10.3969/j.issn.1000-8179.2013.12.016

Objective To optimize human acellular dermal matrix(ADM) and evaluate its biological characters. Methods Human skin was treated with hypertonic saline followed by NaOH maceration(group A), hypertonic saline followed by sodium dodecyl sulfate (SDS) detergent(group B) or Dispase Ⅱ followed by Triton X-100(group C), the resulting ADM were sectioned, and then were stained by special immunohistochemistry method. The cytotoxicity of them were evaluated by methyl thiazolyl tetrazolium (MTT) colorimetry and then cell compatibility was analyzed by cell culture;The optimized ADM resulted was choosen for use. Fibrablasts(FBs)were transfected with adenovirus vector encoding green fluorescent protein gene(Ad-GFP)and the growth of them on the optimized ADM was observed by fluorescent microscopy. Results Collagen and elastic fibers can still be observed in three kinds of ADM. The cells in dermis can be disintegrated both in group A and C, but not in group B. The cytotoxicity scores of the ADM prepared in group A and B were grade 0 or grade 1, while that of group C was more than grade 1.The ADM prepared by NaCl-NaOH maceration had good biocompatibility. There was statistical difference in adhering number of NIH3T3 cells in group A and B. NIH3T3 cells grew well in group A and the resulted ADM was optimized. FBs transfected with Ad-GFP grew well in the optimized ADM. Conclusion The ADM prepared by NaCl-NaOH maceration was a good tissue engineering biomaterial with a little cytotoxicity and rich in resouce.

Objective To study the mechanism of interaction between neuronal nitric oxide syn-thase (nNOS) and inducible nitric oxide synthase (iNOS) following traumatic brain injury (TBI) in rats. Methods A total of 250 male Wistar rats were randomly divided into five groups, ie, sham oper-ation group, trauma group, 7-nitroindazole (7-NI) treatment group, aminoguanidine (AG) treatment group and combined AG and 7-NI treatment group. Severe closed TBI was made by using Marmarou meth-od. Protein expressions of nNOS and iNOS in hippocampus CAI were detected by means of immunohisto-chemical staining at 1,3, 6, 12 hours and at days 1,3, 7 and 14 after TBI. Results The expression of nNOS reached a peak at 6 hour after injury in all groups, with no statistical difference between groups (P > 0. 05), when there was no statistical difference between 7-NI treatment group and trauma group (P > 0. 05) but statistical difference in AG treatment group and combined AG and 7-NI treatment group compared with trauma group at 12 hours after TBI (P <0.05). The expression of iNOS reached maximal level at day 3 after TBI, with lower level in 7-NI group, AG treatment group and combined AG and 7-NI treatment group compared with trauma group (P < 0.05). Conclusions After TBI, nNOS interacts with iNOS by means of the feedback of nitric oxide. The enhanced expression of nNOS is initial factor for increase of iNOS expression, which can down regulate the expression of iNOS.

Objective To study the mechanism of dermal fibroblasts as a feeder layer to support the growth of human keratinocytes. Methods Human dermis fibroblasts were isolated and cultured and then treated with mitomycin-C. The expression of type Ⅰand type Ⅲ precollagen mRNA and relevant protein in feeder layer were examined by RT-PCR and Immunohistochemistry. KCs were cultured both on FB and NIH3T3 feed layer as control, the adhering numbers and the time of fusion were recorded. Results RT-PCR showed an increase of type Ⅰprecollagen mRNA in FB feeder layer as compared with that of normal fibroblasts (P

Objective To explore the mechanism of neurotoxic effects of neuron nitric oxide synthase(nNOS)and inducible nitric oxide synthase(iNOS),and the therapeutic effects of 7-nitroindazole(7-NI)and aminoguanidine(AG),in traumatic brain injury(TBI)rats.Methods Two hundred and fifty adult male Wistar rats were randomly divided into 5 groups:sham operation group,traumatic group,AG group,7-NI group and AG+7-NI group.Animals in each group were divided into 8 subgroups according to the time after trauma(1,3,6,12 hours and 1,3,7,14 days).Severe diffused brain injury model was made with Marmarou method.Expressions of nNOS and iNOS in hippocampus CA1 region were determined by immunohistochemistry,nerve cells apoptosis in hippocampus CA1 region was observed by TUNEL methods,and the relationship between apoptosis and NOS was observed by double staining.Results In trauma group,the expression of nNOS in hippocampus CA1 region peaked at 6h post-trauma,the expression of iNOS and apoptosis of nerve cells in hippocampus CA1 region both peaked at 3d post-trauma,while the apoptosis was alleviated in AG group,7-NI group and AG+7-NI group.The number of both TUNEL staining and nNOS immunostaining positive cells increased at 6h post-trauma in trauma group,significantly higher than that in 7-NI group.The number of both TUNEL staining and iNOS immunostaining positive cells increased at 3d post-trauma in trauma group,significantly higher than that in AG group.Conclusions The over-expression of nNOS and iNOS has toxic effects to neural tissues of brain,serves as one of the factors inducing nerve cell apoptosis in hippocampus CA1 region.7-NI and AG can inhibit the expression of nNOS and iNOS,reduce the nerve cell apoptosis,and play an important role in neuroprotective effect.

Objective To explore the influence of injury severity on transplantation of embryonic neural stem cells (NSCs) after traumatic brain injury (TBI).Methods The NSCs were isolated from the hippocampus of fetal rats aged at from 12-14 days.The cells were cultured and proliferated in the serum-free medium and identified in vitro.The animals received transplants in the bilateral hippocampal areas at day 3 following mild or moderate TBI separately.Conventional histology,TUNEL and immunohistology were examined to detect BrdU,NSE,GFAP,GalC,NGF and BDNF at day 14 post-implantation.Results BrdU-labeled positive cells in the bilateral hippocampus in the mild TBI group were more than those in the moderate TBI group at day 14 post-implantation.Significant differentiation of the astrocytes recognized as GFAP positive cells in the bilateral hippocampus was found at day 14 post-implantation.The expression of NGF and BDNF proteins was increased following TBI,the most evident in the mild TBI group.Conclusion The influence of injury severity on transplantation may be associated with the change of the microenvironment after TBI.

Objective To observe the efficacy of pulsed dual-wavelength 595 and 1064 nm laser in treating ulcerated infantile hemangioma.Methods Lesions from 40 patients were treated with the laser.The distribution of lesion sites was in buttuck and perianal area (13 patients),vulvar lips (7),groin and inside thigh (5),scrotum (4),neck (3),scalp (2),lip (2),trunk and limbs (4).All lesions including ulcer and non-ulcer were conducted with 595 nm plused dye leaser at the first therapy.Later,595 nm pulsed dye leaser was continued or performed with Group 1 or Group 2 parameter of 595 and 1064 nm multiplex mode until the treatment was finished according to the thickness of the lesion.The frequency of treatment was one to six times.The interval of the treatment was four weeks.Results The ulcer from 39 lesions healed gradually in 1 to 2 weeks (average 9.5 days) after the first treatment.These ulcer lesions were healed four weeks after the first time of therapy.The recovery rate of ulcerated lesion was 97.5 ％.For the whole lesion,after 1 to 6 treatments,13 lesions showed excellent result,19 lesions showed good result,and the effective rate was 80 ％.Pain relieved 2 to 7days (average 3.5 days) after the first laser therapy in 35 patients who accompanied with pain.Pain score decreased from 1.875 to 0.125 before and after treatment,with significant difference(P＜0.05).The tolerance of the treatment was good and no side effect was observed.Conclusions 595and 1064 nm dual-wavelength laser is a safe and effective tool for treating ulcerated infantile hemangioma.

AIM: To investigate the effect of extracellular signal-regulated kinase 1/2 signaling pathway after severe diffuse brain injury (DBI) in rats, and to provide base for treatment. METHODS: Male Sprague-Dawley rats were randomly divided into four groups: control group, traumatic group, low dose of inhibitor U0126 treatment group and high dose of inhibitor U0126 treatment group. DBI rat model was established according to the description of Marmarou's diffused brain injury. At 30 min and 1 h, 6 h, 24 h, 48 h and 72 h after injury, morphological changes were observed under light and electronic microscopes. The ERK1/2 phosphorylation and c-Fos were measured by Western blotting. Apoptosis was measured with TUNEL method. Learning and memory function were performed with Morris water maze from 3 days to 7 days after injury. RESULTS: After trauma, some neurons displayed histopathologic changes of necrosis and apoptosis, axon myelin sheath internalization and disconnection. ERK1/2 phosphorylation protein was apparently increased at 30 min after injury, approached peak at 6 h and continued to 24 h. c-fos protein was markedly increased at 30 min after injury, approached peak at 6 h and returned to bottom at 24 h. The number of apoptotic nerve cells increased at 6 h after and approached peak at 72 h. Latencies of searching safety island prolonged. Rats treated with U0126 had reduction in ERK1/2 activity, c-Fos protein, neuronal apoptosis and searching safety island latencies. CONCLUSION: The activated ERK1/2 signaling pathway plays an important role in processing of nerve cell apoptosis after severe DBI.

OBJECTIVE: To investigate the relation betwe en necrosis and apoptosis in the hippocampus of exogenous bFGF on this process. METHODS: With Marmarou's method we produced a severe diffuse brain injury and studied the changes in the hippocampus by adapting a modified T dT-mediated dUTP-biotin nick end labeling (TUNEL) method. At the same time we observed the effect of exogenous bFGF on neuronal necrosis and apoptosis. RESULTS: We found that together with cell necrosis there was an increase in the number of apoptotic neurons in the hippocampus CA2-3 sectors a s early as 4 h after injury, with numbers reaching a maximum at 7 d. Exogenous b FGF resulted in a definite reduction in the amount of necrosis and apoptosis. CONCLUSIONS: Neuronal necrosis and apoptosis occur in combinati on after brain injury and that one of the causes may be the insufficience expres sion of the bFGF gene in the hippocampus after severe injury. Exogenous bFGF and similar substance may prove clinically useful after brain injury by reducing ce ll necrosis and apoptosis.

Objective To determine the clinical value of pulsed dual-wavelength 595 and 1064 nm laser in treating superficial infantile hemangioma. Methods The treatments were conducted in 260 patients with 270 lesions. The location of the lesions were 41 cases in scalp, 88 in face and neck, 79 in trunk and 62 in arms and legs. All lesions were treated several times with 595 nm (Group 1) and/or 1064 nm (Group 4) multiplex mode until the treatment was finished. The frequency of treatment was one to six times. The interval of the treatment was four weeks. Results The total effective rate was 95.56 %, with atrophic scar (8.15 %) but without severe ulcer. No significance was found as compared Group 1 parameter with Group 4 in formation of atrophic scars (x2 = 1. 870, P＞0.05). Significant difference in the incidence of atrophic scars was found among different location of lesions (x2 = 15. 743, P＜0. 01) : lesion in face had the highest probability. No obvious difference was found between different location of lesion and the frequence of treatment (x2 = 21.164, P＞0.05), and so did with the efficacy (x2 = 11. 597, P＞0.05). The size and the thickness of lesion had significant difference in the time of treatment (x2 = 58. 171, P＜0. 01 and x2 = 11. 583, P＜0.05, respectively).Conclusions 595nm/1064 nm dual-wavelength laser is a safe and effective tool for treating superficial infantile hemangioma. Choice of treatment parameter in the mode depends on the features of vascular lesions.