Dedifferentiation Of HUC-MSCs Participates In HIBD Nerve Repair Through HSDF-1alpha

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Identification of the basic characteristics of De-hUC-MSCs and the therapeutic effect after De-hUC-MSCs transplantation in the HIBDObjectives:To identify the basic characteristics of De-hUC-MSCs,hUC-MSCs were dedifferentiated into De-hUC-MSCs.To confirm the neurological repair effect of De-hUC-MSCs,which were transplanted in HIBD rats.Gene chip was used to analyze the whole gene expression pattern of hUC-MSCs and De-hUC-MSCs,and to explore the possible mechanism of De-hUC-MSCs to play a role in nerve repair.Methods:hUC-MSCs were induced to dedifferentiation by MNM with a neural induction method.Morphology of hUC-MSCs and De-hU C-MSCs were observed under inverted microscope;the cell-surface mar kers of HLA-ABC,CD34,CD45,HLA-DR,CD29,CD44,CD90 and C D105 and were detected by flow cytometry;cell proliferation ability wa s detected by CCK-8 kit;potential differentiation ability of osteogenesis,adipogenesis,and cartilage of hUC-MSCs and De-hUC-MSCs were det ected;RT-PCR and Western Blot were used to detect the two groups of cell neural markers and stem cell genes.The HIBD model was establis hed in 7d SD rats.5 days later,the rats were divided into PBS group(n=15),hUC-MSCs transplantation group(n=19)and De-hUC-MSCs tra nsplantation group(n=14).The rats in each group were anesthetized,int racerebroventricular injection was caried out with the help of stereotaxi c apparatus.Each rat was given 5 ?L of cell suspension(containing 2×105 cells),and the HIBD control group was given sterile PBS;and the left side brain tissues were collected 2 days after cell transplantation,an d then stained with CD31 and BrdU double-labelling immunofluorescen ce.One month after cell transplantation,the function of learning and m emory,and the capacity of novel object exploration were detected by M orris water maze test and Object-in-place task.The gene expression of hUC-MSCs and De-hUC-MSCs was detected by Affymetrix(?)Human Genome U133 Plus 2.0 array.Results:(1)hUC-MSCs can be successfully induced into De-hUC-MSCs,and both cells exhibited spindle shaped or needle morphology.(2)Using flow cytometry to analysis hUC-MSCs and De-hUC-MSCs surfac e marker:hUC-MSCs and De-hUC-MSCs had expressed CD29,CD44,CD90,CD105,HLA-ABC,while not expressed HLA-DR,CD34 and C D45.(3)The proliferation curves of hUC-MSCs and De-hUC-MSCs we re similar,with growth period of 1-10 days,3-6 days of logarithmic gr owth phase,after 10 days into decline.(4)hUC-MSCs and De-hUC-MSCs can differatiate into osteogenic and adipogenic,chondrogenic.(5)The ex pressions of MAP2,NSE,Tau and ?-tubulin? in De-hUC-MSCs were significantly higher than those in hUC-MSCs group(***P<0.001,*P<0.05,*P<0.05,*P<0.05),Western Blotting verified the expression of MAP2 and NSE proteins.(6)The expressions of C-MYC and NANOG genes in De-hUC-MSCs were significantly higher than those in hUC-M SCs(**P<0.01,*P<0.05),but there was no difference in the express ion of Oct4 and SOX2 genes.Western Blotting results showed that C-MYC and NANOG in De-hUC-MSCs expressed higher than those in h UC-MSCs.(7)According to the immunofluorescence results,the number of endothelial cells displaying BrdU/nestin double-labelled in De-hUC-MSCs group significantly increased.(8)On the first day,according to t he Morris water maze test results,no significant difference in the distan ce and time was observed between those three groups(P>0.05),after 2-5 day's hidden platform training,all of the groups showed decrease in t he escape latency.The De-hUC-MSCs group had the shortest time to fi nd the platform,and the difference was statistically significant(***P<0.001).In the 6d platform exploration experiment,the number of platfor ms found in the De-hUC-MSCs group was significantly higher than tha t in the hUC-MSCs transplantation group(**P<0.01).According to the Morris water maze experiment,on the first day,no differences in the p ath length or the escape latency was observed between those 3 groups(*P>0.05).After 2-5 days hidden platform training,all 3 groups were decreaseing in the escape latency.The De-hUC-MSCs group had the sh ortest time to find the platform(***P<0.001).On the probe trial tests(Day 6),De-hUC-MSCs transplantation group exhibated more times of crossing the platform quadrant than hUC-MSCs transplantation group(**P<0.01).(9)Object-in-place experiment found that De-hUC-MSCs trans plantation group(n=9)spend much more time to explore new things th an PBS group(n=8)(*P<0.05).(10)The gene expression patterns of hU C-MSCs and De-hUC-MSCs were similar.The hSDF-la was the most d ifferentially expressed genes between De-hUC-MSCs and hUC-MSCs;t he expressions of hSDF-la in De-hUC-MSCs were detected by RT-PC R.Conclusion:(1)De-hUC-MSCs exhibit the morphological characteris tics and surface marker of hMSCs;has strong proliferation ability;has t he potential to differentiate into osteoblasts,Adipogenic and chondrocyt es;high expression of neural markers and stem cell genes.(2)De-hUC-MSCs transplantation can promote the regeneration of brain tissue and spatial learning and memory and the exploration of new objects in HIB D rats compared with hUC-MSCs transplantation.(3)The whole gene ex pression pattern of hUC-MSCs and De-hUC-MSCs was similar,and hS DF-la was the most differentially expressed gene between De-hUC-MS Cs and hUC-MSCs.Part ? De-hUC-MSCs participate in the neurological repair of HIBD model rats by hSDF-laObjective:To further reveal the role of De-hUC-MSCs in the treat ment of HIBD,and to investigate the effect of hSDF-la on HIBD nerv e repair.Methods:(1)Three short hairpin RNA sequences targeting the cod ing region of hSDF-la gene and one pair of oligonucleotides carrying hSDF-la gene were designed.De-hUC-MSCs were infected by the lentiviral a nd purified by puromycin.According to the fluorescent protein in the 1 entiviral vector,the expression of hSDF-1? mRNA and protein were det ected by RT-PCR and ELISA.(2)HIBD rats were randomly divided into five groups:De-hUC-MSCs(n=6),GFP(n=6),AdhSDF-la(n=6),SihSDF-1?(n=6)and hSDF-1?(n=6).The left side brain tissues were collected 2 days after cell transplantation,and then stained with haematoxylin-eos in(HE).The blood vessels and nerve formation in the five groups were detected by BrdU/CD31 and BrdU/Neun double-label immunofluorescen ceand.(3)One month after cell transplantation,the learning and memory function of De-hUC-MSCs(n=8),GFP(n=10),AdhSDF-la(n=9),SihSDF-1 a(n=9)and hSDF-1?(n=7)were detected by Morris water maze test.No vel object exploration capacity of De-hUC-MSCs(n=5),GFP(n=5),AdhS DF-la(n=5),SihSDF-1?(n=5)and hSDF-1?(n=5)were detected by Object-in-place task.Results:(1)The lentivirus can successfully infected De-hUC-MSCs.Establishment of AdhSDF-la and SihSDF-la stable cell line:accordin g to the fluorescence microscope,cells infected with AdhSDF-la,GFP and SihSDF-lalentivirus shown as green fluorescent.Identification of A dhSDF-la,SihSDF-1? stable cell line:RT-PCR results showed that the e xpression level of hSDF-la in SihSDF-1? group was significantly lowe r than that in GFP group and De-hUC-MSCs,the difference was statisti cally significant(*P<0.05).The expression level of hSDF-la in AdhSDF-1? group was significantly increased in GFP group and De-hUC-MSCs group(**P<0.01).The ELISA results showed that the expression of hS DF-1? protein in SihSDF-1? group was lower than that in GFP group a nd De-hUC-MSCs group(*P<0.05),and the expression of hSDF-la prot ein in AdhSDF-1? group higher than that in GFP group and De-hUC-MSCs group(***P<0.001).(2)The pathological changes of brain tissue after De-hUC-MSCs,GFP,AdhSDF-la,SihSDF-1? and hSDF-1? transp lantation in HIBD rats:according to the HE staining,the cells in the hi ppocampus were more closely arranged in the AdhSDF-la group,and the nuclear condensation,cell swelling and vacuoles were significantly r elieved;SihSDF-la group arranged disorder,the structure is not clear,cell swelling,loose,the nuclear condensation is empty.The effect of De-hUC-MSCs,GFP,AdhSDF-la,SihSDF-la and hSDF-la transplantation o n angiogenesis in hippocampus:according to the immunofluorescence re sults,the number of endothelial cells with BrdU/CD31?BrdU/NeuN do uble-labelled was significantly increased in the AdhSDF-1? group.(3)C hange of the spatial learning and memory function of HIBD rats after c ells transplantation:Morris water maze experiment shows:on the first d ay,there were no differences in the path length or the escape latency b etween De-hUC-MSCs,GFP,AdhSDF-1?,SihSDF-la and hSDF-1?(P>0.05).After 2-5 day's hidden platform training,all of the groups sho wed decrease in the escape latency.AdhSDF-la group revealed much more time to find the platform than SihSDF-1? group(*P<0.05).On t he probe trial tests(Day 6),AdhSDF-la transplantation group exhibate d more times of crossing the platform quadrant than other four groups(**P<0.01,AdhSDF-1? vs.De-hUC-MSCs,hSDF-1?,***P<0.001,AdhSDF-la vs.SihSDF-1?;*P<0.05,GFP vs.SihSDF-la,hSDF-1?).Change of novel object exploration capacity of HIBD rats after De-hU C-MSCs,GFP,AdhSDF-1?,SihSDF-1? and hSDF-la transplantation:Obje ct-in-place experiment found that AdhSDF-1? transplantation group spe nd much more time to explore new things than other four group(****P<0.0001)·Conclusion:(1)The stable cell lines of AdhSDF-la,GFP and SihS DF-1? were established successfully.(2)The De-hUC-MSC secreted hS DF-1? can promoted neurological repair in HIBD rats.