| Objectives: Rat model of closed traumatic brain injury(TBI) by the wayof fluid-percussion impact was established and accepted human cordmesenchymal stem cells(hUC-MSCs) transplantation to observe its effect oncell survival, migration, differentiation as well as recovery of neurologicaldefect. It lay a basis for its further application.Methord: The umbilical cords (UCs) from healthy neonatal withfull-term pregnancy cesarean section were used. It uses the method of enzymedigestion and sidewall for primary cell of3~5generation. The harvestingcells were collected and flow cytometry was adopted to analyze the expressionof CD45-PE, CD90-PE, CD105-PE, CD73-PE, CD11b-PE,HLA-DR-PEandCD34-FITC, CD19-FITC, and mouse IgG1-PE/mouse IgG1-FITC ascontrols.Adult male Sprague-Dawley (SD) rats in good condition (the weight isbetween260and320gram) were selected and made TBI models by the wayof fluid-percussion impact. Estimated by modified neurological severity score(mNSS)24hours later (Table1),50rats with a score of7to12(moderateinjury) werer randomly divided into there groups:⑴hUC-MSCstransplantation groups(n=20): it detects cell survival before cell transplantswith thiazole blue [3-(4,5)-dimethylthiahiazol-2-y1-2,5-diphenytetrazolium bromide, determined by MTT]. Rat accepted P5hUC-MSCs transplantation in the area of injury.⑵Phosphate BufferedSaline(PBS) group (n=20):Each rat received PBS injection into the damagedbrain.⑶simple TBI group (n=10): Each rat only accepted brain injurytreatment hUC-MSCs solution was not injected into the damaged area.Another10rats were puted into the sham operation group randomly, just thecranial bone was removed compared with the TBI group, and not giving hUC-MSCs transplantation solution injection.The neural function was estimated by the mNSS at1,7,14,21and28days after transplantation. After functional outcome mesurement the rats weresacrificed and anatomized. It detected to compare with the expression ofglial-fibrillary-acidic-protein（GFAP）, neuron-specific-enolase (NSE), Nestinand myelin-basic-protein (MBP) situation by RT-PCR technique.Results:A few of cells began to be stuck to a surface4～6hours after primarculture and more were found12hours later. When reached to80%～90%confluent after7days of culture, hUC-MSCs were subculture at the rate of1:2or1:3. The cells proliferated quickly and were fairly uniform3～4days later,it can be subcultured again. Flow cytometry showed that hUC-MSCsexpressed surface antigen of MSCs include CD73，CD90, CD105, did notexpress surface antigen of Hematopoietic precursor cells (HPCs) as CD34,yetnot express surface antigen CD19, CD11b, CD45or HLA-DR. It indicatedthat these cells were MSCs. Cell vitality was examined with3-(4,5)-dimethylthiahiazol-2-y1-2,5-diphenytetrazolium bromide(MTT) beforemigration,and the vitality is more than95%.28days after transplantation, only2rats died in the hUC-MSCs groupand the mortality was10.0%;6rats died in the PBS group, the mortality was30.0%;4rats died in simple TBI group that the death rate was40.0%.Compared the mortality, the hUC-MSCs group was lower than the simple TBIgroup, the difference was statistically significant（P<0.05）.The outcomes of mNSS (as table3showed) demonstrated that the scorechanged with time neural functional recovery in the hUC-MSCs group wasbetter than the simple TBI group, the difference was statistically significant（P<0.05）.hUC-MSCs transplant in28days. The nerve function score was over.We taked the fresh brain tissue to extract total RNA. Application of RT-PCR,we proved neural stem cells survive and expressed GFAP,NSE, Nestin anddid not express MBP in rat TBI injury from molecular biology. Conclusion:1hUC–MSCs transplantation following traumatic brain injury improvedthe neural functional recovery in rats.2hUC–MSCs could survive and expressed GFAP, NSE, Nestin and notexpress MBP. |
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