Objective To construct green fluorescent protein (GFP)/Akt fusion gene vector for observing the expression and localization of GFP/Akt in rats bone marrow-derived mesenchymal stem cells （MSCs）. Stem cell factor （SCF） effected expression of c-kit, Akt and VEGF mRNA and protein in MSCs transfected by pEGFP-C1/Akt through PI3-Akt pathway.Methods Akt recombined GFP vector by restriction enzymes, MSCs was transfeced by GFP/Akt and GFP through cationic liposomes, and then veritied by restriction endonuclease assay and sequence analysis. Transfection and localization of GFP were evaluated by fluorescene microscopy. The expressions of c-kit, Akt and VEGF mRNA and protein were examined by RT-PCR and Western blot after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. SCF effected the expression of c-kit, Akt and VEGF mRNA and protein after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. Results Restriction endonuclease assay and sequence analysis verified that thesuccessfulconstructionoftherecombinantvectorpEGFP-C1/AktandefficienthighexpressionofpEGFP-C1/Akt fusion protein in the MSCs of rats. Under fluorescent microscence, green flurescence was seen homogeneously distributed in the entire cell of the cells transfected by the recombinant vector pEGFP-C1, and diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C1/Akt. The expression of Akt and VEGF mRNA and protein were significantly higher in MSCs transfected by pEGFP-C1/Akt (plt;0.05). The expression of c-kit, Akt and VEGF mRNA and protein were significantly higher in experiment group (SCF+pEGFP-C1/Akt) and control group (SCF+pEGFP-C1), plt;0.05. In experiment group， SCF stimulation enhanced expression of Akt and VEGF mRNA and protein (plt;0.01). Conclusion GFP/Akt fusion gene vector is successfully construted and the fusion protein expressed in the MSCs of rats induces the expression of Akt and VEGF mRNA and protein. SCF stimulation enhanced expression of c-kit, Akt and VEGF mRNA and protein through PI3/Akt pathway.
Objective To investigate the protection on the intrahepatic cholangiocyte mediated by hypoxic preconditioning (HP) after liver transplantation and the role of vascular endothelial growth factor (VEGF). Methods The model of autologous liver transplantation was established, and the rats were divided into 3 groups: autologous liver transplantation group, hypoxic preconditioning before operation group (HP group) and sham operation group. At 6, 12, 24, 48 h after operation, blood samples were collected for examination of the serum total bilirubin (TBIL), direct bilirubin (DBIL) and alkaline phosphatase (ALP), and the expression of VEGF was detected by immunohistochemical method. The pathological changes of cholangiocytes were observed by light microscope. Results As compared with autologous liver transplantation group, the levels of seurm TBIL, DBIL and ALP in HP group were lower (P＜0.05), while the expression of VEGF in HP group was higher at the whole process (P＜0.05). The degrees of billiary epithelium damage and inflammatory infiltration in autologous liver transplantation group were more severe than those in HP group. Conclusion HP has protective effect on cholangiocytes after liver transplantation, in which VEGF may play an important role.
【Abstract】Objective To evaluate the status of vascular endothelial growth factor (VEGF) expression in breast carcinoma and benign disease and define the relationship with age,menopause, tumor size，clinical stage，distant metastasis and lymph node metastasis. Methods Seventy cases of invasive ductal breast carcinomas,30 benign breast diseases and 7 adjacent nonneoplastic specimens were assessed for VEGF protein expression by immunohistochemistry LSAB method. Results VEGF were expressed more frequently in breast cancer than in benign diseases.VEGF was significantly correlated with axillary lymph node metastasis and distant metastasis,whereas no statistical correlation with other factors. Conclusion VEGF status has certain value to make differential diagnosis between malignant and benign breast diseases and predict the possibilities of distant and lymph node metastasis.
ObjectiveTo observe the effect of Delta-like ligand 4 (Dll-4) on the pathological structure of retina in early diabetic rats (DM) and its relationship with vascular endothelial growth receptor-2 (VEGFR-2).MethodsA total of 70 male Sprague-Dawley rats were randomly divided into normal group and DM group, with 10 and 60 rats in each group, respectively. The rats of DM group was induced by intraperitoneal injection of streptozotocin to established DM model. The rats with blood glucose recovery and death were excluded, and the final 60 rats were included in the statistics. Rats in the normal group were injected with an equal volume of citric acid-sodium citrate buffer. Rats in the DM group were divided into DM 1 month (DM 1m) group, DM 2 months (DM 2m) group, DM 3 months (DM 3m) group and DM 3m + Anti group, DM 3m + phosphate buffer solution (PBS) group by random number table method, and 10 rats in each group. In the DM 3m+Anti group, 4 μl of anti-Dll-4 polyclonal antibody was injected into the vitreous cavity, and the antibody concentration was 0.25 mg/ml. The DM 3m+PBS group was intravitreally injected with an equal volume of PBS. Five days after the injection, the rats were sacrificed. Rats in the DM 3m group and the normal group were not treated, and were sacrificed 3 months after the model was established. The structure and microvascular changes of the retina were observed by hematoxylin-eosin staining, and the total thickness of the retina was measured. The expression of Dll-4 and VEGFR-2 in the retina was detected by immunohistochemistry and fluorescence quantitative polymerase chain reaction (PCR). One-way analysis of variance was used to compare the expression of Dll-4 and VEGFR-2 in the retina of each group. The least significant difference t test was used to compare the two groups.ResultsLight microscopy showed that the retinal ganglion cells layer in the DM 3m group were obviously edematous, the inner and outer nuclear layers were thinner, the number of cells was reduced, the arrangement was disordered, the edema of outer plexiform layer was obvious, and the microvessels were abnormally dilated. In the DM 3m+Anti group, the edema of outer plexiform layer was lessened than that of the DM 3m group, and the other layers were not significantly different from the DM 3m group. Compared with the normal group, the total retinal thickness of the DM 3m group, the DM 3m+Anti group and the DM 3m+PBS group increased (t=5.596, 3.290, 4.286; P=0.000, 0.008, 0.002). Immunohistochemical staining showed that a small amount of Dll4 was positively expressed in the retinal ganglion cell layer of the normal group; a small amount of VEGFR-2 was positively expressed in the ganglion cell layer and the inner and outer nuclear layers. The positive expression of Dll-4 and VEGFR-2 in retinal vascular endothelial cells of DM 3m group increased significantly. The expression of Dll-4 was significantly decreased in the retinal layers and vascular endothelial cells of DM 3m+Anti group, while the expression of VEGFR-2 was significantly increased. There was no significant difference between the positive expression of Dll4 and VEGFR-2 in the DM 3m+PBS group and the DM 3m group. The results of real-time PCR showed that the relative expression of Dll-4 and VEGFR-2 mRNA in the DM 3m group was significantly higher than that in the normal group (t=6.705, 20.871; P＜0.05). Compared with DM 3m group, the relative expression of Dll-4 mRNA in DM 3m+Anti group decreased, and the relative expression of VEGFR-2 mRNA increased (t=2.681, 3.639;P＜0.05). The relative expressions of Dll-4 and VEGFR-2 mRNA in the DM 3m+PBS group and DM 3m group were not statistically significant (t=0.513, 0.657; P＜0.05).ConclusionsThe expression of Dll-4 in retinal vascular endothelial cells is gradually increased during the early retinopathy of DM rats. The expression of Dll-4 is inhibited, the expression of VEGFR-2 is up-regulated, and the plexus edema is alleviated.
【摘要】 目的 研究过氧化酶增殖因子活化受体γ（peroxisome proliferator-activated receptor gamma， PPARγ）和血管内皮生长因子C（vascular endothelial growth factor C， VEGF-C）在胃癌组织中表达的相关性，分析其表达与临床病理因素之间的关系。 方法 2005年3月-2009年3月间采用逆转录-聚合酶链反应方法检测36例胃癌手术标本中PPARγ和VEGF-C mRNA的表达，同时选取相同患者的胃正常组织作为对照。 结果 PPARγ mRNA在胃癌中的表达量高于癌旁正常组织，两者的差异有统计学意义（P=0.007）；VEGF-C在胃癌中的表达量高于癌旁正常组织，两者的差异有统计学意义（P=0.004）；PPARγ的表达与VEGF-C表达无关联性（r=0.135，P=0.414）；PPARγ表达与胃癌组织中浸润程度有关（χ2=4.620，P=0.032）、淋巴结转移有关（χ2=15.753，P=0.000）和临床病理分期有关（χ2=4.610，P=0.032）；VEGF-C表达与胃癌组织中淋巴结转移有关（χ2=4.729，P=0.030）、远处转移有关（χ2=4.064，P=0.044）和临床病理分期有关（χ2=6.300，P=0.012）。 结论 PPARγ和VEGF-C可能在胃癌新生淋巴管形成中起重要作用，两者的表达水平与胃癌患者的病情判断及预后评价密切相关。【Abstract】 Objective To investigate the significance of expressions of peroxisome proliferator-activated receptor gamma (PPARγ) and vascular endothelial growth factor C (VEGF-C) in gastric carcinoma as well as their correlation, and to study the relationship between the expressions and clinicopathologic characteristics of gastric carcinoma. Methods Thirty-six pairs of normal mucosa and cancer specimens were obtained from patients who had undergone gastric operation for primary gastric carcinoma and subjected to reverse transcription-ploymerase chain reaction (RT-PCR) for PPARγ and VEGF-C mRNA detection. Results The positive rate and level of PPARγ mRNA expression were higher in gastric cancer tissues than those in normal gastric mucosa (P=0.007). The positive rate and level of VEGF-C mRNA expression were also higher in gastric cancer tissues than those in normal gastric mucosa (P=0.004). Simultaneously, the expression of PPARγ was not correlated with that of VEGF-C (r=0.135, P=0.414), while the highly productions of PPARγ and VEGF-C in gastric carcinomas were both associated with the lymph node metastasis and the clinical stage (Plt;0.05). Conclusion PPARγ and VEGF-C may play an important role in the lymphangio-genesis of gastric cancer, and united detection of PPARγ and VEGF-C expressions may be correlated with making diagnosis, evaluating prognosis in patients with gastric cancer at the same time.
Objective To investigate the effect of caveolion-1 on the growth and proliferation in human breastcancer MCF7 cell in vitro and in vivo and approach its mechanisms. Methods The plasmid pCI-neo-cav-1 and its corres-ponding empty vector （pCI-neo） were transfected into MCF7 cell line. The expressions of caveolin-1 and vascular endoth-elial growth factor （VEGF） in transfectants were subsequently confirmed by Western blot analysis. A pair of monoclonal cell lines， MCF7/cav-1 and MCF7/vec， were chose for future studies. The colony formation ability of tumor cells was detected by anchorage-independent growth assay. Xenograft tumor models in nude mice were established. Immunohisto-chemistry was used to characterize Ki-67 and VEGF levels in the tumors tissues. Results Caveolin-1 expression was up-regulated in the MCF7/cav-1 cell as compared with the MCF7/vec cell （P＜0.01） and the MCF7 cell （P＜0.01）. Caveolin-1 overexpression markedly reduced the capacity of the cells to form colonies in soft agar （P＜0.01）. Compared with the MCF7/vec cell， VEGF protein expression reduced in the MCF7/cav-1 cell （P＜0.01）. In vivo experiments in nude mice， the overexpression of caveolin-1 resulted in significant growth inhibition of the xenograft breast tumors. The Ki-67 staining was weak and the VEGF staining was negative by immunohistochemistry that indicated the caveolin-1 inhibited the proliferation in human breast cancer MCF7 cell. Conclusion The caveolin-1 might inhibit the growth and proliferation in human breast cancer MCF7 cell through suppressing activation of VEGF signaling pathway.
Objective To investigate the effects of simvastatin on pulmonary function and vascular endothelial growth factor ( VEGF) levels in induced sputumof patients with COPD exacerbation( AECOPD) .Methods Thirty-eight patients with AECOPD were divided into two groups randomly, ie. a routine medical treatment( RT) group( n =30) and a routine + statin medical treatment( RST) group( n =28) . The VEGF levels in serumand induced sputum were detected by ELISA on the first day and after a week treatment in hospital, respectively. Meanwhile, the pulmonary function measurements were performed. Results There were no significant differences in the pulmonary function ( FEV1% pred and FEV1 /FVC) and VEGF levels in induced sputumbetween the two groups before treatment( P gt;0. 05) . The RT group showed no significantchanges in any parameters before and after a week treatment( P gt; 0. 05) . FEV1% pread, FEV1 /FVC and VEGF levels in induced sputum in the RST group after a week treatment significantly increased compared with those before treatment and the RT group( P lt;0. 01, P lt;0. 01, P lt;0. 05) . But There were no significant differences in serumVEGF levels between the two groups before and after a week treatment. The VEGF levels in induced sputum were positively correlated to FEV1% pread and FEV1 /FVC after a week treatment( r =0. 430, P lt;0. 05; r = 0. 388, P lt; 0. 05) . Conclusions Simvastatin may reduce the decline in pulmonary function and decrease the levels of VEGF in induced sputum of patients with AECOPD. Improvement in pulmonary function may be related to down-expression of lung VEGF