Study Of Inducing By ICAM-1 And Regulating By NF-KB Hepatic Stem Cells To Directional Transfer

PrefaceOVCs are a kind of adult stem cells which possess self-renewing and multipotent differentiation ability. Peterson et al found that OVCs may originate from hemopoietic stem cells (HSCs). OVCs can not only proliferate for a long time but also differentiate into bile duct cells and hepatic parenchymal cells. Under certain condition, they can differentiate into intestinal tract epithelial cells and pancreas cells. OVCs play an important role in the physiological and pathophysiological process of liver cell development, growth, proliferation, and carcinomatous change. There is few hepatic cells to split and proliferate during the quiescence period, but OVCs will be activated when liver is badly damaged and differentiate into hepatic parenchymal cells. However, OVCs can also differentiate into liver cancer cells under certain surroundings. Studies have shown that OVCs existed not only in Hering ducts but in hepatic lobules when liver was damaged by 3'-methyl-dimethylaminoazobenzene (3'-Me-DAB) and was cancerized. Why did they exist in hepatic lobules? What maked that? It was found that ICAM-1 was expressed in HSCs and it induced HSCs to adhere to marrow stroma and inpelled HSCs to migrate, and other experiments about the relation between ICAM-1 and HCC showed that cancer cells expressedICAM-1, while the ICAM-1 expression of normal hepatocytes was negative. Those studies indicated that ICAM-1 played an important role during the period of occurrence and development of HCC. What is that? We presumed: (1) OVCs could express ICAM-1 because OVCs originated from HSCs which expressed ICAM-1,shown in some experiments; (2) OVCs may be induced to adhere to marrow stroma and impelled to directional migrate by ICAM-1 just as HSCs were done. This study was designed on the basis of above-related theories and assumptions to explore the ICAM-1 role during the period of occurrence and development of hepatocirrhosis and HCC. ObjectiveTo establish animal model of damaged liver and OVCs proliferation, observe the distributing of ICAM-1 during various injuring phases; to isolate OVCs and discuss the ICAM-1 expression of OVCs and the regulation of NF-kB to the expression; and to discuss further the role of IC AM-1 and NF-kB in the directional transfer of OVCs, explore an approach of preventing and curing liver cirrhosis and HCC. Methods1. Establishing animal model Sixty Wistar rats, 90-1 lOg, were fed on feedstuff with 0.06% 3'-methyl-dimethylaminoazobenzene (3'-Me-DAB) after fed on routine feedstuff for a week. Then liver tissue specimens were gathered and fixation with 10% formalin at 2nd, 4'\ 6th > 8th, 10l\ 12th week.2. Orientation of ICAM-1 in liver tissue specimens The over-related specimens were treated for these following experiments:1) to stain paraffin sections from rat livers with H&E, and observe the distribution of OVCs with microscope;2) to observe OVCs' morphology and the adhesion of OVCs to the adjacent organic by transmission electromicroscrope;3) to perform immunochemistry to demonstrate ICAM-1 distribution in liver.3 > Expression of ICAM-1 Density ladder centrifugation was used to isolate and culture OVCs, and immunocytochemistry were performed to detect the expression of ICAM-1;4> Regulation of NF-kB to the expression of ICAM-1 OVCs were grouped into A> B> C. Group A contained OVCs without intervening factor, group B with TNF-a, activator of NF-kB, group C with TGF-Pi, inhibitor of NF-kB, then all groups were subdivided into L II > IIL IV subgroups. All the groups were used to do these following experiments:1) to detect the growth of OVCs by microscrope in group A> B and C, and take count of OVCs;2) to perform reverse transcription-polymerase chain reaction(RT-PCR) semiquantitative assay to compare the amount of ICAM-lmRNA in OVCs cultured with TNF-a and TGF-pi;5 > OVCs and Ito cells co-culture without intervening factor detected the adhesion of OVCs to Ito cells by microscrope . Results1 > In initial stage of damaged liver, ICAM-1 mostly distributed in duct portal area, and then gradually increased and expanded toward hepatic lobule;2^ Observing OVCs morphology by transmission electromicroscrope showed that OVCs were infantile and undifferentiated with big nucleus, clear nucleolus, large nucleoplasm-ratio, small mitochondria, and little endoplast;3 > Immunocytochemistry indicated that ICAM-1 expression was positive;4n Compared with that in control group, the proliferation of OVCs was promoted in TNF-a group (.P<0.01)while it was affected non-significantly in TGF-f3i group (P>0.05);5, ICAM-1 expressions were up-regulated and down-regulated in TNF-a group and TGF-Pi group respectively and the gray scale values of the electrophoretic bands were 0.480> 0.752 and 0.403 in control group, TNF-a group and TGF-pi group respectively;6> OVCs and Ito cells were co-cultured, OVCs got together round Ito cells in the second day and in the third day many OVCs adhered to Ito cells. Conclusion1-, The distribution of ICAM-1 accorded with that of OVCs in liver specimens;2> OVCs expressed ICAM-1 in damaged liver tissue;3, ICAM-1 expressions were regulated through activiating NF-kB and inhibited NF-kB respectively;4> ICAM-1 induced OVCs to directional transfer, and the induction was regulated by NF-kB;5n ICAM-1 was likely to induce the cancer cells to directional transfer in liver during the occurrence and development of HCC.