Objective Using nerve growth factor ( NGF) and anti-NGF microspheres injected directly into the asthmatic rat adrenal gland, to explore the possible role of anti-NGF microsphere treatment in asthma.Methods 32 male SD rats were randomly divided into a normal control group, an asthma group, a NGF microspheres group, and an anti-NGF microspheres group. The behavior of rats, lung function testing, light microscopy of lung biopsy, electron microscopy of adrenal medulla cell ultrastructure changes, NGF and phenylethanolamine N-methyltransferase ( PNMT) expressions in the adrenal gland were assayed by immunohistochemistry method, and serum NGF, cortisol, corticosterone, epinephrine and norepinephrine concentrations were detected by ELISA. Results Behavior in the asthma rats showed varying degrees of sneezing, runny nose, wheezing, scratching the head and face, irritability holes, incontinence, increased aggression and other acts, while in the anti-NGF rats showed relatively slighter symptoms. The rats in the asthma, anti-NGF and NGF groups showed significant airway hyperresponsiveness, while RL value reduced and Cdyn value increased in the anti-NGF group compared with the asthma group. HE staining of lung tissue revealed obvious bronchoconstriction, inflammatory cell infiltration around small vessels and alveolar spaces and in interstitum, bronchial epithelial cells desquamation in the asthma group. In anti-NGF group, tracheal epithelium was relatively complete, inflammatory exudation, bronchoconstriction and inflammatory cell infiltration were milder compared to the asthma group. Electron microscopy showed vacuolated changes of adrenal medulla cells, uneven distribution of chromaffin granules in the asthma group and the NGFgroup, and the quantity and concentration of chromaffin granules were significantly lower than normal. There were villous clubbing processes on the adrenal medulla cell membrane in the NGF group. While the anti-NGF group had no significant vacuolar changes in chromaffin granules and the concentration was close to normal. Image analysis showed that mean gray values of PNMT and NGF in the anti-NGF group were significantly different fromthe asthma group. The ELISA results showed that: ( 1) The average concentrations of epinephrine in each group were as follows, ie. the control group gt; anti-NGF group gt; asthma group gt; NGF group. ( 2) The average concentrations of norepinephrine in each group were as follows, ie. the NGF group gt; asthma group gt; anti-NGF group gt; control group. ( 3) There was no significant difference among the groups in the average concentration of cortisol. ( 4) The average concentrations of norepinephrine in each group were as follows, ie. , the control group gt; anti-NGF group gt; asthma group gt; NGF group. Conclusions Local embedding of anti-NGF microspheres can alleviate inflammatory infiltration in lung tissue and improve lung function of rat model with asthma. The mechanismmay be the anti-NGF antagonists the NGF receptor and reverse adrenal medulla cell transdifferentiation process primined by NGF.
Objective To investigate the effect of methylprednisolone sodium succinate (MP) and mouse nerve growth factor (mNGF) for injection in treating acute spinal cord injury (ASCI) and cauda equina injury. Methods Between December 2004 and December 2007, 43 patients with ASCI and cauda equina injury were treated, including 33 males and 10 females with an average age of 43 years (range, 32-66 years). Injured vertebral columns were C2 in 1 case, C4 in 5 cases, C5 in 7cases, C6 in 3 cases, T8 in 1 case, T10 in 1 case, T11 in 2 cases, T12 in 3 cases, L1 in 9 cases, L2 in 5 cases, L3 in 3 cases, L4 in 1 case, and L5 in 2 cases. All the patients had sensory disturbance and motor dysfunction at admission. The Frankel scale was used for assessment of nerve function, 5 cases were rated as Grade A, 12 as Grade B, 22 as Grade C, and 4 as Grade D before operation. In 43 patients, 23 cases were treated with MP and mNGF (group A), 20 cases with MP only (group B). There was no significant difference in general data between 2 groups (P gt; 0.05). All the patients were admitted, received drug treatment within 8 hours of injury, and were given spinal canal decompression, bone transplantation, and internal fixation within 48 hours. The neurological function score systems of American Spinal Injury Association (ASIA) were used for neurological scores before treament, at 1 week and 2 years after treatment. The scores of the activity of daily l iving (ADL) were evaluated and compared. Results All the patients achieved heal ing of incision by first intention. Forty-three cases were followed up 24-61 months with an average of 30 months. Bone graft fusion was achieved after 6-17 months, 11 months on average with stable fixation. No death and compl ications of osteonecrosis and central obesity occurred. There was no significant difference in neurological function scores and ADL scores between 2 groups before treatment (P gt; 0.05); however, the neurological function scores and ADL scores at 1 week and 2 years after treatment were higher than those before treatment (P lt; 0.01) in 2 groups. Group A had higher neurological function scores and ADL scores than group B (P lt; 0.01). At 1 week and 2 years after treatment, the improvement rates of neurological function of group A (47.8%, 11/23 and 91.3%, 21/23) were significantly higher (P lt; 0.01) than those of group B (30.0%, 6/20 and 70.0%, 14/20). Conclusion MP and mNGF play an important role in improving the neurological function in patients with ASCI and cauda equina injury.
Abstract To observe the effect of exogenous high molecular weight nerve growth factor (HMW-NGF) mixed with bletilia striata gelatin (BSG) in the promotion of healing, the experiment was performed as follow: (1) In serumfree medium, the normal saline, BSG, HMWNGF, and BSG+HMW-NGF were added separately, and then, the chick embryo root ganglions (DRGs) were cultivated in the above prepared media and the axonal growth was observed. (2) 40 SD rats were divided into 4 groups. A wound of 2cm×2cm was made on the back of every rat. No treatment was given in group one. In other groups, BSG, HMW-NGF, andBSG+HMW-NGF were given separately to the wounds once daily. After 3 and 10 days, the wound area of every rat was measured, cells in the wounds were observed under light microscope and were calculated, and the time of healing was recorded. The results showed that BSG, HMW-NGF, especially BSG+HMW-NGF could promote wound healing.
Objective To observe whether apoptosis was involved in cells of aspiration fluid from vitrectomy for proliferative vitreoretinopathy(PVR),and whether there was an association with expression of Fas antigen(Fas )and Fas ligand (FasL). Methods Cytocentrifuge slides of 11 fresh vitreous specimenｓ of PVR were prepared to be stained by TUNEL met hod for detection of apoptosis and by immunohistochemical technique for detection of Fas,FasL，and cytokeratin (CK),a cell-type specific antigen. Results Fas and FasL were expressed in normal human retina.Fas,FasL,CK，and apoptosis were found in all preparations.TUNEL-positive cells were 20.53% in total cells.70.35%,51.58%，and 82.97% of cells highly expressed Fas,FasL，and CK,respectively.The linear correlation coefficient of Fas and apoptosis was 0.99(Plt;0．001). Conclusion Vitrectomy specimens of PVR showed expression of Fas，FasL，and apoptosis.Prominent Fas and FasL expressions may be associated with apoptosis of proliferating retinal pigment epithelial cells in the vitreous of PVR. (Chin J Ocul Fundus Dis,1999,15:78-80)
This study demonstrates that nerve growth factor (NGF) plays a protective role in myocardial infarction and early reperfusion by reducing the myocardial cell apoptosis and by improving ventricular remodeling and seeks to assess the effects and mechanisms of NGF on late reperfusion after myocardial infarction. The models of late reperfusion were established by ligating the left main coronary artery and then cutting the suture 2 hours after coronary artery ligation. The rats in NGF treatment group were injected 10μL Ad-NGF (by constructing the adenovirus vector Ad-NGF containing NGF gene) at four locations around infarction. The rats in adenoviral vector (Adv) group were injected 10μL adenoviral cector as the NGF group. The late reperfusion group and the sham group were given normal saline as above, and the sham group underwent thracotomy without coronary ligation. On the 3rd, 7th, 14th and 28th day after operation, we investigated the role of NGF on late reperfusion by recording cardiac structure and function with echocardiography, by examining the expression of NGF andⅧfactor with immunohistochemical method, and by evaluating the myocardial cell apoptosis with terminal dUTP nick end-labeling method (TUNEL). We found that the NGF group had higher expression of NGF protein (P < 0.01) and lower apoptosis index (AI) (P < 0.01 or P < 0.05) compared to the late reperfusion group and Adv group on all time points. The NGF group had remarkably higher level of neovascularization compared to the late reperfusion group on the 14th day (P < 0.01) and the 28th day (P < 0.05). The NGF group also had higher LVEF and FS levels compared to the late reperfusion group on the 14th day (P=0.006, P=0.006) and on the 28th day (P=0.000, P=0.000). Whereas the NGF group had lower LVEDD, LVESD (P=0.038, P=0.000) and lower LVEDV, LVESV (P=0.001, P=0.000) on the 28th day compared to late reperfusion group. In this experiment, the NGF gene carried by adenovirus vector had been trans-fected and obviously increased the expression of NGF protein in NGF group. NGF may help postpone the myocardial remodeling and improve the heart function by promoting the myocardial neovascularization and inhibiting myocardial apoptosis.
Objective To explore the effects of human urine-derived stem cells (hUSCs) and hUSCs combined with chondroitinase ABC (chABC) on the expressions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the spinal cord injury (SCI) of rats, and to investigate the underlying mechanism. Methods hUSCs were cultured from human urine, and their phenotypes were detected by flow cytometry. The SCI model of rats were made via Allen method. Sixty Sprague Dawley rats were divided into 5 groups (n=12): the sham operation group (group A), SCI group (group B), SCI+hUSCs group (group C), SCI+chABC group (group D), and SCI+hUSCs+chABC group (group E). Basso, Beattie, Bresnahan (BBB) score was used to measure the lower extremity motor function of rats in each group at 10, 20, and 30 days after operation. Real-time fluorescent quantitative PCR was used to detect the relative mRNA expressions of NGF and BDNF at 30 days. Meanwhile, the protein expression of NGF and BDNF were confirmed by immunohistochemistry staining. The relative protein expressions of Bax and Bcl-2 were detected by Western blot. Results The hUSCs were identified to have multipotential differentiation potential. At 10, 20, and 30 days, BBB score was significantly lower in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05). Real-time fluorescent quantitative PCR and immunohistochemistry staining demonstrated that the expressions of NGF and BDNF were significantly lower in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05); but there was no significant difference between groups C and D (P>0.05). Western blot results indicated that the protein expression of Bax was significantly higher in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05). Meanwhile, the protein expression of Bcl-2 was significantly lower in group B than in groups A, C, D, and E, in groups C, D, and E than in group A, in groups C and D than in group E (P<0.05). Conclusion hUSCs can protect SCI and this positive effect can be enhanced by chABC; this neuro-protective effect may depend on promoting the expressions of NGF and BDNF, and suppressing the neuronal apoptosis.
Nuclear receptors are transcriptional regulators involved in almost all biological processes such as cell growth, differentiation, apoptosis, substance metabolism and tumor formation, and they can be regulated by small molecules that bind to them. Autophagy is a special way of programmed cell death and it is a highly conserved metabolic process. Once autophagy defects or excessive autophagy occur, the disease will develop. In recent years, numerous studies have shown that nuclear receptors are related to autophagy. Therefore, this paper mainly reviews the research progress on nuclear receptors involved in the regulation of autophagy, and focuses on the mechanism of several nuclear receptors involved in the regulation of autophagy, aiming at understanding the molecular basis of how nuclear receptors participate in regulating autophagy, as well as providing possible ideas and strategies for the treatment of corresponding diseases.
ObjectiveTo evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro.MethodsNGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% (W/V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D.ResultsThe chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 μm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group (P<0.05). In vitro sustained release ratio of NGF showed that when compared with PLGA microspheres in group A, double-walled microspheres in groups B, C, and D released NGF at a relatively slow rate, and the sustained release ratio decreased with the increase of TPP concentration. Except for 84 days, there was significant difference in the sustained release ratio of NGF between groups B, C, and D (P<0.05). The bioactivity of NGF results showed that the percentage of PC12 cells with positive axonal elongation reaction in groups B, C, and D was significantly higher than that in group A1 (P<0.05). At 7 and 28 days of culture, there was no significant difference between groups B, C, and D (P>0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B (P<0.05), and there was no significant difference between groups C and D (P>0.05).ConclusionNGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.
ObjectiveTo investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis.MethodsBMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model (n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR.ResultsAt 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B (P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days (P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A (P<0.05).ConclusionSilencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.
ObjectiveTo describe the research progress of silk-based biomaterials in peripheral nerve repair and provide useful ideals to accelerate the regeneration of large-size peripheral nerve injury. Methods The relative documents about silk-based biomaterials used in peripheral nerve regeneration were reviewed and the different strategies that could accelerate peripheral nerve regeneration through building bioactive microenvironment with silk fibroin were discussed. Results Many silk fibroin tissue engineered nerve conduits have been developed to provide multiple biomimetic microstructures, and different microstructures have different mechanisms of promoting nerve repair. Biomimetic porous structures favor the nutrient exchange at wound sites and inhibit the invasion of scar tissue. The aligned structures can induce the directional growth of nerve tissue, while the multiple channels promote the axon elongation. When the fillers are introduced to the conduits, better growth, migration, and differentiation of nerve cells can be achieved. Besides biomimetic structures, different nerve growth factors and bioactive drugs can be loaded on silk carriers and released slowly at nerve wounds, providing suitable biochemical cues. Both the biomimetic structures and the loaded bioactive ingredients optimize the niches of peripheral nerves, resulting in quicker and better nerve repair. With silk biomaterials as a platform, fusing multiple ways to achieve the multidimensional regulation of nerve microenvironments is becoming a critical strategy in repairing large-size peripheral nerve injury. Conclusion Silk-based biomaterials are useful platforms to achieve the design of biomimetic hierarchical microstructures and the co-loading of various bioactive ingredients. Silk fibroin nerve conduits provide suitable microenvironment to accelerate functional recovery of peripheral nerves. Different optimizing strategies are available for silk fibroin biomaterials to favor the nerve regeneration, which would satisfy the needs of various nerve tissue repair. Bioactive silk conduits have promising future in large-size peripheral nerve regeneration.