| Background and Objectives: Cerebral infarction has the characteristics of high incidence rate,high disability rate and high mortality rate.It is lack of effective treatment for patients with cerebral infarction after hyperacute stage.However the new treatment of regenerative medicine provides a new treatment for cerebral infarction.BMSCs have the ability of self-renewal and multi-directional differentiation,and can secrete a variety of neurotrophic cytokines,which makes it possible for BMSCs to treat cerebral infarction.To investigate whether there is a difference in the therapeutic effect of BMSCs transplantation via carotid artery and stereotactic pathway in acute and chronic cerebral infarction rat models,and to explore the possible reasons and mechanisms of the difference.Methods:1: BMSCs were isolated,purified and amplified by whole bone marrow adherent method;BMSCs were identified by flow cytometry with cell surface markers CD90,CD29,CD106,CD11 b,CD45 and CD34;BMSCs were transfected by lentivirus mediated EGFP to detect the transfection rate and the effect on the growth and proliferation of BMSCs.Firstly,BMSCs were transplanted into the cerebral infarction rat models by carotid artery and stereotactic pathway on the 24 hours after modeling(acute stage of cerebral infarction);Secondly,the neurological functions of the model rats were scored on the 3rd,7th,21 st and 28 th day after BMSCs transplantation.The neurological scoring methods included m NSS score,adhesion test and limb asymmetry application test.3: BMSCs were transplanted into the cerebral infarction rat models by carotid artery and stereotactic pathway on the 28 th day after modeling;then the neurological functions of the model rats were scored on the 7th,14 th,21st and 28 th day(the 35 th,42th,49 th and 56 th day after modeling)after BMSCs transplantation.4: On the 28 th day after transplantation,the cerebral infarction model rats were killed;TTC staining was used to detect the change of infarct volume;bielshowsky’s-Luxol fast blue double staining was used to detected the regeneration of nerve fibers;the pathological changes of brain tissue after BMSCs treatment were detected by HE staining;the number of EGFP positive cells in the focus area of cerebral infarction was detected by direct immunohistochemical staining;the expressions of VEGF,NSE,Ki-67,GFAP,SYN,Nogo-A and the thickness of Glial scar in the focus area of cerebral infarction were detected by immunohistochemistry.Results:1: In the first 24 hours of primary culture,all the cells showed round like suspension growth,and a small number of short fusiform adherent cells could be seen at the bottom of the culture flask from 24 to 48 hours,and then fusiform adherent cells proliferated radially like colonies,and 80-90% of the culture flask could be covered in about 8-10 days;with the fluid changes and passages,the suspension cells continued to be removed,and the growth rate of BMSCs gradually increased;The positive rates of CD90,CD29,CD106,CD11 b,CD45 and CD34 in BMSCs were 92.3+2.76%,87.1+2.34%,54.7+1.29%,12.1+0.25%,2.3+0.18%,1.2+0.21%,respectively;Lentivirus mediated EGFP(MOI = 200 and 400)transfection had harmful effects on the growth and proliferation of BMSCs,while MOI = 100,50 and 25 transfection had no harmful effect on the growth and proliferation of BMSCs;the positive expression rate of EGFP in BMSCs was89.0+7.6% after 96 hours of lentivirus mediated EGFP(MOI = 100)transfection,92.1 +5.4% after 1 week,and 87.2+8.1% after 2 weeks.2:BMSCs treatment of acute cerebral infarction model rats:(1)neurological function score: the m NSS score of the arterial transplantation group was significantly lower than that of the control group on the 7th,14 th,21st and 28 th day after BMSCs transplantation(P<0.05),and the m NSS score of the stereotactic transplantation group was significantly lower than that of the control group on the14 th,21st and 28 th day after BMSCs transplantation(P<0.05),The m NSS score of the arterial transplantation group decreased significantly on the 21 st and 28 th day after BMSCs transplantation compared with that of stereotactic approach group(P<0.05);the time to tear off the sticky paper and the percentage of application of the right limb in the arterial transplantation group decreased significantly on the 14 th,21st and 28 th day after BMSCs transplantation compared with that of the control group(P<0.05),and the time to tear off the sticky paper and the percentage of application of the right limb in the stereotactic approach group decreased significantly on the 21 st and 28 th day after BMSCs transplantation(P<0.05)Compared with the control group,and the time to tear off the adhesive paper and the percentage of application of the right limb in the arterial approach group decreased significantly on the 28 th day after transplantation compared with the stereotactic approach group(P < 0.05);(2)There was no significant difference in the volume percentage of cerebral infarction among carotid artery transplantation group,stereotactic transplantation group and control group(P > 0.05);(3)The number of EGFP positive cells in the cerebral infarction focus area of stereotactic transplantation group was significantly higher than that of the carotid artery transplantation group(P < 0.05),However,in the stereotactic group,many EGFP positive giant multinucleated cells were found in the lesion area;(4)The area of corpus callosum on the infarcted side in the carotid artery and stereotactic transplantation groups was significantly larger than that in the contralateral side of the same group(P < 0.05).The area of corpus callosum on the infarcted side in the carotid artery and stereotactic transplantation groups was significantly larger than that in the control group(P < 0.05),Meanwhile,the area of corpus callosum on the infarcted side in carotid artery transplantation group was significantly different from that in stereotactic transplantation group(P < 0.05).There was no significant difference in the area of contralateral corpus callosum among the three groups(P >0.05);(5)Compared with the control group,the number of Ki-67,VEGF,GFAP,syn and Nogo-A positive cells in the lesion area of carotid artery transplantation group and stereotactic approach transplantation group were significantly different(P <0.05),and there were also statistical differences between carotid artery transplantation group and stereotactic approach transplantation group(P < 0.05)There was no significant difference of the number of NSE positive cells in the lesion area among the three groups(P > 0.05);the thickness of glial scar in the lesion area of carotid artery transplantation group was significantly different from that of stereotactic transplantation group and control group(P < 0.05).BMSCs treatment of chronic cerebral infarction model rats:(1)Neurological function score: compared with the control group,the m NSS score,the time to tear off the sticky paper and the application percentage of the right limb of the rats in the stereotactic approach transplantation group decreased significantly on the 14 th,21st,28 th day after BMSCs treatment(P < 0.05),but there was no significant difference in the neurological function score between the carotid approach transplantation group and the control group(P > 0.05),Meanwhile,the m NSS score of the stereotactic approach transplantation group was significantly better than that of the carotid artery transplantation group at 21 and 28 days after BMSCs transplantation(P <0.05);(2)There was no significant difference in the volume percentage of cerebral infarction among the three group(P > 0.05);(3)The number of EGFP positive cells in the focal area of cerebral infarction in the stereotactic approach group was significantly higher than that in the carotid approach group(P < 0.05);At the same time,there were no EGFP positive giant multinucleated cells in the lesion area in the stereotactic transplantation group;(4)The area of corpus callosum on the infarcted side in the stereotactic transplantation groups was significantly larger than that in the contralateral side of the same group(P < 0.05).The area of corpus callosum on the infarcted side in the stereotactic transplantation group was significantly larger than that in the carotid artery transplantation group and the control group(P<0.05),Meanwhile,the area of corpus callosum on the infarcted side in carotid artery transplantation group was significantly different from that in stereotactic transplantation group(P < 0.05).There was no significant difference in the area of contralateral corpus callosum among the three groups(P > 0.05);(5)The number of Ki-67,VEGF,GFAP,SYN and Nogo-A positive cells in the lesion area of the stereotactic approach transplantation group was significantly different from that of the carotid approach transplantation group and the control group(P < 0.05).There was no significant difference in the number of NSE positive cells and the thickness of glial scar among the three groups(P > 0.05).Conclusions1: In this study,BMSCs were successfully isolated and purified by whole bone marrow adherent method,BMSCs were identified by the flow cytometry to express CD90 and CD29 highly,CD106 moderately and CD45,CD34 and CD11 b lowly;lentivirus mediated EGFP transfected BMSCs with MOI = 100 had high positive rate,and EGFP could stably and persistently express.2:This study confirmed that BMSCs transplantation via carotid artery and stereotactic approach can significantly improve the neurological function score of acute cerebral infarction model rats.Moreover,it may be enhance endogenous cell regeneration,angiogenesis,nerve fiber regeneration and astrocyte proliferation.At the same time,the study found that the effect of BMSCs transplantation via carotid artery may be better than that via stereotactic approach.3:This study confirmed that BMSCs transplantation via stereotactic approach can significantly improve the neurological function score of chronic cerebral infarction model rats,and the mechanism of effect was similar to that of acute cerebral infarction;However,it was found that the carotid artery transplantation of BMSCs may have no therapeutic value for chronic cerebral infarction rat model. |
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