| Objective To establish a human-rat chimeric liver model,to explore the survival and changes of hUCMSC in the liver at different time points after birth,to detect the change of peripheral blood T lymphocyte ratio,and to explore the formation of immune tolerance in chimeric state.The possible mechanism provides more theoretical support and experimental basis for the clinical application of umbilical cord mesenchymal stem cells.Methods1.Isolation,culture and identification of hUCMSC:The micro-tissue block was repeatedly attached to the wall,and the cells were isolated from the umbilical cord micro-tissue block with DMEM-F12 medium containing 10%FBS,and the morphological characteristics of the cultured cells were observed by microscope.The immunophenotype of the cultured cells was identified by flow cytometry,and the differentiation potential of the cultured cells was identified by in vitro differentiation.2,Marking of hUCMSC:co-culture with hUCMSC using lentiviral vector carrying green fluorescent protein gene(GFP),and then confirming the labeling of GFP by fluorescence microscopy.3.Injection of hUCMSC in SD fetus:The gestational age of SD rats was determined by Yin suppository observation.The uterus was exposed to open uterus and hUCMSC was directly injected into the liver of each fetus.After the birth of the fetus,the liver tissue and peripheral blood were collected at the appropriate time.For identification analysis.4.Chimera liver identification:After birth,the liver tissue was harvested at appropriate time to make slices.The frozen sections were observed under fluorescence microscope to observe the distribution of the implanted cells.Paraffin sections were prepared and the differentiation of the implanted cells was identified by immunohistochemical staining.5.HE staining analysis of liver:After birth,the liver tissue was harvested at appropriate time and sectioned and stained with HE.The histological changes were observed under a microscope.6.Determination of T cell subset ratio:After birth,the peripheral blood was collected at the appropriate time to detect the change of the proportion of T cell subsets by flow cytometry.Results1.Using the technique of repeated adherent culture of tissue blocks,using DMEM-F12 medium containing 10%FBS for 10-12 days,it can be observed that the umbilical cord tissue around the cut is grown and attached.Fibroblast-like cells that grow like spirals,and fibroblast-like cells climb out 2-3 days after the tissue block is attached again.When subcultured to the third passage,the isolated cell phenotypes CD 105,CD 13,CD90 and CD29 were highly expressed,and almost no CD34 was expressed.Specific in vitro differentiation experiments confirmed that isolated cells can induce differentiation into fat,cartilage and bone tissue.2.According to the transfection complex(MOI)of 50,the GFP-bearing lentiviral transfection solution and hUCMSC were co-cultured for 72 h,and bright green fluorescence was observed under the fluorescence microscope,indicating that hUCMSC was successfully labeled as GFP-hUCMSC..3,Male and female rats mating combined with vaginal plug observation can determine the pregnancy age of SD rats.The uterus of pregnant mice was exposed to laparotomy under mild or mild anesthesia,and 106 GFP-hUCMSCs suspended in 0.1-0.2 mL saline were directly injected into the peritoneal cavity or liver of each fetal rat.The natural delivery rate of fetal rats was over 60%..4.Rat liver tissues were collected for 45 days,75 days,and 120 days,and frozen sections were prepared.The distribution of green fluorescence signals was observed under a fluorescence microscope,but only a small amount of green fluorescence was observed in the 120-day rats.It was not even observed at all.In the corresponding liver paraffin sections,the expression of human hepatocyte-associated proteins HNF4 a,HNF3 ? and ALB was detected in liver tissues at birth for 45 days and 75 days,and almost 120 days of rat liver tissue was born.No expression of related proteins was detected.The distribution of GFP signal and HNF4 ?,HNF3 ? and ALB showed a tendency to decrease as the growth time of the rats prolonged.5.No obvious inflammatory cell infiltration,hepatocyte necrosis and other histological changes were found in the liver tissues of rats born 45 days,75 days,120 days.6.Peripheral blood of rats born 45 days,75 days,120 days were collected,and the proportion of CD lymphocyte subsets CD3+,CD4+,CD8+,Treg(CD4+CD25+Foxp3+)was detected.The results showed that three groups of injected cells The proportion of T lymphocyte subsets in peripheral blood of rats was significantly different from that of the control group.The proportion of CD3+cells in the rats at 45 days,75 days and 120 days after birth and the control group were:44.08±3.02,58.09±3.39,48.18±4.75 and 37.25±3.21,the proportion of CD4+cells was:29.57±1.38,36.61±2,51,32.64±2.58 and 24.19 ± 0.96,and the ratio of CD8+cells was:16.56±1.00,24.76±1.60,18.04±1.19 and 11.03±1.05,Treg cells.The ratios are:2.81±0.18,0.99±0.20,3.59 ± 0.23,and 1.48±0.24.The trend of the proportion of T lymphocyte subsets was related to the growth time of neonatal rats.Conclusions1.Using the tissue repeated patch method,hUCMSC can be isolated from human umbilical cord and co-cultured with GFP-containing lentiviral vector solution,which can successfully achieve GFP transfection marker.2.The human-rat chimeric liver model can be established by injecting GFP-hUCMSC into the abdominal cavity or even the liver of SD fetuses under 'open laparotomy.After 75 days of growth in neonatal rats,the presence of GFP signal and the expression of human hepatocyte-associated proteins HNF4 ?,HNF3 ?,and ALB were detected in the liver,but gradually decreased with increasing growth time.3.In the embryos born after GFP-hUCMSC injection,the proportion of T lymphocyte subsets in peripheral blood changes with the growth of rats,which may be a combination of immune tolerance-related regulatory factors,leading to intrahepatic chimerism.The human source cells are gradually decreasing. |
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