| Objective:1,Search for the culture optimum density of umbilical cord mesenchymal stem cells(UC-MSCs), its biological and phenotype character.2,Study the proliferation and apoptosis effect of UC-MSCs to HepG2cells,and the possible mechanism.Methods:1,Revive the passage3UC-MSCs and culture in10%fetal bovine serum,DMEM/F12,hand down to passage7,indentify ther phenotype of the two passages,compare their differences.take the passage7cells colture with HepG2in experiment.2,ount the UC-MSCs,colture at the density of8xl06/hole,4xl06/hole,2xl06/hole,1x106/hole,5x105/hole,2.5x105/hole respective,observe and compare their growing,take the right cell density to experiment.3,Digest and collect the UC-MSCs, culture in6plat at the density of4x106/hole,2x106/hole,1x106/hole,5x105/hole,2.5x105/hole respective,breed the HepG2cells at the density of1x106/hole on the Transwell membrane after24hours.coculture for24hours,48hours,72hours,detect the apoptosis of HepG2cells with tunell and the protein of AFP,Bc12,Survivin in the HepG2cells on flow cytometer(FCM). culture in6plat at the density of1.5x106/hole,1.25x106/hole, lx106/hole,7.5xl05/hole,5xl05/hole respective, breed the HepG2cells at the density of5X105/hole on the Transwell membrane after24hour, culture for24hour,48hour,72hour,decet the cell proliferation with MTT.4,Digest and collect the UC-MSCs, colture in6plat at the density of 1.5xl06/hole,1.25xl06/hole, lxl06/hole,7.5xlO5/hole,5xl05/hole respective, breed the HepG2cells at the density of1x106/hole on the Transwell membrane after24hours,coculture for72hours, decet the gene expression with RT-PCR.Results:1,After freezing and reviving, UC-MSCs growing at parallel arrangement and whirlpool with shape homogeneous.2,The Immunophenotyping of passage3and passage7show express the MSCs surface marker CD29,CD44,CD90, CD105positive,and the hemopoietic stem cell surface marker CD34,CD45negative.3,The result of culture the different density of UC-MSCs show that after culturing24hours,the cells at the density of8x106/hole,4x106/hole completely overgrow, observed the8x106/hole and4x106/hole have hight density.the cells can not completely stretch,lots of the8x106/hole cells can not adhere. After culturing24hours,The density of2xl06/hole,lxl06/hole,5xl05/hole,2.5xl05/hole can ahhere completely in good condition,but can not overgrow completely.After72hours,8x106/hole,4xl06/hole,2xl06/hole completely overgrow, only observing8x106/hole cells which were not adherent were dead,the other densities were no dead cells.The size of all cells was amplified with the culture time, and put in order gradually.4,The apoptosis rate of HepG2cells was increased with the density of UC-MSCs and the time of UC-MSCs after coculturing. Statistics has difference (P<0.01),the apoptosis rate of72hours was53.4%.5,The positive rate of AFP,Bcl2,Survivn in the HepG2cells was decreased with the density of UC-MSCs. Statistics has difference (P<0.01),and decreased with the time of UC-MSCs on HepG2cells,beside the control group (P>0.05),the other density statistics has difference (P<0.01)6,The HepG2cells proliferation inhibition ratio has increased with the density of UC-MSCs and the time of UC-MSCs after coculture, statistics has difference (P<0.001) 7,The HepG2cells'genes AFP,Bc12,Survivin rate was decreased with the density of UC-MSCs,statistics has difference (P<0.01)Conclusion:Coculture UC-MSCs and HepG2cells can promote the HepG2cells to apoptosis,suppress the proliferation of HepG2cells,this effect was relate to AFP> Bc12,Survivn in the HepG2cells. |
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