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ABSTRACT: BACKGROUND: Various tissue engineering strategies have been developed to facilitate axonal regeneration after spinal cord injury. This study aimed to investigate whether neural stem cells (NSCs) could survive in poly(L-lactic-co-glycolic acid) (PLGA) scaffolds and, when cografted with Schwann cells (SCs), could be induced to differentiate towards neurons which form synaptic connection and eventually facilitate axonal regeneration and myelination and motor function. METHODS: NSCs and SCs which were seeded within the directional PLGA scaffolds were implanted in hemisected adult rat spinal cord. Control rats were similarly injured and implanted of scaffolds with or without NSCs. Survival, migration, differentiation, synaptic formation of NSCs, axonal regeneration and myelination and motor function were analyzed. Student's t test was used to determine differences in surviving percentage of NSCs. One-way analysis of variance (ANOVA) was used to determine the differences in the number of axons myelinated in the scaffolds, the mean latency and amplitude of cortical motor evoked potentials (CMEPs) and Basso, Beattie & Bresnahan locomotor rating scale (BBB) score. The χ(2) test was used to determine the differences in recovery percentage of CMEPs. RESULTS: NSCs survived, but the majority migrated into adjacent host cord and died mostly. Survival rate of NSCs with SCs was higher than that of NSCs without SCs ((1.7831 ± 0.0402)% vs. (1.4911 ± 0.0313)%, P < 0.001). Cografted with SCs, NSCs were induced to differentiate towards neurons and might form synaptic connection. The mean number of myelinated axons in PLGA + NSCs + SCs group was more than that in PLGA + NSCs group and in PLGA group ((110.25 ± 30.46) vs. (18.25 ± 3.30) and (11.25 ± 5.54), P < 0.01). The percentage of CMEPs recovery in PLGA + NSCs + SCs group was higher than in the other groups (84.8% vs. 50.0% and 37.5%, P < 0.05). The amplitude of CMEPs in PLGA + NSCs + SCs group was higher than in the other groups ((1452.63 ± 331.70) µV vs. (428.84 ± 193.01) µV and (117.33 ± 14.40) µV, P < 0.05). Ipsilateral retransection resulted in disappearance again and functional loss of CMEPs for a few days. But contralateral retransection completely damaged the bilateral motor function. CONCLUSIONS: NSCs can survive in PLGA scaffolds, and SCs promote NSCs to survive and differentiate towards neurons in vivo which even might form synaptic connection. The scaffolds seeded with cells facilitate axonal regeneration and myelination and motor function recovery. But regenerating axons have limited contribution to motor function recovery.
Chinese medical journal 03/2013; 126(5):909-917. · 0.86 Impact Factor
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ABSTRACT: Direct or ex vivo BMP9 adenoviral gene therapy can induce massive bone formation at the injection sites and clearly promote spinal fusion. A comprehensive analysis of the osteogenic activity indicated that BMP9 was one of the most potent inducers of osteogenic differentiation both in vitro and in vivo among 14 types of human BMPs. However, genetic variations and whether they correlated with OPLL were not considered. We have sequenced the complete BMP9 gene in 450 patients with OPLL and in 550 matched controls. Analyses were performed on single markers and haplotypes. Single marker tests identified 6 SNPs, among which the minor alleles of rs7923671 (T>C; P=0.0026; OR: 1.33, CI: 1.10-1.60), rs75024165 (C>T, Thr304Met; P<0.001; OR: 1.76, CI: 1.47-2.12) and rs34379100 (A>C; P<0.001; OR: 1.52, CI: 1.27-1.82) were associated with OPLL. Logistic regression analysis showed that the additive model of rs75024165 (TT vs. CT vs. CC; P<0.001; OR: 1.74) and rs34379100 (CC vs. AC vs. AA; P=0.003; OR: 1.95) retained statistical significance when adjusted for clinical and demographic characteristics. Linkage disequilibrium (LD) analysis identified one 3 kb block of intense LD in BMP9 and one specific haplotype, CTCA (P<0.001; OR: 2.37), that contained the OPLL-associated risk alleles and was a risk factor for OPLL. This haplotype is associated with increased severity of OPLL, as shown by the distribution of ossified vertebrae in patients with OPLL (P=0.001). In summary, in the Chinese population studied, SNPs in the BMP9 gene appear to contribute to the risk of OPLL in association with certain clinical and demographic characteristics. The severity of OPLL seems to be mediated predominantly by genetic variations in a 3kb BMP9 locus with the specific haplotype CTCA.
PLoS ONE 01/2012; 7(7):e40587. · 4.09 Impact Factor
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ABSTRACT: Previous genome-wide microarray analysis of candidate genes involved in the ossification of the posterior longitudinal ligament (OPLL) of the spine resulted in the identification of a novel, clinically relevant gene encoding bone morphogenetic protein 4 (BMP4) but was defined only by its expression patterns. The complete genomic BMP4 coding DNA from 450 patients with OPLL and 550 matched controls were sequenced and compared. We identified 18 SNPs, among which the minor alleles of SNP8 (C>T; p < 0.001; OR: 1.58), SNP13 (rs17563C>T; p < 0.001; OR: 1.76), and SNP14 (rs76335800A>T; p < 0.001; OR: 1.68) were associated with OPLL. Logistic regression analysis showed that the additive model of SNP8 (p < 0.001; OR: 3.48), SNP13 (p < 0.001; OR: 2.22), and SNP14 (p < 0.001; OR: 1.99) retained statistical significance. Linkage disequilibrium (LD) analysis identified a 3-kbp block of intense LD in BMP4 and 1 specific haplotype, TGGGCTT (p < 0.001, OR: 2.54), which was associated with OPLL-associated risk alleles and increased severity of OPLL, as shown by the distribution of ossified vertebrae in patients with OPLL (p = 0.002). Novel mutations in the BMP4 gene and a specific haplotype TGGGCTT appear to contribute to the risk of developing OPLL. Also the severity of OPLL seems to be mediated predominantly by genetic variations in this specific BMP4 gene region, but might be associated with other certain clinical and demographic characteristics in the Chinese population studied.
Journal of Orthopaedic Research 11/2011; 30(5):748-56. · 2.81 Impact Factor
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ABSTRACT: The most important objective of transplant studies in the injured spinal cord has been to provide a favorable environment for axonal growth. Moreover, the continuing discovery of new grafts is providing new potentially interesting transplant candidates. Our purpose was to observe the morphological and functional repair effects of the co-transplantation of neural stem cell (NSC), Schwann cells (SCs) and poly lactide-co-glycolide acid (PLGA) on the spinal cord injury of rats.
A scaffold of PLGA was fabricated. NSCs and SCs were cultured, with the NSCs labeled with 5-bromodeoxyuridine, and the complex of NSC/PLGA or NSC + SCs/PLGA were constructed. Thirty-six Wistar rats were randomly divided into three groups: group A (transplantation of PLGA), group B (transplantation of NSC/PLGA) and group C (transplantation of NSC + SCs/PLGA). The 3 mm length of the right hemicord was removed under the microscope in all rats. The PLGA or the complex of PLGA-cells were implanted into the injury site. Basso-Beattie-Bresnahan (BBB) locomotion scores, motor and somatosensory evoked potential of lower limbs were examined to learn the rehabilitation of sensory and motor function at 4 weeks, 8 weeks, 12 weeks and 24 weeks after injury. All the recovered spinal cord injury (SCI) tissues were observed with HE staining, immunohistochemistry, and transelectronmicroscopy to identify the survival, migration and differentiation of the transplanted cells and the regeneration of neural fibres at 4 weeks, 8 weeks, 12 weeks and 24 weeks after injury.
(1) From 4 weeks to 24 weeks after injury, the BBB locomotion scores of cell-transplanted groups were better than those of the non-cell-transplanted group, especially group C (P < 0.05). The amplitudes of the somatosensory evoked potential (SEP) and motor-evoked potential (MEP) were improved after injury in groups B and C, but the amplitude of SEP and MEP at 4 weeks was lower than that at 12 weeks and 24 weeks after injury. Compared with group B, the amplitude of SEP and MEP in group C was improved. The amplitude of SEP and MEP was not improved after injury in group A. (2) HE staining revealed the volume of the scaffold decreased and the number of cells in the scaffold increased. Newly-grown capillaries also could be seen. Immunohistochemistry staining showed the transplanted NSCs could survive and migrate until 24 weeks and they could differentiate into neurons and oligodendrocytes. The regenerated axons were observed in the scaffold-cell complex with transelectronmicroscopy. The above manifestations were more extensive in group C.
The transplanted NSC can survive and migrate in the spinal cord of rats up to 24 weeks after injury, and they can differentiate into various neural cells. Co-transplantation of cells/PLGA can promote the functional recovery of the injured spinal cord. The effect of co-transplanting NSC + SCs/PLGA is better than transplanting NSC/PLGA alone.
Chinese medical journal 09/2010; 123(17):2424-31. · 0.86 Impact Factor
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ABSTRACT: It is generally known that low-intensity pulsed ultrasound (LIPUS) accelerates peripheral nerve tissue regeneration. However, the precise cellular mechanism involved is still unclear. The purpose of this study was to determine how the Schwann cells respond directly to LIPUS stimuli. Thus, we investigated the effect of LIPUS on cell proliferation, neurotrophin-3 (NT-3), and brain-derived neurotrophic factor (BDNF) mRNA expression in rat Schwann cells. Schwann cells were enzymatically isolated from postnatal 1-3 day rat sciatic nerve tissue and cultured in the six-well plate. The ultrasound was applied at a frequency of 1 MHz and an intensity of 100 mW/cm(2) spatial average temporal average for 5 minutes/day. The control group was cultured in the same way but without the administration of ultrasound. Immunohistochemistry demonstrated that more than 98% of the experimental and control cells were positive for S-100, NT-3, and BDNF. With 5-bromo-2'-deoxyuridine (BrdU) assay, the stimulated cells also exhibited an increase in the rate of cell proliferation on days 4, 7, 10, and 14. Further investigation found that mRNA expression of NT-3 was significantly upregulated in experimental groups compared with the control 14 days after the LIPUS stimulation (the ratio of NT-3/beta-actin was 0.56 +/- 0.13 vs. 0.41 +/- 0.09, P < 0.01), whereas the mRNA expression of BDNF was significantly downregulated in experimental groups compared with the control (the ratio of BDNF/beta-actin was 0.51 +/- 0.05 vs. 0.60 +/- 0.08, P < 0.05). These results demonstrated that the application of LIPUS promotes cell proliferation and NT-3 gene expression in Schwann cells, and involved in the alteration of BDNF gene expression.
Microsurgery 04/2009; 29(6):479-85. · 1.61 Impact Factor
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ABSTRACT: To investigate the recovery of rat transected spinal cord injury after implantation of Schwann cells combined with poly (lactide-co-glycolide) (PLGA).
Schwann cells were expanded, co-cultured with PLGA for 9 days in vitro, and then analyzed with scanning electron microscope (SEM). Rat spinal cord at the level of T(9) was transected. Schwann cells labeled with BrdU and PLGA scaffold were implanted to injury site. After 1, 3, 6 months, BrdU/MBP immunohistochemistry double staining, semi-thin sections stained thionin and ultra-thin section were performed to investigate myelin renew. BBB open field locomotion, motor evoked potential (MEP), compound muscle action potential (CMAP) and somatosensory evoked potential (SEP) were recorded.
Schwann cells grew well on PLGA under SEM. BrdU/MBP double positive cells would been seen, remyelination was thin and formed by Schwann cells at 6 months later under electron microscope (EM). BBB behavioral tests revealed no significant difference in recovery comparing with experiment group and control group. The results of MEP, CMAP and SEP showed no significant improvement in the conduction of spinal cord.
There are the compatibility between Schwann cells and PLGA. Although remyelination was found in morphology, function conduction of spinal cord failed to be established.
Zhonghua wai ke za zhi [Chinese journal of surgery] 07/2007; 45(12):843-6.
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ABSTRACT: To investigate whether there is neogenesis of myelin sheath and neuron after transplantation of Schwann cells into cerebral hemorrhage lesion.
Schwann cells were expanded, labeled with BrdU in vitro and transplanted into rat cerebral hemorrhage with blood extracted from femoral artery and then injected into the basal nuclei. Double immunohistochemistry staining and electron microscopy were used to detect the expression of BrdU/MBP and BrdU/GAP-43 and remyelination.
BrdU/MBP double positive cells could be seen at 1 week up to 16 weeks after transplantation of Schwann cells. Thin remyelination was observed under electron microscope. GAP-43 positive cells appeared after 12 weeks and were found more in Hippocamp.
Grafted Schwann cells participate in remyelination and promoter nerve restore in rat cerebral hemorrhage.
Biomedical and Environmental Sciences 03/2007; 20(1):47-51. · 1.35 Impact Factor
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ABSTRACT: To analyze the upstream region of radiation-induced junB gene.
Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/beta-actin measured by quantitative Northern blot hybridization.
CAT mRNA expression containing 900 bp and 1560 bp junB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation.
590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.
Biomedical and Environmental Sciences 07/2006; 19(3):210-3. · 1.35 Impact Factor
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ABSTRACT: To study the protective effect of volatile anesthetics, isoflurane and sevoflurane, on ischemic neurons after cerebral ischemia-reperfusion in rats and its possible molecular mechanism.
Rat cerebral ischemia-reperfusion model was developed by occlusion of the middle cerebral artery (MCA) and bilateral common carotid arteries (CCAs) 1 h after reperfusion. Using flow cytometry (FCM) and Northern blot hybridization, we calculated the number of apoptotic bodies and detected the expression of bcl-2 mRNA and interleukin-1beta converting enzyme (ICE) mRNA.
The apoptotic bodies in hippocampus analyzed by FCM peaked at appeared 24 h after reperfusion, and decreased about 54% and 40%, respectively, after treatment with isoflurane and sevoflurane, as compared with ischemic group. There was no significant difference in the expression of bcl-2 mRNA and ICE mRNA between the inhaled anesthetic groups and ischemic group in hippocampus 24 h after MCA/CCAs occlusion.
Isoflurane and sevoflurane partially inhibit apoptosis but have no significant effect on the expression of bcl-2 and ICE genes.
Biomedical and Environmental Sciences 05/2006; 19(2):143-6. · 1.35 Impact Factor
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Analytical Biochemistry 07/2005; 341(2):369-71. · 3.00 Impact Factor
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ABSTRACT: To explore JunB gene expression in spleen cells of mice after the whole body irradiation as well as in normal hematopoietic and leukemia cells in the primary culture after different dosages of X-ray irradiation.
Spleen cells were isolated from the mice irradiated with 3 Gy X-rays. Primary cultured cells from mice were incubated in different intervals after X-irradiation at different dosages. Total RNA was extracted from the cells and the fluctuation of JunB mRNA level was assessed by the RNA ratio of JunB/beta-actin measured by quantitative Northern blot hybridization.
After the mice were exposed to 3 Gy X-rays irradiation, JunB expression in spleen cells was remarkably and rapidly increased, and reached its peak 0.5 h later in C3H/He mice and 1 h later in Balb/c mice. In the primary culture of normal spleen and leukemia cells, JunB mRNA levels increased 30 min after irradiation. The enhanced levels of JunB mRNA were returned to a normal level within 240 min after irradiation.
JunB gene is responsive to ionizing irradiation and is induced at immediate-early phase after the stimulation. This suggests that the JunB gene plays an important role in the early process of the cells against radiation.
Biomedical and Environmental Sciences 10/2004; 17(3):327-32. · 1.35 Impact Factor
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ABSTRACT: The murine genome has about 1,000 copies of DNA elements for the intracisternal A-particle (IAP) that resembles a retrovirus. We previously reported that the genomic DNA of the cells from radiation-induced acute myeloid leukemia (AML) lines derived from C3H/He inbred mice was frequently rearranged by the integration of the IAP element. In this study, 8 IAP elements from the characteristic integration sites in 6 cell lines of radiation-induced AML from different mice were characterized and compared in structure with 114 IAP elements isolated from the normal C3H/He genome. One of the 8 elements was a full-length type I IAP, and 7 were of type-I Delta 1 with a common deletion site. Although the type I Delta 1 form is a minor population accounting for about 6% of total genomic IAP elements, it is predominantly retrotransposed in the AML cells from different C3H/He mice. This indicates that limited populations of the IAP elements contribute to the unique retrotransposition in AML cells.
Journal of Radiation Research 04/2004; 45(1):25-32. · 1.68 Impact Factor
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ABSTRACT: To explore the possibility of Schwann cells transplantation to promote the repair of injured brain stem reticular structure in rats.
Schwann cells originated from sciatic nerves of 1 to 2-day-old rats were expanded and labelled by BrdU in vitro, transplanted into rat brain stem reticular structure that was pre-injured by electric needle stimulus. Immunohistochemistry and myelin-staining were used to investigate the expression of BrdU, GAP-43 and new myelination respectively.
BrdU positive cells could be identified for up to 8 months and their number increased by about 23%, which mainly migrated toward injured ipsilateral cortex. The GAP-43 expression reached its peak in 1 month after transplantation and was significantly higher than that in the control group. New myelination could be seen in destructed brain stem areas.
The transplantation of Schwann cells can promote the restoration of injured brain stem reticular structure.
Biomedical and Environmental Sciences 10/2003; 16(3):212-8. · 1.35 Impact Factor
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ABSTRACT: Immediately after X-irradiation, monocytic cells can express the gene for interleukin (IL)-1beta, which enhances inflammation and contributes to radioprotection in mice. In order to analyze the mechanism(s) for the immediate-early induction of IL-1beta after X-irradiation at 20 Gy in cultured murine macrophages, we examined the molecules that bound to the DNA fragments corresponding to the upstream region of 10 kb of the mouse IL-1beta gene using an electrophoretic mobility-shift assay. Three DNA fragments corresponding to the 8,500, 8,000 and 2,500 bases upstream of the gene showed an unique binding site with the nuclear extract. Specific binding activity with these DNA fragments was observed in the nuclear extract from non-irradiated cells, and disappeared upon a pretreatment of the extract with proteinase K. The binding activity was not detected in the nuclear extract from irradiated cells. This shows that protein(s) specifically binding to the far-upstream regions of the IL-1beta gene disappear immediately after X-irradiation in the nuclei of macrophage cells, and that the event is potentially related to the immediate-early response of IL-1beta gene expression.
Journal of Radiation Research 07/2003; 44(2):117-23. · 1.68 Impact Factor
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ABSTRACT: To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs).
The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and beta-tubulin, GalC, hoechst 33342 and GFAP labellings were detected.
In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were beta-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiated and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died.
The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.
Biomedical and Environmental Sciences 04/2003; 16(1):90-4. · 1.35 Impact Factor
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ABSTRACT: To explore the factors which induce differentiation of embryonic neural stem cells.
Rat embryonic neural stem cells were co-cultured with newborn rat Schwann cells in serum-free medium. The phenotype and specific-markers including tubulin-beta, glial fibrillary acidic protein (GFAP) and galactorcerebroside (GalC), were demonstrated by phase contrast microscopy and double immunofluorescence staining.
Overall, 80% +/- 5% of neural stem cells protruded several elongated processes and expressed tubulin-beta antigen at high levels, while 20 +/- 3% of them protruded several short processes and were GalC or GFAP positive.
The factors secreted by Schwann cells could induce rat embryonic neural stem cell to differentiate.
Chinese medical journal 04/2003; 116(3):428-31. · 0.86 Impact Factor
