Effect of gene transfection at different time on bone mineral density and strength of newly formed bone in mandibular distraction gap in rabbit / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of gene transfection at different time on bone mineral density and strength of newly formed bone in mandibular distraction gap in rabbit, so as to explore the optimal time for gene therapy and enhance the therapeutic effect.</p><p><b>METHODS</b>48 New-Zealand rabbits were employed to receive mandibular osteotomy and implantation of distraction devices bilaterly. Then the rabbits were randomly divided into 4 groups as group A, B and C and D. The animals in group A, B, and C were transfected with recombinant plasmids pIRES-hBMP2-hVEGF165 via electroporation-mediated approach at latency period, distraction period, consolidation period respectively. Group D was used as control group without gene transfection. After 3 days of latency period, the distraction devices were activated at the rate of 0.8 mm per day for 10 days. Three rabbits in each group were sacrificed at 1 wk, 2 wk, 4 wk and 8 wk of consolidation respectively. The mandibles were harvested and the left mandible received X-ray examination for bone healing, and quantitative computed tomography (QCT) dectection for the bone mineral density (BMD) of newly formed bone in the distraction gap. The biomechanical properties of the new generation bone at 4 th and 8 th week of consolidation in each group were detected by three point bending test.</p><p><b>RESULTS</b>The bone mineral density and the biomechanical strength of newly formed bone increased along the length of consolidation in each group. After 1 week of consolidation, there was no significant difference in BMD among group A (83.43 +/- 9.96), group B (92.29 +/- 11.25), group C (89.93 +/- 14.15), P > 0.05. But the BMD of group A, B and C was higher than that of group D (70. 31 +/- 3.30), P < 0.05. After 2wk, 4 wk and 8 wk of consolidation, the BMD of group B (137.54 +/- 7.20,492.93 +/- 17.57, 790.48 +/- 12.19) was significantly higher than those of group A (121.44 +/- 9.27, 396.15 +/- 15.70, 603.39 +/- 16.46), C (125.06 +/- 7.24, 464.15 +/- 15.45, 764.15 +/- 17.28), and D (98.86 +/- 8.13, 336.45 +/- 11.95, 577.89 +/- 18.43), P < 0.05. The biomechanical parameters were also higher in group B than those of group A, C and D after four and eight weeks of consolidation (P < 0.05).</p><p><b>CONCLUSION</b>It is better to transfect gene at the beginning of distraction (distraction period) than at other stages of DO. In this way, more remarkable effect could be obtained on new bone formation. It suggests that the distraction stage is the optimal time for gene therapy.</p>

Effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit.</p><p><b>METHODS</b>Bilateral mandibular osteotomy was performed in 45 New-Zeland rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/day for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups. 2 microg (0.1 microg/microl) of pIRES-hVEGF165-hBMP2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES and the same volume of normal saline (NS) was injected into the distraction area in each group, respectively. After injection, electroporation was performed in every group. Three animals in each group were sacrificed at 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations. The expression of cyclins A, D1 ,E in positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with the single factor analysis of variance and q test.</p><p><b>RESULTS</b>Cyclins A, D1, E staining was mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues around the new bone. The expression reached to the peak at 7th day of consolidation, and decreased at 14th day, and weak at 28th day. Image analysis results showed that, at 7th day, the expression absorbance A in group C (0.59 +/- 0.14) was the strongest, compared to group A (0.41 +/- 0.13), B (0.38 +/- 0.14), D (0.34 +/- 0.12) and E (0.31 +/- 0.10), showing a significant difference (P < 0.05, P < 0.01). There was no significance difference between group A and B (P > 0.05), but the difference between group A/B and group D/E (P < 0.05). At 14th and 28th day, there was no significant difference among group A (0.39 +/- 0.11), B (0.34 +/- 0.10) and C (0.33 +/- 0.09) (P > 0.05), but there was significant difference between group A/B/C and group D (0.19 +/- 0.12) or E (0.14 +/- 0.04) (P < 0.05 or P < 0.01).</p><p><b>CONCLUSIONS</b>Electroporation-mediated gene transfection can promote cyclins A, D1, E expression effectively, which may promote cell differentiation and proliferation, stimulate extracellular matrix synthesis and new bone formation in distraction gap.</p>

Influence of serum from rats with scald on the cytoskeleton of colonic smooth muscle cells of rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the influence of serum from scalded rats on the cytoskeleton of colonic smooth muscle cells (CSMC) of rats cultured in vitro, and to probe the possible mechanism of gastrointestinal motility disorder after burn.</p><p><b>METHODS</b>CSMC isolated from healthy adult Wistar rat were cultured and divided into scald serum group (SS) and normal serum group (NS) according to the random number talbi. Two normal Wistar rats were used, one of which was inflicted with deep partial-thickness scald. Serum was obtained from blood collected from these two rats respectively and diluted to 20% in concentration. Serum from scald and normal rats were respectively added to the culture of CSMC in SS and NS groups. The expression of actin and the relative content of β-tubulin in CSMC was respectively determined with flow cytometry and Western blot at post treatment hour (PTH) 1, 3, 6, and 12 (with 10 samples in each group at each time point). Data were processed with t test.</p><p><b>RESULTS</b>Fluorescence intensity of actin in SS group at PTH 1, 3, 6, and 12 was respectively 59 ± 4, 26 ± 6, 39 ± 6, and 42 ± 6, all significantly lower than those in NS group (95 ± 10, 91 ± 10, 102 ± 9, and 97 ± 9, with t value respectively 10.528, 18.069, 18.748, 16.647, P < 0.05 or P < 0.01). In SS group, the fluorescence intensity decreased to the nadir at PTH 3, and then increased persistently at PTH 6 and 12. (2) Relative content of β-tubulin in SS group at PTH 1, 3, 6, and 12 was respectively 14.44 ± 0.26, 8.61 ± 0.19, 11.76 ± 0.31, and 12.13 ± 0.29, all significantly less than those in NS group (22.37 ± 1.15, 21.87 ± 1.79, 23.24 ± 1.55, and 21.99 ± 2.02, with t value respectively 21.176, 23.365, 23.000, 15.273, P values all below 0.01). In SS group, the relative content of β-tubulin decreased to the nadir at PTH 3 and increased slowly at PTH 6 and 12.</p><p><b>CONCLUSIONS</b>The reduction of CMSC content which has the tendency of increasing later, can be attributed to the influence of scald serum in initial stage. This may be related to the tolerance and adaptation to scald serum and self-repair of CMSC.</p>

The effect of electroporation mediated gene therapy on bone mineral density and strength of new-formed bone in mandibular distraction gap in rabbit / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the effect of electroporation mediated gene therapy on bone mineral density and strength of new-formed bone in mandibular distraction gap, so as to enhance the osteogenesis and shorten the distraction term.</p><p><b>METHODS</b>New-Zealand rabbits were employed. The distraction began after 3 days of latency period at the rate of 0. 8 mm per day for 7 days. After distraction, the rabbits were randomly divided into 5 groups to receive injection in the distraction gap with recombinant plasmid 2 microg (0.1 microg/microl) pIRES-hVEGF165-hBMP2 in group A, with recombinant plasmid pIRES-hBMP2 in group B, with recombinant plasmid pIRES-hVEGF165 in group C, with pIRES in group D, and with normal saline (NS) in group E. After injection, electroporation was performed in all the groups. After 1 week, 2 weeks, 4 weeks and 8 weeks of consolidation, all the animals underwent X-ray and quantitative computed tomography (QCT). The new-formed bone in distraction gap was selected as regions of interest (ROI) to measure the bone mineral density(BMD). Then the rabbits were sacrificed and the new-formed bone samples were harvested to detect 3-point crushing strength.</p><p><b>RESULTS</b>BMD of newly formed bone in group A, B and C was markedly higher than that in group D and E (P < 0.01). After 2 weeks of consolidation, BMD in group A was much higher than that in the other groups, but there was no difference between group B and C. After 4 weeks of consolidation, BMD in group A and B was markedly higher than that in group C, D and E (P < 0.01). After 8 weeks of consolidation, BMD in group A was markedly higher than that in the other groups. While the BMD was not significantly different between group B and C, but the BMD in group B and C was higher than that in group D and E (P < 0.01). After 4 weeks of consolidation, the 3-point crushing strength of newly formed bone in group A was markedly higher than that in group B,C, D and E (P < 0.01), which was still the same after 8 weeks of consolidation. And the crushing strength in group B was higher than that in group C, D and E (P < 0. 05).</p><p><b>CONCLUSIONS</b>Electroporation-mediated transfection of recombinant plasmid pIRES-hVEGF165-hBMP2 could greatly enhance osteogenesis and calcification. A combination of VEGF and BMP may promote osteogenesis and angiogenesis simultaneously, so as to magnify the effect of each growth factor, resulting a synergetic effect.</p>

Establishment of animal model for electroporation-mediated gene therapy in distraction osteogenesis of rabbit mandible / 中华整形外科杂志

<p><b>OBJECTIVE</b>To evaluate the feasibility of electroporation-mediated transfection of recombinant plasmid to mandibular distraction area of rabbit in vivo.</p><p><b>METHODS</b>New-Zeland rabbit were employed. The mandible was distracted 3 days after operation at a rate of 0.8 mm per day for 7 days. The rabbits were randomly divided into 3 groups as group A (recombinant plasmid pIRES-VEGF165-EGFP), group B (recombinant plasmid plRES-VEGF165-EGFP) and group C (normal saline). The rabbits were sacrified at 3 hours, 1, 3, 7 and 14 d after injection respectively. The tissue at the distraction area was taken out for frozen section. The gene expression was assessed by the detection of expression of green fluorescence protein (GFP) using fluorescence microscope. The liver and kidney function test (ALT, AST, BUN, Scr) and the histological examination of heart, liver and kidney were also performed.</p><p><b>RESULTS</b>GFP was seen in the distraction area in group A and group B 3 hours after injection, which increased at the 1st day, reached peak value at the 3rd day, decreased at the 7th day and was very lower at 14th day. The GFP expression was much stronger in group A than in group B. GFP was not expressed in group C. There was no statistical difference in the concentration of ALT, AST, BUN and Scr in serum of rabbits among the three groups.</p><p><b>CONCLUSIONS</b>Electroporation-mediated transfection of recombinant plasmid can be expressed in the distraction area of rabbits, and there was no toxicity to the liver and kidney of rabbits. Electroporation could obviously improve transfection efficiency in vivo. It indicates that electroporation-mediated transfection of recombinant plasmid to distraction area tissue of rabbits is feasible.</p>

An experimental study on the prevention and treatment of postburn intestinal injury and bacterial translocation by Sijunzi decoction in scalded rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effects of traditional Chinese herbal medicine Sijunzi decoction on amelioration of postburn intestinal injury in scalded rats.</p><p><b>METHODS</b>One hundred and eighty Wistar rats were randomly divided into 3 groups, i.e. scald and treatment (T), scald control (S) and normal control (C) groups. The rats in T group were gavaged with the decoction consisting of tangshen, tuckahoe, large head atractylodes rhizome, glycyrrhizic and rhubarb in a dose of 2 ml twice daily, while the rats in C group were just gavaged with the same amount of distilled water. The rats were sacrificed according to the scheduled postburn observation timepoints. The contents of TNF, NO, MDA and ATPase activity in rat plasma and the intestinal mucosa and the S-IgA content in the intestinal mucus were determined respectively. The changes in histopathology of intestinal mucosa were observed. The samples from internal organ tissue and blood were obtained for bacterial culture.</p><p><b>RESULTS</b>The contents of TNF, NO and MDA in the intestinal mucosa tissue and the rat plasma in scalded rats were lowered significantly by Sijunzi decoction. Furthermore, S-IgA secretion from intestinal mucous cells was maintained by Sijunzi decoction. T cell count was recovered and intestinal mucous barrier injury were lessened, and the bacterial positive rate in the internal organs was decreased.</p><p><b>CONCLUSION</b>Traditional Chinese herbal medicine Sijunzi decoction might be helpful in alleviation of postburn intestinal injury and in the prevention of intestinal bacterial translocation.</p>

xperimental study on the effect of Naogongfude on the aging of the rat brain / 第三军医大学学报

Objective　To evaluate the effect of Naogongfude (NGFD, a traditional Chinese medicine orally administrated) on learning and memory and the expressions of synaptophysin (SY) and Tau-protein in cerebral cortex and hippocampus in aged rats and to explore the possible mechanism. Methods　Rats were divided into normal and neural disturbance groups according to the outcomes of active avoidance reaction (AAR) test, and then each group was randomly divided into control and NGFD-treated experimental groups. Animals were orally fed with NGFD for 2 months (5 ml/d) in experimental group or routinely fed in control and taken AAR and passive avoidance reaction (PAR) tests. After the rats were sacrificed, the synaptosome count, the expression of SY and Tau-protein, and the neuron apoptosis in cerebrum were examined. Results　The rats after 2-month NGFD administration had an increased AAR acquisition, obviously delayed AAR extinction and prolonged step through latency (STL) of PAR. The number of synaptosomes was raised and the immunoreactive intensity of synaptophysin was increased remarkedly, while Tau-protein immunoreactivity and apoptotic cells were decreased in cerebrum. Conclusion　NGFD does have the effect of improving brain function and putting off the aging of rat brain according to the results of behavior study and morphological observation.

xperimental study on the effect of Naogongfude on the aging of the rat brain / 第三军医大学学报

Objective　To evaluate the effect of Naogongfude (NGFD, a traditional Chinese medicine orally administrated) on learning and memory and the expressions of synaptophysin (SY) and Tau-protein in cerebral cortex and hippocampus in aged rats and to explore the possible mechanism. Methods　Rats were divided into normal and neural disturbance groups according to the outcomes of active avoidance reaction (AAR) test, and then each group was randomly divided into control and NGFD-treated experimental groups. Animals were orally fed with NGFD for 2 months (5 ml/d) in experimental group or routinely fed in control and taken AAR and passive avoidance reaction (PAR) tests. After the rats were sacrificed, the synaptosome count, the expression of SY and Tau-protein, and the neuron apoptosis in cerebrum were examined. Results　The rats after 2-month NGFD administration had an increased AAR acquisition, obviously delayed AAR extinction and prolonged step through latency (STL) of PAR. The number of synaptosomes was raised and the immunoreactive intensity of synaptophysin was increased remarkedly, while Tau-protein immunoreactivity and apoptotic cells were decreased in cerebrum. Conclusion　NGFD does have the effect of improving brain function and putting off the aging of rat brain according to the results of behavior study and morphological observation.