ABSTRACT
Objective To study the role of autophagy in proliferation,migration and tube formation of retinal vascular endothelial cells (RVECs) under the condition of hypoxia in vitro.Methods Mouse RVECs were isolated from 50 C57BL/6J mice primarily cultured using explant culture method,and the cells were identified by immunofluorescence of CD34.Well cultured RVECs were divided into normal control group,hypoxia control group and hypoxia+ 3-methyladenine (3-MA) group.The cells in the normal control group were cultured in the normal environment for 24 hours,and the cells in the hypoxia control group were cultured 1％ O2 environment for 24 hours,and the cells in the hypoxia+3-MA group were pretreated with 5 mmol/L 3-MA for 4 hours and then exposed to 1％ O2 environment for 24 hours.Microtubule-related protein 1 light chain 3 (LC3-Ⅱ/LC3-Ⅰ) and Beclin-1 protein contents in the cells were detected by Western blot analysis;the ultrastructure of autophagosome was examined under the transmission electron microscope.The proliferation,migration and tube formation of the cells were detected by Click-iTEdU kit,scratch assay and matrigel,respectively.Results Primarily culture cells grew well with the cobblestone-like appearance 5-7 days after culture and showed positive response for CD34.The autophagosome number was increased in the hypoxia control group compared with the hypoxia+3-MA group and normal control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.243 ± 0.030,0.658 ± 0.032 and 0.405 ± 0.095;the relative expression of Beclin-1 protein in the cells was 0.260±0.040,0.650±0.071 and 0.461±0.089;the proliferation rate of the cells was (45.93± 6.39) ％,(22.74± 2.35) ％ and (24.12 ± 3.59) ％;the scratch healing rate of the cells was (36.02 ± 5.84) ％,(57.26±11.98) ％ and (18.16±9.73) ％;the number of tube formation was 29.20±6.10,41.40±4.04 and 22.00± 2.92 in the normal control group,hypoxia control group and hypoxia + 3-MA group,respectively,with significant differences among the groups (F =35.86,23.53,34.28,21.12,23.27;all at P＜0.01).Compared with the normal control group,the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein,scratch healing rate and the number of tube formation were significantly increased,and the proliferation rate was significantly reduced in the hypoxia control group (all at P ＜ 0.05).Compared with the hypoxia control group,the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein,scratch healing rate and the number of tube formation were significantly decreased in the hypoxia + 3-MA group.Conclusions Hypoxia environment activates autophagy of mouse RVECs,which enhances the migration and tube formation abilities of the cells.3-MA inhibits the angiogenesis abilities of mouse RVECs.
ABSTRACT
Objective In this study,we explored the role of combination of autophagy inhibition and anti-VEGF in proliferation,migration and tube formation of mouse retinal vascular endothelial cells(RVECs). Methods Well cultured mouse RVECs were randomly divided into four groups:autophagy inhibition group(add-ing autophagy inhibitor 3-MA),anti-VEGF group(adding anti-VEGF-A neutralized antibody),autophagy inhibi-tion+anti-VEGF group(adding the two reagents)and the control group.All cells were then cultured in the hypoxic condition. The cell proliferation,migration and tube formation were detected by EdU,transwell and matrigel as-say,respectively. Results The cell proliferation rate,number of migrated cells and number of tube formation of the other three groups decreased when compared with the control group.These data above in autophagy inhibition+anti-VEGF group were all significantly less than 3-MA group and anti-VEGF group. Conclusion Combination of autophagy inhibition and anti-VEGF may be more effective than simple anti-VEGF in inhibition of retinal neovascu-larization.
ABSTRACT
Objective To study the expressions of Chemerin in hepatocellular carcinoma (HCC) and HCC cell lines,and to demonstrate the relationship between the expressions and prognosis.Methods The expressions of Chemerin protein in normal hepatocellular tissues,HCC and their paired tissues were detected by immunohistochemical staining of SABC; the expressions of Chemerin protein in HCC,cell lines and immortalized hepatocyte cell lines were detected by RT-PCR.Results The positive expression rates of Chemerin were 56.67％,90.00％,100％ in HCC,HCC cell line and normal hepatocellular tissues respectively,and the differences were significant (P＜0.05).The expressions of Chemerin mRNA in HCC paired tissues were higher than in HCC (P＜0.05).There was a higher expression of Chemerin mRNA in immortalized hepatocyte cell line LO2 ; but a lower expression in HCC cell lines.The expressions of Chemerin were related to lymph node metastasis,portal vein tumour thrombi,differentiation and TNM stage but not related to sex,age,tumour size,HBsAg and AFP.Conclusions Down-regulated expression of Chemerin may play an important role in the development,progression and metastasis of HCC.It may be a molecular marker for prognosis of HCC.