| Objective:Cerebral palsy(CP)is a kind of non-progressive brain injury.Due to its high disability rate,it brings a heavy burden to the family and society.The current therapeutic means only treat the symptoms of CP and the effect yet unsatisfactory.Currently,stem cell therapy is widely used in clinical treatment and research of a number of diseases.It is considered to be one of the most important potential therapies in the field of medical treatment.It is expected to treat CP from the level of central nervous system injury.However,the current clinical trials are few and the sample size is small,the control group is not standardized,and the research conclusions are inconsistent.Therefore,in this paper,systematic review and meta-analysis were used to evaluate the efficacy and safety of stem cell therapy for CP,and subgroup analysis was conducted on the basis of which to screen the cell types with the best effect of stem cell therapy for CP.The therapeutic effect of this cell type was verified in cell and animal models and the possible mechanism of action was discussed,which provided evidence-based medicine and molecular biology basis for the clinical application of stem cell therapy,and also provided a new idea and direction for stem cell therapy of CP.Methods:Part 1:efficacy and safety analysis of stem cell therapy for cerebral palsyThe medical subject headings and entry terms of stem cell and cerebral palsy were formed into a search strategy to conduct a comprehensive literature search.Data sources included PubMed/Medline,Web of Science,EMBASE,Cochrane Library,and the search period is limited to the database establishment until January 2,2022.The literature was screened according to the exclusion and inclusion criteria of the Population,Intervention,Comparison,Outcome,and Study design(PICOS).The main outcome index was the Gross Motor Function Measure(GMFM),and the secondary outcome index included the scores of the Comprehensive Function Assessment(CFA),the Gross Motor Performance Measure(GMPM),the Bayley Scales of Infant and Toddler Development(BSID-II)and the Functional Independence Measure for Children(WeeFIM),and the adverse events as safety assessment indicators.ROB2 was used to evaluate literature quality,and RevMan5.4 was used to comprehensively analyze the extracted outcome indicators.The included studies were grouped according to cell type and follow-up time for subgroup analysis.Part 2:acquisition and identification of hUCMSCFive fresh umbilical cord specimens were collected from the Department of Obstetrics and Gynecology of First Affiliated Hospital of Gannan Medical University.They were placed in sterile glass bottles with normal saline and transferred to the ultra-clean table of cell room under 4℃.Wharton’s jelly was extracted from the umbilical cord,and primary human umbilical cord mesenchymal stem cells(hUCMSC)were obtained by tissue adhesion method.The primary cells were passaged by pancreatic enzyme digestion method,and the hUCMSC obtained were identified by cell morphology,flow cytometry,induction of lipogenic osteogenic differentiation and proliferation ability.Part 3:NogoA/NgR/Rho pathway was involved in hUCMSC to improve the neurobehavioral state and the process of brain injury in hypoxia/ischemia CP rat modelsPublished brain RNA sequencing data sets of CP rats and control rats were downloaded from GEO database,GEO2R was used to screen differentially expressed genes,and GO was used to annotate their main enriched functions.The expressions of RTN4/Nogo in each group were extracted from the data expression matrix and analyzed statistically.Mice hippocampal neuron cell line(HT22)was treated with hypoxia for 3h to establish the model of hypoxia injury.After hUCMSC was co-cultured with the cell model for 24h,the protein expression changes of TUBB3,NogoA,NgR,RhoA,Rac1 and CDC42 in neuron cells were detected by Western Blot(WB).The expressions of TUBB3 and NogoA were observed by immunofluorescence.The CP rat model was established by ligation of the left common carotid artery and hypoxia for 2.5 h.After model identification,CP rats were randomly divided into CP group,CP+PBS group and CP+hUCMSC group,the control group received sham operation.The control group and CP+PBS group were injected with PBS via caudal vein,once a week,four times.CP+hUCMSC group was injected hUCMSC 1×106 by caudal vein for 4 times a week.In the CP group,only empty needles were used to enter the caudal vein without injecting any fluid.One week after the modeling and cell injection,the neurobehavioral status of CP rats in each group was determined by open field test,suspension test and oblique board test respectively.Pathological brain injury of CP rats was observed by HE staining and Nissl staining.The mRNA and protein expressions of NogoA,NgR,RhoA,Racl and CDC42 in brain tissues of each group were detected by RT-qPCR and western blot.Results:Part 1:efficacy and safety analysis of stem cell therapy for cerebral palsyA total of 798 literatures were retrieved,and 9 studies were finally included after screening.The results of the comprehensive analysis showed that,compared with the control group,the scores of GMFM,CFA,GMPM and WeeFIM were significantly increased in the stem cell treatment group,except BSID-Ⅱ.Subgroup analysis results showed that improvements in GMFM scores were more skewed toward mesenchymal stem cells(MSC)than cord blood(UCB).In addition,time subgroup analysis showed no difference in GMFM scores between the stem cell treatment group and the control group at 1 month after receiving cell injection,and significantly higher scores in the stem cell treatment group than the control group at 3,6 and 12 months.Importantly,there was no significant difference in the incidence of adverse events between the stem cell group and the control group.Part 2:Acquisition and identification of hUCMSCIn this experiment,hUCMSC adherent primary cells were successfully obtained,with typical long spindle shape and whirlpool arrangement.The surface markers identified by flow cytometry showed that CD44,CD73,CD90 and CD 105 were positive,and CD34,CD45 and HLA-DR were negative.Lipid droplets and mineral nodules were observed after induction of lipogenesis and osteogenesis.In addition,there was no significant difference in cell proliferation ability between 3 and 7 generations.Part 3:NogoA/NgR/Rho pathway was involved in hUCMSC to improve the neurobehavioral state and the process of brain injury in hypoxia/ischemia CP rat modelsAccording to the biogenic analysis of GSE23317 data,compared with the sham operation group,the gene expression in the cortex tissue of rats with ischemia and hypoxia was different,and a total of 798 differential genes were selected.The GO annotation results showed that the differential genes were closely related to the biological processes of neuronal death and apoptosis as well as the regeneration of neurons and glial cells.The expression of RTN4/Nogo in ischemic anoxic rats was significantly higher than that in control group.In HT22 cells treated with hypoxia,the relative protein expression of TUBB3,Rac1,CDC42 was significantly lower than that of control group and hypoxia+hUCMSC,while the relative protein expression of NogoA,NgR,RhoA was significantly higher than that of control group and hypoxia+hUCMSC.After ischemia and hypoxia modeling,CP rats showed obvious abnormal motor function and pathological injury.Compared with CP group,hUCMSC transplantation can significantly improve the neurobehavioral status of CP rats and reduce brain pathological injury.The mRNA and protein relative expressions of NogoA,NgR and RhoA in brain tissue of CP group were significantly higher than those of control group and CP+hUCMSC group.The relative expressions of Racl and CDC42 mRNA and protein in brain tissue of CP group were significantly lower than those in control group and CP+hUCMSC group.Conclusion:Stem cell therapy for CP is safe and effective.According to the results of subgroup analysis,the therapeutic effect of stem cells can be observed at least one month later,and the effect of MSC is superior to that of UCB.The morphology,surface molecular expression,lipogenic and osteoblastic differentiation ability and proliferation ability of primary cells obtained in this experiment were all in line with the definition of MSC,so it was determined that hUCMSC was successfully isolated.This study confirmed that down-regulation of NogoA/NgR/Rho pathway was involved in the process of hUCMSC improving neurobehavioral state and alleviating brain injury in hypoxia/ischemia CP rat models. |
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