Involvement of gene expressions in apoptosis of vascularendothelial cells induced by rattlesnake venom

Formation of apoptotic bodies is a typical character ofaPoptotic cell death, but how the processes are controlledis not known. In this study, we compared two apoptosisinducing systems in vascular endothelial cells (VEC). Wefound that the formation of aPoptotic bodies during apop-tosis induced by rattlesnake venom, which is an unique andspecific aPoptosis inducer to vascular endotheliaI cells, wasmuch faster than that induced by deprivation of survivalfactors (aFGF and serum). When we blocked the synthesisof mRNAs in cells treated with rattlesnake venom by DRB(5, 6- dichloro- 1 -β- D- rib ofur anosylb enzimidazole ), an in-hibitor of transcription, the formation of aPoptotic bodieswas dramatically inhibited. We examined the expressionof Psa gene and found that its expression was much higherin apoptosis induced by rattlesnake venom than that inaPoptosis induced by deprivation of aFGF and serum. Ourresults suggest that gene expression is important and P53gene may play a major role in inducing the formation ofapoptotic bodies in VEC.

Effects of Shenggu Injection (生骨注射液) on mRNA Expression of Vascular Endothelia Growth Factor in Rat Osteoblasts in vitro

Objective： To study the effects of Shenggu injection （生骨注射液,SGI） on mRNA expression of vascular endothelia growth factor （VEGF） in rat osteoblasts in vitro and to explore its possible molecular mechanisms in promoting fracture healing. Methods： Rat osteoblasts cultured in vitro were stimulated with SGI according to the protocol. The expression levels of VEGF mRNA in the cells in every group were examined by reverse-transcriptase ploymerase chain reaction （RT-POR）. Results： When osteoblasts were stimulated with different concentrations of SGI for 5 days, the expression of VEGF mRNA peaked with 1 mg/ml SGI on the 5th day. When treated with 1 mg/ml SGI from the 1st to the 5th day, the expression of VEGF mRNA increased gradually with the increase of culturing time. Conclusion： SGI could promote significantly the expression of VEGF mRNA in rat osteoblasts in vitro. The levels of expression of VEGF mRNA changed along with different concentrations and stimulating time of SGI.

Glioma-conditioned Medium Blocks Endothelial Cells' Apoptosis Induced by Hypoxia and Promotes Its Angiogenesis via Up-regulation of u-PA/u-PAR

Objective： To investigate the role of urokinase-type plasminogen activator/urokinase-type plasminogen receptor（u-PA/u-PAR） system in glioma angiogenesis under hypoxic conditions, we studied the effect of glioma-conditioned medium on the hypoxia induced changes in human endothelial-like ECV304 cells proliferation, apoptosis, cord formation in vitro and u-PA/u-PAR expression. Methods： MTT assay was used to examine the changes in cell proliferation. Cell apoptosis was analyzed by Hoechst 33258 staining. Matrigel cord-like formation assay was used to evaluate the angiogenesis ability of ECV304 cells in vitro. Expressions of u-PA/u-PAR mRNA were detected by quantitative real-time RT-PCR. Results： Hypoxia inhibited ECV304 cells proliferation and induced cell apoptosis. Hypoxic conditioned medium（H-CM） while not normoxic conditioned medium（N-CM） of U251 glioma ceils partially blocked the effect of hypoxia on ECV304 cells proliferation and apoptosis. H-CM of U251 glioma ceils also promoted the cord formation of ECV304 cells seeded on matrigel. When u-PA or u-PAR monoclonal antibodies were added into ECV304 cells culturing medium, cord formation ability was partially inhibited. H-CM of U251 glioma cells induced uPA and uPAR expression in ECV304 cells. Conclusion： These suggest that u-PA/u-PAR system is involved in glioma angiogenesis trigged by hypoxic microenviroment.

Cloning and Identification of A Novel Variant of Human Vascular Endothelial Growth Factor

A novel variant of human vascular endothelial growth factor (h'VEGF165) cDNA was amplified by nested PCR method from the HL60 1 cells and was cloned into a eukaryotic expressing vector pcDNA 3 to construct a recombinant plasmid pCD-h'VEGF165. The amplified h'VEGF165 cDNA fragment was identified by enzyme digestion and DNA sequencing methods. Also, wild-type hVEGF165 cDNA was obtained, identified and cloned into a eukaryotic expressing vector pcDNA3 by using the same methods. The results of DNA sequencing showed that h'VEGF165 cDNA cloned from HL60 1 was 600 bp in size with 8 % of the base sequence in h'VEGF165 cDNA being changed as compared with the base sequence in the wild-type hVEGF165 cDNA. The results of sequencing of hVEGF165 which was cloned from HL60 by us were consistent with the reports completely.

Estimation of the density of neural,glial,and endothelial lineage cells in the adult mouse dentate gyrus

The dentate gyrus subregion of the mammalian hippocampus is an adult neural stem cell niche and site of lifelong neurogenesis.Hypotheses regarding the role of adult-born neuron synaptic integration in hippocampal circuit function are framed by robust estimations of adultborn versus pre/perinatally-born neuron number.In contrast,the non-neurogenic functions of adult neural stem cells and their immediate progeny,such as secretion of bioactive growth factors and expression of extracellular matrix-modifying proteins,lack similar framing due to few estimates of their number versus other prominent secretory cells.Here,we apply immunohistochemical methods to estimate cell density of neural stem/progenitor cells versus other major classes of glial and endothelial cell types that are potentially secretory in the dentate gyrus of adult mice.Of the cell types quantified,we found that GFAP^(+)SOX2^(+)stellate astrocytes were the most numerous,followed by CD31^(+)endothelia,GFAP-SOX2^(+)intermediate progenitors,Olig2^(+)oligodendrocytes,Iba1+microglia,and GFAP^(+)SOX2^(+)radial glia-like neural stem cells.We did not observe any significant sex differences in density of any cell population.Notably,neural stem/progenitor cells were present at a similar density as several cell types known to have potent functional roles via their secretome.These findings may be useful for refining hypotheses regarding the contributions of these cell types to regulating hippocampal function and their potential therapeutic uses.All experimental protocols were approved by the Ohio State University Institutional Animal Care and Use Committee(protocol#2016A00000068)on July 14,2016.

Correlation between endothelia cells activation and imbalance of cytokines in pulmonary hypertension of congenital heart disease

Objective To explore the correlation between endothelia cells activation and cytokines (ET-1, NO) levels in patients with pulmonary hypertension (PH), and to discuss their roles in the development of PH. Methods Twenty patients with simple ventricular septal defect (VSD) were chosen as controls, and 30 patients with PH were studied. Plasma levels of ET-1 and NO were measured by radioimmunoassay or colorimetric method. Before cardiopulmonary bypass was established, the specimens from right lung were fixed with formaldehyde solution, embedded with paraffin and stained by SP immunohistochemistry. Intercellular adhesion molecule-1 (ICAM-1) expression was measured through the determination of the light density with computer imaging technology. Results Compared with that of the patients with simple VSD, the light density of ICAM-1 and plasma level of ET-1 increased in patients with PH; but plasma level of NO decreased (P<0.05). Positive correlation was observed between ICAM-1 and ET-1/NO (P<0.05). Conclusion Endothelia cells activation and imbalance of ET-1/NO might play an important role in the development of PH.

Effects of recombinant adeno-associated viruses expressing human vascular endothelial growth factor 165 on spinal cord injury

Numerous studies have confirmed that vascular endothelial growth factor （VEGF） improves the function of neural cells following spinal cord injury （SCI）. However, some studies have also verified that VEGF cannot significantly induce the increase in vascular density at or surrounding the lesion, and that VEGF therapy exacerbated secondary damage following SCI. Based on the dual effects of VEGF on SCI, we constructed the recombinant adeno-associated viruses （rAAV）-hVEGF165-IRES-human recombinant green fluorescent protein （hrGFP） （AAV-VEGF） and rAAV-IRES-hrGFP （AAV-GFP）. Our results suggested that rAAV expressed hVEGFles, and a low dose of VEGF relieved increased vascular permeability, improved microcirculation in the local spinal cord, lessened spinal cord edema, and decreased neuronal apoptosis. These results verified that the releasing effects of the rAAV virus vector had protective effects on the spinal cord.

PPAR-γ甲基化、VEGF在乳腺增生患者中的表达水平及意义

目的 探讨过氧化物酶体增殖物激活受体-γ(PPAR-γ)甲基化、血管内皮生长因子(VEGF)在乳腺增生患者中的表达水平及意义。方法 选取2020年1月至2022年3月在涿州市医院就诊的158例良性乳腺增生患者为观察组,同期确诊的158例早期乳腺癌患者为恶性对照组,另选取同期158例体检健康者为健康对照组。对比3组及观察组不同乳腺影像报告与数据系统(BI-RADS)分级患者的PPAR-γ甲基化水平和VEGF水平。采用Spearman相关系数分析PPAR-γ甲基化水平、VEGF水平与BI-RADS分级的相关性,多因素Logistic回归分析影响BI-RADSⅣ级乳腺增生进展为乳腺癌的危险因素。结果 观察组PPAR-γ甲基化水平和VEGF水平高于健康对照组,但低于恶性对照组,差异均有统计学意义(P<0.05);观察组患者随着BI-RADS分级增加,PPAR-γ甲基化水平、VEGF水平呈升高趋势(P<0.05);PPAR-γ甲基化水平、VEGF水平均与BI-RADS分级呈正相关(r=0.836、0.763,均P<0.001);校正体重指数、初产年龄、绝经、乳房周期性疼痛、每天体育锻炼时间、月经规律、定期体检因素后,PPAR-γ甲基化水平及VEGF高表达仍是BI-RADSⅣ级乳腺增生进展的危险因素。结论 乳腺增生患者PPAR-γ基因甲基化水平升高,且VEGF呈高表达,二者与乳腺病变的发生、发展密切相关。

人脑胶质瘤侵袭微生态系统的形态学观察

目的 探讨人脑胶质瘤侵袭微生态系统的形态学特征。 方法 利用光镜及透射电镜观察 12例手术切除的脑胶质瘤标本中微血管内皮细胞和基底膜及其与肿瘤细胞的相互关系。 结果 光镜下可见侵袭性生长的瘤细胞可在血管周形成巢状结构。电镜下不同胶质细胞起源的胶质瘤中的微血管内皮细胞基本上为无孔型 ,偶见窗孔形成。内皮细胞下基质完整 ,基底膜呈局限性或广泛性增厚 ,有的伴有多层基板样结构形成。基底膜靠近瘤细胞侧常出现大小不等、数量不一的虫蚀样空洞 ,含有较大空洞的基底膜常突向瘤细胞侧。有的血管外腔扩张 ,腔内可见瘤细胞以单个形式侵袭 ,少见瘤细胞伸出伪足突入基底膜及穿透内皮细胞间隙现象 ,瘤细胞与基底膜相互接触处呈光滑平整的界面。 结论 无孔型内皮细胞、肿瘤微血管基底膜结构的局限性或广泛性增厚和虫蚀样空洞的改变 ,及少见瘤细胞伸出伪足突入其间 。

野百合碱诱导实验性肺动脉高压病理形态观察

目的 :30只Wistar大鼠一次性腹腔注射野百合碱 (monocrotaline ,MCT) (6 0mg kg体重 ) ,建立肺心病模型 ,观察野百合碱诱导肺动脉高压的机理。方法 :利用注射野百合碱方法建立肺动脉高压的动物模型 ,通过光学显微镜及电子显微镜观察野百合碱对肺动脉血管内皮细胞的损伤 ,和导致肺动脉血管平滑肌增生及右心室的变化 ,并分析病变之间的关系。结果 :野百合碱注射可使肺动脉血管内皮细胞损伤 ,并诱导肺动脉血管壁的重建 ,特别是平滑肌明显增生 。

腰椎间盘中血管内皮生长因子的表达及其意义

目的 :观察并探讨腰椎间盘中内源性血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)的表达分布规律及其意义。方法 :采用免疫组织化学技术检测 1 6例胎儿期、8例生长期、1 2例成熟退变期和 42例突出椎间盘中VEGF的表达。结果 :胎儿椎间盘脊索细胞和纤维环外层血管内皮细胞出现阳性免疫染色 ,阳性率为 87 5 % ;生长期和成人期椎间盘未见阳性表达 ;退变突出组总阳性率为 83 3 %。VEGF表达主要出现于破裂型和游离型椎间盘突出 (P <0 0 1 ) ,其细胞来源主要是突出椎间盘组织中毛细血管内皮细胞、髓核内的类软骨细胞及单核巨噬细胞。年龄与VEGF表达阳性率关系不大 (P >0 0 5)。病程超过 1年者VEGF阳性率明显低于 1年以内者 (P <0 0 5)。结论 :胎儿期和破裂型 (包括游离型 )突出椎间盘组织可产生内源性VEGF ,突出椎间盘中VEGF的阳性表达与病程。

低G预适应对重复+10Gz应激大鼠左心室收缩功能和心肌内皮素与前列环素含量的影响

目的进一步验证低G预适应对 +Gz应激致心肌损伤的防护效应。方法 42只雄性Wistar大鼠随机分为 3组 (每组n =1 4) :A( + 1Gz)组、B( + 1 0Gz)组和C(低G预适应 )组。B组实验条件为 :+ 1 0Gz峰值暴露 30s ,5次 /d(间隔 + 1Gz 1min) ,3d/wk ,共 3wk ;A组仅受 + 1Gz作用 5min ;C组在每日+ 1 0Gz暴露前约 1h受 + 2Gz作用 5min ,其余处理同B组。于末次 +Gz实验后次日断头处死大鼠 ,速取心脏离体观察左室收缩压的变化 ;另取心肌组织测定其内皮素与前列环素含量。结果与A组相比 ,B组心肌收缩功能明显降低 ,心肌内皮素含量显著升高 ;而低G预适应使 + 1 0Gz应激大鼠左室收缩压明显增加 ,心肌内皮素含量明显降低。结论低G预适应对重复高G应激大鼠左室功能和心肌血管内皮细胞分泌功能均有一定的保护作用。

蛇葡萄素的血管生成抑制作用

目的：探讨蛇葡萄素对内皮细胞及肿瘤细胞的生长及其血管生成的影响。方法：体外用MTT法对牛主动脉内皮细胞进行增殖抑制实验；用ELISA法、免疫组化染色法以及流式细胞仪观察蛇葡萄素对人肝璃细胞Bel-7402分泌VEGF、bFGF的抑制作用；体内观察蛇葡萄素对人肝癌Bel-7402裸鼠移植瘤的抑制作用。结果：蛇葡萄素对牛主动脉内皮细胞的增殖在6．4～51．2μg/ml呈现浓度依赖性抑制作用，IC50=22.0±4.0μg/ml。ELISA法测定结果显示，12．8μg/ml、25.6μg/ml、38．4μg/ml蛇葡萄素对肝癌Bel-7402细胞分泌VEGF的抑制率分别为14.2％、40．0％及49．6％。免疫组化染色结果显示，12．8μg/ml以卜的蛇葡葡素能使细胞体积缩小，分泌VEGF、hFGF明显减少。流式细胞仪检测VEGF、hFGF结果，25．6μg/ml及38．4μg/ml蛇葡萄索对VGEF的抑制率分别为32．2％及57.4％，对bFGF的抑制率分别为54．9％及62．6％。蛇葡葡素浓度为100、150、200mg／kg时对人肝痛Bel-7402裸鼠移植瘤的抑制率分别可达24．3％、41．4％及45．7％。结论：蛇葡萄素在体外能有效抑制内皮细胞增殖投人肝癌Bel-7402细胞分泌VEGF和bFGF，具有抑制血管牛成的作用；体内能有效抑制人肝癌Bel-7402裸鼠移植瘤的生长，提示蛇葡萄素也许可作为一个肿瘤血管牛成抑制剂用于肿瘤防治。

血管内皮细胞生长因子在烧伤后肉芽组织和增生性瘢痕中的表达

目的观察血管内皮细胞生长因子 (VEGF)在烧伤创面肉芽组织和烧伤后不同时期增生性瘢痕中的动态变化 ,探讨VEGF在烧伤创面修复过程中的表达规律及其作用。方法选择临床手术切除的新鲜烧伤创面肉芽及各个时期的增生性瘢痕 ,制成匀浆液 ,应用ELISA方法检测匀浆液中VEGF的变化。结果VEGF可在烧伤创面肉芽组织中有一定的表达 ,随着增生性瘢痕的发展 ,其表达量进一步增加 ,在 4— 6个月的增生性瘢痕中 ,表达量达到峰值。瘢痕成熟后 ,VEGF的组织表达逐渐下降。

水蛭提取液对凝血酶诱导血管内皮细胞释放凝血因子的影响

目的 观察水蛭提取液对凝血酶诱导人脐静脉内皮细胞（HUVECs）释放血管性血友病因子（vWF）、纤溶酶原激活物抑制剂-1（PAI-1）、组织型纤溶酶原激活物（t-PA）的影响,探讨水蛭抗凝的作用机制.方法 体外培养 HUVECs,将生长良好的2～3代细胞分为对照组、凝血酶刺激组及水蛭提取液高、中、低剂量组5组.将生药浓度600、300、150 mg/L的水蛭提取液加入到10 kU/L凝血酶刺激的HUVECs培养12 h,对照组加等量RPMI 1640培养液.用酶联免疫吸附法（ELISA）检测细胞培养上清液中vWF、PAI-1、t-PA的含量.结果 与对照组比较,凝血酶刺激组细胞培养上清液中vWF、PAI-1（μg/L）含量显著升高[vWF：（11.01±0.54）%比（7.05±0.49）%;PAI-1：72.26±0.85比55.89±1.26,均P〈0.01],t-PA（μg/L）含量显著降低（15.15±1.41比34.29±1.22,P〈0.01）.与凝血酶刺激组比较,水蛭提取液高、中、低剂量组vWF、PAI-1含量[vWF：（9.48±0.64）%、 （8.57±0.50）%、 （7.97±0.44）%;PAI-1：58.30±0.90、 62.69±1.01、 67.47±1.28]显著降低,t-PA含量（32.59±1.46、 26.40±0.82、 21.06±1.13）显著升高（均P〈0.01）.说明水蛭提取液能显著减弱凝血酶诱导HUVECs释放vWF、PAI-1,增强凝血酶促进HUVECs表达t-PA.结论 水蛭具有明显的抗凝作用,其作用机制可能为参与调节内皮细胞释放vWF,调节纤溶平衡及保护受损的血管内皮细胞.

脑出血急性期内皮祖细胞形态学和功能变化及其意义

目的探讨高血压脑出血急性期患者外周血来源的内皮祖细胞动态形态学和功能变化及其意义。方法分离、诱导分化高血压脑出血急性期患者和高血压患者外周血内皮祖细胞,动态观察两组内皮祖细胞形态学变化,检测两组患者内皮祖细胞集落和细胞计数、黏附、增殖及迁移能力并进行对比研究。结果高血压脑出血组内皮祖细胞集落计数为(5.87±0.55),细胞计数为(74.73±11.38),黏附能力为(26.13±5.13),增殖能力为(0.273±0.060),迁移能力为(15.83±2.02);高血压组内皮祖细胞集落计数为(1.73±0.24),细胞计数为(56.70±9.72),黏附能力为(19.97±3.07),增殖能力(0.192±0.058)而迁移能力为(10.33±2.30)。两组相比,脑出血组内皮组细胞功能明显增强,具有显著性统计学差异(P<0.05或P<0.01)。与高血压组相比,脑出血组内皮祖细胞和集落出现较早,集落体积较大和典型,细胞密度较大。结论高血压脑出血急性期患者内皮祖细胞数目增多,黏附、增殖和迁移功能增强。这可能有助于血管损伤的修复和再生。

力学环境影响下肿瘤内外血管生成的数值模拟

目的考察肿瘤内外不同的力学环境对肿瘤血管生成过程中内皮细胞运动的影响,数值生成肿瘤内外血管网络。方法肿瘤外正常组织和肿瘤组织设为线性粘弹性体。肿瘤外正常组织内皮细胞扩散系数是常数;肿瘤内,肿瘤细胞的活性下降导致组织的黏附力降低,内皮细胞扩散系数设为离中心的幂次函数。建立内皮细胞运动的扩散方程,数值生成肿瘤内外血管网络。结果当考虑肿瘤内外不同力学环境对内皮细胞运动的影响时,可以获得相对真实的分层毛细血管网络:在肿瘤外层富含毛细血管,越靠近肿瘤中心,血管越稀少。在忽略力学环境的影响时,无法得到实验观测到的分层网络。结论肿瘤内外不同的力学环境对肿瘤血管生成的网络结构有着重要影响。

糖皮质激素瘤内注射治疗婴幼儿血管瘤机理的研究

目的:探讨糖皮质激素治疗婴幼儿血管瘤的机理.方法:对50例不同组别血管瘤(治疗显效组18例,无效组17例,未治疗组15例)及16例正常皮肤标本进行:①凋亡研究:光镜及透射电镜下观察组织形态学变化;TUNEL法检测血管内皮细胞(VEC)的凋亡水平;IHC法检测凋亡调控基因bcl-2,c-myc的表达;②增殖水平研究:IHC法检测PCNA.结果:①组织形态学变化:光镜下各组均无明显炎症及坏死征象,透射电镜观察显效组有较多VEC凋亡征象.②VEC增殖及凋亡:显效组VEC的凋亡水平显著高于无效组、未治疗组及正常皮肤组(TUNEL:47.86±3.14,22.05±3.93,19.20±2.82,35.51±3.81,P<0.01),而增殖水平明显低于上述三组(PCNA:10.25±3.89,44.33±5.01,56.32±4.12,17.91±3.76,P<0.01).③凋亡调控情况:显效组bcl-2表达显著低于无效组和未治疗组(21.28±4.70,49.96±5.33,52.51±7.69,P<0.01);c-myc表达也明显低于后两组(25.20±5.01,51.81±5.68,56.28±6.92,P<0.01).结论:①糖皮质激素可能通过诱导VEC凋亡及抑制其增殖而促进血管瘤消退.②糖皮质激素对VEC凋亡的影响可能与其下调bcl-2和c-myc的表达水平有关.

虫草提取物对高糖诱导的脐静脉内皮细胞损伤模型的保护作用

目的研究虫草提取物对人脐静脉内皮细胞损伤模型的影响,探讨其保护内皮功能的机制。方法原代培养人脐静脉内皮细胞,高糖(40mmol/L)孵育,建立细胞损伤模型。实验分组:空白组、高糖组、冬虫夏草组、蛹虫草组。MTT法检测细胞增殖;SA-β-半乳糖苷酶(SA-β-gal)染色法检测SA-β-gal染色阳性率;流式细胞术测定细胞周期及细胞内活性氧;实时荧光定量PCR检测衰老相关的p16、p21、沉默信息调节因子1(SIRT1)、Bcl-2mRNA表达水平。结果与空白组比较,高糖组细胞增殖明显下降,细胞内活性氧增加(P<0.05)。与高糖组比较,蛹虫草组及冬虫夏草组细胞增殖增加,S期细胞明显增多,SIRT1mRNA水平显著升高,细胞内活性氧和p21mRNA水平均显著降低(P<0.05)。结论虫草提取物对损伤内皮具有保护作用,其机制可能与促进SIRT1mRNA表达,减轻细胞氧化应激损伤,抑制p21mRNA表达,促进内皮细胞生长增殖有关。

SD大鼠血管内皮细胞损伤模型的制作方法

目的 :建立SD大鼠血管内皮细胞损伤动物模型。方法 :SD大鼠随机分为正常对照组和模型组。模型组大鼠皮下注射稀释 1.5倍的肾上腺素 0 .0 5 / 10 0 g ,连续 5天。于第 4天起 ,每二次给药中间时间将大鼠置于 0℃冰水中 5min ,共 4次。正常对照组则皮下注射等量 0 .9%NS。于第 6天 ,二组大鼠自颈总动脉取血 ,分别测定CEC计数、t PA、PAI活性、6 Keto PGF1α含量及血小板最大聚集率。结果 :模型组较正常对照组大鼠CEC计数、PAI活性、血小板最大聚集率明显升高 (P <0 .0 0 1～ 0 .0 1) ,t PA活性、6 Keto PGF1α含量降低 (P <0 .0 1～ 0 .0 5 )。结论 :大剂量肾上腺素加冰泳刺激确能造成SD大鼠血管内皮细胞损伤。