人内皮抑素基因克隆及在大肠杆菌中的表达

目的：获得具有抑制血管内皮细胞生长活性的重组人内皮抑制素蛋白。方法：从人胎肝中分离总RNA,经反转录聚合酶链反应（RT－PCR）得到endostatin全基因。用原核细胞表达载体构建pBV220/endostatin重组质粒,经DNA测序确认后,将其转化大肠杆菌DH5a进行表达,初步纯化及活性测定。结果：经SDS－PAGE电泳分析表达产物分子量约20Kda,薄层扫描显示表达产物可达细菌总蛋白的30％。结论：经生物学活性测定,表达蛋白能够抑制bFGF(碱性成纤维细胞生长因子）对牛毛细血管内皮细胞（BCEC）的增殖作用。     

TIPSS术中胆汁漏出对内皮细胞NOS活性影响的研究

目的：探讨经颈静脉肝内门腔分流术（TIPSS）中胆汁漏出对内皮细胞一氧化氮合酶（NOS)及一氧化氮（NO）合成的影响,进一步了解胆汁对支架内皮化的影响。方法：取人脐静脉内皮细胞进行体外培养,加入不同浓度（5％、10％、15％、20％、25％）胆汁干预,观察内皮细胞生长状况,并测条件培养液中NO及细胞NOS活性。结果：含5％、10％、15％胆汁的细胞生长状况与不含胆汁者相似,含20％、25％胆汁的细胞明显减少并显幼稚;各浓度胆汁的细胞NOS活性较不含胆汁者明显降低;条件培养液中NO含量均无明显差异。结论：胆汁抑制内 皮细胞的生长,并抑制内皮细胞NOS活性。     

碱性成纤维细胞生长因子促进人内皮细胞αv整合素及MMP-1表达的研究

创伤修复是体内多种因子和细胞参与的复杂过程，碱性成纤维细胞生长因子（bFGF）可影响创伤修复的整个过程，深入了解其作用机制有重要意义。笔者通过研究经bFGF刺激后人脐静脉内皮细胞（HUVEC）αv整合素表达及基质金属蛋白酶-1（MMP-1）分泌的情况，从而分析bFGF促进内皮细胞迁移，血管新生的可能机制，为进一步研究bFGF促进创伤修复作用提供依据。     

bFGF对辐射诱导的血管内皮细胞凋亡的作用

目的：研究碱性成纤维细胞生长因子(bFGF)对辐射诱导的血管内皮细胞凋亡的作用。方法：以^60Coγ射线照射法建立猪主动脉内皮细胞株PAE(porcine aortic endothelial cells)和原代培养的人脐带静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)调亡模型。以流式细胞术[膜联蛋白(annexin)-V-FTTC PI标记]评价细胞凋亡率，观察不同浓度的bFGF对细胞凋亡的影响。结果和结论：bFGF对辐射诱导的血管内皮细胞凋亡有明显的抑制作用。     

碱性成纤维细胞生长因子及胰岛素样生长因子-1对体外兔关节软骨细胞的生物学效应

目的：了解胰岛素样生长因子－1（IGF－1）及碱性成纤维细胞生长因子（bFGF)对兔关节软骨细胞分裂增殖及功能代谢的影响，为组织工程方法修复关节软骨缺损提供实验依据。方法：取生长良好的兔正常关节软骨细胞，在含体积分数为10％新生小牛血清的DMEM条件下体外单层培养，细胞贴壁后随机分组，并分别加入不同浓度的bFGF和（或）IGF－1；培养液不加任何因子为对照组，以四唑盐（MTT）法检测细胞相对数，二苯胺显色法测各瓶细胞DNA含量，咔唑硫酸法测基质中糖醛酸含量，以间接反映蛋白多糖含量。并采用流式细胞技术进行细胞周期亚时相分析。结果：在实验浓度范围内，两种因子均促进细胞增殖，DNA合成及增加胞外基质中葡萄糖醛酸含量，且呈剂量－效应依赖关系。当IGF－1浓度≥10ng/ml,bFGF浓度≥1ng/ml时，其促进效果较显著（P＜0．05，0．01）。bFGF的促细胞增殖作用更为明显，最大为对照组的1．32倍，而IGF－1对细胞外基质葡萄糖醛酸含量的影响更显著，最大为对照组的1．78倍，bFGF能明显缩短DNA合成前期（G1期）和分裂前期及分裂期（G2M期）时间；bFGF IGF-1作用后，同样缩短G1期和G2M期时间，显著缩短G1期时间；而IGF－1仅缩短G2M期时间。结论：在本实验条件下，bFGF及IGF－1均以剂量依赖性方式影响细胞，刺激细胞增殖及功能代谢，两种因子的协同作用仅表现在对细胞的增殖作用上，对细胞功能代谢没有明显影响。两种因子促进细胞增殖是通过缩短细胞周期不同亚时相而实现的。     

体外培养的贲门癌组织对神经节突起的促生长和诱向作用

目的：探讨体外培养条件下肿瘤组织和原代培养肿瘤细胞对鸡胚神经节神经元的影响。方法：（１）贲门癌组织块和原代培养的肿瘤细胞与鸡胚背根神经节共培养;（２）贲门癌组织／原代细胞培养液与鸡胚背根神经节和交感神经节共培养;（３）贲门癌组织／原细胞培养液中加入神经生长因子（ｎｅｒｖｅ ｇｒｏｗｔｈ ｆａｃｏｔｒ,ＮＧＦ）抗体后与鸡前根神经节和交感神经节共培养。结果：（１）贲门癌组织块及原代培养的肿瘤细胞对神经     

BMP-2和bFGF在强直性脊柱炎活动期病理性表达及意义

目的：探讨骨形态发生蛋白2（BMP-2）和碱性成纤维细胞生长因子（bFGF）在强直性脊柱炎（AS）活动期的病理性表达及意义。方法：通过原位杂交技术检测活动期AS患者与对照组（骨盆骨折患者）骶髂关节滑膜组织中BMP-2、bFGF的表达,用图像分析系统检测阳性细胞与阴性细胞灰度值。结果：在活动期AS患者骶髂关节滑膜组织中BMP-2和bFGF阳性表达,而对照组骶髂关节滑膜组织中BMP-2和bFGF阴性表达,图像灰度值比较有统计学差异（P（0.01）。结论：BMP-2和bFGF均为强直性脊柱炎病理性成骨过程中重要的成骨因子,它们与强直性脊柱炎骶髂关节成骨硬化过程密切相关。控制BMP-2、bFGF的表达可能阻断AS的病理性成骨硬化,为治疗强直性脊柱炎提供新的思路。     

一次性大面积切痂防治烧伤早期脏器损害的临床研究

目的：探讨一次性大面积切痂防治烧伤早期脏器损害的作用及机制．方法：大面积烧伤病人６０例，分为一次切痂组（３５例）和常规分次切痂组（２５例）．一次性切痂组病人在漂浮导管监测下将Ⅲ度焦痂一次性全切除，同时观察血流动力和氧合指标、血浆内毒素和肿瘤坏死因子（ＴＮＦ）含量，以及病人血清对体外培养的内皮细胞的损伤作用．结果：与常规切痂组比较，一次性切痂组脏器损害和多器官功能障碍综合征（ＭＯＤＳ）的发生率均显著减少，治愈率显著提高．血浆内毒素和ＴＮＦ及内皮细胞培养液中乳酸脱氢酶（ＬＤＨ）和６－酮－ＰＧＦ１α含量明显下降；培养的内皮细胞基本维持原有形态和排列．结论：一次性大面积切痂通过遏制全身炎症反应综合征和减轻内皮细胞损伤，可有效地防治烧伤早期脏器损害和ＭＯＤＳ．     

恒河猴骨髓基质细胞体外诱导分化成神经干细胞的实验研究

为研究恒河猴骨髓基质细胞（BMSC）体外培养及诱导分化为神经干细胞的可行性，分离恒河猴BMSC，在体外应用神经干细胞培养液和细胞因子进行诱导分化，利用免疫细胞化学方法进行鉴定。结果发现，恒河猴BMSC能在体外培养中增殖，分化和表达干细胞特异性抗原神经巢蛋白（Nestin)，并最终能分化为胶质细胞样细胞和神经元样细胞；免疫细胞化学检测可见有神经元特异性烯醇化酶（NSE）和胶质原性纤维酸性蛋白（GFAP）抗原表达；未发现维甲酸（RA），表皮生长因子（EGF）和碱性成纤维细胞生长因子（bFGF)对骨髓基质干细胞的诱导分化具有明显影响作用。提示恒河猴BMSC是具有较强的自我更新能力和多分化潜能的细胞，在一定条件下，可分化为表达神经系细胞（神经元和神经胶质细胞）抗原的细胞，可作为神经细胞的种子细胞。     

比较不同诱导体系体外诱导华通胶间充质干细胞分化为内皮细胞的研究

目的比较诱导因子和非接触共同培养2种方法诱导人脐带华通胶间充质干细胞（human umbilical cord Wharton′s jelly mesenchymal stem cells,HUW MSCs）分化为内皮细胞的优劣。方法剖宫产取脐带用酶消化和组织块贴壁2种方法获得HUW MSCs。实验分诱导因子组、共同培养组和单纯培养组。诱导因子组用血管内皮细胞生长因子、碱性成纤维细胞生长因子、表皮生长因子联合诱导培养;共同培养组HUW MSCs与人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）非接触共同培养;单纯培养组只加培养液培养。培养周期为14 d。第7天检测CD309,第10天进行Dil标记乙酰化低密度脂蛋白（Dil labeled acetylated low density lipoprotein,Dil Ac LDL）吞噬实验;第14天检测CD31、血管假性血友病因子（von Willebrand factor,vWF）、内皮素 1(endothelin 1,ET 1）和一氧化氮（nitric oxide,NO）。结果各组细胞形态较实验前有显著变化,但均未见典型铺路石样改变。3组细胞表达CD309上调;诱导因子组和单纯培养组表达CD31下降,而共同培养组CD31明显上调。NO及ET 1含量检测均较HUVECs低,但差异无统计学意义（P>005）,3组之间比较差异同样无统计学意义（P>005）。vWF及Dil Ac LDL各组反应呈弱阳性。结论 HUW MSCs在诱导因子和内皮细胞旁分泌作用下均具有向内皮细胞分化倾向,使用诱导因子较非接触共同培养方法诱导HUW MSCs向内皮分化的效果更好。     

低氧预处理脐带间充质干细胞的旁分泌对成骨细胞功能的影响

目的：研究低氧预处理人脐带间充质干细胞（ umbilical cord mesenchymal stem cells,UCMSC）旁分泌作用对人成骨细胞（ MG－63）增殖、迁移及成骨的影响。方法分别于低氧及常氧条件下培养UCMSC,获取两种UCMSC条件培养基。实验分为3组：低氧培养基组、常氧培养基组、DMEM对照组,分别用3种不同的培养基常温培养MG－63。于培养1、3、5 d后用四氮唑盐（ mosmann tetrazoline colorimetry, MTT）比色法检测细胞增殖情况,记录各组的光密度值进行统计学分析;用划痕法检测细胞在不同培养条件下的迁移能力;于培养21 d后进行茜素红染色检测各组成骨钙结节的形成。酶联免疫吸附法（ ELISA）检测低氧及常氧条件培养基中血管内皮生长因子（ vascular endothelial growth factor,VEGF）的含量。结果 MTT法检测结果表明,低氧及常氧培养基组的MG－63细胞在培养1、3、5 d后的增殖能力均大于DMEM对照组,差异有统计学意义（P＜0．05）,且低氧组大于常氧组,差异有统计学意义（P＜0．05）;划痕法结果为此两组细胞迁移能力大于DMEM对照组,且低氧组细胞迁移能力大于常氧组;钙结节染色结果显示低氧组成骨钙结节形成最多,常氧组次之,DMEM对照组最少。 ELISA法检测低氧组VEGF含量高于常氧组,差异有统计学意义（P＜0．01）。结论低氧预处理UCMSC可增强其旁分泌功能,促进成骨细胞MG－63的增殖、迁移及成骨。     

VEGF、bFGF联合应用对胎鼠大脑皮质神经干细胞分化的影响

目的观察血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)联合应用对胎鼠大脑皮质神经干细胞(NSCs)分化的影响。方法取孕14dSD大鼠,收集胎鼠大脑皮质NSCs,采用无血清培养基传代培养,取P3代细胞,分别加入200μg/LVEGF和(或)20μg/LbFGF进行诱导分化,并设对照组(不加VEGF和bFGF),7d后采用免疫细胞化学方法检测巢蛋白(Nestin)、β微管蛋白-Ⅲ(β-tubulin-Ⅲ)和胶质纤维酸性蛋白(GFAP)的表达情况。结果从胎鼠大脑皮质中分离的细胞在体外悬浮生长,形成致密的球形细胞团,传代后能形成新的细胞球,免疫细胞化学染色显示细胞球nestin表达阳性。诱导分化后的细胞部分表达β-tubulin-Ⅲ,部分表达GFAP。与对照组比较,VEGF和(或)bFGF均可使NSCs向神经元方向分化的比例增加,其中VEGF+bFGF联合作用后NSCs的β-tubulin-Ⅲ阳性率(80.3%)最高,GFAP阳性率(18.6%)最低。bFGF作用后NSCs的β-tubulin-Ⅲ阳性率(60.4%)和GFAP阳性率(38.5%)与VEGF作用后(分别为60.3%、38.7%)比...     

Studies on mRNA expression of basic fibroblast growth factor in wound healing for wound age determination

We investigated the mRNA expression of basic fibroblast growth factor (bFGF) for wound age determination during dermal, cerebral, hepatic and renal wound healing in mice. The bFGF mRNA expression in the injured skin peaked at 1 h and was detected in epidermal cells, fibroblasts, endothelial cells and neutrophils. In the injured cerebrum the expression increased from 1 h and peaked at 48 h. In the intact cerebrum, bFGF was detected exclusively in the endothelial cells, whereas it was also detected in astrocytes during wound healing. Time-dependent expression of bFGF mRNA in skin and cerebrum was considered to be useful for wound age determination. On the other hand it was suggested that bFGF mRNA in astrocytes could be a vital sign of the acute phase. In hepatic and renal injuries, however, bFGF mRNA expression increased slightly in endothelial cells at 24 h, in neutrophils of the liver and in the glomeruli of the kidneys.     

Development of a biologically active Guglielmi detachable coil for the treatment of cerebral aneurysms. Part I: in vitro study

BACKGROUND AND PURPOSE: Stronger cellular adhesion on the surface of endovascular devices promotes accelerated healing of aneurysms. The purpose of this in vitro study was to study the cellular interaction on the surface of bioactive Guglielmi detachable coils (GDCs) after using the surface-modification technology, ion implantation. METHODS: Polystyrene (PS) dishes and platinum plates were used to simulate a GDC surface. They were treated with either simple collagen coating or collagen coating followed with ion implantation. Bovine endothelial cells (2-2.5 x 10(4) cells in 1 mL) were suspended in medium supplemented with 10% fetal bovine serum on the PS dishes or platinum plates. Five days after cell seeding, the strength of cell adhesion was evaluated by trypsin treatment and flow shear stress. The cell detachment from the PS and platinum surfaces was observed microscopically. RESULTS: Five days after cell seeding, both simple collagen-coated surfaces and collagen-coated ion-implanted surfaces showed uniform endothelial proliferation. After trypsin treatment, or under flow shear stress, stronger cell adhesion against chemical and flow shear stress was observed on the ion-implanted collagen-coated surface. In contrast, the endothelial cells were detached easily from the non-ion-implanted collagen-coated surface. CONCLUSION: Ion implantation in combination with protein coating improves the strength of surface cell adhesion when exposed to flow shear stress and proteolytic enzymes. Strong endothelial cell adhesion is reported to be important to achieve earlier endothelialization across the neck of an embolized aneurysm with bioactive GDCs. This new technology may improve long-term anatomic outcome in cerebral aneurysms treated with GDCs.     

EGFP标记的VEGF165重组质粒的构建及其在骨髓间充质干细胞中的表达

目的构建pIRES2-EGFP-VEGF165真核表达质粒,并在大鼠骨髓间充质干细胞内表达。方法应用DNA重组方法构建pIRES2-EGFP-VEGF165,并将其转染到大鼠骨髓间充质干细胞内,采用ELISA和MTT法检测转染后细胞培养上清液中VHGF165的含量及生物活性。结果成功构建了pIRES2-EGFP-VHGF165,将其转染骨髓间充质干细胞后,VEGF蛋白表达水平明显增高．转染后的细胞培养上清具有促使内皮细胞增殖的生物活性。结论成功构建了真核表达质粒pIRES2-EGFP-VEGFl65．将其转染骨髓间充质干细胞后可高水平地表达具有生物学活性的VEGF蛋白。     

The blood-tissue barrier of human brain tumors: correlation of scintigraphic and ultrastructural findings: concise communication

Through the first 2 hr, uptake of [Tc-99m]pertechnetate and of Co-57 bleomycin were assessed in 29 brain tumors and were correlated with the ultrastructure of the tumor's capillary endothelium. No difference in uptake was found between the two tracers. Permeability of brain tumors to these agents was found to be governed by the same ultrastructural features that determine permeability in experimental brain tumors: the type of junction between contiguous endothelial cells in the capillaries. Meningiomas, which showed very high uptake of the radiotracers, demonstrated open or punctate junctions with short fusion of apposed membranes. They also showed a large number of pinocytotic vesicles and fenestrae. Capillaries of tumors without uptake had a small number of short tight junctions (less than 0.25 mu) between adjacent endothelial cells and a relatively large number of long junctions (greater than 0.5 mu). In intracerebral tumors that showed relatively high uptake, the reverse was true: most of the junctions were short and only a few long junctions were found. That uptake of [Tc-99m]pertechnetate and of Co-57 bleomycin depends on tumor capillary ultrastructure (which determines the permeability) suggests the possibility of the use of radiopharmaceuticals as in vivo indicators of tumor permeability. Brain scintigraphy may help to asses brain-tumor availability to non-lipid-soluble chemotherapeutic drugs.     

MEBT/MEBO对血管内皮细胞粗面内质网影响的实验研究

目的:本课题旨在进一步探讨烧伤湿性医疗技术(MEBT/MEBO)对皮肤溃疡局部血管内皮细胞粗面内质网结构的影响,以期可以从不同侧面揭示MEBT/MEBO的改善微循环、祛腐生肌、促进创面再生与修复作用的分子生物学机制.方法:将40只SPF级SD雄性大鼠随机分为美宝湿润烧伤膏(MEBO)治疗组和贝复济对照组,每组20只,造模后于次日开始用药,观察用药后治疗组和对照组溃疡创面愈合情况、肉芽组织生长情况,并记录创面愈合时间.在造模后第8天,取两组同时相点创面组织,用电镜观察标本的超微结构,主要观察血管内皮细胞中粗面内质网的结构形态.结果:MEBO组大鼠的皮肤溃疡创面愈合情况明显好于贝复济组,创面愈合时间明显短于贝复济组,有显著性差异(P<0.05).组织病理学检测MEBO组治疗的创面,造模后第8天标本中血管内皮细胞中粗面内质网结构形态多于贝复济组.结论:MEBO较贝复济能明显的缩短实验性SPF级SD雄性大鼠体表皮肤溃疡模型皮肤慢性难愈合创面的修复愈合时间,具有促进创面愈合,减少瘢痕愈合的作用.其部分愈合机制:促进新生毛细血管的增殖等以加速肉芽组织形成.     

转录因子KLF4在内皮祖细胞分化过程中的表达及意义

目的探讨转录因子KLF4在内皮祖细胞分化过程中的表达及意义。方法以Ficoll密度梯度离心法从SD大鼠骨髓分离单个核细胞,用含20%胎牛血清的DMEM/F12培养基培养,以DiI-ac-LDL、FITC-UEA-I双荧光染色和FITC标记的CD133、CD34进行鉴定。细胞免疫化学法检测原代培养7d内皮祖细胞KLF4的表达,RT-PCR法检测原代培养5、10、15d后细胞内KLF4及一氧化氮合酶(eNOS)mRNA的表达。结果经DiI-ac-LDL、FITC-UEA-I双荧光染色鉴定证实获得的细胞为内皮祖细胞。细胞免疫化学检测发现KLF4表达于细胞核内;RT-PCR检测结果显示,5、10、15d大鼠内皮祖细胞的KLF4mRNA表达(分别为0.830±0.017,0.980±0.014,1.090±0.014)逐渐增强(P<0.01),eNOSmRNA表达(分别为0.310±0.008,0.500±0.011,0.650±0.010)也逐渐增强(P<0.01)。结论在内皮祖细胞分化为内皮细胞的过程中,KLF4的表达逐渐增强,并与成熟内皮功能相关。     

Transformation by radiation of rat foetal glial cells

Purpose: To establish and characterize an in vitro model of radiation-induced transformation of normal glial cells. Materials and methods: During the last week of gestation, pregnant Sprague-Dawley rats were either irradiated at 3.5Gy (0.022Gy h -1) with a 60 Co source or sham irradiated. On day 21 of gestation, cortical nerve cells from foetuses were isolated, and then maintained in culture for about 100 passages, in presence of 10 -9 g/ml of tetradecanoyl phorbol acetate (TPA). To follow transformation, various parameters: cell type, proliferation, clonogenicity, karyotypes and tumorigenicity, were studied at different passages. Results: As the number of passages increased, control cells lost their glial morphology and were immortalized. They kept on expressing specific markers of type 2 astrocytes (glial fibrillary acid protein (GFAP) and A2B5). Karyotypes remained near diploid. At all passages tested, they were not tumorigenic in nude mice. Irradiated cells expressed the 2A progenitor cell specific markers: GFAP, vimentin and A2B5. Karyotypes evolved toward polyploidy and cells displayed an iso 7 and a marker. These changes were synchronous with modifications in tumorigenicity. Metastases were even observed in nude mice. Conclusions: Cells from irradiated animals were fully transformed, while cells from sham irradiated animals were only immortalized.