Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The ...Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories： （1） MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h, 24 h, 48 h and 72 h; （2） MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4.7 μg/ml linoleic acid, 1×ITS, 10^-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 （100 ng/ml） and HGF （20 ng/mL）. Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; （3） 0.2-0.3 ml of MNCs with a cell density of 2×10^7/ml were transplanted into prepared recipient mice [n=12, injected with 0.4 ml/kg （20%） CCl4 and 150 ng/kg 5-fluorouracil （5-Fu） prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results： （1） After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin （ALB）, α-fetal protein （AFP） and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cuhured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. （2） In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. （3） In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion： MNCs from human umbilical cord blood could convert into hepatocyte-like ceils in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.展开更多
基金Supported by the Shenzhen Science & Technology Planning Program (No. 200204109, No. JH200505270412B)
文摘Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories： （1） MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h, 24 h, 48 h and 72 h; （2） MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4.7 μg/ml linoleic acid, 1×ITS, 10^-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 （100 ng/ml） and HGF （20 ng/mL）. Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; （3） 0.2-0.3 ml of MNCs with a cell density of 2×10^7/ml were transplanted into prepared recipient mice [n=12, injected with 0.4 ml/kg （20%） CCl4 and 150 ng/kg 5-fluorouracil （5-Fu） prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results： （1） After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin （ALB）, α-fetal protein （AFP） and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cuhured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. （2） In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. （3） In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion： MNCs from human umbilical cord blood could convert into hepatocyte-like ceils in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.
