HUCMSCs Survival,Distribution,Proliferation,Differentiation,and Effects Of Tongxinluo In MCAO Mice By Intravascular Transplantation

Objective:To investigate the in vivo and ex vivo differences in the survival,distribution,proliferation,and differentiation of HUCMSCs transplanted via intravenous and arterial routes in MCAO mice,to determine the optimal cell transplantation pathway and the effects of the traditional Chinese medicine Tongxinluo intervention,and carry out related mechanisms the study.Methods:In vitro experiments:Construct a luciferase-enhanced green fluorescent protein lentiviral vector(LV-EF1a-lucifase-2A-eGFP-Puro-WPRE),transfect HUCMSCs and culture the transfected HUCMSCs.In vivo experiments:C57BL/6 mouse MCAO models were made using the modified suture method and randomly divided into 6 groups:18 in the sham operation group,18 in the model group,18 in the vein transplantation group,and 18 in the vein transplantation+Chinese medicine group.There were 18 rats in the arterial transplantation group and 18 rats in the arterial transplantation+Chinese medicine group.Among them,the sham operation group was only exposed and separated from the right cervical,external and internal carotid arteries under anesthesia,without blocking blood flow and other operations.The rest of the groups had the same modeling method;Longa score was used.Neural function scores of mice in each group were measured before modeling,1,3,and 7 days after modeling,and TTC staining was used to determine the cerebral infarct area of mice in each group at 1,3,and 7 days after modeling;using living organisms Optical molecular imaging technology traces the differences in survival,distribution,migration of HUCMSCs in mice at 1,3,and 7 days after transplantation and the effects of Tongxinluo intervention;immunofluorescence dual-label technology was used to detect 1,3,and 7 after transplantation Expression of eGFP+GFAP,eGFP+NeuN around ischemic focal points,Werstern blot and Elisa methods to detect post-transplantation inflammation(ICAM1,VCAM1,E-selectin;MCP-1),nerve regeneration and angiogenesis(CXCR4;SDF1α)Changes in the expression of related factor proteins,clarify the mechanism of migration,proliferation,and differentiation of transplanted HUCMSCs and the role of Tongxinluo.Results:1.Successfully constructed a luciferase-enhanced green fluorescent protein lentiviral vector(LV-EF1a-lucifase-2A-eGFP-Puro-WPRE),transfected HUCMSCs and cultured the transfected HUCMSCs for dynamic observation transplantation The survival,distribution,and migration of HUCMSCs in MCAO mice prepared experimental basis.2.HUCMSCs can significantly improve the neurological function of mice 7 days after intravenous and arterial transplantation,and the Chinese medicine intervention can significantly reduce the neurological score of mice,suggesting that the traditional Chinese medicine Tongxinluo has neuroprotective effect on MCAO mice after HUCMSCs transplantation;After 1,3,and 7 days after HUCMSCs transplantation,compared with the model group,the area of cerebral infarction in mice after arterial and intravenous transplantation was reduced.Seven days after HUCMSCs transplantation,the area of cerebral infarction in the arterial transplantation group was significantly smaller than that in the arterial transplantation group(p<0.05),suggesting that arterial transplantation can effectively reduce the area of cerebral infarction in mice and that the traditional Chinese medicine Tongxinluo has a promoting effect.3.In vivo bio-optical molecular imaging results found that the fluorescence signal intensity of HUCMSCs in each group showed a trend of attenuation on days 1,3,and 7.The fluorescence signal intensity of HUCMSCs in the arterial transplantation group was higher than that in the vein transplantation group,and the vein transplantation The fluorescence signal intensity of HUCMSCs in the group was significantly reduced on the 7th day;the fluorescence signal intensity after the intervention of Chinese medicine was higher than that of the simple cell transplantation group,suggesting that the traditional Chinese medicine Tongxinluo could promote the distribution and survival of transplanted HUCMSCs in cerebral ischemia mice And migration.4.The results of double-labeled immunofluorescence assay showed that in the ischemic penumbra zone,eGFP-labeled HUCMSCs had different proliferation and differentiation abilities at various time points after transplantation.At 1,3,and 7 days after transplantation,the number of eGFP+ positive cells was in the arterial transplantation group and the vein transplantation group,and the number of eGFP+positive cells gradually increased with time.Among them,the arterial transplantation group had the most eGFP+GFAP expression on day 7 and responded to eGFP.The proliferative capacity of labeled HUCMSCs in mice;the differentiation of HUCMSCs in the ischemic penumbra zone was dominated by eGFP+GFAP,with only a small number of eGFP+NeuN-positive cells on day 7.The expression of eGFP-labeled HUCMSCs positive cells at various time points after Chinese medicine intervention was higher than that of the simple cell transplantation group,indicating that the traditional Chinese medicine Tongxinluo could promote the proliferation and differentiation of transplanted eGFP-labeled HUCMSCs in mice.5.Werstern blot results showed that the protein expressions of inflammation-related factors ICAMI,VACM1,and E-Selectin in all groups showed a decreasing trend at 1,3,and 7 days after transplantation,and the decline was particularly obvious at 3 and 7 days,and at 7 Less expression at day;CXCR-4 protein expression that promotes nerve regeneration and angiogenesis shows a downward trend at 1 and 3 days after transplantation,and starts to increase at day 7;at day 7 after transplantation,inflammation-related protein expression in the arterial transplantation group Significantly reduced,significantly increased the expression of proteins that promote nerve regeneration and angiogenesis,and promoted by the traditional Chinese medicine Tongxinluo;Elisa results showed that the expression of inflammation-related factors MCP-1 in each group showed a downward trend at 1,3,and 7 days after transplantation The expression of SDF1α related to the promotion of nerve regeneration and angiogenesis generally showed an upward trend.Among them,the expression of inflammation-related factor MCP-1 in the arterial transplantation group was significantly reduced on the 7th day after transplantation,which promoted nerve regeneration and angiogenesis-related factors.The expression of SDF1α was significantly increased,and the Chinese medicine Tongxinluo had a promoting effect after intervention.ConclusionCompared with intravenous transplantation,arterially transplanted HUCMSCs have stronger survival,distribution,proliferation,and differentiation capabilities in MCAO mice,and have a better effect on MCAO mice.Tongxinluo intervention can enhance their survival and distribution,Proliferation,differentiation ability,its mechanism may be related to regulating the expression of related inflammatory factors after cerebral ischemia and promoting nerve repair and vascular regeneration.