| Objectives: To study the effect of human umbilical cord mesenchymal stem cells(Huc-MSCs)transfected with Notch1 gene on the recovery of neurological function in ischemic cerebrovascular disease of SD rats by tail vein transplantation,in order to provide the experimental basis for further clinical treatment of the ischemic encephalopathy.Methods:Huc-MSCs were cultured by tissue explant adherent method,and the vector plasmid DNA(pcDNA3.1-EGFP-AMP)with Notch1 gene was transfected into Huc-MSCs for use before transplantation and the fluorescence expression of transfected Huc-MSCs was observed under the inverted fluorescence microscopy.From one month after the transplantation,the expression of Notch1 mRNA and the protein in the rats’ brain tissue was detected respectively by quantitative real-time PCR and Western blot.The model of middle cerebral artery occlusion(MCAO)was established in healthy male SD rats by improved suture method;and the TTC method was used to determine whether the site and extent of cerebral ischemia are consistent with the artery blood supply areas in the brain.According to the Zea Longa neurological function score,the rats were graded,and those 40 SD rats of cerebral ischemia model which conformed with the standard were randomly divided into four groups [group transfected with Notch1 gene(transplanted group A),human mesenchymal stem cell group(transplanted group B),empty plasmid group(transplanted group C),negative control group].After 7 days of the model establishment of the transplanted groups,Huc-MSCs were transplanted into the rats’ body by tail vein method,and the negative control group was not treated.The neurological impairment score(mNSS)was used to detect the recovery of rats’ neurological function on the 3rd,7th,14 th and 30 th day after the transplantation.Results:1.The primary human umbilical cord mesenchymal stem cells were isolated and cultured by tissue explant adherent method,and after the detection by flow cytometry,the results have shown that those cultured cells expressed CD44 and CD105 explicitly,whileCD45 and CD34 were not expressed,which confirmed that the cultured cells were consistent with the characteristics of the morphology and surface markers of human umbilical cord mesenchymal stem cells;therefore,we may infer that the cultured cells are human umbilical cord mesenchymal stem cells.This indicates that human umbilical cord mesenchymal stem calls can be passaged and proliferated in vitro,and have the ability of healthy self-renewal and the potential of multi-directional defferentiation.2.After the MCAO model was established by improved suture method,the neurological function of rats have been obviously deficit.TTC has detected that the right side of the striatum and cortex area were pale,that is to say,the modeling was successful.3.By observing the peaks in the charts of the comparison of the sequencing results and the standard sequence and the verified figure of double digests,the results have shown that the Notch1 gene was constructed into the vector plasmid DNA(pcDNA3.1-EGFP-AMP).4.At the 7th,14 th and30th day after the transplantation of Notchl gene,the mNSS score of transplanted group A was lower than that of negative control group,which had the statistical significance(P<0.05);the difference between the mNSS score of translplanted group B and translplanted group C and that of negative control group has no statistical significance(P>0.05);at the same period,the mNSS score of transplanted group A was lower than that of transplanted group B and translplante group C,which was statistically significant(P<0.05).5.After one month of transplantation,the Notch1 mRNA and Notch1 protein of the transplanted group A were respectively detected by quantitative real-time PCR and Western blot,and both their expressions have been increased,which had statistical significance when comparing with that of the control group(P <0.05).Conclusions: Huc-MSCs transfected with Notch1 gene can survive and express in the host brain by tail vein transplantation,and can improve the recovery of rat’s behavioral function and the deficiency of rat’s neurological function. |
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