The Research Of The Function Of Pigment Epithelium-derived Factor On Gastric Carcinoma SGC7901and HUVEC In Vitro

Objective: In the present study, we designed the SGC7901infected withrecombinant pigment epithelium-derived factor adenovirus (Ad-PEDF) in vitroand investigated the expression efficiency of PEDF, Proliferation migrationassay and tube formation of SGC7901and humanumbilical veinendothelialcells(HUVEC) was used to evaluate the biological activity of PEDF. and toevaluate the antiangiogenic property of PEDF and tumor growth or metastasis.also explore a new method for gastric cancer gene therapy.Methods:(1) Human Embryonic Kidney293cells were transfected byAd-PEDF and Ad-GFP recombinants, the lysate of the cells was collected.(2)The expression of PEDF mRNA or protein in HEK293cells was determinedby RT-PCR or Western Blot, The virus titer was determined by TCID50.(3) The SGC7901infected by the optimal multiplicity of infection (MOI) ofamplified virus, meanwhile, make the gastric cancer cell line SGC7901group excluding adenovirus but culture medium as a blank control group, andcollect conditioned medium from each group of cells after72h incubation, thenuse Western blot to identify the PEDF protein expression of cells of eachgroup.(4) The proliferation and viability tests via CCK8kit were executed toevaluate the inhibition activities of PEDF on the growth of both SGC7901cells and HUVECs.(5) The Transwell tests were carried out to investigate the retardation of invasion and migration of SGC7901cells and HUVECs byPEDF.(6) The tests of capillary-like tube formation were acted to assess thethe bioactivity of PEDF in different conditioned media on HUVECs. Thisexperiment adopts entirely random single factor analysis of variance (ANOVA)methods to statistically analyze each group’s testing result, and uses LSDinspection to do pairwise comparison to check the difference.Results:(1) Recombinant adenovirus vector were amplified largely in HEK293cell after being infected with it.(2) By amplifying Ad-PEDF as per designedPEDF primer sequence PCR, one can observe PEDF gene segment of about1.25kb long. Western blot result shows that Ad-PEDF group infected withHEK293cell generates an immune hybridization stripe of50kDa moleculeweight; while no target hybrid stripe exists in Ad-GFP group or other groupfree of HEK293infection. It’s authenticated by TCID50methods, the virustiter of amplified Ad-PEDF and Ad-GFP is relatively high—6.3×107pfu/mLand6.3×108pfu/mL.(3) Recombinant virus can get infected with gastriccancer cell effectively. In Western blot analysis the gastric cancer cell infectedin different conditioned medium and culture supernatant, PEDF objectiveprotein expression is visible in but bot transfected by Ad-PEDF/gastric cancer7901; while it’s invisible in Ad-GFP/cancer7901.(4) Use CCK8kit to analyzerecombined PEDF’s proliferative effect on gastric cancer cell and umbilicusveins endothelial cells. The result shows, compared with negative group andcontrol group, Ad-PEDF group’s influence over the proliferation of gastric cancer cell line SGC7901has no obvious difference, nor statistical meaning(P>0.05); in the proliferation experiment of umbilicus veins endothelial cells,the OD value of Ad-PEDF/cancer SGC7901group is0.3534±0.0098, whichhas obvious statistical significance (P<0.01) compare with untransfectedgastric cancer SGC7901and Ad-GFP/cancer SGC7901group.(5) The resultof the experiment on the invasion and migration of gastric cancer cell andumbilicus veins endothelial cells shows, Ad-PEDF can obviously restrain theinvasion and migration of gastric cancer cell line SGC7901, Ad-PEDF/gastriccancer cell line SGC7901can prevent the invasion and migration of umbilicusveins endothelial cells, the difference has significant statisticalmeaning(P<0.01).(6) It can be observed in the experiment result of simulatingin vitro vascularization on matrigel, untranfected gastric cancer SGC7901andAd-GFP/gastric cancer SGC7901group produce plenty of tubules, whileAd-PEDF/gastric cancer SGC7901forms less, which has statisticalsignificance (P<0.01).Conclusion: The experiment verifies that PEDF can restrain thedevelopment and transfer of gastric cancer by the means including:alternatively and directly prevent gastric cancer cell from invading andmigrating; on the other side, indirectly restrain the proliferation of vascularendothelial cell, preventing it from invasion and migration, and restrainingangiogenesis, so as to hold back the development and transfer of gastric cancer,thus providing a new mentality for gene therapy of gastric cancer.