Advances in the differentiation of pluripotent stem cells into vascular cells

Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Temporal alterations in pericytes at the acute phase of ischemia/reperfusion in the mouse brain

Pericytes,as the mural cells surrounding the microvasculature,play a critical role in the regulation of microcirculation;however,how these cells respond to ischemic stroke remains unclear.To determine the temporal alterations in pericytes after ischemia/reperfusion,we used the 1-hour middle cerebral artery occlusion model,which was examined at 2,12,and 24 hours after reperfusion.Our results showed that in the reperfused regions,the cerebral blood flow decreased and the infarct volume increased with time.Furthermore,the pericytes in the infarct regions contracted and acted on the vascular endothelial cells within 24 hours after reperfusion.These effects may result in incomplete microcirculation reperfusion and a gradual worsening trend with time in the acute phase.These findings provide strong evidence for explaining the“no-reflow”phenomenon that occurs after recanalization in clinical practice.

Migraine attack restores the response of vascular smooth muscle cells to nitric oxide but not to norepinephrine

AIM:To clarify whether the vasoconstrictory response is impaired and to study vascular function in patients with migraine during the headache attack.METHODS:We studied vascular reactivity in the resistance arteries by using the forearm perfusion technique associated with plethysmography.We measuredforearm blood flow by strain-gauge plethysmography during intra-brachial infusion of acetylcholine,sodium nitroprusside or norepinephrine in 11 controls and 13patients with migraine,11 of them(M) in the interval between the migraine attacks and 4 during a headache attack(MH).Written informed consent was obtained from patients and healthy controls,and the study was approved by the Ethics Committee of the University Federico Ⅱ.RESULTS:Compared to healthy control subjects,in patients with migraine studied during the interictal period,the vasodilating effect of acetylcholine,that acts through the stimulation of endothelial cells and the release of nitric oxide,was markedly reduced,but became normal during the headache attack(P<0.05by analysis of variance).The response to nitroprusside,which directly relaxes vascular smooth muscle cells(VSMCs),was depressed in patients with migraine studied during the interictal period,but normal during the headache attack(P<0.005).During norepinephrine infusion,forearm blood flow decreased in control subjects(-40% ± 5%,P<0.001).In contrast,in patients with migraine,either when studied during or free of the headache attack forearm blood flow did not change compared to the baseline value(-3%±13% and-10.4%±15%,P>0.05).CONCLUSION:In migrainers,the impaired relaxation of VSMCs is restored during the headache attack.The vasoconstrictory response is impaired and remains unchanged during the migraine attack.

EFFECT OF QUERCETIN ON CULTURED HUMAN VASCULAR ENDOTHELIAL CELLS

Objective To study the effects of quercetin (Que) on the release of endothelin-1 (ET-1) and prostacylin(PGI2) by normal human vascuiar endothelial cell (VEC). Methods Radioimmunoassay(RIA) was used to assess the amount of ET-1 and PGI2 produced by VEC. VEC prollferation was assessed by tetrazolium(MTT) assay. Results Que increased the normal VEC prollferation at the concentration or 5, 2o, 4o, 8o, 1oompol/L and increased the production of PG12 and inhibits the release of ET by the normal VEC at the concentratiou or 5, 2o and 8ompol/L. Que at the concentration of 5, 2o and 8omol/L had no direct effect on morphology of the normal VEC. ConcIusion Que can stimulate the proliferation of VEC and inhibit tbe reIease of ET-1 and increase the formation of PGI2. The data suggest that Que might be beneficial for the prevention and treatment of vascular endothelial injury-related cardiovascular diseases, such as atherosclerosls and thromboembolism diseases.

榛蘑多糖对乙醇所致大鼠血管内皮细胞损伤的保护作用

探究榛蘑多糖对乙醇所致大鼠血管内皮细胞损伤的保护作用。将40只SD大鼠随机分成4组(正常对照组、损伤组、榛蘑多糖低剂量组、榛蘑多糖高剂量组)。除正常对照组外的其余各组均按10 mL/kg mb灌胃体积分数40%乙醇诱导血管内皮细胞损伤。榛蘑多糖低、高剂量组分别以100、400 mg/kg mb灌胃榛蘑多糖,其余组以等体积生理盐水代替,实验共进行4周。使用苏木精-伊红(hematoxylin-eosin,HE)染色观察颈动脉组织病理变化;检测血清甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,T-CHO)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、内皮型一氧化氮合酶、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、一氧化氮(NO)、内皮素1(endothelin 1,ET-1)、超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)水平。此外,以600μmol/mL乙醇诱导人脐静脉内皮细胞损伤模型,分析不同剂量(100、400μg/mL)榛蘑多糖对细胞活性氧(reactive oxygen species,ROS)、线粒体跨膜电位、细胞凋亡以及凋亡相关蛋白B淋巴细胞瘤2(B cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)和Caspase-3表达的影响。结果表明:榛蘑多糖减轻乙醇所致的血管内膜损伤,降低T-CHO、TG、LDL-C、iNOS、NO、ET-1和MDA水平,提高HDL-C和SOD活性;在体外条件下,榛蘑多糖降低细胞ROS水平,抑制乙醇所致的线粒体跨膜电位的下降和细胞凋亡,并提高Bcl-2/Bax,下调Cleaved caspase-3表达水平。综上,榛蘑多糖对乙醇诱导的大鼠血管内膜损伤有保护作用,机制可能与其降脂、抗氧化和抗凋亡作用有关。

支气管肺发育不良模型小鼠肺周细胞变化及对肺血管内皮细胞成管的影响

背景:肺周细胞位于肺血管连接凹陷处,与肺血管的形成和稳定有着密切关系。然而,在支气管肺发育不良发病过程中肺周细胞如何影响肺血管内皮细胞活动的研究较少。目的:分析支气管肺发育不良不同时期肺周细胞亚群与内皮细胞间数目变化关系,探讨血小板源性生长因子受体β、蛋白聚糖NG2、α-平滑肌肌动蛋白三阳性(PDGFR-β^(+)NG2^(+)α-SMA^(+))肺周细胞对肺血管内皮细胞早期成管活动的影响。方法:①动物实验:取新生C57BL/6小鼠12只,于出生24 h内采用随机数字表法分为常氧组及高氧组,每组6只,高氧组小鼠暴露于体积分数85%O_(2)环境下喂养,建立支气管肺发育不良动物模型;常氧组小鼠置于同一室内空气中喂养。出生后7,14 d取小鼠肺组织,苏木精-伊红染色观察肺组织病理改变,流式细胞术检测肺周细胞3种亚群与血管内皮细胞数量。②细胞实验:将小鼠第3代PDGFR-β^(+)NG2^(+)α-SMA^(+)肺周细胞与小鼠肺血管内皮细胞共培养(实验组),细胞比例为1∶4,以单独培养的小鼠肺血管内皮细胞为对照组。培养15 h后,分析两组血管内皮细胞成管差异。结果与结论:①动物实验:苏木精-伊红染色显示,7 d时,常氧组小鼠肺组织结构规则,存在明显肺泡结构,大小均匀;高氧组小鼠肺组织肺泡数量较少,肺泡形态不规则。14 d时,常氧组小鼠肺泡逐渐发育成熟,肺泡结构逐步规整,大小均匀,肺泡密度逐渐增加;高氧组小鼠肺组织结构相对紊乱,肺泡形成滞后体积逐渐增大,肺泡结构简单化。流式细胞术检测显示,与常氧组相比,高氧组7,14 d肺组织中的PDGFR-β^(+)NG2-α-SMA^(+)和PDGFR-β^(+)NG2^(+)α-SMA^(+)肺周细胞数量增加(P<0.01),PDGFR-β^(+)NG2^(+)α-SMA-肺周细胞与肺血管内皮细胞数量减少(P<0.01,P<0.05)。②细胞实验:对照组肺血管内皮细胞排列成条索状向四周延伸,部分区域中形成类似管腔样结构;实验组未观察到PDGFR-β^(+)NG2^(+)α-SMA^(+)肺周细胞及其伪足,肺血管内皮细胞形成的不规则网格状结构较对照组明显减少,内皮细胞以团块样聚集为主。③结果表明:支气管肺发育不良小鼠肺组织以α-SMA^(+)周细胞亚群为主,PDGFR-β^(+)NG2^(+)α-SMA^(+)肺周细胞则能直接抑制肺血管内皮细胞成管活动,可能参与了支气管肺发育不良发生过程中肺微血管发育异常的过程。

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P【0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.

Adhesion molecule and proinflammatory cytokine gene expression in hepatic sinusoidal endothelial cells following cecal ligation and puncture

INTRODUCTIONMultiple organ dysfunction syndrome (MODS) isthought to be a frequent consequence of sepsis[1-3].Despite substantial advances in our knowledge and understanding of the basic pathophysiologic mechanisms[4-7], in critically ill patients infections and sepsis are still associated with a high mortality[8,9].

黄芪甲苷对氯吡格雷致大鼠胃黏膜损伤的治疗作用及机制

目的 探讨黄芪甲苷对氯吡格雷致大鼠胃黏膜损伤的治疗作用及机制。方法 将50只SD大鼠随机分为对照组、模型组及黄芪甲苷低、中、高剂量组,每组10只,分别予生理盐水1 mL/100 g、氯吡格雷15.6 mg/kg、氯吡格雷15.6 mg/kg+黄芪甲苷15、30、60 mg/kg灌胃,每日1次,干预1周后脱颈处死大鼠。用损伤指数评分评价胃黏膜损伤情况,用免疫组化SABC染色法检测胃黏膜组织中血管内皮细胞生长因子(VEGF)、磷酸化血管内皮细胞生长因子受体2(p-VEGFR2)蛋白表达。结果 与对照组比较,模型组胃黏膜损伤指数评分高,VEGF、p-VEGFR2蛋白表达低(P均<0.05);与模型组比较,各黄芪甲苷组胃黏膜损伤指数评分均低,VEGF、p-VEGFR2蛋白表达高(P均<0.05);黄芪甲苷低、中、高剂量组胃黏膜损伤指数评分逐渐降低,VEGF、p-VEGFR2蛋白表达逐渐升高(P均<0.05)。结论 黄芪甲苷可减轻氯吡格雷所致胃黏膜损伤,其作用机制可能与上调VEGF、p-VEGFR2表达有关。

基于Ang1/Tie2信号通路研究周细胞调控腹膜血管新生的作用机制

目的:本研究旨在通过观察血管周细胞与血管内皮细胞间的相互作用,探讨周细胞调控腹膜血管新生的作用机制。方法:使用转化生长因子β1(TGF-β1)干预人微血管周细胞(HMPCs)24、48和96 h,Western blot检测HMPCs中血管生成素1(Ang1)的表达。收集TGF-β1干预HMPCs后的上清液作为条件培养基(T-HMPCs-CM),用T-HMPCs-CM处理人腹膜血管内皮细胞(HPVECs)构建间接共培养模式;将TGF-β1处理过的HMPCs与HPVECs共同接种于基质胶上,构建直接共培养模式;以未经TGF-β1处理的HMPCs作为阴性对照组,以外源性Ang1处理的HPVECs作为阳性对照组,以人血管生成素受体Tie2抑制剂处理的HPVECs作为抑制剂组。采用CCK-8法、细胞划痕实验、Transwell实验、成管实验和细胞膜荧光探针染色法观察HPVECs增殖、迁移、血管生成情况,以及HPVECs与HMPCs共定位情况;Western blot检测HPVECs中Tie2蛋白及其磷酸化水平。结果:TGF-β1处理96 h,HMPCs中Ang1蛋白表达水平显著升高(P<0.01)。与阴性对照组相比,T-HMPCs-CM能够抑制HPVECs迁移、血管新生,并增加Tie2磷酸化以稳定血管状态,其作用类似于外源性Ang1;而使用Tie2抑制剂阻断Ang1/Tie2信号通路后,上述作用一定程度上被逆转。结论:TGF-β1可刺激HMPCs分泌Ang1,进而激活HPVECs的Tie2信号通路,从而抑制HPVECs血管新生,发挥维持腹膜血管稳定的作用。

视网膜微血管周细胞来源的外泌体对糖尿病小鼠视网膜血管功能障碍的影响

目的探讨视网膜微血管周细胞(RMVPCs)来源的外泌体(Exos)通过调控血管内皮细胞的生物学功能进而对糖尿病视网膜血管功能障碍的影响。方法通过超速离心法提取并鉴定人视网膜微血管周细胞(HRMVPCs)来源的Exos(HRMVPCs-Exos),并对Exos进行PKH67染色后,与人脐静脉血管内皮细胞(HUVECs)孵育进行内吞实验,此后,体外实验分为Control组(5.55 mmol·L^(-1)葡萄糖)、高糖(HG)组(30.00 mmol·L^(-1)葡萄糖)和HG+HRMVPCs-Exos组,各组HUVECs进行相应处理后,应用CCK-8、细胞迁移、流式细胞术以及成管实验等方法检测HUVECs的增殖、迁移以及血管的生成。体内实验分为NC组、链脲佐菌素(STZ)组和STZ+HRMVPCs-Exos组,每组5只健康雄性C57BL/6J小鼠,各组小鼠进行相应处理后,进行眼底照相、OCT、荧光素眼底血管造影(FFA)、视网膜血管密度以及血管渗漏检测。结果HRMVPCs可分泌Exos,可以进入HUVECs中。与Control组相比,HG组和HG+HRMVPCs-Exos组HUVECs的存活率、迁移率以及血管生成数均显著减少(均为P<0.05);与HG组相比,HG+HRMVPCs-Exos组HUVECs的存活率、迁移率以及血管生成数均显著升高(均为P<0.05)。STZ+HRMVPCs-Exos组小鼠视网膜结构破坏严重,并出现大量渗出物以及荧光素渗漏,而STZ组小鼠视网膜出现少量渗出物和微血管异常,NC组小鼠视网膜无明显病变。与NC组相比,STZ组和STZ+HRMVPCs-Exos组小鼠视网膜血管密度均显著下降(均为P<0.01),血管渗漏面积均显著增加(均为P<0.01)。与STZ组相比,STZ+HRMVPCs-Exos组小鼠视网膜血管密度显著下降(P<0.01),血管渗漏面积显著增加(P<0.01)。结论HRMVPCs-Exos进入HUVECs中,通过加强HG下HUVECs的生物学功能,最终加重糖尿病小鼠视网膜血管功能障碍。

Inhibitive effects of glucose and free fatty acids on proliferation of humanvascular endothelial cells in vitro

OBJECTIVES: To investigate the effects of glucose and free fatty acids (FFAs) on the proliferation and cell cycle of human vascular endothelial cells in vitro, and to examine whether the combined presence of elevated FFAs and glucose may cross-amplify their individual injurious effects. METHODS: Cultured human vascular endothelial cells (ECV304) were incubated with various concentrations of glucose and/or FFAs (palmitate and/or oleate) for 24 - 96 h. Morphologic alterations were observed using a phase contrast microscope and an electron microscope. Inhibition of proliferation was measured by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell viability was determined using trypan blue exclusion. Distribution of cells along phases of the cell cycle was analyzed by flow cytometry. RESULTS: Glucose 15 or 30 mmol/L, palmitate (PA) 0.25 or 0.5 mmol/L, and oleate (OA) 0.5 mmol/L inhibited proliferation and accelerated death of endothelial cells in a dose-and-time-dependent manner. After treatment with elevated glucose and/or FFAs, the G(0)/G(1) phase cells increased, whereas S phase cells decreased, suggesting that high glucose and/or FFAs mainly arrested endothelial cells at G(0)/G(1) phase. The inhibitive rates of proliferation and population of dead cells in endothelial cells incubated with glucose plus FFAs (glucose 30 mmol/L + PA 0.25 mmol/L, glucose 30 mmol/L + OA 0.5 mmol/L, glucose 30 mmol/L + PA 0.25 mmol/L + OA 0.5 mmol/L) increased more markedly than those treated with high glucose or FFAs (PA and/or OA) alone. CONCLUSION: Both high ambient glucose and FFAs can inhibit proliferation and accelerate death of endothelial cells in vitro. These changes were cross-amplified in the combined presence of high levels of glucose and FFAs.

同型半胱氨酸对培养内皮细胞损伤的研究

目的 :本研究拟探讨同型半胱氨酸对培养人脐静脉内皮细胞的损伤效应 ,以揭示同型半胱氨酸致动脉粥样硬化的可能机制。方法 :将人脐静脉内皮细胞暴露于不同浓度的同型半胱氨酸 2 4h后 ,观察细胞形态并测定细胞的乳酸脱氢酶释放率、总蛋白含量、凋亡及脂质过氧化的程度。结果 :(1)同型半胱氨酸不仅可诱导细胞凋亡而且在较高浓度时可致细胞坏死 ;(2 )同型半胱氨酸有较强的促脂质过氧化的效应 ,并呈量效关系 ;(3)同型半胱氨酸的上述效应因加入LDL而增强 ,示同型半胱氨酸与LDL有协同作用。结论 :同型半胱氨酸可能是通过氧化应激机制导致内皮细胞出现坏死 。

氧化苦参碱对肿瘤诱导血管内皮细胞增殖的抑制作用

目的 探讨氧化苦参碱 (Oxy)对肺癌和胃癌细胞诱导血管内皮细胞 (VEC)增殖的抑制作用。方法 用MTT法检测不同浓度 Oxy对 VEC、人肺腺癌 SPC- A - 1细胞、人低分化胃癌 MKN- 45细胞增殖及 SPC- A- 1和MKN- 45细胞诱导 VEC增殖的抑制。结果 Oxy浓度为 2 .5～ 10 m g/ ml时 ,对 VEC增殖的抑制率为 4.6 %～36 .4% ;Oxy浓度为 0 .15 6～ 10 mg/ m l时 ,对肺癌 SPC- A- 1和胃癌 MKN- 45细胞增殖的抑制率分别为 3.7%～93.9%和 2 .1%～ 93.6 % ;经浓度为 1.2 5～ 10 m g/ ml Oxy作用后的肺癌 SPC- A - 1和胃癌 MKN- 45细胞条件培养液对 VEC增殖的抑制率分别为 11.1%～ 37.0 %和 8.7%～ 39.1% ;Oxy浓度为 1.2 5～ 10 mg/ ml时 ,对肺癌 SPC-A- 1和胃癌 MKN- 45细胞条件培养液诱导 VEC增殖的抑制率分别为 13.8%～ 5 8.6 %和 15 .9%～ 79.4%。结论 Oxy对肺癌和胃癌细胞诱导的 VEC增殖具有抑制作用。

硫酸多糖对体外人脐静脉内皮细胞损伤的保护作用

研究表明，硫酸多糖体外对多聚阳离子和氧自由基损伤的人脐静脉内皮细胞有保护作用。肝素、硫酸软骨素Ａ抗多聚阳离子损伤作用比同浓度低分子肝素和甘糖酯强。肝素、硫酸软骨素Ａ、甘糖酯抗氧自由基损伤作用优于同浓度低分子肝素。结果显示硫酸多糖有保护血管内皮的作用，其作用可能与所带阴离子基团有关。

内皮抑素治疗人喉癌裸鼠模型的实验研究

目的 研究内皮抑素对裸鼠喉鳞状细胞癌皮下移植模型的抑瘤作用,探讨其抑瘤机制及人喉癌生物治疗新方法。方法 建立人喉癌Hep-Ⅱ细胞株裸鼠皮下接种模型。应用内皮抑素治疗,观察肿瘤生长情况,对用药后肿瘤组织进行病理学检查,通用型二步法免疫组化检查及电镜观察。计数微血管密度(microvessel density,MVD),计算增殖细胞核抗原(proliferationg cell nuclear antigen,PCNA)和血管内皮生长因子(vascuoar endothelial growth factor,VEGF)阳性率。结果 在实验组与对照组之间,鼠净重、瘤重、瘤体积及瘤重/鼠净重,经t检验P值<0.05或<0.01,差异均有显著性意义,抑瘤率为45.9％。病理学检查及电镜观察表明,实验组应用内皮抑素治疗肿瘤生长受到抑制,细胞分裂少见,可见肿瘤细胞的坏死凋亡,新生血管明显减少。MVD、PCNA及VEGF表达在实验组明显低于对照组,经t检验P值分别为<0.01、<0.05、<0.05,差异均有显著性意义。结论 内皮抑素可显著抑制人喉癌裸鼠模型肿瘤的生长和发展,其可能机制为抑制肿瘤血管生成。

血管组织工程中单根主动脉培养的内皮细胞和平滑肌细胞形态学和生长增殖

目的:探索体外培养及扩增同一来源内皮细胞和平滑肌细胞的有效方法,为研究内皮细胞和平滑肌细胞相互关系和体外构建组织工程血管提供理论和实践基础。方法:在体外用酶消化法和组织块贴壁法分别建立内皮细胞和平滑肌细胞的原代,并应用胰蛋白酶和乙二胺四乙酸钠传代培养。应用光镜、透射电镜和细胞计数对内皮细胞和平附肌细胞的形态和增殖进行研究。结果:酶消化法和植块培养法可以有效的建立起内皮细胞和平滑肌细胞的原代。讨论:内皮细胞和平滑肌细胞作为血管构成的基本细胞,对于它们的形态学、体外培养和扩增以及相互关系的研究具有重要的意义。

葡萄糖诱导人血管内皮细胞凋亡及其对bax和bcl-2表达的影响

目的 探讨糖尿病 (DM)状态下葡萄糖 (Glu)对人脐静脉内皮细胞 (HUVEC)凋亡的影响及其分子机制。 方法 细胞凋亡用吖啶橙 (AO)荧光染色、原位末端标记 (TUNEL)法、流式细胞仪分析检测。同时行bcl 2和bax免疫细胞化学染色和定量分析。 结果 高浓度Glu( 2 0mmol/L、4 0mmol/L)能够诱导HUVEC凋亡 ,且呈浓度和时间依赖性。高糖 ( 2 0mmol/L)培养HUVEC 72h后bax表达的MOD×area值由 387± 97升至 136 9± 2 2 5 (P <0 .0 1) ,对照组和高糖组bcl 2染色的MOD×area分别为 15 0± 36和 186± 76 ,两者间差异无显著意义 (P >0 .0 5 )。 结论 DM状态下高血糖能诱导内皮细胞凋亡 ,其机制可能与bax基因表达上调有关。