| ObjectiveTo establish a human-goat chimeric liver and observe the human umbilical cord mesenchymal stem cells(hUCMSC)of surviving and changing rules,provide experimental animal models for the study of immune tolerance and liver disease.Method1.hUCMSC isolation,identification;Under the aseptic condition collect caesarean baby umbilical cord by adherent culture method to separate hUCMSC.microscopy method is used to observe the cell morphological characteristics,cellular immune phenotype is indentificated by flowcyte analysis.hUCMSC induced differentiation potential—into fat,osteogenesis and cartilage cells is made by vitro method appraisal.2.hUCMSC markers:lentiviral vectors carrying green fluorescent protein gene(GFP)was co cultured with hUCMSC to label hUCMSC,through fluorescence microscope detection method to confirm Mark Success.3.Fetal liver positioning injection GFP marked hUCMSC:Under the ultrasound guide the hUCMSC labeled fluorescent protein(GFP)were transplanted into the fetal liver parenchyma,After the birth of the sheep,through the open surgery method to cut partial liver tissue to assay the seeded cells.(1)GFP positive cells observation:the liver tissue was harvested from the lamb and frozen sections were made.The presence and distribution of GFP positive cells in liver tissue were observed under fluorescence microscope.(2)Identification of SRY gene expression:a small amount of liver tissue of newborn lamb was collected,respectively.DNA was extracted and amplified by quantitative PCR to identify the expression of SRY gene.(3)FISH identification and analysis:a small amount of liver tissue was collected to make paraffin sections,and SRY sequences were used to make gene probes for in-situ hybridization to confirm the presence of implanted cells.4.Functional identification of human-derived cells in the liver tissue of lamb:a study on functional identification of human-derived cells by excision of partial liver tissue after birth of lamb;(1)RT-PCR detection and analysis of human hepatocyte-related genes:After the lamb was born in 1 month the lamb part of the liver tissue were used to extract total RNA,the expression of human hepatocyte related genes was detected by RT-PCR.(2)Immunohistochemical staining analysis of human hepatocyte-related proteins:a small amount of liver was collected to make paraffin sections,and immunohistochemical staining was used to detect the expression of human liver cell related proteins.(3)Detection of peripheral bloody albumin(ALB):collection of neonatal lamb peripheral blood,separating serum and detection of serum ALB content by human albumin ELISA kit.5.Long-term observation on the survival of human cells in lamb liver;9 months after the birth of the lamb,Cut partial lamb liver tissue to identify the presence and change of human cells by immunohistochemical staining,FISH and ELISA.Results1.By means of tissue block method,using the DMEM-F12 medium containing 10%fetal bovine serum to directly culture the male umbilical cord tissue which had been sheared,the cells of fibroblast-like cells with vortex-like adherent cells were successfully isolated,and the flow cytometry showed that they expressed CD29,CD90,CD 105 and did not express CD34.In vitro induction,It can be induced to differentiate into adipocytes,osteocytes and chondrocytes,which can be judged as hUCMSC,the passage to the 5th generation can obtain sufficient number of cells for experimental study.2.The bright green fluorescence could be observed under a fluorescent microscope after the transfection of lentiviral transfer of GFP transfected with hUCMSC 72h,according to the 100 dose of transfection.3.Under the guidance of B-ultrasound,the fetal liver position of 2-4-month-old goat can be accurately located,and accurate puncture and accurate injection of GFP labeled hUCMSC to fetal goat liver parenchyma region,once injection of 108 hUCMSC will not cause abortion of fetal goat.4.Identification of human-goat chimeric liver:(1)The frozen sections observed under fluorescence microscopy injected cells in liver tissue with green fluorescence distribution,some tubular structure rules,and more standard injection of mesenchymal stem cells in liver tissue,lamb had more GFP positive tubular structure.Frozen Section observation:After the lamb born lmonth,under the fluorescence microscope may observe the lamb liver tissue to have the green fluorescence distribution,some assumes the regular tubular structure,some disperses in the distribution,the more injection the hUCMSC to the lamb liver tissue,the more GFP positive signal will be detected.The GFP-positive signal was not detected by negative controls that did not receive cell injections.(2)Identification of SRY gene expression:the expression of SRY gene can be detected in the liver tissue of the lamb in the newborn 1 months after injection of male hUCMSC,including the female newborn lamb,and the SRY gene expression was not detected by the male lambs without cell injection.(3)FISH analysis appraisal:the FISH method can detected SRY gene expression in liver tissue 1 month after injection of male hUCMSC lamb,including female newborn lamb,negative control male lamb without injected cells did not detect SRY gene expression,It is confirmed that the male hUCMSC do exist.5.Functional identification of human source cells in the liver tissue of lamb;(1)Expression of human hepatocyte-related genes:the expression of human hepatocyte-related gene AFP,HNF4?,HNF3?,CK18 and ALB gene was detected by RT-PCR in all newborn lambs after 1 months with hUCMSC injection.The expression of human hepatocyte-related genes was not detected by a negative control that received no hUCMSC injection.(2)Detection of human hepatocyte-related proteins:the expression of human hepatocyte-related protein hnf4a and ALB was detected in the liver tissue of the lamb in all newborns after 1 months who had injected male hUCMSC,HNF4a mainly around the central meridians of the liver.ALB mainly scattered in the liver lobules.No human hepatocyte-related proteins was detected with negative control of no cell injection.(3)Determination of peripheral bloody albumin:By ELISA,ALB can be detected in the peripheral blood of the lamb in 1 months of the newborn with a male hUCMSC(average 41.7972ng/ml).6.Long-term observation of the survival of human-derived cells in chimeric liver:A 9-month-old lamb liver tissue was collected,and the fluorescent microscope still observed a green fluorescence distribution in the liver tissue of a frozen section injected with hUCMSC,PCR and fish analysis confirmed the presence of SRY genes,RT-PCR and Immunohistochemical analysis showed ALB and HNF4a positive,and human ALB could be detected in peripheral blood(average 4.548057ng/ml).Conclusion1.Using tissue patch method,containing 10%FBS-DMEM/F12 medium,Cells can be separated from the male umbilical cord expansion to the vortex-dense growth of hUCMSC.In accordance with the MOI value of 100 of the dose of the green fluorescent protein gene(GFP)of lentivirus co-culture,can be successfully labeled hUCMSC.2.Under the guidance of ultrasound,it can realize the positioning injection of hUCMSC in the liver parenchyma of the fetal goat in 2-4 months of age,and it will not result in the abortion of the pregnant goat in a single injection of 108 hUCMSC.3.A human-goat chimeric liver can be formed by injecting GFP-labeled male hUCMSC into fetal liver.The presence of GFP positive cells or the formation of tubular structures can be observed in the liver tissue of lambs born 1 months old.The expression of human SRY gene was found in the liver tissues of female lambs born one months after PCR and fish analysis,RT-PCR and Immunohistochemical analysis showed that the expression of hepatocyte HNF4? and ALB in the mRNA and protein levels in chimeric liver was mainly located around the central meridians of the liver lobules,The latter mainly scattered in the hepatic lobules.More importantly,the presence of human albumin can be detected in the peripheral blood of the lamb after birth,the presence of implanted cells and the expression of human hepatocyte-related proteins can be maintained at least 9 months after the birth of the sheep,indicating the successful establishment of the human-goat chimeric liver,hUCMSC has acquired the function of Hepatocyte biology in the process of survival and evolution of fetal liver. |
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