| Objective To supply more evidence for the differentiation of hepatocyte from transplanted human umbilical cord blood stem cells, to testify its plasticity and lay a foundation for cord blood stem cells applied to the final hepatic disease, Experimental study was conducted in BALB/C nude mice.Methods Umbilical cord blood (UCB) samples were collected by blood bags and were separated by hydroxyethyl starch (HES) method and Ficoll method. Nuclear cells , CD34~+ cells and trypan blue dye viability were compared between HES and Ficoll method.30 BALB/C nude mice were randomly divided into experimental group and control group, Each group was 15,then experimental group was randomly divided into group A, B and C , each group was 5. 30 BALB/C nude mice were exposed to 350 cGy radiation under the sublethal condition. After 3 hours, fresh umbilical cord blood nuclear cells were injected into the mice for experimental group via tail vein, 1.0×10~7 , 2.0×10~7 and 3.0×10~7 every mouse in group A,B,C respectively, and the equal volume normal sodium (NS) was injected into control group. White blood cells in peripheral blood for experimental group and the control group were detected at7,14,21,28,56 day after transplantation. HuCD34 + and huCD45 + in peripheral blood for the experimental group were detected by using flow cytometry analysis on 4, 6 and 8 week after transplantation in order to know the human-mouse chimeras. After one month, carbon tetrachloride (CCI4) diluted to 100 microliters with corn oil by 0.4 ml per kg was injected into experimental group A, B and control group, and the equal volume normal sodium were injected into experimental group C . Liver injured by CCU could induce umbilical cord blood stem cells back to liver and to differentiate into the liver cell ,then a human-mice chimeras model that human umbilical cord blood differentiate into hepatocyte in nude mice was established. Transplantation was conducted after two month (liver was damaged after one month), livers for mice were examined by immunohistochemistry and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), the expression of human cytokeratin 18(CK18), cytokeratin 19 (CK19)and albumin were detected.Results 1 .Collecting of human umbilical cord blood and separating of nuclear cells . Ten samples of human umbilical cord blood (UCB) were collected, the volumes were between 65 ml and 124 ml, average is 87 ml. Improved HES as compared with Ficoll, there was no difference in the volume of umbilical cord blood, the density and the number of the nuclear cells before separation(P>0.05). But after separation, the number of nuclear cells and CD34+ cells, rate of recollection for nuclear cells(the percentage of the number of nuclear cells of post-separation and pre-separation) of HES separation was higher than those of Ficoll separation (P<0.05). There was no difference in the rate of refusing trypan blue dye between HES and Ficoll separation .The resultof bacterium cultured between HES and Ficoll was negative.2.After human umbilical cord blood stem cells were transplanted,main indictors were detected as follow.?Survival of the nude mice. No mice died in the experimental group , 4 mice in control group died after irradiation and 3 mice died after CCU injected.?Change for the number of white blood cells.Difference of measuring time and nuclear cells injected in different groups had influence on the number of white blood cells (F=722.146, P=0.000) . There was no difference in the number of white blood cells of experimental groups and control group in pre-transplantion and on 8 week of post-transplantion(.P>0.05),but the number of white blood cells of experimental groups and control group isn't all the same on 1,2,3,4 week of post-transplantion (P<0.05) .As compared with pre-transplantion, the number of white blood cells of post-transplantion at different time in experimental groups and control group was decreasing , the lowest was on 1 week ,then went up and reached the level of pre-transplantation on 4 week in experimental groups and on 8 week in control group .?Detection of huCD34+ and huCD45+ cells. huCD34+ and huCD45+ in peripheral blood for nude mice appeared on 4 week, but the total level was low. The number of CD34+> CD45+ cells wasn't all the same among the experimental group A ,B and C at different time in the same group((F=43.181,F=0.000;F=67.08,P=0.000)), and the number of CD34\CD45+ cells wasn't all the same at same time in the different group too( (F=10.351, P=0.002; F=186.26, P=0.000) . The number of CD34\ CD45+cells in experimental group A was lower than those in experimental group B and C at all the stage (PO.05) ,but there was no difference between experimental group B and C(P>0.05).There was obvious correlation between huCD34+ and huCD45+ on 4 week (r=0.903, .PO.05), but this correlation didn't appear on 6 and 8 week(P>0.05), which might be related to the size of sample.?GVHD. After the mice were killed , there was no obvious change in liver ,skin and small intestines of mice in experimental group by using the pathology examination.(5) Expression of AFP,CK18, CK19 and albumin. Brown-yellow lamellar or focal area didn't appear in control group and experimental group without CCU injected after UCB transplantion when AFP,CK18, CK19 and albumin were detected by using liver tissue for nude mice under the Immunohistochemistry examination, However, brown-yellow lamellar or focal area appeared in experimental group with CCU injected after UCB transplantion,and those cells with brown- yellow granule in their plasms allocated as flakes or clusters. The results of picture analysis suggested that the special expression of CK18,CK19 and albumin in experimental group B was stronger than that of experimental group A (PO.05) ,but there was no difference among CK18, CK19 and albumin in the same experimental group , and expression of AFP was negative. Special strap appeared after the mRNAof hu CK18, hu CK19 and ALB in liver tissue of the experimental group with CC14 was detected under the RT-PCR , but there was no this phenomenon in the experimental group without CC14 and control group. The results of picture analysis suggested that the special expression of CK18 , CK19 and albumin in experimental group B was stronger than that of experimental group A (PO.05) , but there was no difference among CK18 , CK19 and albumin in the same experimental group(/>>0.05),which suggested there were human mature hepatocyte and cholangiocyte/bile duct cells in liver injured by CC14 after UCB transplantation.Conclusions ?The quantity of blood stem/progenitor cells collected by using improved HES separation is great and its quality is high, and the operation is simple and convenient, the speed of separating is quick, the opportunity of pollution is small and final volume is small, Therefore, this method of improved HES may be applied to the transplant of USB and clinical practice.(2)The establishment of BALB/C nude mice immune system is not perfect, immune reaction is weak, so it may be an ideal experiment animal model of human-mouse chimeras xenograft.?USB can form the human-mouse chimeras after it was transplanted in the nude mice , moreover, the rate of GVHD is low. At a certain degree, the speed of white blood cell recovery in peripheral blood and level of the human-mouse chimeras after transplantation will be improved accompany with the nuclear cells increasing.?Liver injured by CC14 can successfully induce the stem cell back to liver tissue, and make it differentiate into hepatocyte under the special condition of liver injured and express albumin, and the level of its express was directly related to the number of UCB.
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