Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
Objective To develop a new method for a tissue engineered vascular graft by combining endothelial cells and an acelluarized allogenic matrix. Methods Acellularized matrix tubes were obtained by a 0.1% trypsin and 0 02% EDTA solution for 24 hours and 1% Triton X 100 for 176 hours, respectively. Endothelial cells were isolated from alloaorta and expanded in vitro. Finally, the inner surface of acellularized matrix was reseeded with endothelial cells. Acellularity and reseeding were analysed by light microscopy and scanning electron microscopy. Results The acellularization procedure resulted in an almost complete removal of the original cells and the loose three-dimensional (3D) matrix. The acellular matrix could be reseeded with expanded endothelial cells in vitro, and endothelial cells had the potential of spread and proliferation. Conclusion Acellular matrix produces by Tritoon X-100 and trypsin possesses satisfactory biocompatibility for allogenic endothelial cell. Vascular grafts can be generated in vitro by a combination of endothelial cells and allogenic acelluarized matrix.
Objective To detect the expression of thromhospondin-1 （TSP-1） in gastric cancer and metastaticlymph node tissues， and to study its relationship of TSP-1 to clinicopathologic parameters or tumor angiogenesis. Methods The TSP-1 and vascular endothelial growth factor （VEGF） expressions and microvessel density （MVD） were evaluated by immunohistochemistry in 72 specimens obtained by gastric resection from patients with gastric cancer， including corres-ponding adjacent normal gastric mucosa tissues （distant from cancer ≥5 cm） and lymph nodes surrounding cancer. A semiquantitative scoring system was used for evaluating the staining. The relationship of TSP-1 to VEGF expression， MVD， or clinicopathologic parameters was analyzed. Results ① TSP-1 positive expression rate was 45.8% （33/72） in the primary gastric cancer tissues， 90.3% （65/72） in the corresponding adjacent normal gastric mucosa tissues， and 50.8% （30/59） in the metastatic lymph nodes tissues. The expressions of TSP-1 in the primary gastric cancer tissues and metastatic lymph nodes tissues were significantly lower than those in the adjacent normal gastric mucosa tissues （χ2=32.710，P=0.000；χ2=25.298， P=0.000）. The expression of TSP-1 had no statistical significance in the primary gastric cancer tissues as compared with in the metastatic lymph nodes tissues （χ2=0.327， P=0.568）. ② The expression of TSP-1 in the metastatic lymph nodes tissues was significantly lower than that in the non-metastatic lymph nodes tissues （Z=-2.573， P=0.010）. ③The expression of TSP-1 in the primary gastric cancer tissues and metastatic lymph nodes tissues suggested a negative correlation with VEGF （rs=-0.309， P=0.008；rs=-0.269， P=0.040） and MVD （rs=-0.348， P=0.003；rs=-0.272， P=0.037）. Conclusions TSP-1 expression is down-regulated and has a negative correlation with VEGF and MVD in the primary gastric cancer and the metastatic lymph nodes tissues. According to the present results， it seems likely that TSP-1 is a tumor angiogenesis inhibitor.
目的 建立重组人内皮抑素（恩度）联合顺铂一线治疗肿瘤进展的小鼠模型，继续应用内皮抑素联合紫杉醇二线治疗，研究内皮抑素协同紫杉醇抗肿瘤的作用及其机制。 方法 建立小鼠Lewis 肺癌移植瘤动物模型，内皮抑素联合顺铂治疗后观察肿瘤生长情况，遴选出肿瘤进展小鼠16只，随机留取1只，余15只小鼠随机分成紫杉醇组和联合用药组治疗，观察疗效。另取上述肿瘤进展小鼠1只，剥离肿瘤组织，重新接种，将成瘤小鼠随机分成生理盐水组，紫杉醇组及联合用药组治疗，观察疗效。治疗结束后24 h处死所有小鼠，采用免疫组织化学CD31单克隆抗体标记检测微血管密度（MVD），采用原位末端转移酶（TUNEL）检测细胞凋亡。 结果 只肿瘤进展小鼠中，联合用药组相比紫杉醇组生存时间无明显延长，但肿瘤体积增长较慢；而在重新接种成瘤的小鼠中，联合用药组较其余各组微血管密度明显减低（P＜0.05），凋亡指数明显增加（P＜0.05），肿瘤体积抑制明显。 结论 在内皮抑素联合顺铂治疗肿瘤进展的小鼠模型中，继续应用内皮抑素治疗与紫杉醇有较明显的协同抗肿瘤作用。
Objective To investigate the influence of cationic liposomemediated endostatin gene on colorectal cancer liver metastasis. Methods Animal model for colorectal carcinoma liver metastasis were established. The plasmid expressing endostatin genelipofectAMINE were injected in vein. Results After cationic liposomemediated endostatin gene were injected in vein, the incidence of liver metastasis and mean numbers of liver tumors were decreased, survival time of animal was significantly longer. Conclusion Intravenous injection of cationic liposomemediated endostatin gene can control the development of colorectal cancer liver metastasis effectively.
Objective To investigate the serum levels of endostatin and vascular endothelial growth factor ( VEGF) at different therapy stages of mouse Lewis lung carcinoma, and elucidate the relation to the progress and prognosis of tumor. Methods Forty-four Lewis lung carcinoma-bearing C57BL/6 mice were randomly divided into 4 groups, ie. a non-therapy group, a chemotherapy group, a gene therapy group, and a combination therapy group ( chemotherapy plus gene therapy) . Eleven healthy mice were included as normal control group. Serum was collected on the 0th, 5th, 19th day after therapy for measurement of endostatin and VEGF by ELISA. The correlations were analysed between endostatin and VEGF levels in each group. Results ( 1) The serum endostatin levels had no significant difference in all groups on the 0th day,but increased significantly on the 5th day in the gene and combination therapy groups than those in other three groups ( all P lt;0. 01) . Then the endostatin level decreased on the 19th day in the gene and combination therapy groups, but still higher than those in the chemotherapy group and the normal group. ( 2 ) On the contrary, serum VEGF levels of the gene and combination therapy groups decreased significantly on the 5th day and increased little on the 19th day, which were both significant lower than those in chemotherapy group on the 5th and 19th day( all P lt; 0. 05) . There were significant diferences between the three therapy groups and the non-therapy group( all P lt;0. 05) . ( 3) Negative correlations between VEGF and endostatin levels were revealed in the gene and combination therapy groups ( r = - 0. 77 and - 0. 761 respectively) .Correlation was not found in the non-therapy and chemotherapy groups. Conclusion The serum levels of endostatin and VEGF might be used as monitoring indices of antiangiogenesis therapy.
Objective To investigate the effects of Hep-A and Hep-B on vascular endothelial growth factor (VEGF)-induced breakdown of blood-retinal barrier. Methods The mice were subcutaneously injected vehicle, Hep-A or Hep-B 10 mg/kg twice a day for 5 days. Then, 1 μl of 10-6mol/L VEGF were intravitreous injected. After 6 hours, 13.7×104Bq/g3H-mannital were injected intraperitoneally. The mice were sacrificed and the retinas, lungs, kidneys were removed and examined for radioactivity. The result were analyzed using SPSS software to calculate and compare retina/lung and etina/kidney leakage ratio among groups of different treatment. Result The retina/lung and retina/kidney leakage ratio were 0.38±0.04 and 0.21±0.03 respectively in normal mice; increased significantly to 1.05±0.11 and 0.46±0.04 respectively in model mice, both Plt;0.01 compared to those in normal mice; decreased to 0.59±0.06 and 0.32±0.03 respectively in mice treated with Hep-A, both Plt;0.01 compared to those in model mice; decreased 0.54±0.04 and 0.35±0.03 in mice treated with Hep-B,both Plt;0.01 compared to those in model mice. Conclusion Hep-A and Hep-B can significantly reduce VEGF-induced breakdown of blood-retinal barrier in mice. Chin J Ocul Fundus Dis，2004，20：352-354)
Objective To investigate clinical significance of serum VEGF-C level and C-erbB-2 protein expression in patients with breast cancer. Methods Sixty-two female patients with breast invasive ductal cancer and breast benign lesion were respectively selected. Serum VEGF-C level was detected by enzyme-linked immunosorbent assay (ELISA) before operation and at one month after operation, and C-erbB-2 protein expression in tissues of breast cancer was detected by immunohistochemistry. Then, the relationship between serum VEGF-C level and clinicopathologic characteristics and C-erbB-2 protein expressions wereas analyzed. Results The serum VEGF-C level before operation in breast cancer patients〔(279.65±17.34) pg/ml〕 was significantly higher than that in breast benign lesions patients 〔(167.26±12.15) pg/ml〕, P＜0.01. In breast cancer patients, the serum VEGF-C level before operation was higher than that at one month after operation 〔(209.45±15.23) pg/ml〕, P＜0.01. The serum VEGF level was related to tumor stage (P＜0.05) but not to patient age, tumor size, menopause status , lymph node metastasis or not and ER and PR expression (Pgt;0.05). The positive expression rate of C-erbB-2 protein in breast cancer patients (54.84%, 34/62) was significantly higher than that in breast benign lesion patients (11.29%, 7/62), P＜0.01. Moreover, the positive expression rate of C-erbB-2 protein in breast cancer patients with axilla lymph node metastasis (69.44%) was significantly higher than that without axilla lymph node metastases (34.62%), P＜0.05. The serum VEGF level increased with increasing expression intensity of C-erbB-2 protein and there was positive correlation between them (r=0.813，P＜0.05). Conclusions The serum VEGF-C level in breast cancer may be conducted as an assisted marker to differential diagnosis of breast tumor. C-erbB-2 is related to lymph node metastasis of breast cancer patients. There is synergistic effect between VEGF-C and C-erbB-2 in the lymph node metastasis way of breast cancer.
Abstract: Objective To evaluate the significance of expression of vascular endothelial growth factor-C （VEGF-C） and cytokeratin 19 （CK19） in patients with stage I non-small cell lung cancer （NSCLC）. Methods A total of 269 patients with NSCLC who underwent standard lobectomy and lymph node dissection by the same surgical team in our hospital from January 2004 to June 2005 were included in this study. All the clinical data and follow-up results were complete, and all the pathological specimens were well kept. No preoperative or postoperative adjuvant therapy such as radiotherapy and chemotherapy was administered to those patients. Expressions of VEGF-C in cancer tissues was detected by immunohistochemical streptavidin-peroxidase （S-P） method, and CK19 was marked to examine micrometastasis in hilar and mediastinal lymph nodes. Clinical outcomes, pathological results and follow-up data were analyzed in combination with VEGF-C and CK19 expression. Results VEGF-C expression was not statistically different between different category in sex（Hc=1.722，P=0.084）, age （Hc=0.914，P=0.360）, smoking （Hc=2.440，P=0.295）, pathology type （Hc=5.668，P=0.058）or tumor size （Hc=0.165，P=0.920） . VEGF-C expression was statistically different between different groups of pathological differentiation （Hc=29.178，P=0.000). CK19 expression was not statistically different between different category in sex（χ2=0.000，P=0.999）, age （χ2=0.005，P=0.999）, smoking （χ2=2.294，P=0.317）, pathology type （χ2=0.573，P=0.289）, tumor size（χ2=0.006，P=0.999）, and pathological differentiation （χ2=2.927，P=0.231）. Five-year survival rate was statistically different between different grade of VEGF-C expression （χ2=37.318，P=0.000), and was also statistically different between positive group and negative group of CK19 （χ2=39.987，P=0.000). There was statistical difference between different grade of VEGF-C expression and positive rate of CK19 （χ2=25.954，P=0.000). Conclusion Expression of VEGF-C and CK19 is closely related to postoperative 5-year survival of patients with stage I NSCLC. Detection of VEGF-C and CK19 is of great clinical significance as it is helpful to predict patient prognosis and choose proper postoperative adjuvant therapy.
Objective To understand the value of pre-coating in artificial vessel endothelialization. Methods Literature concerning precoating in artificial vessel endothelialization was extensively reviewed. Results Pre-coating included chemical coatings(collagen, fibronectin, laminin, poly-l-lysin, gelatin andextracellular matrix), pre-clotting(plasma, blood, serum and fibrin glue), chemical bonding (heparin, RGD and lectins) and surface modification. Most of them could enhance the adhesion of the endothelial cells. Conclusion Pre-coating couldimprove endothelialization, but further research is needed to search for the appropriate concentration and incubation time.