Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells

After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.

Gene Expression Analysis of Human Vascular Endothelial Cells Treated by Ouabain in Pathological Concentration

Objectives To study the geneexpression of human vascular endothelial cells (HU-VEC) treated by ouabain in pathological concentra-tion. Methods The response of endothelial cells toouabain of 1.8 nmol/L was explored with a comple-mentary DNA microarray representing 8 464 differenthuman genes. Results The results of mRNA profilesanalysis indicated that 129 of the genes were differ-ently expressed, 26 were upregulated. ConclusionsThe pathological role of ouabain on HUVEC may beinvolved in the controlling of DNA transcription、pro-tein translation、metabolism and signal transduction.

Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation

To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.

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.

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.

Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

Objectives To construct a recombinant plasmid carrying enhanced green fluore- scent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expre- ssion in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP- C1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluoresce- nce microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection. Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

Transplantation of endothelial progenitor cells transfected with VEGF165 to restore erectile function in diabetic rats

The present study investigated the effect of transplanting endothelial progenitor cells （EPCs） transfected with the vascular endothelial growth factor gene （VEGF165） into the corpora cavernosa of rats with diabetic erectile dysfunction （ED）. A rat model of diabetic ED was constructed via intraperitoneal injection of streptozotocin. After streptozotocin treatment, pre-treated EPCs from each of three groups of rats were transplanted into their corpora cavernosa. Our results, following intracavernosal pressure （ICP） monitoring, showed that ICP increased significantly among rats in the trial group when compared to the results from rats in the blank-plasmid and control groups during basal conditions and electrical stimulation （P〈O.01 for both comparisons）. Histological examination revealed extensive neovascularisation in the corpora cavernosa of rats in the trial group. Fluorescence microscopy indicated that many of the transplanted EPCs in the trial group survived, differentiated into endothelial cells and integrated into the sites of neovascularisation. Based on the results of this study, we conclude that transplantation of VEGF165-transfected EPCs into the corpora cavernosa of rats with diabetic ED restores erectile function.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.

Effect of Composite Salviae Dropping Pill on Endothelin Gene Expression in Circulating Endothelial Cells of Patients with Coronary Heart Disease

Objective: To explore the effect of composite Salviae droping pill(CSDP) on endothelin (ET-1) gene expression in circulating endothelial cells. Mothods: Seventy cases of stable angina pectoris were randomly divided into two groups, the CSDP group and the isosorbide dinitrate (ID) group. They were treated with CSDP and ID respectively,their ET-1 gene expression in endothelial cells of peripheral circulation was measured before and after treatment by reverse transcriptase-polymerase chain reaction(RT-PCR) and compared between the two groups as well as with that of healthy subjects. Results: Electrophoresis banding of 546 bp cDNA procured from 59 cases of 70 patients was positive, while no positive banding was obtained from the healthy subjects. Six cases from the 29 patients treated with CSDP had their banding turned to negative,while in the ID group,no one turned to negative after treatment. And ET-1 PCR product (absorbed optic density) in the CSDP group was markedly lower than that in the ID group, P＜0.05. Conclusion: DSP could directly inhibit ET-1 gene expression in endothelial cells of peripheral circulation.

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells

Objective: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). Methods: Two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. Results: These analyses have identified both novel and known genes whose expression is influenced by LPS. The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. Conclusions: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.

Differential mRNA Expressions in HCMV infected HUVECs

Objective The aim was to identify the gene expressions of human cytomegalovirus(HCMV)-infected human umbilical vein endothelial cells(HUVECs)and to study its possible pathogenic mechanism on atherosclerosis using microarray technology.Methods The gene expression differences in HCMV AD169 strain-infected HUVECs were studied by the microarray technology to explore the potential molecular mechanism of HCMV infection.The qPCRs were performed to verify the transcriptome results.Results A total of 2,583 differentially expressed genes,including 407 down-regulated genes and 2,176 up-regulated genes,were detected by the systematic bioinformatics analysis.The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses showed that the significantly differentially expressed genes were mainly involved in regulating protein kinase activity,inflammatory response,ubiquitination,protein phosphorylation,cell metabolism,and exosomes,among which 12 genes had significant changes and were screened by protein-protein interaction(PPI)analysis and verified by qPCR.The experimental qPCR results were consistent with the microarray results.Conclusion The GO and KEGG analyses revealed that the regulation of protein kinase activity,inflammatory response,ubiquitination,protein phosphorylation,and cell metabolism played important roles in the process of endothelial cell infection.Furthermore,12 genes were involved in the process of HCMV infection of endothelial cells and contributed to the current understanding of the infection and pathogenic mechanisms of atherosclerosis.

Molecular regulation mechanisms of lymphangiogenesis

Lymphangiogenesis, the growth of new lymphatic vessels, has long been regarded as a putative efficient pathway to neoplastic metastization. Recent results have shown the necessity of lymphatic molecular markers and growth factors for lymphangiogenesis. Importantly, lymphatic endothelial receptor tyrosine kinase VEGFR-3 and its ligands VEGF-C and VEGF-D play crucial roles in promoting lymphatic vascular growth both during development and in pathological conditions. Isolation of pure cultures of lymphatic and blood vascular endothelial cells and systematic characterization of their transcriptomes provide useful cell culture models and novel potential vascular markers and offer further insights into the lymphatic vascular biology. Ectopic expression of the lymphatic endothelial specific homeobox transcription factor Prox1 in blood endothelial cells results in a shift in the gene expression profile towards the lymphatic endothelial phenotype. It demonstrates the plasticity of endothelial cells and offers the possibility of transcriptional reprogramming of vascular endothelial cells as future putative therapeutic applications.

PEG3、STMN1基因在非小细胞肺癌的表达及其与临床病理特征、血管生成相关性研究

目的 探讨父系表达基因3(PEG3)、抑微管装配蛋白1(STMN1)基因在非小细胞肺癌(NSCLC)的表达及其与临床病理特征和血管生成的关系。方法 收集宁夏医科大学总医院肿瘤医院2021年1月—2023年1月经病理证实的96例NSCLC患者癌组织及癌旁组织,采用实时荧光定量聚合酶链反应(qRT-PCR)检测和免疫组织化学检测癌组织与癌旁组织PEG3、STMN1、VEGF及CD105 mRNA的表达;比较不同临床病理特征NSCLC患者癌组织PEG3、STMN1的阳性表达率;采用Pearson法分析PEG3、STMN1与VEGF及CD105关系;采用受试者工作特征(ROC)曲线分析PEG3、STMN1对NSCLC的诊断价值。结果 与癌旁组织比较,PEG3在NSCLC组织中表达降低(P <0.05),STMN1、VEGF及CD105在NSCLC组织中表达升高(P <0.05);NSCLC组织中STMN1、VEGF及CD105阳性率分别为62.50%、69.79%和72.92%,分别高于癌旁组织5.21%、10.42%和13.54%(P <0.05),NSCLC组织中PEG3阳性率为8.33%低于癌旁组织73.96%(P <0.05);不同年龄、性别及肿瘤类型NSCLC患者的PEG3及STMN1表达水平比较,差异无统计学意义(P>0.05),不同TNM分期、淋巴结转移及分化程度NSCLC患者的PEG3及STMN1表达水平比较,差异有统计学意义(P <0.05);NSCLC组织的PEG3与STMN1、VEGF及CD105均呈负相关(P <0.05),NSCLC组织的STMN1与PEG3呈负相关(P <0.05),与VEGF及CD105均呈正相关(P <0.05);PEG3诊断NSCLC的曲线下面积为0.750(95%CI:0.453,0.936)、敏感性为73.66%(95%CI:0.650,0.937)、特异性为79.62%(95%CI:0.590,0.956);STMN1诊断NSCLC的曲线下面积为0.796(95%CI:0.540,0.942)、敏感性为80.30%(95%CI:0.744,0.978)、特异性为81.12%(95%CI:0.612,0.996);PEG3+STMN1联合诊断的曲线下面积为0.935(95%CI:0.753,0.995)、敏感性为92.33%(95%CI:0.751,0.930)、特异性为77.12%(95%CI:0.735,0.948)。结论 NSCLC组织PEG3降低、STMN1升高,与肺癌TNM分期、分化程度相关,其可以加快肿瘤血管生成,能够一定程度提高疾病诊断价值。

长链非编码RNA-MEG3在人心脏微血管内皮细胞缺氧损伤中的作用

目的研究长链非编码RNA-MEG3在人心脏微血管内皮细胞(HCMEC)缺氧损伤中的作用。方法将HCMEC分为对照组、缺氧/复氧(H/R)组及缺氧/复氧联合MEG3敲减(H/R+siMEG3)组。荧光定量PCR检测MEG3及炎性因子的表达情况[白介素1β(IL-β)、IL-6、IL-8、IL-10、肿瘤坏死因子(TNF-α)];CCK-8检测HCMEC细胞的增殖活性;流式细胞术分析细胞凋亡情况;Western blotting检测PI3K/AKT/eNOS信号通路表达变化,ELISA检测一氧化氮(NO)、超氧化物歧化酶(SOD)、血管内皮生长因子(VEGF)和活性氧(ROS)表达水平。结果利用qPCR检测对照组及H/R组细胞MEG3表达,结果发现:H/R组MEG3相对表达倍数[(6.87±0.239)倍]较对照组MEG3表达倍数(1.00±0.026)倍显著升高(n=3,t=42.32,P<0.0001),提示MEG3可能在心脏微血管内皮细胞IRI中起到重要作用。H/R组细胞与对照组比较,细胞增殖活性显著减弱(P<0.01);而H/R+siMEG3组与H/R组比较,细胞增殖活性进步受到抑制(P<0.01)。H/R组较对照组PI3K、AKT及eNOS磷酸化水平显著减低,而H/R+siMEG3组细胞较H/R组磷酸化水平更低(P<0.05)。H/R组与对照组比较,NO、SOD及VEGF显著减低,而ROS水平升高(P<0.05);而H/R+siMEG3组与H/R组比较,NO、SOD及VEGF水平进步下降,ROS升高显著。利用qPCR检测各处理组细胞相关炎症基因表达,结果发现:H/R组较对照组基因表达显著升高(P<0.05);H/R+SiMEG3组较H/R组基因表达进一步升高(P<0.01)。结论长链非编码RNA-MEG3在心脏微血管内皮细胞H/R损伤过程中起到重要保护作用,靶向提升MEG3水平有望成为减缓心脏微血管IRI的潜在治疗靶点。

丹参酮ⅡA对猪主动脉内皮细胞受血管紧张素Ⅱ作用时产生NO及eNOS基因表达的影响

目的探讨丹参酮ⅡA对血管内皮细胞的保护作用。方法采用硝酸还原酶法、逆转录聚合酶链式反应(RT-PCR)和免疫组织化学法,分别检测不同作用时间(1h、6h、24h)的血管紧张素Ⅱ(AngⅡ)以及在AngⅡ作用的不同时间点(0h点为A组、6h点为B组)加入丹参酮ⅡA对培养的猪主动脉内皮细胞产生NO及其内皮型一氧化氮合酶(eNOS)的蛋白和mRNA表达。结果(1)随着AngⅡ作用时间的延长,血管内皮细胞NO的产生及eNOS的表达呈显著下降(P<0.01),表现出时间依赖性的负性作用。(2)丹参酮ⅡA可抑制AngⅡ对内皮细胞分泌NO及eNOS基因表达的负性作用(P<0.01)。(3)在丹参酮ⅡA作用1h、6h,A组的抑制效应明显强于B组(P<0.05);随着作用时间延长至24h,两组比较差异无显著性。结论丹参酮ⅡA可抑制AngⅡ对血管内皮细胞分泌NO以及细胞eNOS基因表达的负性作用。

通心络对同型半胱氨酸损伤的内皮细胞的基因表达谱的影响

目的:探讨通心络对同型半胱氨酸(Hcy)损伤的人脐静脉内皮细胞(HUVECs)的基因表达谱的影响。方法:原代培养的HUVECs,随机分为对照组、Hcy组和通心络组,提取总RNA并纯化,反转录合成荧光分子(Cy3/Cy5)标记的cDNA探针,采用高通量Affymetric人类全基因组寡核苷酸微阵列芯片(含47 000个基因或基因片段)杂交,杂交信号经扫描和数字化处理,通过生物信息学数据分析推测通心络对同型半胱氨酸损伤的HUVECs细胞功能的影响以及可能的信号通路。结果:与对照组相比,Hcy组有4 343个基因表达上调,316个基因表达下调;与Hcy组相比,通心络组有3 409个基因表达上调,121个基因表达下调。这些共同的差异基因生物功能涉及转录因子调控、激酶、细胞骨架与蛋白合成、转运功能、细胞因子、细胞-细胞受体相互作用和细胞黏附因子等。参与的信号通路主要是与血管新生、凋亡、氧化应激、凝血纤溶和炎症等相关的信号通路,如mTOR信号通路、MAPK信号通路和Toll样受体信号通路。结论:通心络能导致同型半胱氨酸损伤内皮细胞基因表达谱的改变,这些基因改变可能参与了细胞凋亡、氧化应激和凝血纤溶等过程。

川芎内酯A预处理对心肌微血管内皮细胞缺氧/复氧损伤保护作用及机制研究

目的:探讨川芎内酯A预处理对心脏微血管内皮细胞缺氧/复氧(H/R)损伤的保护作用及作用机制。方法:采用体外培养的大鼠乳鼠心脏微血管内皮细胞,空白组不加任何处理;单纯H/R组缺氧4 h/复氧2 h;单纯预处理组低氧25 m in/复氧30 m in,然后缺氧4 h/复氧2 h,各给药组分别加1×10-7,1×10-9,1×10-11mol.L-1的川芎内酯A孵育55 m in,然后缺氧4 h/复氧2 h;实验结束后取细胞上清液测一氧化氮(NO),一氧化氮合酶(NOS),内皮素(ET),取造模后的且固定在盖玻片的细胞,用原位杂交法检测iNOSmRNA和ETmRNA的基因表达。结果:H/R组细胞存活数降低明显,细胞培养液中NO和NOS活性显著降低,ET的活性明显升高,iNOSmRNA表达量减少,ETmRNA表达量增加;川芎内酯A 3个剂量组与H/R组比较,细胞存活数均明显增加,细胞培养液中NO和NOS活性增加,ET活性降低,iNOSmRNA表达量增加而ETmRNA表达量则减少。结论:川芎内酯A预处理能减轻内皮细胞损伤,对心脏微血管内皮细胞缺氧/复氧损伤可能具有保护作用,其作用机制可能与上调内皮细胞中iNOSmRNA的表达和下调ETmRNA的表达有关。