【摘要】 目的 探讨骨形成蛋白-2（bone morphogenetic protein-2，BMP-2）对室管膜前下区（anterior subventricular zone，SVZa）神经干细胞DLX5表达的影响。 方法 体外培养SVZa神经干细胞，用BMP-2及其拮抗剂Noggin诱导SVZa神经干细胞，分别用免疫荧光染色和逆转录-聚合酶链反应（RT-PCR）检测DLX5表达变化。 结果 BMP-2组SVZa神经干细胞DLX5蛋白表达和DLX5mRNA表达水平明显高于对照组（Plt;0.05），且该效应能被其拮抗剂Noggin特异性地抑制。 结论 BMP-2是DLX5上游调节基因，可促进SVZa神经干细胞DLX5的表达。【Abstract】 Objective To investigate the effect of bone morphogenetic protein-2 (BMP-2)on expression of DLX5 of neural stem cells in anterior subventricular zone (SVZa). Methods The neural stem cells of SVZa were separated and cultured in vitro, which were induced by BMP-2 and Noggin.Immunofluorescence staining and RT-PCR were employed to assay the expression of DLX5. Results The percentages of expression of DLX5 protein and DLX5 mRNA in BMP-2 group were much higher than those in the control group (Plt;0.05). And this induction could be specifically blocked by Noggin. Conclusion BMP-2 is an upstream gene of DLX5; BMP-2 can promote the expression of DLX5 of the neural stem cells of SVZa.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To study the effects of the human keratinocyte growth factor 2 (hKGF-2) on the survival and differentiation of human neural stem cells (hNSCs). Methods The hNSCs at 17 passages preserved in liquid nitrogen were resuscitated and cultured for 7 days with normal methods to form neural spheres. The specific Nestin antigen and differentiated cells antigen were identified using immunohistochemistry technology. Some concentrated hNSCs were incubated in 12-well culture plate with 1 mL basic medium [(DMEM/F12 + N2 (1 ∶ 100) + epidermal growth factor (EGF) (20 ng/mL)] and divided into 7 groups, 6 wells each group. hKGF-2 (0, 10, 30, 60, 90, and 120 ng/mL) and bFGF (10 ng/mL) were added in groups A (control), B, C, D, E, F, and G, respectively. The neurospheres and the cell number were recorded for analyzing growth and multiplication of neural spheres. Some concentrated hNSCs were incubated in 6-well culture plate (cover glass coated with polylysine) with 3 mL DMEM/F12 medium and divided into 4 groups, 6 wells each group. N2 (1 ∶ 100), N2 (1 ∶ 100) + hKGF-2 (90 ng/mL), FBS (1 ∶ 20), and FBS (1 ∶ 20) + hKGF-2 (90 ng/mL) were added in groups A1, B1, C1, and D1, respectively. Then, the growth and multiplication of neural spheres were observed during culture; the separated neural spheres was identified and analyzed with indirect immunofluorescence and flow cytometry. Results Reanimated hNSCs could form neural spheres containing a lot of Nestin antigen; differentiated cells by induction expressed the specific antigens of neurofilament 200 (NF- 200) and glial fibrillary acidic protein (GFAP). At 7 days after culture, enlarged neural spheres were observed in each group. The neurospheres and the cell number of hNSCs increased with increased concentration of hKGF-2, showing a gradually increasing tendency; they were significantly higher in groups E, F, and G than that in groups A, B, C, and D (P lt; 0.05); significant differences were found among groups B, C, and D (P lt; 0.05), but no significant difference between groups A and B, and among groups E, F, and G (P gt; 0.05). After induction in vitro, the cell growth showed a progressive increase, significant difference was found among groups (P lt; 0.05); the percentage of NF-200 positive cells in group B1 was significantly higher than that in the other 3 groups (P lt; 0.05); the percentage of GFAP positive cells in group B1 was significantly lower than that in the other 3 groups (P lt; 0.05), but no significant difference among groups A1, C1, and D1 (P gt; 0.05). At 14 days after culture, cell growth reached the peak, which were mainly astero-cells. Conclusion The hNSCs are pure after incubated to 17 passages in vitro. hKGF-2 can promote the clone and the growth of differentiated cells, and increase the proportion of neuron.
【Abstract】 Objective To review the progress in the treatment of spinal cord injury (SCI) by graft of neuralstem cells (NSCs) or bone marrow mesenchymal stem cells (BMSCs) as well as immune characteristics of two stemcells. Methods Different kinds of documents were widely collected, and then immunologic characteristics of NSCs andBMSCs were summarized. The therapy of SCI by stem cell transplantation was reviewed. Additionally, some problems intreatment were analyzed. Results Experimental study showed that graft of NSCs and BMSCs can promote the functionalrecovery of the injured spinal cord in animals. Due to immunologic properties of two stem cells, rejection reaction oftransplantation could produce a harmful effect on SCI treatment. Conclusion Transplantation of NSCs or BMSCs might bean effective measure for SCI treatment, but immunologic rejection reaction must be considered.
Objective To compare single cell suspension of neural stem cells (NSCs) with neurospheres transplantation for spinal cord injury (SCI) so as to explore the therapeutic effectiveness of two NSCs transplantation methods for SCI. Methods The NSCs were isolated from the spinal cord of adult Sprague Dawley (SD) rats, purified and cultured. At passage 3, the cells were identified by Hoechst33342, Nestin staining, and gl ial fibrillary acidic protein staining for differentiated cells. Sixty adult SD rats (weighing 230-250 g) were made the SCI models at T10 level with modified Allen method and randomlydivided into 3 groups (20 rats in each). The injury sites were treated by injecting 5 μL sal ine (group A), 5 μL single cellssuspensions of NSCs at passage 3 (group B), and 5 μL neurospheres cell suspensions at passage 3 (group C). At preoperation and 3, 7, 14, 21, and 28 days after operation, the locomotor functions of each group were assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale. HE staining was applied to observe the morphology of spinal cord. Subsequently immunofluorescence staining was used to observe microtubule-associated protein 2 (MAP-2). Results The cells cultured were NSCs by morphological observation and immunofluorescence staining. After 3 days of modeling surgery, BBB score significantly decreased when compared with preoperative score, and there was no significant difference among 3 groups at 3 and 7 days (P gt; 0.05). BBB score increased in different degrees with time; at 14, 21, and 28 days, BBB score of groups B and C was better than that of group A, and group C was better than group B, showing significant differences (P lt; 0.05). HE staining showed that spinal cord structure of group C was more clear than that of groups A and B, and had less scar. There was no significant difference in the number of MAP-2 positive cells among 3 groups at 3 and 7 days (P gt; 0.05). At 14, 21, and 28 days, the number of MAP-2 positive cells of groups B and C was significantly more than that of group A, and group C was more than group B, showing significant differences (P lt; 0.05). Conclusion Transplantation of neurospheres suspension compared with single cell can significantly promote NSCsto differentiate into neurons and is conducive to recover the lower extremity function after SCI.
Objective To explore the effects of Neurogenesin 1 (Ng1) gene on functional recovery after spinal cord injury (SCI) and its mechanism. Methods Thirty-six rats (aging 4 months, weighing 230 g and being male or female), were randomly divided into two groups: experimental group (n=18) and control group (n=18). After spinal cord contusive injury at T10 level was made in all these rats using modified Allen’s method, Ng1 recombinant plasmid and blank plasmid were transfectedinto the damaged areas of exprimental group and control group respectively by Alzet pumps. At 1 day, 1 week, 2 weeks, 3 weeks, and 4 weeks after SCI, Basso-Beattle-Bresnahan (BBB) Rating Scale was used to observe the recovery of motor function. At 1 week after injury, the expressions of Ng1 mRNA and protein in injured spinal cord were detected by RT-PCR and Western blot techniques. And at 2 and 4 weeks, double immunofluorescence and histopathologic examinations were performed to study the prol iferation of the adult endogenous neural stem cells and pathological change after SCI. Results At 1-4 weeks after SCI, the BBB scores in the exprimental group was significantly higher than that in control group (P lt; 0.05), and at 4 weeks the BBB score of the experimental group (16.80 ± 1.79) was significantly higher than that of the control group (9.60 ± 1.67), (P lt; 0.01). RTPCR and Western blot showed that the mRNA and protein expressions of Ng1 were observed in the exprimental group and no expression was seen in the control group. Histologic observation showed that the morphology of spinal cord and neurons in the exprimental group was better than that in the control group and was close to the normal tissue. The mean number of Nestin+/ BrdU+ newborn endogenous neural stem cells in the exprimental group was significantly more than that in control group (P lt; 0.05). Conclusion Ng1 gene could promote the prol iferation of endogenous neural stem cells and protect the injured neurons, which enhances the repair of the motor function after SCI.
Objective To investigate the division, prol iferation and differentiation abil ities of nestin+/GFAP+cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs). Methods Twelvemale SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group inwhich the spinal cord injury model was establ ished by aneurysm cl ip compression method, and control group in which no processing was conducted. At 5 days after model ing, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cellsuspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was appl ied to induce differentiation. Immunohistochemistry detection and flow cytometry were appl ied to observe and analyze the type of cells and their capabil ity of division, prol iferation and differentiation. Results At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 ± 0.71 and 1.12 ± 0.38, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Concerning cell cycle, the proportion of S-phase cell and prol iferation index of the model group (15.49% ± 3.04%, 15.88% ± 2.56%) were obviously higher than those of the control group (5.84% ± 0.28%, 6.47% ± 0.61%), indicating there were significant differences between two groups (P lt; 0.01). In the model group, primary cells gradually formed threedimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multi ple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of β-tubul in III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ ol igodendrocyte, β-tubul in II+ neuron and GalC+ cell body and protruding were observed. Conclusion Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the abil ity of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
Objective To review the fundamental research and the experimental study in the nerve tissue engineering of self-assembl ing peptide nanofiber scaffold (SAPNS). Methods The l iterature concerning basic and experimental studies on SAPNS in the nerve tissue engineering was extensively reviewed. Results SAPNS can promote the neural stem cell adhesion,prol iferation, differentiation and neuron axon outward growth and extension, promote extracellular matrix synthesis and inhibit gl ial cell adhesion and differentiation, and simulate the environment of a cell in the body. Conclusion SAPNS is an ideal matrix material and provides a new way for the repair of nerve tissue injury.
To explore the expression of Wnt-1 during the process of inducing neural stem cells (NSCs) into neurons by using all-trans-retinoic acid (ATRA) in vitro and the effect of Wnt-1 on NSCs differentiation. Methods NSCs isolated from cerebral cortex of SD rat embryo (12-16 days’ gestation) were cultured. The concentration of cells at passage 3 were adjusted to 1 × 106 cells /mL and treated with ATRA at 0.5, 1.0, 5.0 and 10.0 μmol/L, respectively. Differentiation ratio of NSCsinto neurons in each group was detected by double-labelling immunofluorescence technique and flow cytometry, and 1.0 μmol/ L was selected as the best concentration for ATRA to promote NSCs differentiation. In experimental group, NSCs at passage 3 were cultured with ATRA at 1.0 μmol/L in vitro, and expression of Wnt-1 was detected by immunocytochemistry staining, realtime flurescent quantitive PCR and Western blot at 3, 5, 7 and 9 days after culture, respectively. The cells at passage 3 receiving no ATRA served as control group. Results Immunocytochemistry staining: in the control group, there was l ittle Wnt-1 protein expression; in the experimental group, peak expression of Wnt-1 and numerous positive cells occurred at 3 days after culture, the positive expression of Wnt-1 was still evident at 5 days after culture, and there was significant difference between two groups in integrated absorbance (IA) value at 3 and 5 days after culture(P lt; 0.05), obvious decrease of positive expression of Wnt-1 was evident, and no significant difference was evident between two groups in IA value at 7 and 9 days (P gt; 0.05). Real-time fluorescence quantitative PCR: the relative expression of Wnt-1 mRNA in the control group was 0.021 7 ± 0.072 1; the relative expression of Wnt-1 mRNA in the experimental group at 3, 5, 7 and 9 days was 0.512 2 ± 0.280 0, 0.216 4 ± 0.887 0, 0.038 5 ± 0.299 4 and 0.035 5 ± 0.309 5, respectively, indicating the value decreased over time, and there were significant difference between two groups at 3 and 5 days (P lt; 0.05), and no significant difference at 7 and 9 days (P gt; 0.05) . Western blot detection: specific and visible staining band was noted; in the control group, Wnt-1 protein expression was 0.005 1 ± 0.558 3; in the experimental group, Wnt-1 protein expression at 3, 5, 7 and 9 days was 0.451 7 ± 0.071 3, 0.311 7 ± 0.080 5, 0.007 3 ± 0.052 7 and 0.004 7 ± 0.931 4, respectively, suggesting the value decreased over time; there were significant differences between two groups at 3 and 5 days (P lt; 0.05), and no significant differences at 7 and 9 days (P gt; 0.05). Conclusion With the induction of ATRA at 1.0 μmol/L, Wnt-1 and NSCs differentiation in early stage are positively correlated. Its possible mechanism may rely on the activation of such signals as classic Wnt-1 signal pathway, indicating Wnt-1 relates to the differentation of NSCs into neurons.
To summarize Notch, basic hel ix-loop-hel ix (bHLH) and Wnt gene signal transduction pathways in the process of differentiation and development of neural stem cells. Methods The l iterature on the gene signal transduction pathway in the process of differentiation and development of neural stem cells was searched and then summarized and analyzed. Results The formation of Nervous System resulted from common actions of multi-signal transduction pathways. There may exist a fixed threshold in the compl icated selective system among Notch, bHLH and Wnt gene signal transduction pathways. Conclusion At present, the specific gene signal transduction pathway of multi pl ication and differentiation of neural stem cells is still unclear.