenzhen518110China School of Life Sciences University of Science and Technology of ChinaHefei230027China Joint School of Life Sciences Guangzhou Institutes of Biomedicine and HealthChinese Academy of SciencesGuangzhou Medical UniversityGuangzhou511436China

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by flk-1 deficiency in mouse.

关键词： Blastocyst complementation Interspecies chimera Intraspecies chimera flk-1 Vascular endothelial cell Hematopoietic cell

Inhibition of choroidal neovascularization by lentivirusmediated PEDF gene transfer in rats

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International Journal of Ophthalmology(English edition) 2016年第8期9卷 1112-1120页

作者： Ya-Jie Yu Bin Mo Lu Liu Yan-Kun Yue Chang-Li Yue Wu Liu Beijing Tongren Eye Center Beijing Tongren HospitalCapital Medical University Beijing Ophthalmology & Visual Sciences Key Laboratory Department of Ophthalmology Fuxing Hospital Capital Medical University Department of Pathology Beijing Tongren Hospital Capital Medical University

AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and flk-1.

关键词： pigment epithelium-derived factor choroidal neovascularization lentivirus vascular endothelial growth factor flk-1

金鱼雌核发育单倍体胚胎血液循环障碍产生机制

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水生生物学报 2022年第1期46卷 126-136页

作者：张琼宇唐甲卉彭娟唐鹏程孙远东永州职业技术学院公共基础学部永州425100 湖南省中医药研究院附属医院长沙410006 湖南科技大学生命科学学院湘潭411201

为研究鱼类单倍体血液循环障碍产生机制,人工诱导获得金鱼(Carassius auratus)雌核发育单倍体胚胎并进行活体观察及邻联茴香胺染色,结果显示金鱼雌核发育单倍体胚胎存在不同程度的血液循环不良和红细胞生成缺陷。为进一步探讨其发生的... 详细信息

为研究鱼类单倍体血液循环障碍产生机制,人工诱导获得金鱼(Carassius auratus)雌核发育单倍体胚胎并进行活体观察及邻联茴香胺染色,结果显示金鱼雌核发育单倍体胚胎存在不同程度的血液循环不良和红细胞生成缺陷。为进一步探讨其发生的分子机制,利用反义RNA整胚原位杂交技术比较分析了原始造血和血管发生关键基因scl(Stem cell leukemia)、gata-1(Gata binding protein-1)和flk-1(Fetal liver kinase-1)在雌核发育单倍体和自交二倍体胚胎中的表达模式。原始造血关键基因scl和gata-1的检测结果显示,在金鱼单倍体胚胎中,后侧板中胚层(Posterior lateral-plate mesoderm,PLM)不能正常迁移至胚体中线融合形成原始造血关键部位中间细胞群(Intermediate cell mass,ICM);同时,scl和gata-1的表达沿单倍体胚胎前后轴缩短,并在躯干区域逐渐减弱。血管内皮标记基因flk-1的检测结果显示,单倍体胚胎原始体轴血管结构紊乱,节间血管发育不良或者缺失,没有形成完整的血管系统。此外,活体观察及gsc(Goosecoid)和N-cadherin(Neural cadherin)原位杂交检测结果显示,金鱼雌核发育单倍体胚胎原肠胚期存在细胞运动缺陷。以上研究结果提示,细胞运动迁移异常导致ICM和原始体轴血管发育缺陷,同时红细胞分化减少及外周血管发育不全,可能是造成金鱼雌核发育单倍体胚胎血液循环障碍的重要原因。

关键词：雌核发育单倍体综合症血液循环障碍 scl gata-1 flk-1 细胞运动金鱼

A DNA vaccine against extracellular domains 1-3 of flk-1 and its immune preventive and therapeutic effects against H22 tumor cell in vivo

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World Journal of Gastroenterology 2004年第14期10卷 2039-2044页

作者： FanLü Zhao-YinQin Wen-BinYang Yin-XinQi Yi-MinLi DepartmentofGeneralSurgery SecondHospitalofXi'anJiaotongUniversityXi'an710004ShaanxiProvinceChina DepartmentofGeneralSurgery SecondHospitalofXi'anJiaotongUniversityXi'an710004ShaanxiProvinceChina

AIM: To construct a DNA vaccine against extracellular domains 1-3 of fetal liver kinase-1(flk-1), and to investigate its preventive and therapeutic effect against H22 cell in vivo. METHODS: flk-1 DNA vaccine was produced by cloning extracellular domains 1-3 of flk-1 and by inserting the cloned gene into pcDNA3.1(+). Fifteen mice were divided into 3 groups and inoculated by vaccine, plasmid and saline respectively to detect specific T lymphocyte response. Thirty Mice were equally divided into preventive group and therapeutic group. Preventive group was further divided into V,P,andS subgroups, namely immunized by vaccine,pcDNA3.1(+) and saline, respectively, and attacked by H22 cell. Therapeutical group was divided into 3 subgroups of V,P and S, and attacked by H22, then treated with vaccine, pcDNA3.1(+) and saline, respectively. The tumor size, tumor weight, mice survival time and tumor latency period were compared within these groups. Furthermore,intratumoral microvessel density (MVD) was assessed by immunohistochemistry. RESULTS: DNA vaccine pcDNA3.1(+)flk-1-domains 1-3 was successfully constructed and could raise specific CTL activity. In the preventive group and therapeutic group,tumor latency period and survival time were significantly longer in vaccine subgroup than that in P and S subgroups (P＜0.05);the tumor size, weight and MVD were significantly less in vaccine subgroup than that in P and S subgroups (P＜0.05). The survival time of therapeutic vaccine subgroup was significantly shorter than that of preventive vaccine subgroup (P＜0.05);the tumor size, and MVD of therapeutic vaccine subgroup were significantly greater than that of preventive vaccine subgroup (P＜0.05).CONCLUSION: DNA vaccine against flk-1 domains 1-3 can stimulate potent specific CTL activity; and has distinctive prophylactic effect on tumor H22, and also can inhibit the tumor growth in vivo. This vaccine may be used as an adjuvant therapy because it is less effective on detectable tumor.

关键词： DNA疫苗细胞外功能区1-3 flk-1 免疫学预防性治疗作用反向作用 H22肿瘤细胞

小鼠胚胎AGM区flk-1^+细胞的造血特性

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中国实验血液学杂志 2013年第2期21卷 446-450页

作者：周帆李专刘兵安徽医科大学解放军307医院临床学院安徽合肥230032 军事医学科学院附属医院 307-青藤转化医学中心北京100071

本研究旨在考察flk-1分子在胚胎期10.5 d(E10.5)的主动脉-性腺-中肾区(AGM区)不同类型造血前体细胞的表达情况。通过流式细胞术分析发现低于10%的flk-1+细胞与CD41、CD45/Ter119有共表达,继而分选出CD31+CD45-Ter119-flk-1+CD41+(R1,代... 详细信息

本研究旨在考察flk-1分子在胚胎期10.5 d(E10.5)的主动脉-性腺-中肾区(AGM区)不同类型造血前体细胞的表达情况。通过流式细胞术分析发现低于10%的flk-1+细胞与CD41、CD45/Ter119有共表达,继而分选出CD31+CD45-Ter119-flk-1+CD41+(R1,代表不成熟的造血前体细胞)和CD31+CD45-Ter119-flk-1+CD41-(R2,代表内皮细胞)两群细胞,利用造血集落培养和OP9共培养体系分别考察其体外的髓系与淋系潜能。结果表明:仅R1组细胞能在造血集落培养体系中形成典型的造血集落,但与OP9共培养7-9 d后,两组细胞均能产生丰富的造血前体细胞(CD45+c-Kit+)、髓系细胞(Gr-1+/Mac-1+)、红细胞(Ter119+)和B淋巴细胞(CD19+)。结论:E10.5的小鼠胚胎AGM区flk-1仍然在幼稚的造血前体细胞以及更早期的生血内皮细胞表达,但是此时的造血干细胞前体和造血干细胞是否表达flk-1仍需通过体内功能实验明确。

关键词：主动脉-性腺-中肾区造血前体细胞内皮细胞淋巴细胞 flk-1

dhfr基因过表达对乙醇致斑马鱼胚胎心血管发育异常的效应研究

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中国当代儿科杂志 2020年第8期22卷 916-922页

作者：孙淑娜蒋璆陆定桂永浩复旦大学附属儿科医院心内科上海201102 复旦大学附属中山医院心血管病研究所上海200032 上海市第八人民医院儿科上海200235

目的观察二氢叶酸还原酶基因(dhfr)过表达对乙醇致斑马鱼胚胎心脏和血管发育异常的作用,并初步探讨其机制。方法体外转录dhfr mRNA并将其显微注射入斑马鱼受精卵内使dhfr基因过表达,并进行过表达的效率验证。将野生型斑马鱼分为正常对... 详细信息

目的观察二氢叶酸还原酶基因(dhfr)过表达对乙醇致斑马鱼胚胎心脏和血管发育异常的作用,并初步探讨其机制。方法体外转录dhfr mRNA并将其显微注射入斑马鱼受精卵内使dhfr基因过表达,并进行过表达的效率验证。将野生型斑马鱼分为正常对照组、乙醇处理组(400 mmol/L乙醇溶液)、乙醇+dhfr mRNA组(显微注射6 nL dhfr mRNA),观察各组胚胎的整体发育情况。按照上述分组,利用红色荧光蛋白标记心脏的转基因斑马鱼系Tg(cmlc2:mcherry)观察胚胎心房和心室的发育状况;利用荧光显微造影观察野生型斑马鱼胚胎心脏流出道及血管发育情况;通过心率和心室收缩指数评估心功能情况。构建基因探针并通过胚胎整体原位杂交及Real-time PCR法,检测胚胎nkx2.5、tbx1、flk-1的基因表达情况。结果与乙醇处理组相比,乙醇+dhfr m RNA组斑马鱼胚胎发育异常百分率明显下降,存活百分率显著上升(P1、flk-1 m RNA的表达较对照组下降(P1、flk-1 m RNA的表达较乙醇处理组上调,但仍未达到正常对照组水平(P1、flk-1水平降低有关。

关键词： dhfr基因乙醇心脏血管 nkx2.5 tbx1 flk-1 斑马鱼

差速贴壁分离法:获得小鼠骨髓内皮祖细胞诱导分化培养的最佳贴壁时间

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生理学报 2011年第6期63卷 574-580页

作者：杨娜娜焦鹏李大伟王孟赞姚树桐宗传龙秦树存泰山医学院动脉粥样硬化研究所泰安271000 泰山医学院附属聊城市人民医院心血管内科聊城252004

本文旨在比较差速贴壁方法分离的不同时间点贴壁的小鼠骨髓单个核细胞诱导分化为内皮祖细胞的生物学特性,探讨最适宜贴壁时间。Ficoll密度梯度离心分离小鼠骨髓单个核细胞,接种于预先铺有纤维连接蛋白的培养板上,定义为1d贴壁细胞组,取1... 详细信息

本文旨在比较差速贴壁方法分离的不同时间点贴壁的小鼠骨髓单个核细胞诱导分化为内皮祖细胞的生物学特性,探讨最适宜贴壁时间。Ficoll密度梯度离心分离小鼠骨髓单个核细胞,接种于预先铺有纤维连接蛋白的培养板上,定义为1d贴壁细胞组,取1d非贴壁细胞再接种为3d贴壁细胞组,继续培养2d取非贴壁细胞再接种为3d非贴壁细胞组,继续培养20d后,分别检测3组诱导分化内皮祖细胞亚群表面标志、粘附能力和成血管能力。结果显示,1d贴壁细胞中CD34+、flk-1+、CD34+/flk-1+细胞数明显多于3d贴壁细胞和3d非贴壁细胞(P1);1d贴壁细胞粘附能力、成血管能力均显著高于3d贴壁细胞和3d非贴壁细胞;1d贴壁细胞和3d贴壁细胞内皮型一氧化氮合酶(endothelial NO synthase,eNOS)表达量无显著差异,但均显著高于3d非贴壁细胞(P1);3d贴壁细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达水平显著高于1d贴壁细胞和3d非贴壁细胞(P1)。以上结果提示,小鼠骨髓单个核细胞1d贴壁诱导分化的内皮祖细胞生物学功能显著高于3d贴壁细胞和3d非贴壁细胞;3d贴壁细胞较1d贴壁细胞和3d非贴壁细胞表达较多的VEGF,1d贴壁细胞和3d贴壁细胞eNOS表达高于3d非贴壁细胞。小鼠骨髓内皮祖细胞诱导分化培养最适宜贴壁时间为1～3d。

关键词：内皮祖细胞差速贴壁小鼠 CD133 flk-1

当归对大鼠缺血性脑损伤后Flt-1、flk-1mRNA表达的影响

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中国应用生理学杂志 2006年第4期22卷 385-389页

作者：郑婵娟廖维靖范明杨万同蒙兰青武汉大学中南医院康复医学科湖北武汉430071 军事医学科学院基础医学研究所北京100850

目的:研究大鼠缺血性脑损伤后不同时间点Flt-1、flk-1mRNA的表达及当归对其表达的影响。方法:雄性Wistar大鼠,随机分为缺血损伤组和当归治疗组。采用线栓法制作大鼠短暂性大脑中动脉阻断(MCAO)与再灌模型,治疗组腹腔注射当归注射液(剂量... 详细信息

目的:研究大鼠缺血性脑损伤后不同时间点Flt-1、flk-1mRNA的表达及当归对其表达的影响。方法:雄性Wistar大鼠,随机分为缺血损伤组和当归治疗组。采用线栓法制作大鼠短暂性大脑中动脉阻断(MCAO)与再灌模型,治疗组腹腔注射当归注射液(剂量5g/kg)。36只大鼠(每组各18只)在脑缺血/再灌后1d、3d、7d神经行为学评分完成后被处死,取大脑行氯化三苯四唑(TTC)染色以测脑梗死比;另取72只大鼠(每组各36只)在脑缺血/再灌后3h、6h、12h、1d、3d、7d分别被处死,应用半定量逆转录聚合酶链反应(RT-PCR)技术检测缺血侧Flt-1、flk-1mRNA的表达。结果:在同时间点神经功能缺损评分比较,缺血损伤组明显高于当归治疗组(P1)。RT-PCR检测表明,缺血损伤组Flt-1、flk-1mRNA在缺血/再灌后3h即开始表达增强,于3d达高峰,后逐渐降低;当归治疗组Flt-1、flk-1mRNA表达比缺血损伤组明显增加,于3d达到高峰后缓慢降低至第7d仍保持较高水平。缺血损伤组和当归治疗组中Flt-1mRNA与flk-1mRNA的表达呈正相关性,相关系数为r=0.957(P1)。结论:当归可增强缺血性脑损伤后Flt-1、flk-1mRNA表达。Flt-1、flk-1mRNA的表达紧密相关。

关键词：缺血性脑损伤 Flt-1 flk-1 当归注射液