Sorting And Preliminary Study Of CD90 Positive Cells In Human Hepatocellular Carcinoma Cell Line 7721

Objective:To isolate CD90 positive cells and CD90 negative cells from human hepatocellular carcinoma cell line SMMC-7721, for it stem cells characteristic research.Methods:Isolate CD90 positive cells by magnetic microbeads cell sorting. Dilute it to 10～15 cells per milliliter Dulbecco's Modified Eagle Media:Nutrien t Mixture F-12 for monoclonal cultivation. Other goal cells make each group t o the same density, place it to culture bottle. Detect the expression rate of ste m cell markers CD34, CD45, CD90 and CD133 of each group, Real-time fluo rescence quantitative PCR to detect the disparity of gene expression. The candi date gene include AFP,CCNA2,CLDN10,PTEN,SPINT,TFDP1 et al. Det ect the disparity of protein(AFP,CK18,CK19) expression between each group cells by immunocytochemistry. SMMC-7721 did not undergo MACs and CD9 0 negative cells which collected from cell sorting for control.Result:Four times cell sorting we were successful in isolating the purpose cell, But the resulting purpose cell number and purity is low, CD90 expression for 0.8-1.2% before the four sorting. The number and purity of purpose cell were 1.37X 104; 7.5%,1.23×104; 9.0%,1.42×104; 30.7%,2.85×104; 27.2% respectively. Later, we added low concentration(0.5μM) of ethylene diamine tetraacetic acid and 2% fetal bovin serum in phosphate buffer solution to prevent cells agglomerate, and give a sufficient combination of antibodies and purpose cells. So the sorting purity raised to 30.7%, but, there's no apparent elevation of the purpose cells number.There have difference of morphology among some monoclonal cultivated cells, but whith the generation cultivation, these difference get ambiguouse gradually. Majority cells have the capacity to proliferation and differentiation into cells which express other stem cell markers, only minority cells of three groups necrosis after transient proliferation. Even in the same cultivate cell, there have cells necrosis and well growth.The result of fluorescence activated cell sorting shows that P0 generation cells expression rate of CD90 in CD90 positive Group:69.9%; CD90 negative group: 0.4%;7721 group:5.2%, and the expression rate of CD90 in three groups from P1 to P4 were 3.0%,2.3%,0.7%; 1.1%,0.1%,1.0%; 8.1%,4.4%,8.3%; 6.7%,3.7%,7.4%。. In CD90 positive group expression rate of CD34 were 0.3-7.8%; CD45: 33.1%-96.0%; CD133:2.0%～59.1%; expression rate of each marker in 7721 group were0.8%～11.2%;25.7%～53.2%; 3.6%～13.6%; and an appropriate rate of 0.9%～12.3%; 19.5%～70.0%;2.5%～11.1% in CD90 negative group. This result indicated that expression rate have variance among each group, but with cells differentiation, the expression rate of various stem cell markers returned to the level of SMMC-7721 gradually.And the outcome of immunocytochemistry stain indicated that expression of AFP, Gpc3, CK18 and PCNA there's no difference between CD90 positive and negative group cells. But CK19 and Vimentin negative stain in CD90 positive group while positive stain in SMMC-7721 and CD90 positive group.CD90 positive cells group and CD90 negative cells group.The gene expression testing results take rank sum test for analysis, results of analysis indicated that expression variance of gene PTEN between CD90 positive group and negative group from monoclonal cell culture are significantly. (p=0.044), CD90 negative group have a high expression of this cancer inhibit gene than CD90 positive group cells. While, seven other gene ANGPT, AFP, APC of monoclonal cultivation and E2F1, HGF, P16 et al seventeen gene of two groups from serial subcultivation expression variance have no statistical significance (p> 0.05)Conclusion:This study indicated that, (1) indirect marked microbeads cell sorting have a strictly request of temperature and protect from light. Need an experienced manipulator, a tiny negligence can take gross influence to efficiency of sorting. (2) Not only CD90 positive cells, but also CD90 negative cells of hepatocellular carcinoma can self renew and proliferation consistently. They also have the capacity of multidifferentiation. (3) Result of Real-time fluorescence quantitative test and immunocytochemistry indicated that have variance of protein and gene expression between CD90 positive and negative group cells in different degree. So, it need a further study to varificate if these variance of different clone result in different invasity and carcinogenisis of them.