Evaluation Of The Efficacy Of HGF Gene-modified Mesenchymal Stem Cells In Neonatal Mice With Hypoxic-ischemic Encephalopathy

Objective: Hypoxic-ischemic encephalopathy(HIE)can lead to neonatal death,and survivors often suffer from severe neurological dysfunction.At present,bone marrowderived mesenchymal stem cell(BMSCs)transplantation for the treatment of HIE is a very potential therapeutic strategy.Many researchers use BMSCs as exogenous gene carriers,by means of transfection,it can efficiently overexpress a certain gene or cytokine to improve the therapeutic effect of BMSCs.In this paper,from in vivo and in vitro experiments,hepatocyte growth factor(HGF)gene-modified BMSCs transplantation was used to evaluate the therapeutic effect of HIE and explore its mechanism,in order to open up a new path for the clinical treatment of HIE.Methods:(1)BMSCs were extracted and purified.Then BMSCs were transfected with recombinant adenovirus vector,and the level of HGF m RNA transcription and cell proliferation in BMSCs were detected after transfection.(2)Construct HIE animal model,transplant HGF gene-modified BMSCs into neonatal mouse HIE model.Detect cerebral infarct volume by TTC staining.Immunofluorescence staining was used to detect HGF expression in brain tissue.RT-q PCR was used to detect HGF m RNA and Western bolt was used to detect HGF protein expression.Detect the expression of p-ERK and Bcl-2after BMSCs-HGF transplantation by Western bolt.(3)Construct an oxygen-glucose deprivation(OGD)cell model in vitro,detect cell apoptosis by TUNEL staining,and detect the expression of Bcl-2 by Western bolt,and so as to systematically explore the neuroprotection of HGF gene modified BMSCs.Results:(1)The results of recombinant adenovirus vector transfection showed that compared with the control group,the p DC316-m CMV-EGFP-HGF(Ad-HGF)group promoted the proliferation of BMSCs(p<0.05),while the p DC316-m CMV-EGFP(AdGFP)group did not promote the proliferation of BMSCs(p>0.05).(2)The results of HIE treatment in vivo showed that compared with the control group,the volume of cerebral infarction in BMSCs-HGF group and BMSCs-GFP group was significantly reduced(p<0.001);The infarct volume of the BMSCs-HGF group was further significantly reduced compared with the BMSCs-GFP group(p<0.001).The implantation of BMSCsHGF increased the expression levels of p-ERK and Bcl-2,while the implantation of BMSCs-GFP had relatively little effect on the expression levels of p-ERK and Bcl-2.(3)Exploration of the mechanism in vitro showed that: TUNEL staining results suggested that compared with BMSCs-GFP co-cultured with cortical neurons,HGF gene-modified BMSCs co-cultured with cortical neurons enhanced the protective effect against OGD;Inhibition of ERK phosphorylation eliminated the neuroprotective effect of BMSCs-HGF on OGD.Conclusions: HGF gene transfection can promote the proliferation of BMSCs and enhance the protective effect of BMSCs on HIE;BMSCs-HGF can increase the expression of HGF,p-ERK and Bcl-2 in brain tissue;BMSCs-HGF can protect neurons from OGD-induced apoptosis;The ERK/Bcl-2 pathway is involved in the underlying neuroprotective mechanism.