| Objective: To explore the possible therapeutic effect and mechanism of transplantation of bone marrow mesenchymal stem cells(BMSCs)carryingthe ApoJ gene on apoptosis of intracerebral hemorrhage in rats.Methods: 1.Rat BMSCs were isolated,cultured and purified in vitro by "adherence screening method".2.Recombinant green fluorescent protein pEGFP-N1-ApoJ was transferred into BMSCs in vitro,transfection efficiency of recombinant plasmid was observed under the fluorescence microscope,and the transfection group with the highest transfection rate was screened out.3.The intracerebral hemorrhage(ICH)models were established by stereotaxic brain locator and improved two-steps injection method of autologous blood.144 male SD of intracerebral hemorrhage in rats,Rats were randomly divided into three groups: ApoJ gene modified BMSCs transplantation group(ApoJ/BMSCs group),BMSCs transplantation group(BMSCs group)and saline group(NS group),48 rats in each group.4.After 24 hours of successful modeling,The three groups rats were respectively transplanted with the same volume of transfected cell suspension,BMSCs suspension and saline to the site of cerebral hemorrhage in rats.Each group was divided into 1d,3d,5d and 7d four subgroups,with 12 rats in each subgroup.5.The modified neurological severity score(mNss)was used to evaluate the neurological function of each subgroups rats at different time points;Water content of brain tissue was measured by dry-wet weight method;HE staining to observe the pathological changes of the brain tissue around hematoma.The expression of Omi/HtrA2 mRNA was observed by reverse transcription-polymerase chain reaction(RT-PCR),The different expression levels of Omi/HtrA2 protein were detected by immunohistochemical staining and western blot technique.Results: 1.Applying the whole bone marrow adherent method separates and develops BMSCs with good activity and high purity successfully,and can be applied to the next experimental study.2.The recombinant plasmid pEGFP-N1-apoJ was successfully transfected into BMSCs.ApoJ gene modified Bone Mesenchymal Stem Cells were able to expression apolipoprotein J and the growth of BMSCs in large quantities.3.On the 1d,3d,5d and 7d after transplantation,The mNSS of ApoJ/BMSCs group was significantly lower than that of BMSCs group and NS group at the same time point(P < 0.05),and BMSCs group was lower than that of NS group at same time point(P<0.05).4.The results of brain water content showed that the brain water content of ApoJ/BMSCs group was significantly lower than that of BMSCs group and NS group at the same time point on 1d,3d,5d and 7d(P < 0.05),and BMSCs group was also lower than NS group(P < 0.05).5.The results of HE staining showed that the edema,necrosis,inflammatory cells and erythrocyte aggregation infiltration of neurons around hematoma in ApoJ/BMSCs group and BMSCs group were less than those in NS group on 1d,3d,5d and 7d,what's more the ApoJ/BMSCs group was the least amount of inflammatory cell infiltrationthe,lightest cell edema degree,and the degree of damaged nerve structure was the best.6.RT-PCR results showed that the expression of mitochondrial apoptotic protein Omi/HtrA2 mRNA in ApoJ/BMSCs group was significantly lower than that in other two groups at some time points(P < 0.05),and BMSCs group was lower than NS group at the same time point(P < 0.05).7.The results of Western blot showed that the expression level of Omi/HtrA2 protein in ApoJ/BMSCs group was significantly lower than that of the BMSCs group and the NS group at the each time point after cell transplantation(P<0.05),and BMSCs group was lower than NS group at the same time point(P < 0.05).8.The results of immunohistochemical staining showed that the expression of Omi/HtrA2 protein in ApoJ/BMSCs group was significantly lower than that in the other two groups at each time point after transplantation(P < 0.05),and BMSCs group was lower than NS group at same time point(P<0.05).Conclusion: ApoJ gene modified BMSCs transplantation is is more effective in therapeutic effect than simple BMSCs transplantation on ICH in rats.Exogenous ApoJ may alleviate brain edema,promote the recovery of nerve function and alleviate secondary brain damagafter cerebral hemorrhage by inhibiting Omi/HtrA2 mediated apoptotic pathway. |
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