| Part Ⅰ Preliminary identification of the basic characteristics of hUC-MSCsObjective:To investigate the basic characteristics of hUC-MSCs, hUC-MSCs were cultured in vitro, the surface marker and differentiation potential were identified.Methods:hUC-MSCs were cultured in vitro, morphology of hUC-MSCs at passages 2,10 and 15 were observed under inverted microscope; To detect the chromosomal karyotype in the different passages of hUC-MSCs, the cytogenetic analysis was used to determine the karyotypic stability of increasing cell passages; The cell-surface antigen of CD29, CD44, CD90, CD 105, HLA-ABC, CD34, CD45 and HLA-DR were detected by flow cytometry; To obtain the differentiation and redifferentiation hUC-MSCs, cells were pre-induced in all-trans-retinoic acid (ATRA) (1 μmol/L) for 24 hours, and the undifferentiated hUC-MSCs were induced using MNM for 24 hours to achieve neuronal differentiation status cells (represented by Dif). The MNM was replaced with complete culture medium for 24 hours following neuronal differentiation. Cells were then transferred to MNM for another 24 hours to achieve neuronal redifferentiation status cells (represented by Re-Dif). Changes in morphology following neuronal MNM-induced differentiation and redifferentiation of hUC-MSCs were observed; The neural markers of NSE, MAP2, β-tubulinⅢ were detected by Real-time PCR and Western Blotting.Results:(1) Culture and expansion of hUC-MSCs:The hUC-MSCs exhibited a spindle-shaped fibroblast-like morphology and were arranged in a spiral. The cells at passages 10 and 15 hold high proliferation capacity and demonstrated no apparent morphological differences compared to the cells at passage 2. (2) Cytogenetic analysis of hUC-MSCs:Chromosomal aberration of the hUC-MSCs was not observed during 15 generations, suggesting that these hUC-MSCs maintained a normal chromosomal structure for at least 15 passages. (3) Flow cytometry analysis of surface marker expression in hUC-MSCs:hUC-MSCs positively expressed CD29, CD44, CD90, CD 105 and HLA-ABC but not CD34, CD45 or HLA-DR. (4) Changes in morphology following neuronal MNM-induced differentiation and redifferentiation of hUC-MSCs:Following neuronal differentiation (Dif group), the cells exhibited morphology similar to hUC-MSCs. However, the cells subjected to neuronal redifferentiation (Re-Dif group) displayed a dramatically neuron-like morphology, with a round, bright cell body and neurite-like extensions. The neural induction rate was nearly 80%-90%. (5)The mRNA and protein expression levels of neural-specific markers in undifferentiated, differentiated and redifferentiated hUC-MSCs:The mRNA expression levels of NSE, MAP2 and β-tubulinⅢ were gradually increased following the induction of differentiation and redifferentiation. In response to the redifferentiation treatment, we found that the levels of the three markers were sharply increased (**P< 0.01,***P< 0.001, one-way ANOVA). The changes in the protein expression levels of these three neural-specific markers were completely consistent with the changes in their mRNA expression.Conclusions:(1) hUC-MSCs in the present study exhibit the morphological characteristics of MSCs, express MSCs-related surface markers and hold strong proliferation ability. (2) hUC-MSCs keep excellent biological stability, chromosomal aberration was not observed during 15 generations. (3) hUC-MSCs in the present study have the potential of neural differentiation and redifferentiation in vitro, the redifferentiation cells express higher neural markers than differentiation cells.Part Ⅱ Neuroprotection of hUC-MSCs, neuronal differentiation and redifferentiation hUC-MSCs transplantation in the HIBD ratsObjective:To identify the neuroprotective potential of hUC-MSCs, neuronal differentiation and redifferentiation hUC-MSCs in vivo.Methods:134 rats were randomly divided into Sham operation group (the Sham group, n=27) and HIBD model group (the HIBD group, n=107); According to the method of Rice, the healthy SD rats in the HIBD group at age of seven days were suffered to the operations that double ligation and dividing the left common carotid artery, and then treated with hypoxia for 2.5 hours.While the pups in the Sham group were only dissected and exposed the left common carotid artery; Five days after HIBD modeling, the rats in HIBD group were divided into PBS group (n=34) and cell transplantation group (n=73). The cell transplantation group consisted of hUC-MSCs group (n=34), neuronal differentiation hUC-MSCs group (Dif group, n=21) and neuronal redifferentiation hUC-MSCs group (Re-Dif group, n=18); The rats were anesthetised and fixed, intracerebroventricular injection was carried out under the guidance of stereotaxic apparatus,2×105 cells suspended in 5 μL of sterile PBS were transplanted into the cell transplantation group rats, the HIBD control group was injected with the same volume of sterile PBS; The pups were sacrificed two days after cell transplantation, and the left side brain tissues were collected and stained with haematoxylin-eosin (HE); One month after cell transplantation, the Morris water maze test and Object-in-place task were performed to detect the learning and memory function and novel object exploration capacity individually; In addition, fEPSPs in the hippocampal CA1 region of brain slices were recorded.Results:(1) Histopathological changes in the brain tissue surrounding the lateral ventricle after hUC-MSCs transplantation:The periventricular brain tissue displayed uneven, diffuse staining and nuclear condensation, and the cell morphology in the HIBD pups was altered compared to the Sham rats. In contrast, hUC-MSCs transplantation partially alleviated these changes. (2)The effect of hUC-MSCs transplantation on the spatial learning and memory function of HIBD rats:In the Morris water maze test, on the visible platform tests (Day 1), no difference in the escape latency or the path length was detected in the Sham group(n=9), HIBD group(n=18) and hUC-MSCs group(n=17) (P>0.05, one-way ANOVA), which indicated that the HIBD model and cell transplantation did not affect the movement or visual ability of the rats. During the hidden platform training (Day 2-5), all of the groups demonstrated gradual decreases in the escape latency. The HIBD group exhibited a longer escape latency than the Sham group (*P< 0.05,**P<0.01,***P<0.001, Sham vs. HIBD, one-way ANOVA), whereas the escape latency was significantly reduced in the HIBD group transplanted with hUC-MSCs (#P<0.05, HIBD vs. hUC-MSCs, one-way ANOVA). On the probe trial tests (Day 6), the hUC-MSCs-treated group entered the platform quadrant significantly more than the HIBD group, although less than the Sham group (***P<0.001, Sham vs. HIBD;*P< 0.05, HIBD vs. hUC-MSCs, Sham vs. hUC-MSCs). (3) The treatment potential of hUC-MSCs transplantation in novel object exploration capacity: The Object-in-place task was performed in the three groups (Sham group (n=9), HIBD group(n=8) and hUC-MSCs group(n=8)), the HIBD rats spent less time investigating the two objects at the new location than the Sham rats (***P<0.001, Sham vs. HIBD); however, the novel object exploration time was significantly increased following hUC-MSCs treatment (*P<0.05, HIBD vs. hUC-MSCs). (4) The influence of hUC-MSCs transplantation on hippocampal slice electrophysiological function of HIBD rats:The basal level of fEPSPs during the 20 min pre-HFS and the 60 min post-HFS were recorded in the Sham (n=6), HIBD (n=5) and hUC-MSCs groups (n=6). At 30 min post-HFS application, the incremental mean slope of the fEPSPs in response to HFS in the HIBD group (110.1%±11.7% of baseline, n=5) was lower than that in both the hUC-MSCs (153.1%±17.5% of baseline, n=6) and Sham groups (167%±21.8% of baseline, n=6) (*P<0.05, HIBD vs. hUC-MSCs;***P<0.001, HIBD vs. Sham). The fEPSP level induced by HFS remained constant until the end of the experimental recording (60 min) in the three groups. (5) Evaluation of the spatial learning and memory function of the differentiated and redifferentiated hUC-MSCs transplanted into HIBD rats:On the Morris water maze test, no significant difference in the escape latency or the path length during the Day 1 test was detected between the undifferentiated (n=17), Dif (n=12) and Re-Dif (n=10) groups (P> 0.05, one-way ANOVA). During the hidden platform acquisition training phase (from Day 2-5), the escape latency of all three groups gradually decreased, demonstrating that the rats in each group showed spatial memory for the underwater platform, and no difference between the hUC-MSCs, Dif and Re-Dif groups was detected (P> 0.05, one-way ANOVA). During the probe trial (Day 6), although the number of platform quadrant entries displayed a slight increase in both the Dif and Re-Dif groups, no significant difference between the three groups was detected (P > 0.05, one-way ANOVA). (6) Discrimination between the novel and familiar objects in the undifferentiated (n=8), Dif (n=9) and Re-Dif groups (n=8):On the Object-in-place task, the three groups exhibited similar memory for the novel objects, and no significant differences were observed (P>0.05, one-way ANOVA).Conclusions:(1) hUC-MSCs transplantation alleviated histopathological changes in the brain tissue. (2) hUC-MSCs treatment improved spatial learning and memory function and novel objects configuration ability in HIBD rats. (3) hUC-MSCs transplantation enhanced hippocampal synaptic plasticity in HIBD rats. (4) Similar to undifferentiated hUC-MSCs, both differentiated and redifferentiated hUC-MSCs promote neural function recovery in HIBD rats; however, the Dif and Re-Dif cells did not confer greater therapeutic advantages than the undifferentiated hUC-MSCs, even though they expressed higher levels of neuronal markers in vitro.Part III hUC-MSCs improve learning and memory function in HIBD rats via IL-8Chapter I The differences in the gene expression profiles among the undifferentiated, neuronal differentiation and redifferentiation hUC-MSCs in vitroObjective:To clarify the molecular mechanism underlying the process of neuronal differentiation and redifferentiation, and then to reveal the possible mechanism involving in the therapeutic effect of hUC-MSCs.Methods:Affymetrix(?) Human Genome U133 Plus 2.0 array was used to detect the whole gene expression in undifferentiated, differentiation and redifferentiation hUC-MSCs; Genome cluster analysis was employed to analyse the differential genes expression pattern; GO biological analysis software were applied to explore the biological function of differential genes; KEGG-pathway analysis was applied to determine the possible pathways that participate in the neural differentiation and redifferentiation process; The expression levels of various cytokines were analysed according to the gene expression profiling chip assay data.Results:(1) The whole gene expression in undifferentiated, differentiation and redifferentiation hUC-MSCs:According to the genome cluster analysis and the scatter plot, the Dif and Re-Dif groups displayed similar gene expression patterns, which were very different from that of the undifferentiated hUC-MSCs. (2) Cluster analysis of consistently altered genes:We screened the consistently altered genes in the three groups (consistently increased or decreased) and selected 483 genes that displayed significantly different expression according to the cluster analysis. Following neural differentiation and redifferentiation, the Dif group exhibited little change, and the Re-Dif group showed the opposite gene expression pattern to the undifferentiated hUC-MSCs group. (3) Gene ontology analysis of the 483 differentially expressed genes:The predominant biological functions were signal transduction, development, negative regulation of cell proliferation, cell cycle, and nervous system development. (4) KEGG pathway analysis of the 483 differentially expressed genes:Many signalling pathways participated in the neural differentiation and redifferentiation process, such as cytokine-cytokine receptor interaction, p53 signalling pathway, MAPK signalling pathway and TGF-β signalling pathway. Of the many possible signalling pathways, the cytokine-cytokine receptor interaction pathway displayed the most significant difference. (5) The cytokine genes expressed in hUC-MSCs based on the Affymetrix U133 GeneChip data:hUC-MSCs expressed high levels of many cytokine genes, such as TIMP-1, IL-8, CXCL1, TIMP-2, IL-1β, MCP-1,VEGF.Conclusions:(1) The gene expression pattern in undifferentiated hUC-MSCs was very different from that of the Dif and Re-Dif groups, while the Dif and Re-Dif hUC-MSCs exhibit the similar gene expression patterns. (2) The induction of differentiation and redifferentiation in hUC-MSCs is a gradual process that results in more mature and terminal gene expression in the Dif and Re-Dif hUC-MSCs. (3) The cytokine-cytokine receptor interaction pathway is primarily involved in the process of neuronal differentiation and redifferentiation. (4) TIMP-1, IL-8, CXCL1, TIMP-2, IL-1β, MCP-1, VEGF and other cytokines may play a role in the treatment advantage of hUC-MSCs.Chapter Ⅱ IL-8 secreted by hUC-MSCs regulates angiogenesis in the hippocampus of HIBD rats via the JNK signalling pathwayObjective:To explore the mechanism of IL-8 secreted by hUC-MSCs involved in the nerve repair function of HIBD rats.Methods:Three different groups were set:the normal control group (the Sham group, n=6), the PBS intracranial injection group (the HIBD group, n=6) and the hUC-MSCs transplantation group (the hUC-MSCs group, n=6); Two days after intracranial injection, brain tissue in the three groups were collected for the following test; To validate the result of microarray, ELISA was used to detect the expression of IL-8, TIMP-1, MCP-1, CXCL1 in the culture supernatant of hUC-MSCs; ELISA were employed to investigate the hIL-8 and VEGF secretion, and Western Blotting were used to examine the IL-8 and CD31 protein expression levels in brain tissue from the Sham, HIBD and hUC-MSCs groups individually; The number of endothelial cells in hippocampal blood vessels displaying CD31 and BrdU double-labelling in the three groups was determined via immunofluorescence; And then, Western Blotting was performed to determine the protein expression levels of p-JNK and JNK in rat brain tissue from the Sham, HIBD and hUC-MSCs groups; Through the siIL-8 and GFP lentivirus transfection, the siIL-8-hUC-MSCs and GFP-hUC-MSCs stable cell line were established; Expression levels of IL-8 mRNA and protein in the siIL-8-hUC-MSCs and GFP-hUC-MSCs stable cell lines were determined by Real-time PCR and Western Blotting; The siIL-8-hUC-MSCs(n=14) and GFP-hUC-MSCs(n=14) stable cell line were transplanted into HIBD rats, the protein expression level of IL-8 and CD31, and the hIL-8 and VEGF secretion were measured by Western Blotting and ELISA; Through CD31 and BrdU double-labelling immunofluorescence, the effect of siIL-8-hUC-MSCs transplantation on angiogenesis was clarified; p-JNK and JNK protein expression in rat brain tissue from the GFP-hUC-MSCs and siIL-8-hUC-MSCs groups were measured by Western Blotting; Futhermore, the learning and spatial memory in siIL-8-hUC-MSCs (n=8) and GFP-hUC-MSCs (n=8) transplantation groups were investigated with Morris water maze test.Results:(1) The levels of different cytokines in the culture supernatants of hUC-MSCs:The hUC-MSCs expressed high levels of IL-8, TIMP-1, MCP-1 and CXCL1, while IL-8 was the cytokine that showed the highest level of secretion by hUC-MSCs(***P<0.001, IL-8 vs. TIMP-1, one-way ANOVA). (2) The expression level of IL-8 in rat brain tissue in the Sham, HIBD and hUC-MSCs groups:Both the levels of hIL-8 secretion and IL-8 protein expression were significantly increased in the hUC-MSCs-transplanted brain tissue (*P<0.05, HIBD vs. hUC-MSCs, Sham vs. hUC-MSCs, one-way ANOVA). (3) The CD31 and VEGF expression level in the three groups:The levels of CD31 protein expression and VEGF secretion in the hUC-MSCs group were significantly higher than those in the HIBD group (**P<0.01, HIBD vs. hUC-MSCs;**P<0.01, Sham vs. HIBD). (4) The effect of hUC-MSCs transplantation on angiogenesis:The immunofluorescence results revealed that the number of endothelial cells displaying CD31 and BrdU double-labelling was significantly increased in the hUC-MSCs group compared to the HIBD group (*P<0.05, HIBD vs. hUC-MSCs, Sham vs. HIBD, one-way ANOVA). (5) The possible mechanism of promoting angiogenesis after hUC-MSCs treatment:The level of p-JNK protein expression was clearly increased in the HIBD brain tissue following hUC-MSCs transplantation compared to the Sham and HIBD brain tissue, even though no significant difference in the level of total JNK protein expression was detected between the three groups. (6) Establishment of siIL-8-hUC-MSCs stable cell line: By inverted fluorescence microscope, cells infected with GFP and siIL-8 lentivirus expressed green fluorescent persistently. Compared with uninfected hUC-MSCs, the infection rate was more than 85%. What’s more, the cell viability and proliferation ability were not affected by virus infection. (7) Identification of siIL-8-hUC-MSCs stable cell line:Gene expression of IL-8 in siIL-8-hUC-MSCs group was significant lower than GFP-hUC-MSCs group, the difference was statistically significant (***p< 0.001, Student’s t test). IL-8 protein expression level was similar with the gene expression level. (8) IL-8 protein expression in the brain tissue after siIL-8-hUC-MSCs transplantation:The levels of hIL-8 secretion and IL-8 protein expression in rat brain tissue were statistically significantly lower in the siIL-8-hUC-MSCs group than in the GFP-hUC-MSCs group(***P< 0.001, Student’s t test). (9) CD31 protein expression and VEGF secretion level in the brain tissue after siIL-8-hUC-MSCs transplantation:During subsequent follow-up, the levels of VEGF secretion and CD31 protein expression were significantly reduced in the siIL-8-hUC-MSCs group compared to the GFP-hUC-MSCs group (*P<0.05, Student’s t test). (10) The effect of siIL-8-hUC-MSCs transplantation on angiogenesis in hippocampus:Immunofluorescence results revealed that the number of endothelial cells displaying CD31 and BrdU double-labelling was significantly decreased in the siIL-8-hUC-MSCs group (*P<0.05, Student’s f test). (11) The mechanism of IL-8 secreted by hUC-MSCs involved in the angiogenesis:The levels of p-JNK protein expression clearly decreased following siIL-8-hUC-MSCs transplantation, although total JNK protein expression was not significantly different. (12) IL-8 secreted by hUC-MSCs participated in the repairment of learning and memory function in HIBD rats: On the Morris water maze test, the siIL-8-hUC-MSCs group exhibited a longer escape latency, and spent less time in the platform quadrant than the GFP-hUC-MSC group (*P<0.05, Student’s t test).Conclusions:(1) IL-8 secreted by hUC-MSCs may regulates angiogenesis in the hippocampus of HIBD rats via the JNK signalling pathway. (2) The stable cell lines of siIL-8-hUC-MSCs and GFP-hUC-MSCs were established successfully. (3) The decrease in hUC-MSC-secreted IL-8 can attenuate angiogenesis and treatment benefits. |
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