Effects Of TSA Preconditioning On HUC-MSCs Neural Differentiation And Stem Cell Therapy For TBI

Currently,the repair of nerve function is the focus in the field of regenerative medicine.Traumatic brain injury(TBI)is a disease that the nerve function has been severely damaged.Because of the limited repair function of the neural system,the damage to the nervous system caused by TBI often leads to disability and even death.Neural differentiation of stem cells was used as an important strategy for to repair damaged function in regenerative medicine.However,stem cell research is often affected by ethical controversy and the source of the restrictions,which to some extent,hindered its application in the study of functional recovery of diseases.Therefore,we used human umbilical cord derived mesenchymal stem cells(hUC-MSCs)as the research object,hUC-MSCs has the potential to differentiate into neural cells,and they have advantages of extensive sources and fewer disputes,valuable for clinical application.The use of stem cell transplantation for the treatment of TBI has achieved some success,however,the number of stem cells in the lesion area is less and the efficiency of differentiation is low,which greatly hindered the clinical application of stem cell replacement therapy to treat TBI.Therefore,how to improve stem cell migration and differentiation efficiency have become the key to stem cell therapy for TBI.Migration of stem cell to the lesion area is mainly mediated by the SDF-1/CXCR4 reaction axis.Study found that histone deacetylase(HDAC)inhibitor could increase CXCR4 expression on the surface of the stem cell.It was also found that histone acetylation could regulate the proliferation and differentiation of stem cells,and the use of HDAC inhibitors could promote the differentiation of neural stem cells.A recent study found that histone H3 lysine residues Yajilai acetylation(H3K9Ac)has beneficial effects in maintaining stem cell pluripotency and differentiation into neural cells.Trichostatin A(TSA)is a broad-spectrum inhibitor of HDAC,many domestic and foreign literatures reported that TSA can inhibit theactivity of HDAC ?and ? and the expression of H3K9 Ac,what's more,TSA can promote the differentiation of stem cells into a variety of other cells.However,whether TSA pretreatment can promote the neural differentiation of hUC-MSCs is unknown.The effect of TSA preconditioning on the repair of TBI mice after transplantation of hUC-MSCs has not been reported.Therefore,we hypothesized that the treatment of TSA with hUC-MSCs may promote the migration and differentiation of hUC-MSCs into the lesion area,and then improve the nerve repair effect of hUC-MSCs transplantation in the treatment of TBI mice.Objectives In vitro study to explore TSA pretreatment on the proliferation,apoptosis,migration and neural differentiation of h UC-MSCs;in vivo experiment to study to the effect of TSA pretreated h UC-MSCs transplantation on the nerve repair of TBI model of C57BL/6J mice,as well as the molecular mechanism of acetylation in the migration and differentiation of h UC-MSCs.Method Part one ? Effects of TSA on proliferation,differentiation,migration and differentiation of h UC-MSCs in vitro1.The experiment was divided into 2 groups: control group(MSCs group)and TSA treatment group(TSA-MSCs group).2.The influence of TSA on cycle and apoptosis of h UC-MSCs was detected by flow cytometry,and expression of cycle and apoptosis related protein Cyclin D1 and Bcl2 via Western Blot detection.3.The expression of CXCR4 on the cell membrane was detected by Western Blot.Transwell SDF-1 was employed to evaluate the migration ability.4.The expression of H3K9 Ac and HDAC1 was detected by Western Blot after TSA treatment;relative expression of NSE and DCX was detected by q RT-PCR after neural differentiation in vitro.Part two?In vivo experiments were conducted to investigate the neural repair effects of TSA treated h UC-MSCs on TBI mice.1.TBI mice model was made on 81 C57BL/6J mice using stereotaxic apparatus,Mice were randomly divided into 3 groups: normal saline group(Veh group),h UC-MSCs group(group MSCs),h UC-MSCs transplantation group,and TSA treatment(TSA+MSCs group),27 rats in each group.2.Expression of SDF-1 in the lesions were detected by q RT-PCR,human cells in the lesions were detected by immunohistochemistry to judge the migration of h UC-MSCs to the lesion.3.The body weight was assessed to analyz the comprehensive condition of the mice;Neurologic deficit score(NDS)and thread test to evaluate the movement function of the mice,sucrose preference test to evaluate the depression degree,novel object recognition(NDR)test was used to evaluate mice cognitive ability,and Morris water maze to evaluate spatial learning memory.4.The water content of the damage lateral of the brain was assessed by the dry-wet proportion;CV staining was used to assess brain injury volume,PI was used for detection of necrosis of neurons in the lesion and the expression of apoptosis related gene,Caspase3-Cleaved,Bcl2 were detected by Western Blot.5.The inflammation related factors,IL-10 IL-4,IL-1 beta,TNF-alpha,and oxidative stress related factors SOD,GSH,GSH-Px and MDA was tested by ELISA.6.The relative expression of acetylation related gene,HDAC1 and H3K9Ac,were detected by Western of Blot,the relative expression of neural markers,DCX and MAP2 and nerve growth factor,BDNF,NGF,and NT3 were detected by q RT-PCR and the expression of DCX and Neu N in brain tissue were detected by immunofluorescence.results1.Western Blot and flow cytometry showed that,compared with the MSCs group,no significant difference in apoptosis of TSA-MSCs group,cells were arrested in G1/S phase(P<0.05);2.Western Blot showed that compared with MSCs group,TSA treated h UC-MSCs expressed more of CXCR4(P< 0.05);Transwell indicated that more TSA-MSCs migrated to the SDF-1(P<0.05);3.The level of acetylation showed that the expression of HDAC1 in the TSA-MSCs group was lower than that in the MSCs group,and the expression of H3K9 Ac was up-regulated(P<0.05);compared with MSCs group,expression of neuronal markers DCX and NSE was significantly increased in the TSA-MSCs group,with statistical difference(P<0.05).Part two?The vivo experimental results1.There was no significant difference of SDF-1expression in the lesion area between different groups,human cells were detected in the TSA+MSCs group and MSCs group.2.Compared with the MSCs group,there were more human cells in the lesion area in the TSA+MSCs.Compared with the Veh group,MSCs group gained more weight,and showed better performance in NDS,hanging test,sugar preference,novel object recognition and water maze behavioral test,indicating significantly improved in athletic ability,learning ability,improve the degree of depression,moreover,better effect can be achieved in TSA+MSCs group(P<0.05).3.Compared with Veh group,mice of ipsilateral hemisphere brain cells transplantation showed decreased water content,damage volume necrosis necrosis cells in the lesion,as well as creased the expression of apoptosis related protein Bcl2,Caspase3-cleaved.The TSA+MSCs group performed better(P<0.05).4.Compared with Veh group,cell transplantation group of inflammatory factors IL-4 and IL-10 expression increased,IL-1 expression level of TNFalpha beta and decreased expression of MDA decreased oxidative stress,free radical scavenging substances SOD,GSH,GSH-Px were Results Part one?The vitro experimental down-regulated,in addition to the IL-1 beta,TSA+MSCs group increased and decreased more significantly(P<0.05).5.Compared with Veh group,the expression of HDAC1 was significantly reduced in TSA+MSCs group and MSCs group,the expression of H3K9 Ac was significantly elevated(P<0.05);nerve growth factor BNDF,NT3,NGF and neural specific markers,DCX,Neu N and MAP2 expression was significantly up-regulated,with TSA+MSCs group increased more significantly(P<0.05).Conclusion1.After treatment with TSA,the acetylation level is increased in the h UC-MSCs,at the same time,the cell cycle was inhibited,apoptosis had no obvious change,TSA can promote the expression of CXCR4 on the h UC-MSCs membrane,increase migration to SDF-1,upregulate of the expression of neuronal cell markers DCX and NSE,promote the differentiation of MSCs into neuron like cells.2.After TSA treatment,more MSCs can migrate to the lesion area in TBI mice,and increase the acetylation level of the lesion,improve the mice behavior ability,alleviate cerebral edema,reduce the lesion area of necrosis cells,reduce the inflammation,promote nerve regeneration in the lesion area,and finally improve the effects of MSCs transplantation on TBI mice nerve repair.