The Study Of Mesanchymal Stem Cells Treating Liver Injury By Trichloropropane Poision

With industry development rapidly, the industry produce poison isincreasing. Trichloropropane (TCP) can made severe liver injury, and theeffects of traditional medicines and man-made liver on liver poison waslimited, patients died with liver failure. Recently, the study showed bonemarrow mesanchymal stem cells can engraft liver and perform recoveryfunctions on patients with liver disease. The study found MSC can reduce livercells death and enhance it recovery. Though the study is preliminary and hasmany problems, there is a good future on MSC treatment liver disease. Wedesigned MSC treat rat liver injury model. To study MSC liver engraftment,repair function, safe dosage and gene transfect MSC.Objective of the experiment:1.MSC is culture and identificationSPF Wistar rats were randomly taken, female MSC were isolated from rat bonemarrow and purified by means of adherent way. The microscope was applied to observethe changes of the cell and the cells had a spindled，fibroblast appearance in culture. Flowcytometry analysis demonstrated that phenotypes of MSCs were CD34-/CD45-/CD105+CD44+.It is demonstrated that MSCs retained their ability to form ostcoblasts andadipocytes at Passages4through differentiation assays,which showing a multipotentplasticity. All the description above is consistent with MSCs.2. the liver injuried model of rats by TCP poisonForty SPF Wistar rats were randomly divided into four group：PBS group(n=10)，TCP40ug/kg group（n=10），TCP60ug/kg group（n=10），TCP80ug/kg group（n=10）.TCPwas injected into abdomen of rats every two days.Animals were drown blood after3、5、6、7times poison. The blood sample was detected including ALT,TP,Alb,TBil,DBil. Theslice liver pathology was observed.The result showed that the TCP60ug/kg group appearsignificant liver poisoning symptom、sign，and pathologicalandbiochemistry change，butthe40ug/kg group had slighter liver injury,and rapid recovery,the TCP80ug/kg group hadsevere liver injury,animal had high mortality rats. 3. The study of CM-DiI marked MSC homing injuried liverThe MSCs marked by CM-DiI from Female rats were infused poisoning and normalrats.the rats were killed and made liver sliver at3,15and30d after transfusion.the resultsshowed MSCs distrabuted into bone marrow and intestines in normal rats.MSCs homedto the liver at15days after infusion.4. MSC treat liver injured by TCP poisonSeventy-five SPF Wistar rats were randomly divided into five group：A: the normalgroup, PBS group(n=15)，; B: TCP60ug/kg plus PBS group(n=15);C:，TCP60ug/kg plus1×10~5MSCs group(n=15)，D: TCP60ug/kg plus1×10~6MSCs group(n=15);.E:PBS wasinjected into abdomen and PBS infusioned by tail vein(n=15).Animals were drown bloodafter3、5、6、7times poison. The blood sample was detected includingALT,TP,Alb,TBil,DBil. The slice liver was observed. The result showed that the C groupappear significant rapid recovey of the ALT,TP,Alb,TBil,DBil than these of B and Cgroup(P<0.05)5.The study of recombinant adenoviral vector with co-expressing KGF andenhanced green fluorescent protein transfected to murine Marrow MesenchymalStem CellsThe target gene KGF was cloned into the shuttle plasmid expressed the report geneEGFP.Then the recombinant shuttle plasmid was transformed into Dh5a bacteria torecombine with backbone vector pAdxsi.Next,the plasmid pAd-EGFP-mKGF wasamplified in H293cells and the viral titer was determined. The MSCs were separated andenriched by using bone marrow adherent culture and identificated in vitro, to observe theefficiency of transfection. It is concluded the recombinant adenoviral vectorAd-EGFP-mKGF was successfully constructed and can transfect MSC effectively andsafly. Conclusion:1、Using adherent screening can successfully separate,purificy and culture MSC from ratbone marrow in vitro..2、the TCP60ug/kg poisoning group was proper liver poison model.3、CM-DiI marked MSCs homed to the liver of rat undergone TCP poison.4、In one hand,chemotaxis and colonization of mesenchymal stem cells are impacted and constraint by its micro-environment.The heavier the liver from the rat was damaged,the more MSCs were engrafted.In the other hand,mesenchymal stem cells can in turn change the micro-environment of the damaged part. Mesenchymal stem cells can effectively reduce liver injury in rat which is caused by TCP poison.1×106MSCs was safe and effective to treat liver injury.5recombinant adenoviral vector Ad-EGFP-mKGF was successfully constructed and can transfect to MSC effectively and safly.