帕唑帕尼对甲状腺癌细胞和血管内皮细胞增殖的影响及其作用机制

目的 研究新型酪氨酸激酶抑制剂帕唑帕尼对甲状腺癌细胞株SW579和血管内皮细胞株HUVEC增殖的影响及作用机制.方法 将SW579和HUVEC暴露于不同浓度（1、2、4、8和16 μmoL/L）的帕唑帕尼至72 h,无药物处理的细胞作为对照.采用细胞活度测定、图像分析技术及分裂细胞荧光免疫染色观察评价帕唑帕尼对细胞增殖的影响,应用流式细胞仪进行细胞周期时相分析.结果 细胞活度测定显示,帕唑帕尼对SW579和HUVEC细胞的生长增殖均有明显的抑制作用,并呈浓度依赖性及时间依赖性,其半数抑制浓度（IC50）分别约为2和4μmol/L.图像分析显示,在IC50作用下,帕唑帕尼使两种细胞的细胞密度降低,分裂指数下降.帕唑帕尼作用48 h时,SW579细胞分裂指数（0.9％）明显低于其对照细胞（2.2％）,差异有统计学意义（P＜0.01）;HUVEC细胞分裂指数（1.9％）亦明显低于其对照细胞（4.0％）,差异有统计学意义（P＜0.01）.细胞周期时相分析表明,帕唑帕尼可使SW579和HUVEC发生细胞周期休止,分别休止于G1期和S期.帕唑帕尼作用24、48 h时,SW579在G1期的比例均明显高于其对照组细胞[（60.4±2.2）％比（49.2±1.6）％,（70.4±1.6）％比（58.6±3.3）％],差异有统计学意义（P＜0.01）;HUVEC细胞在S期的比例均明显高于其对照组细胞[（47.3±2.7）％比（39.5±0.6）％,（43.5±1.6）％比（28.8±4.2）％],差异有统计学意义（P＜0.01）.结论 帕唑帕尼对甲状腺癌细胞和血管内皮细胞的增殖均有明显的抑制效应,其作用是藉细胞周期休止作用而实现,但在两种细胞细胞周期休止时相不同.

血管内皮生长因子-C在肾癌中的表达及意义

目的:探讨血管内皮生长因子-C(VEGF-C)在肾癌发生发展中的作用。方法:应用免疫组织化学SP法检测VEGF-C在肾癌及正常肾组织中的表达情况。结果:肾癌组织VEGF-C的阳性表达率(85.0%)显著高于正常肾组织(P<0.01)。肾癌组织VEGF-C的阳性表达与年龄、性别、分化程度无关(P>0.05),与有无淋巴结转移和肾癌临床分期有关(P<0.05)。结论:VEGF-C可能通过促进淋巴管生成参与肾癌的淋巴结转移,在肾癌的发生发展中发挥重要作用。

血管内皮生长因子-B与眼部新生血管性疾病

异常的新生血管会导致多种眼部疾病,而血管内皮生长因子（VEGF）在新生血管的发生及发展中起重要作用.VEGF-B作为VEGF家族的一员,对血管生成和血管通透性无显著影响,但可通过血管生存作用和细胞凋亡作用对新生血管的生长进行调控,从而抑制新生血管的发生和发展.同时,VEGF-B对心脏和神经元等具有保护作用.抗VEGF治疗作为目前明确有效的治疗新生血管的方法,一直受到广泛关注,而因VEGF-B的两面性,对于抗VEGF-B的药物在治疗眼科新生血管性疾病方面的作用尚需进一步研究.就VEGF-B对机体血管、细胞、神经元及心脏所起的作用及其在眼部新生血管性疾病治疗中的应用进行综述.

Effect of acetyl L-carnitine on human retinal pigment epithelium-19 cells in hypoxic conditions

AIM:To investigate the effect of acetyl-L-carnitine(ALCAR)on cell viability,morphological integrity,and vascular endothelial growth factor(VEGF)expression in human retinal pigment epithelium(ARPE-19)cells using a hypoxic model.METHODS:In the first set of experiments,the optimal CoCl_(2) dose was determined by exposing ARPE-19 cell cultures to different concentrations.To evaluate the effect of ALCAR on cell viability,five groups of ARPE-19 cell culture were established that included a control group,a sham group(200μM CoCl_(2)),and groups that received 1,10 and 100 mM doses of ALCAR combined with 200μM CoCl_(2),respectively.The cell viability was measured by MTT assay.The morphological characteristics of cells were observed by an inverted phase contrast microscope.The levels of VEGF and HIF-1α secretion by ARPE-19 cells were detected by enzyme linked immunosorbent assay(ELISA)assay.RESULTS:ARPE-19 cells were exposed to different doses of CoCl_(2) in order to create a hypoxia model.Nevertheless,when exposed to a concentration of 200μM CoCl_(2),a notable decrease in viability to 83% was noted.ALCAR was found to increase the cell viability at 1 mM and 10 mM concentrations,while the highest concentration(100 mM)did not have an added effect.The cell viability was found to be significantly higher in the groups treated with a concentration of 1 mM and 10 mM ALCAR compared to the Sham group(P=0.041,P=0.019,respectively).The cell viability and morphology remained unaffected by the greatest dose of ALCAR(100 mM).The administration of 10 mM ALCAR demonstrated a statistically significant reduction in the levels of VEGF and HIF-1α compared with the Sham group(P=0.013,P=0.033,respectively).CONCLUSION:The findings from the current study indicate that ALCAR could represent a viable therapeutic option with the potential to open up novel treatment pathways for retinal diseases,particular relevance for age-related macular degeneration(AMD).However,to fully elucidate ALCAR’s application potential in retinal diseases,additional investigation is necessary to clearly define the exact mechanisms involved.

高胆固醇膳食对兔动脉组织及其一氧化氮合成的影响

为研究高脂高胆固醇膳食对动脉壁内皮一氧化氮（ＮＯ）合成的影响，以高脂高胆固醇饲料喂饲雄性纯种新西兰兔８周后，通过定量逆转录多聚酶链式反应（ＲＴ－ＰＣＲ），观察高脂高胆固醇膳食对动脉组织一氧化氮合成酶（ＮＯＳ）基因表达影响。结果表明：高脂高胆固醇膳食８周后，内皮型（ＮＯＳ）ｍＲＮＡ表达下降、总ＮＯＳ活力减弱、血浆一氧化氮代谢产物（ＮＯＰ）水平降低（Ｐ＜０．０５）；内皮素（ＥＴ）水平上升。结果提示：高脂高胆固醇膳食引起高脂血症，可损害动脉内皮，降低ＮＯＳ的基因表达。

Hydroxysafflor Yellow A Promotes Vascular Endothelial Cell Proliferation via VEGF/VEGF Receptor

Aim To study the proliferative effeet of hydroxysaftlor yellow A （HSYA） on cultured canine aortic endothelial cell （VEC） in normoxic （21% O2 ） or hypoxic （10% O2 ） culture and the underlying mechanism. Methods The endothelial cells were scratched from trypsined canine aorta endothelium. HSYA was added to the cells at final concentrations of 1 × 10^-3, 1 × 10^-4 and 1 × 10^-5 mol· L^-1, respectively. VEGF （2.6 × 10^-7 mol· L^-1 ）-treated cells were used as the positive control. The proliferative effect of HSYA on VEC was determined at 48, 72, 96, and 120 h in normoxic culture by MTI＂ assay. Similarly, the proliferation of VEC was determined at 12, 24, 48, and 72 h in hypoxic culture by MTF assay. The effects of HSYA on VEC proliferation and VEGF secretion were investigated by MTr and ELISA assays at the presence of the antibodies to VEGF and VEGF receptors. Results Pretreatment with HSYA at concentrations of 1 × 10^-3 and 1 × 10^-4 mol· L^-1 enhanced VEC proliferation in normoxic culture. The most significant enhancing effect of HSYA on VEC proliferation was achieved at 24, 48, and 72 h in hypoxic culture in concentration-dependent and time-dependent manner. HSYA at 1 × 10^-3 mol·L^-1 showed a potency similar to VEGF at 2.6 × 10^-7 mol·L^-1 . Pretreatment with the antibodies of Flt-1, KDR or VEGF blocked the proliferative effect of HSYA with similar potencies. Antibodies of Fit-1 or VEGF antagonized the promoting effect of HSYA on VEGF secretion. Conclusion HSYA promotes VEC proliferation either in normoxic or hypoxic culture, especially in the latter condition. This effect of HSYA is at least partly mediated by VEGF and VEGF receptor.

抗血管内皮生长因子类药物在新生血管性青光眼及滤过手术抗瘢痕治疗中的应用

新生血管性青光眼（NVG）是一种由多种疾病导致视网膜缺血缺氧而发生的继发性青光眼,研究发现其病因与VEGF有关。目前,抗VEGF药物作为治疗NVG的手段之一,已成为现阶段青光眼研究领域的热点,为提高NVG疗效开辟了新的途径。本文就有关NVG病理生理基础与临床过程,包括Bevacizumab、Ranibizumab、Pegaptanib及Aflibercept在内的4种抗VEGF药物的基础研究,抗VEGF药物在NVG及滤过手术抗瘢痕治疗中应用的国内外研究进展进行综述,以期为临床治疗NVG、提高手术成功率提供参考。

血管内皮生长因子在糖尿病肾病中的作用及其机制

血管内皮生长因子在糖尿病肾病中起重要作用,高血糖引起的蛋白激酶C、氧化应激、缺血缺氧等都能诱导血管内皮生长因子的过表达.大量研究表明血管内皮生长因子可通过多种途径调控肾小球各类细胞及基质的结构和功能,参与糖尿病肾病的病理生理过程,并在疾病进展中起重要作用.这些发现为糖尿病肾病的治疗提供了新的思路.

IMPACTS ON THE EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN ISCHEMIC BRAIN TISSUE TREATED WITH CLUSTER PUNC-TURE ON SCALP ACUPOINTS IN RATS

Objective To probe into the impacts on the expression of vascular endothelial growth factor （VEGF） in ischemic brain tissue treated with cluster puncture on scalp acupoints in rats, Methods 128 rats were randomized into pseudo-operation group, model group, scalp-needling group and cluster-needling group, In scalp-needling group, penetration method was used on the focal side from Bǎihuì （百会 GV20） to Qǔbīn （曲鬓 GB7）, and in cluster-needling group, penetration method was used on both sides from GV20 to GB7, and a common needling on GV 20. Needles were punctured 2 mm in depth, constantly rotated for 10min, retained for 2 h. Immunohistrochemical method was applied to determine VEGF expression and microvessel density （MVD）. Results With the intervention of cluster puncture on scalp acupoints, VEGF： expressions on every time-spot after ischemia were enhanced apparently, superior to those in scalp-needling group. On the three time-spots of the 7^th, 14^th and 21^st days, MVD was increased after cluster puncture on scale acupoints, superior to those in scalp-needling group. Conclusion Cluster puncture on scalp acupoints up-regulated VEGF expression and promoted regeneration of microvessel.

Real-time Quantitative PCR Analysis of Vascular Endothelial Growth Factor Receptor-2(VEGFR-2) Expression at Zebrafish Different Developmental Stages

[Objective]To investigate the expression of zebrafish vascular endothelial growth factor-2（VEGFR-2） at different developmental stages.[Method]Total RNAs were extracted from 12,24,48,72 and 96 hpf stage zebrafish embryos and larvae.Real-time quantitative RT-PCR was performed to examine the expression of VEGFR-2.The data were analyzed by 2^-△△Ct method.[Result]The expression level of VEGFR-2 gene increased gradually from 12 to 72 hpf,and subsequently decreased at 96 hpf.The expression level was lowest at 12 hpf,highest at 72 hpf,and had significant differences when compared with that of other developmental stages.[Conclusion]The expression level of VEGFR-2 increases gradually before blood vessel maturation and decreases as blood vessels mature.

Location of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in Tissues of Natural Cases

[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome （HP-PRRS）. [Method] Antigen location and histopathological observation in natural cases infected by highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） were analyzed by immunohistochemistry and H. E. staining. [Result] The virus antigen mainly existed in epithelial calls, and also a few in mecrophages, lymphocytes and brain nerve cells. [ Conclusion] The cell and tissue tropism of HP-PRRSV strain in natural cases is different from that of previous strains.

关注湿性年龄相关性黄斑变性抗血管内皮生长因子治疗中的无应答病例

抗VEGF药物已经成为湿性AMD患者的一线标准用药，但是仍有部分患者治疗后出现无应答，其原因目前仍不确切，但可能与AMD的类型、黄斑区解剖因素异常、遗传基因型的不同、AMD病变的程度及抗药反应有关。眼科医师应当关注这部分无应答病例并给予正确的处理，最大限度地避免抗VEGF药物的滥用，并积极探索适合患者最佳个性化治疗措施。（中华眼科杂志，2014，50：406-410）

外源性表达VEGF165b对人膀胱癌裸鼠移植瘤生长影响的研究

目的建立人膀胱癌T24细胞裸鼠移植瘤模型,探讨外源性表达VEGF165b对其生长的影响及其机制。方法分别移植未转染和转染VEGF165b表达载体的人膀胱癌T24细胞到裸鼠膀胱内,28d使处死动物,称量肿瘤重量,测定肿瘤体积。Western blot法检测肿瘤VEGF165b、p-VEGFR2、AKT、p-AKT蛋白表达。结果转染pcDNA-VEGF165b组肿瘤重量和体积与未转染组和转染pcDNA3.0组相比明显降低(P<0.05),而未转染组和转染pcDNA3.0组比较无统计学差异(P>0.05)。Western blot结果显示,转染pcDNA-VEGF165b组与未转染组和转染pcDNA3.0组比较,VEGF165b表达增高,p-VEGFR2、p-AKT表达降低,而未转染组和转染pcDNA3.0组间无明显差异。结论外源性表达VEGF165b可通过抑制VEGFR2信号通路传导而明显抑制人膀胱癌裸鼠移植瘤生长。

Expression of Angiopoietin-2 and Vascular Endothelial Growth Factor in Oral Squamous Cell Carcinoma and Its Significance

Objective： To investigate the expression of angiopoietin-2 （Ang-2） and vascular endothelialcell growth factor （VEGF） in oral squamous cell carcinoma （OSCC） and their correlations with clinicopathologic parameters, angiogenesis and vessel maturation of OSCC. Methods： The expression of Ang-2 and VEGF was detected in 41 speciments of human OSCC, 30 adjacent noncancerous oral tissues and 10 specimens of normal oral mucosa by conventional immumohistochemistry. Microvessel density （MVD） and vessel maturation index （VMI） were also assessed by double-labelling immumohistochemistry staining against CD34, a marker of pan-endothelial cells, and that against alpha-smooth muscle actin （α-SMA）, a marker of mural cells （pericytes/smooth muscle cells）. Results： The positive expression rate of Ang-2 and VEGF in 41 OSCC tissues was 51.22% and 63.42%, respectively. The expression of Ang-2 and VEGF was significantly higher in OSCC than in adjacent noncancerous oral tissues （all P〈0.05） and normal oral mucosa （all P〈0.05）. In the clinicopathologic parameters, the Ang-2 expression was closely correlated with tumor lymph node metastasis （P〈0.01） and the VEGF expression was correlated with tumor differentiated degree （P〈0.05）, but there was no significant correlation among the Ang-2 and VEGF expression and patients＇ sex, age and TNM stages （all P〉0.05）. The MVD of OSCC positive for both Ang-2 and VEGF was significantly higher than that of OSCC negative for both Ang-2 and VEGF （P〈0.05）. The VMI of OSCC positive for Ang-2 was significantly lower than that of OSCC negative for Ang-2 （P〈0.05）. When Ang-2 expression was combined with the staus of VEGF expression, MVD of OSCC positive for both Ang-2 and VEGF was the highest （51.08±2.99） as compared with that of other status in patient with OSCC （all P〈0.05）. Conclusion： The overexpression of Ang-2 and VEGF may play a crucial role in the development of OSCC. They are closely associated with angiogenesis and vessel maturation of tumor.

Increased protein and mRNA expression of endostatin in the ischemic brain tissue of rabbits after middle cerebral artery occlusion

Objective To explore the changes of endostatin （a strong anti-angiogenesis factor） and vascular endothelial growth factor （VEGF） in the brain tissues of rabbits following cerebral ischemia induced by middle cerebral artery occlusion （MCAO）. Methods Twenty-four New Zealand white rabbits were randomly divided into 5 groups： control （n = 5）, sham-operation （n = 4）, 2-hour ischemia （n = 5）, 24-hour ischemia （n = 5）, and 48-hour ischemia （n = 5）. The expression of VEGF and endostatin were measured by enzyme-linked immunosorbent assay （ELISA） and immunohistochemistry, respectively. In situ hybridization was used to characterize the expression of mRNA for the endostatin. Results Both the protein （at least 50%, P 〈 0.01） and mRNA （at least 70%, P 〈 0.05） of endostatin increased significantly in the ischemic brain tissues after MCAO compared with the control group. VEGF increased at least 270% in the brain after cerebral ischemia （P 〈 0.05）. Conclusion Cerebral ischemia leads to an up-regulation of endostatin in the brain, which is not associated with the increase of VEGF in the brain. The increase of endostatin may serve as a deleterious mechanism for ischemic injury through blocking angiogenesis.

Preparation of BCEC-Astrocyte Co-culturing Membrane Plate Insert

To prepare a hand-made micropore membrane culture plate insert forco-culture. Methods The plate insert was made using plastic centrifuge tube and micropore membrane.After seeding brain capillary endothelial cells (BCECs) on it (under the effect ofastrocyte-conditioned medium), the plate insert was assessed by analysis of trans-endothelialelectrical resistance (TEER). Results The plate insert has a stability of at least 15 d underculture condition. TEER increased significantly under co-culture condition from (66.1 +- 13.3)Ωcm^2 to (182.2 +- 6.7) Ωcm^2. Conclusion This micropore membrane culture plate insert can beeasily made, on which BCEC culture can be successfully performed. Moreover, it is adjustable andrecyclable. It follows that the plate insert is a useful tool for co-culture and the relatedresearch fields.

Effects of Ghrelin Antisense Inhibition on VEGF and Its Receptor Flt-1 mRNA Expression

[ Objective] This study was to investigate the effect of VEGF and its receptor Fit-1 mRNA expression in Mongolia sheep umbilical vein endothelial cells by ghrelin antisense inhibition. [ Method] Experiments were divided into 4 groups： group Ⅰ （blank control group） ; group Ⅱ （liposome group） ; group Ⅲ （SCON group： 20 μmol/L sense oligonucleotide） ; group Ⅳ （ASCON： 20 μmol/L antisense oligonucleotide）. VEGF and its receptor Fit-1 mRNA expression changes were detected by using real-time fluorescence quantitative detection after 24, 36 and 48 h. [ Result] The expression of VEGF mRNA in group Ⅰ, group Ⅱ were insignificantly different at higher expression levels, and did not change significantly with the time; the expression of VEGF mRNA in group Ⅲ assumed a slight decrease, but there were no significant differences between group I and group Ⅱ （P 〉0.05）, the expression of VEGF mRNA in group Ⅳ（antisense oligonucleotide group ） decreased significantly （P 〈 0.05） ; the expression of VEGF receptor FLT-1 mRNA was similar to that of VEGF. [ Conclusion] Antisense inhibition ghrelin has a downward effect to the expression of VEGF and its receptor Fit-1 the mRNA.

Vascular Endothelial Growth Factor C Expression in Gastric Carcinoma and Its Relationship with Lymph Node Metastasis

Objective: To study the expression of vascular endothelial growth factor C (VEGF-C) in gastric carcinoma and its relationship with lymph node metastasis. Methods: The expression of VEGF-C mRNA in 5 gastric carcinoma cell lines was detected by reverse transcription-polymerase chain reaction (RT-PCR). Simultaneously, the expression of VEGF-C protein in gastric carcinoma tissues, which were obtained from 63 patients who underwent radical gastrectomy, was detected by immunohistochemistry. Results: Three of the 5 gastric carcinoma cell lines, MKN-45, SGC-7901 and AGS, expressed VEGF-C mRNA. VEGF-C protein was expressed in 52.4% (33/63) of patients. VEGF-C protein expression was more frequently found in tumors with lymph node metastasis than in those without (P<0.01). VEGF-C protein expression was also closely related to lymphatic invasion (P<0.01) and TNM stage (P<0.01). However, there was no significant correlation between VEGF-C expression and the age, gender, tumor size, tumor location, Lauren classification, depth of invasion, and vascular invasion. Conclusion: The expression of VEGF-C is closely related to lymph node metastasis of gastric carcinoma, and lymphangiogenesis might be a new target for treatment of gastric carcinoma.

Association of Glu298Asp Polymorphism of the En-dothelial Nitric Oxide Synthase Gene with Essential Hypertension in Elderly People

Objective: To investigate the association of Glu298Asp polymorphism of theeNOS gene with essential hypertension in elderly people. Methods: Ninety-five cases of essentialhypertension were randomly chosen from outpatients and inpatients as the study group, and an equalnumber of sexes, age-matched healthy people as the control group. Their height, weight and bloodpressure were recorded and their fasting plasma lipid concentrations were measured. Glu298Asppolymorphism of the eNOS gene was measured using the methods of PCR and RFLP. Results: Theconstituent ratio of Genotype Glu/Asp in the study group (26.3%) was higher than that in the controlgroup (12.6%, x^2 = 5. 67, P<0.05), the allelic frequency of 298Asp in the study group (13.2%) wassignificantly higher than that in the control group (6.3%, x^2 = 5.06, P<0.05). Conclusion: Glu298Asp variant of the eNOS gene may be an independent predictor in essential hypertension.

The Potential Pathway of L-arginine·L-aspartate for Inhibition of Platelet Function

Aim L-Arginine· L-aspartate, a double salt, has been recently reported toinhibit platelet aggregation and thrombosis, but its action mechanism is not clear yet. This studywas conducted to investigate its effect on FITC-PAC-1, an anti-glycoprotein IIb/IIIa monoclonalantibody binding to activated platelets, and on correlative autacoid levels in plasma or inplatelets in order to explore its potential pathway of inhibiting platelet aggregation andthrombosis. Methods Monoclonal antibody binding to activated platelets was assayed by flowcytometry; NO was assessed by colorimetric method. cAMP, TXB_2 or 6-keto-PGF_(1α) levels wereassessed by radioimmunoassay. Results Gavaged 30 mg·kg^(-1) of L-arginine·L-aspartate increasedboth concentration of NO in plasma and 6-keto-PGF_(1) in incubated supernatant of aortic segment ofrats ex vivo (P < 0.05), but it did not influence cAMP content in platelets and the level of TXB_2or 6-keto-PGF_(1) in plasma of rats, whereas ASA significantly lowered TXB_2 or 6-keto-PGF_(1α) inplasma. Both 100 μmol-L^(-1) of L-arginine ·L-aspartate and ASA inhibited FITC-PAC-1 binding toactivated platelets in vitro. Conclusion The increase in NO and PGI_2 release from endo-thelialcells and consequent inhibition of platelet activation may contribute to the inhibition of plateletaggregation and thrombosis by L-arginine· L-aspartate; whereas arachidonic acid or cAMP metabolicpathway is not closely correlative with the studied effect.