Expression Of Stem Cell Marker CK19and CD90at Different Stages In The Process Of Chemical Carcinogenesis

Objective To detection CK19,CD90expression at different stages of the process of C57BL/6J mice which were induced of liver cancer combined with DEN/CC14/Ethanol To showed that a different regulation of CK19and CD90in the process of hepatocarcinogenesis.And speculated that the possible mechanism of liver cancer stem cells in the process of hepatocarcinogenesis.Methods All mice were divided into experimental and control groups, according to the random number table,were housed separately, for standardized light and free food and drinking water.The experimental group had50C57BL/6J male mice, induced liver cancer through the combined DEN/CCl4/Ethanol chemical method, the control group had50C57BL/6J male mice,only fed them the normal mouse feed pellets,and fleely drank sterilized ordinary water. Observe the general growth, every two weeks randomly collected each five mice from the experimental and control groups, executed and open abdominal, and removed the liver to obtain liver tissue(experimental group was mainly cut liver tissue specimens with suspicious lesions),and detected the changes of CK19,90gene and protein by RT-PCR, Q-RT-PCR,Western blot methods and flow cytometry (FCM), at the same time, pathological changes were observed through HE Stain.Results Using DEN/CC14/Ethanol chemical-induced C57BL/6J mice liver cancer,the experimental group and control group had none of the mice died throughout the carcinogenesis process, to carcinogenesis20th weeks,the mice of experimental group retained tumorigenicity rate was100%.The control grou mice grew well, the weight increased with age, the everage weight was about from16g to30g or so, liver color was red,capsule smooth,soft, the sharp edge,without nodules.After18th weeks, the weight of experimental group was to a downward trend, the average body weight decreased from29g to26g. Before14th weeks, liver tissue capsule was less smooth, dark red, the texture was slightly tough. HE staining pathology results showed that liver congestion and edema, part of the liver celldegeneration and necrosis, inflammatory cell infiltration and part of liver cell proliferation. The stromal had a small amount of fibrous tissue proliferation, It was the performance of drug-induced hepatitis, be to chronic liver injury. It could be seen false lobularformation in the chemical carcinogenesis14th weeks and the interstitial fibrous tissue proliferation, part of the bile duct epithelial hyperplasia. And progress with the carcinogenesis,liver cells arranged in a structure with mild disorders, be to Cirrhosis.Mice which were sacrificed after16th weeks,it’s liver surface appeared graynodules, single or multiple, in diameter1-3mm,microscopically, liver cell proliferation nodules,nodules within the liver cells showed atypical hyperplasia, be to atypical hyperplasia. Liver nodules is about5mm in-diameter and multiple in the18th and20th weeks;Pathology revealed a highly differentiated hepatocellular carcinoma cells, showed that the tumor giant cells and pathologicalmitosis,be to cancer. The results of qualitative by RT-PCR, Western Blot and that of quantitative by FQ-RT-PCR, flow cytometry, showed that between the experimental and control groups and different carcinogenesis cycle,CK19gene and protein were weakly expressed in the first14th weeks of chemical carcinogenesis,expression began to increase in the16th weeks,significantly increased in the20th weeks(P<0.05).The comparison of CD90gene and protein expression between the experimental and control groups,and different carcinogenesis cycle,differences were significantly higher after the16th weeks, was more significant in the20th weeks, and the expression level increased with the time carcinogenesis cycle increased (P<0.05).In summary, comparing with the normal control group, there were dynamic changes in the experimental group of CK19,CD90gene and protein expression in different cancer induced cycle. But CK19and CD90began to increase respectively after14th and16th weeks of chemical carcinogenesis,and both the most obvious in20th weeks.Conclusion1.No mice died throughout the carcinogenesis process,the mice intotumor rate was100%in the20th weeks of carcinogenesis, pathological type showed moderately differentiated or well-differentiated hepatocellular carcinoma.2. CK19gene and protein,qualitative and quantitative detection,expressed lowly after14th weeks.Expression levels increased after16th weeks,significantly to20th weeks.It was significant differences compared with the control group and the early experimental group, It showed that CK19in HCC was differential expression in the development process.3.Qualitative and quantitative detection of CD90gene and protein, expression increased after the first16th weeks, expression level was significantly higher to20th weeks.It was significant differences comparing with the control group and the early experimental group, Tipped that CD90expression was a dynamic change in the process of chemically incuced liver cancer.4.CK19, CD90gene and protein were regulated expression of the time difference in the process of chemically induced liver cancer. Illustrate to play different role in regulation at different stages of liver cancerformation. The next step will be two specific regulatory mechanisms and the signaling pathway in-depth study.