| Liver origins from endoderm and is an important hematopoietic organ in embryo. Hematopoietic stem cells (HSC) migrate to liver from yolk sac in the 6th week of embryo when liver begins its hematopoiesis and up to bloom stage during 15-24 weeks' embryo. Although granulocytes, monocytes, lymphocyte and megakaryocytes in various development stages can be found in fetal liver hematopoiesis, erythrocytes increase and mature in advance. The amount of erythroblast is especially dominant in the system of erythrocyte. Fetal liver hematopoiesis began weaken at 6 months' embryo and is replaced by bone morrow after birth.Vasoactive intestinal peptide (VIP) secreted from endocrine cells of gastrointestinal tract has shown multiple biological functions. When it flushes into liver through portal vein, VIP is bound to its receptor on the surface of hepatocytes. VIP receptor will innerize and degrade it. Finally, VIP will be cleared by lysosome. Therefore, liver is a crucial place to clear and inactivate overwhelming majority of VIP. We are interested in these espects: What effects do VIP and receptor in liver on liver hematopoiesis and migration? And what effects do VIP and receptor in liver on transdifferentiation of HSC in liver? To answer these questions, we studied the direct effect of VIP on HSC biological function and behavior.ObjectivesIn this research,we try to investigate:1. The effects and mechanism of Vasoactive Intestinal peptide(VIP) on proliferation and colony forming unit(CFU) of Hematopoietic Stem Cells(HSC).2. The effect of VIP on differentiation of HSC to blood cells.3. The effect of VIP on differentiation of HSC to hepatic related cells to probe the possibility that VIP affect HSC transdifferentiation.4. Quantitative alteration of VEP and its receptor in hepatic tissue in the course of rat liver development to study the migrative mechanism of fetal liver hematopoiesis.5. Does VIP mediate the function of HSC via the corresponding receptors on HSC? What subtype of VIP receptors localized on HSC?Methods1. Human cord blood were collected, and to isolate mononuclear cells by Ficoll.2. MACS assay was used to purify CD34+ cells from MNC, the purity of the CD34+ cells was evaluated by flow cytometry.3. Colony Forming Unit(CFU) enumeration was used to assess the effect of VIP on proliferation of HSC.4. Amplification culture was counted to judge the effect of VIP on CD34+ cells.5. TNF-a, TGF-beta and AFP in cultured HSC and its supernatant were measured with ELJSA assay.6. Different blood cells in different culture time of CD34+ cells were identified by Flow Cytometry(FCM).7. Liver tissue markers on HSC, AFP, ALB and CK-19, were measured byimmunohistochemistry.8. Western blot assay was used to detect the expression of ALB on HSC.9. Nest RT-PCR was used to detect the expression of AFP mRNA and ALB mRNA on HSC, the product of ALB was chosen to measure the sequence.10. SD rat liver tissues in different development phase were collected.11. Biacore assay was used to detect the expression of receptor for VIP on human HSC and rat liver tissue.12. Radioimmunologic assay was used to detect the concentration of VIP in rat liver development.13. VIPR mRNA on human HSC and rat liver tissue was identified by RT-PCR.Results1. CFUs of CD34+ cells treated with VIP in the concentration range of 10~7 mol/L ~10-12 mol/L were significantly lower than that of control group (inhibition ratio > 26.97 ±13.72%), P<0.05. VIP at 10-8 mol/L showed the highest inhibition. With this concentration, the proliferative folds of CD34+ cells were 11.78±4.39. 16.71 ±2.98. 21.69±3.28 at the day 7, 10 and 14 separaterly. They were greatly lower than those of control group (20.13± 3.32. 25.64±3.81> 28.33±2.61), P<0.05.2. VIP upregulated the concentration of TNF-a in HSC significantly, it was 149.15 pg/ml, 49% higher than control, P<0.05. VIP also markly increased the concentration of TGF-beta1 in HSC, it was 116.10 pg/ml,...
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