| Objective: To investigate the effects of ApoJ-modified rat bone marrow mesenchymal stem cells(BMSCs)on the expression of TNF-? after Intracerebral hemorrhage in rats and its possible mechanism.Methods: 1.The "improved secondary blood injection method" was used to construct a model of Intracerebral hemorrhage in SD rats.2.Rat bone marrow mesenchymal stem cells(BMSCs)were cultured and purified in vitro using the whole bone marrow differential adherent culture method.3.LipofectaminTM 2000-mediated transfection of BMSCs with the recombinant plasmid pEGFP-N1-apoJ.Observe the transfection effect of the recombinant plasmid with a fluorescence microscope.Select the group with the highest transfection efficiency for subsequent experiments.4.144 rats with successful Intracerebral hemorrhage were randomly divided into 3 groups: saline control group(NS group)? BMSCs transplantation group(BMSCs group)and ApoJ-modified BMSCs transplantation group(ApoJ/BMSCs group).48 rats in each group.5.Transplantation.Twenty-four hours after successful modeling,three groups were transplanted with equal volumes of normal saline,BMSCs suspension,and BMSCs suspension transfected with ApoJ gene to the cerebral hemorrhage site of rats,and were sacrificed at 1d,3d,5d,and 7d respectively.Rats were divided into 4 subgroups according to sacrifice time,12 rats in each subgroup,and the neurological recovery of each subgroup was evaluated before sacrifice.Modified neurological severity score(mNSS)was used.6.After scoring,the rats in each subgroup were quickly sacrificed,and the brain tissue water content was measured by dry and wet weight method.The pathological changes of brain tissue after cerebral hemorrhage in rats were observed under HE staining microscope;immunohistochemical staining on TNF-? protein around hematoma.The expression was analyzed semi-quantitatively;the real-time quantitative PCR was used to quantitatively analyze the TNF-? mRNA around the hematoma;determination of TNF-? protein expression in brain tissue around hematoma by western blot.Results: 1.BMSCs with good activity and high purity were cultured in vitro using the whole bone marrow differential adherent culture method.2.Liposome mediated plasmid pEGFP-N1-ApoJ was successfully transfected into BMSCs,and it can stably express a large amount of exogenous ApoJ after transfection.3.mNSS score: The mNSS score of ApoJ / BMSCs on the first day after transplantation was significantly lower than that of the NS group at the same time point(P <0.01).Other than that,the pairwise comparison between the three groups was not different on the first day(P> 0.05).However,the mNSS scores of the ApoJ / BMSCs group at the other time points were significantly lower than at the same time those of the BMSCs group and NS group(P <0.01),while the BMSCs group was lower than that of the NS group at the same time point(P <0.01).4.Brain tissue water content results showed that there was no significant difference between the first day after transplantation(P> 0.05).The brain water content in the ApoJ / BMSCs group on the 3rd,5th,and 7d after transplantation was significantly lower than NS at the same time point.Group and BMSCs group(P <0.05),BMSCs group was lower than NS group at the same time point(P <0.01).5.HE staining results showed that compared with control group NS group,ApoJ / BMSCs group and In the BMSCs group,edema,cell degeneration and necrosis,red blood cell aggregation,and inflammatory cell infiltration were mild in the surrounding tissue at the same time point,and the damaged tissue structure was best repaired in the ApoJ / BMSCs group.6.RT-PCR results showed that the ApoJ / BMSCs group at the various time points after transplantation,the mRNA expression levels of TNF-? were significantly lower than those of the BMSCs and NS groups at the same time point(P <0.01),and that in BMSCs group at the same time point was lower than that in NS group(P <0.01).).7.The results of immunohistochemistry showed that at various time points after transplantation,compared with the ApoJ / BMSCs group,the BMSCs group and the NS group,the expression level of TNF-? protein was significantly reduced(P <0.01),of which 3d?7d after transplantation,the ApoJ / BMSCs group was significantly lower than the BMSCs group(P <0.01).8.Western blot results showed that at various time points after transplantation,compared with the NS control group,the expression of TNF-? protein in the ApoJ / BMSCs group and the BMSCs group was significantly reduced(P <0.01),and the ApoJ / BMSCs group was lower than the BMSCs group at the same time point(P <0.01).Conclusion: ApoJ-modified BMSCs transplantation can reduce inflammatory cell infiltration,reduce tissue edema,and promote nerve cell repair after cerebral hemorrhage in rats,and it has a better effect than BMSCs transplantation alone.ApoJ may play a neuroprotective effect on cerebral hemorrhage by down-regulating the expression of the inflammatory factor TNF-? and reducing secondary damage caused by inflammation. |
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