OBJECTIVE: To investigate the effect of nerve growth factor(NGF) on the burn wound healing and to study the mechanism of burn wound healing. METHODS: Six domestic pigs weighting around 20 kg were used as experimental animals. Twenty-four burn wound, each 2.5 cm in diameter, were induced on every pigs by scalding. Three different concentrations of NGF, 1 microgram/ml, 2.5 micrograms/ml, 5 micrograms/ml were topically applied after thermal injury, and saline solution used as control group. Biopsy specimens were taken at 3, 5 and 9 days following treatment and immunohistochemistry method was used to detect the epidermal growth factor(EGF), EGF receptor (EGF-R), NGF, NGF receptor (NGF-R), NGF, NGF-R, CD68 and CD3. RESULTS: The expression of EGF, EGF-R, NGF, NGF-R CD68 and CD3 were observed in the experimental group, especially at 5 and 9 days, no expression of those six items in the control group. CONCLUSION: NGF can not only act directly on burn wound, but also modulate other growth factors on the burn wound to accelerate the healing of burn wound.
ObjectiveTo construct recombinant adenovirus expressing nerve growth factor (NGF) and myelin associated glycoprotein (MAG) (Ad-NGF-MAG) and to investigate its effect on repair and regeneration of sciatic nerve injury in rats. MethodsNGF and MAG gene sequences were cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK293 cells, the recombinant Ad-NGF-MAG underwent sequence and identification. Thirty-two male Sprague Dawley rats were randomly divided into 4 groups (n=8): control group (normal control), adenovirus vector group (Ad group), Ad-NGF group, and Ad-NGF-MAG group. The sciatic nerve injury model was established by transection of the right sciatic nerve; then, the empty adenovirus vector, Ad-NGF, and Ad-NGF-MAG were injected into the gastrocnemius muscle of the affected limb at a dose of 1×108 PFU every other day for 3 times in Ad group, AdNGF group, and Ad-NGF-MAG group, respectively. The right sciatic nerve was exposed only, and then the incision was closed in the control group. The sciatic nerve function index (SFI) was measured, and neuro-electrophysiology was observed; mRNA and protein expressions of NGF and MAG were detected by RT-PCR and Western blot; and histological examination was performed at 31 days after operation. ResultsRecombinant adenovirus vectors of Ad-NGF and Ad-NGF-MAG were constructed successfully. All rats survived and incision healed by first intension. The SFI, nerve conduction velocity, evoked potential amplitude, and latent period of Ad-NGF-MAG group were significantly better than those of Ad group and Ad-NGF group (P < 0.05). MAG mRNA and protein expressions of Ad-NGF-MAG group were the highest in all the groups (P < 0.05). The expressions of NGF mRNA and protein increased in Ad-NGF group and AdNGF-MAG group when compared with control group and Ad group (P < 0.05). Histological examination showed that the nerve had good continuity in control group; nerve fibers disarranged in Ad group; neurons connections formed in some nerve fibers of Ad-NGF group, but nerve fibers arrange disorderly; and the growth of the nerve were ordered and wellstructured in Ad-NGF-MAG group. ConclusionAd-NGF-MAG can effectively promote the growth of the nerve and inhibit the form of abnormal branches, facilitating the repair of sciatic nerve injury in rats.
Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats. Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups, 10 rats in each group, including normal control group (group A), normal＋balanced salt solution (BSS) group (group B), normal＋netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 sub-groups). DM rats were induced by intraperitoneal injection of streptozocin. Three months after intraperitoneal injection, 10 DM rats in the control group were injected with BSS (group D). Forty DM rats were injected with 5 μl of different concentrate netrin-1, and were divided into DM+netrin-1 10 μg/ml group (group E), DM+netrin-1 50 μg/ml group (group F), DM＋netrin-1 100 μg/ml group (group G), DM＋netrin-1 500 μg/ml group (group H) according to the different concentration. Non-DM rats in group C were injected with netrin-1 500 μg/ml. The expression of occludin was determined by immunohistochemistry for protein, and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level. Retinal vascular permeability was measured by Evans blue infusion. Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71, 8.59;P=0.00, 0.00). However, the retinal vascular permeability increased in group D (t=−42.72,P=0.00). The expression of occluding protein, occludin mRNA and retinal vascular permeability showed significant differences between group D, E, F, G and H (F=146.31, 16.54, 67.77;P=0.00, 0.00, 0.00). Compared the group B with group C, there was no significant differences between the expression of occludin protein, occludin mRNA and the retinal vascular permeability (t=−1.13, 0.93, 1.04;P=0.27, 0.36, 0.31). The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73, 0.81;P=0.00, 0.00), but negative correlation to the vascular permeability (r=−0.61,P=0.00). Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability, which depended on the concentration of netrin-1.
【Abstract】 Objective To construct a recombinant adeno-associated virus (AAV) shuttle vector expressing nervegrowth factor β (NGF-β) gene. Methods By PCR amplification, the structural element of pAAV-multi ple cloning site(MCS) and the functional element of pGenesil-1.1 were obtained and cloned into T-easy vector, respectively; the recombinant T-easy vectors were digested by restriction enzyme, then the target fragments were reclaimed and connected by DNA l igase, so the recombinant AAV shuttle vector pAAV-U6/CMV-enhanced green fluorescent protein (EGFP) containing U6 promoter and CMV promoter was obtained. The vector was transfected into 293 cells. The human Miapaca-2 cell l ine was cultured, and total RNA was extracted, then human NGF-β gene was obtained by RT-PCR. T-easy-NGF-β vector was constructed by cloning human NGF-β gene into T-easy vector and identified by RT-PCR, digestion, and DNA sequencing. As NGF-β gene was cloned into pAAV-U6/CMV-EGFP vector, the recombinant AAV shuttle vector expressing NGF-β gene was obtained and identified by RT-PCR, digestion, and DNA sequencing. Results The bands of 800 bp and 4 250 bp were detected when pAAV-U6/CMVEGFP was digested. The GFP was detected when pAAV-U6/CMV-EGFP was transfected into 293 cells. The bands of 736 bp and 3 015 bp were detected when T-easy-NGF-β was digested; DNA sequencing result of T-easy-NGF-β was fully consistent. The bands of 736 bp and 4 250 bp were detected when pAAV-U6/CMV-NGF-β was digested. DNA sequencing result of pAAV-U6/ CMV-NGF-β showed that sequences were completely correct. Conclusion The AAV shuttle vector pAAV-U6 /CMV-NGF-β is successfully constructed, providing experimental basis for investigation of the repair of spinal cord injury.
Objective To observe in vitro the protective effect of cranial nerve growthine (CNG) on the optic nerve injured by acute ocular hypertension. Methods Thirty white rabbits were divided into five groups,and 6 in each.The acute ocular hypertension(50 mm Hg) models were established by forcing perfusion of normal saline solution into the anterior chamber sustained for 6 h in one eye,and the contral ateral eye of each rabbit was regard as control.Three rabbits in each group were then treated by CNG 0.2 ml intramuscularly every day.The optic nerve and retina was surgically removed at five different time points (lst,3rd,7th,15th and 30th day) after operation.With the HRP orthograde tracing technique and transmitted electron microscope,the effect of CNG on the optic nerve was observed by the changes of axonal transport and ultrastructure of optic nerve. Results Compare with experimental control groups (25.17plusmn;1.03),HRP reactive products of treated groups (39.79plusmn;2.29) markedly increased after seven days (Plt;0.01).The degeneration of axons in treated groups was relatively lighter after fifteen days and some axons recovered after thirty days. Conclusion CNG might improve the axonal transport and the recovery of axons after the optic nerve injured by acute ocular hypertension. (Chin J Ocul Fundus Dis,2000,16:88-90)
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.
ObjectiveTo explore the expressions of nerve growth factor (NGF) and leukemia inhibitory factor (LIF) in both asthmatic mice and respiratory syncytial virus(RSV)-infected mice,explore if there is a same neurogenic mechanism between ashtma and RSV infection,in order to find a new treatment target for asthma. MethodsOne hundred healthy Balb/c inbred mice were randomly divided into a control group,a RSV group,an asthma group,an asthma with RSV group,and a dexamethasone group. The lung tissue pathology was observed by hematoxylin-eosin staining(HE). The quantitative analysis of NGF mRNA and LIF mRNA of lung tissue was detected by RT-PCR. The expression of NGF protein and LIF protein was detected by immunohistochemical method. ResultsUnder light mocroscope,there were alveolar septum widening,alveolar epithelium swelling,and interstitial edema in the RSV group. There were widen alveolar septum,narrowed bronchial lumen,thicken bronchial wall and a large number of inflammatory cells infiltration around the small blood vessels,alveolar and bronchioles both in the asthma group and the asthma with RSV group,with the latter being more serious. Compared with the RSV group,the inflammation was relieved significantly in the dexamethason group. There were mRNA and protein expressions of NGF and LIF in all groups, which were highest in the asthma with RSV group,then the RSV group and the asthma group,and lowest in the dexamethasone group. ConclusionsThe expressions of LIF and NGF in the lung of mice after RSV infection and futher increase when combined with asthma. Dexamethason can inhibit the expression of NGF and LIF to some extent.
Thirty-five SD rats were divided into 3 groups, in which 5 rats were served as control. The other 2 groups, 15 rats in each received either NGF solution or normal saline. The injury at the level of T8 spinal segment of the rat in these two groups were made by dropping a weight of 10 g from a height 2.5 cm after a total laminectomly from T7-11 segments. A thin plastic tube was inserted into the subarachnoid space below the injured segments. NGF was introduced through the tube at intervals of 1, 2, 3, 4, 8, 12, 24, 48 hours in the NGF group, and normal saline in the normal saline group. At 4, 8, 24 hours following surgery, 5 rats in each group were sacrificed and the injured segments were resected for examination. The contents of water and calcium were measured by dry-wet method and atomic absorption spectroscopy. The result showed that total calcium and water contents in normal saline group were markly increased, however, the changes of these two parametere were not so prominent in NGF group. It was suggested that NGF play a role in protecting the spinal cord by maintaining the calcium level of the injured segment.
OBJECTIVE: To study the nerve growth factor (NGF) expression and the influence of IL-1 alpha or IL-1 beta on NGF secretion in newborn rat astrocytes. METHODS: Astrocytes obtained from the brain cortex of newborn rats were cultured and purified, and they were divided into three groups, experimental, control and blank groups. IL-1 alpha or IL-1 beta were added into the experimental group with 25, 50 and 100 U/ml, each group was cultured for 24, 48 or 72 hours, and then the NGF contents in cultured astrocytes suspension media were measured by a two-cite enzymelinked immunoserbent assay (ELISA). RESULTS: Astrocytes could secret NGF by themselves and each concentration of IL-1 alpha or IL-1 beta media at any testing time could enhance NGF secreting in newborn rat astrocytes in certain degrees. The effects of IL-1 beta were ber than IL-1 alpha, the best effect in the unit time was observed in IL-1 beta with 50 U/ml for 24 hours. CONCLUSION: Astrocytes can express NGF, and IL-1 alpha or IL-1 beta can enhance the NGF expression in newborn rat astrocytes.
ObjectiveTo evaluate the effects of nerve growth factor (NGF) on angiogenesis and skeletal muscle fiber remodeling in ischemic hindlimbs, and study the relationship of NGF and vascular endothelial growth factor (VEGF) to angiogenesis. MethodsEighteen mice were randomly allocated to normal control group (n=6), blank control group (n=6), and NGF gene transfection group (n=6). The left hindlimb ischemia model was established by ligating the femoral artery. NGF plasmid (125μg) was injected into the mouse ischemic gastrocnemius in the NGF gene transfection group. The same volume of normal saline (200μL) was injected into the mouse ischemic gastrocnemius in the blank control group. The gastrocnemius of left hindlimb was harvested under the condition of peritoneal cavity anesthesia on the 21th day after operation, and then the mice were sacrificed. The gastrocnemius of three groups were tested by hematoxylin-eosin staining, proliferating cell nuclear antigen (PCNA) and CD34 were determined by immunohistochemistry staining. Skeletal muscle fiber type was tested by myosin ATPase staining. NGF and VEGF protein expression were detected by enzyme linked immunosorbent assay. ResultsOn the 21th day after surgery, compared with the blank control group, the skeletal muscle atrophy degree was weaker, the functional assessment score was significantly lower (P < 0.05), the endothelial cell proliferation index, capillary density, the typeⅠskeletal muscle fiber proportion, NGF and VEGF expression were significantly higher (P < 0.05) in the NGF gene transfection group. ConclusionsNGF gene transfection could promote NGF and VEGF expression and angiogenesis in ischemic hindlimbs, and induce typeⅠskeletal muscle fibers formation in ischemic hindlimbs. The molecular regulation mechanism still needs to be further studied.