Objective To investigate an optimal method for SD rat skeletal muscle decellularization. Methods Sixteen SD rats (male and female) weighing 180-200 g were used. Thirty-six skeletal muscle bundles obtained from 10 rats were randomly divided into 3 groups: normal group (group A, n=4) received non-decellularization; time group (group T, n=16) andconcentration group (group C, n=16) underwent decellularization using hypotonic-detergent method. Concentration of sodium dodecyl sulfate (SDS) was 1.0% for T group, which was subdivided into groups T1, T2, T3 and T4 (n=4 per subgroup) according to different processing durations (24, 48, 72 and 96 hours). Group C was treated for 48 hours and subdivided into groups C1, C2, C3 and C4 (n=4 per subgroup) according to different SDS concentrations (0.5%, 1.0%, 1.5% and 2.0%). The muscle bundles of each group underwent HE staining observation and hydroxyproline content detection in order to get the optimal decellularization condition. Seven of 14 complete skeletal muscle bundles obtained from 6 SD rats were treated with the optimal decellularization condition (experimental group), and the rest 7 muscle bundles served as normal control (control group). The muscle bundles of each group were evaluated with gross observation, Masson staining and biomechanical test. Results HE staining: there was no significant difference between groups T1, T2, C1, C2 and C3 and group A in terms of muscle fiber; portion of muscle fibers in group C4 were removed; muscle fibers in group T3 were fully removed with a complete basement membrane structure; muscle fibers of group T4 were fully removed, and the structure of basement membrane was partly damaged. Hydroxyprol ine content detection: there was no significant difference between group A and groups C1, C2, C3, T1 and T2 (P gt; 0.05); significant difference was evident between group A and groups C4, T3 and T4 (P lt; 0.05); the difference between group C4 and groups T3and T4 was significant (P lt; 0.05); no significant difference was evident between group T3 and group T4 (P gt; 0.05). The optimal decellularization condition was 4 , 1.0% SDS and 72 hours according to the results of HE staining and hydroxyproline content detection. Gross observation: the muscle bundles of the experimental group were pall id, half-transparent and fluffier comparing with the control group. Masson staining observation: the collagen fibers of the experimental group had a good continuity, and were fluffier comparing with control group. Biomechanics test: the maximum breaking load of the experimental group and the control group was (1.38 ± 0.35) N and (1.98 ± 0.77) N, respectively; the maximum extension displacement of the experimental group and the control group was (3.19 ± 3.23) mm and (3.56 ± 2.17) mm, respectively; there were no significant differences between two groups (P gt; 0.05). Conclusion Acellular matrix with intact ECM and complete removal of muscle fibers can be obtained by oscillatory treatment of rat skeletal muscle at 4℃ with 1% SDS for 72 hours.
Objective To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue. Methods The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues. Results The contents of hydroxyproline in the hypertrophic scar(84.10±1.76) and keloid tissue (92.38±2.04) were significantly higher than that of normal skin tissue (26.52 ± 4.10) (P lt; 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 ± 0.63) and keloid tissue (0.84 ± 0.04) were higher than that of normal skin tissue ( 0.09 ± 0.25) (P lt; 0.01). Conclusion Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.
ObjectiveTo explore the effect of exogenous ghrelin on early recovery of rats after subtotal gastrectomy. MethodsTwelve rats undergoing subtotal gastrectomy (B-Ⅰtype) were randomly divided into two groups, and saline or ghrelin was intraperitoneally injected in two groups, respectively. The body weight and daily food intake were measured before operation and on 1-7 d after operation. Rats were killed on day 7 after operation and the expressions of ghrelin mRNA in the fundus of stomach and anastomotic stoma was determined by realtime fluorescent quantitative PCR assay. The anastomotic bursting pressure and hydroxyproline content of anastomotic stoma tissues were also detected. ResultsThere was no significant difference (P＞0.05) in pre and postoperative body weight between two groups. Gradual decrease in postoperative body weight among the rats of saline group was observed which was significantly lower than that before operation (Plt;0.01). Body weight reached it’s lowest on day 1 after operation (Plt;0.01), after which it gradually increased but was still lower than that before operation (Plt;0.01). The postoperative body weight of rats in ghrelin group gradually decreased too, and was also significantly lower than preoperative body weight (Plt;0.01), except for the day 1 after operation (P=0.693). It reached the lowest on day 4 after operation (Plt;0.01), then it gradually increased but was still lower than that before operation (Plt;0.05 or Plt;0.01). The cumulative food intake of rats in ghrelin group was （52.50±6.77） g, which was significantly higher than that in saline group 〔(45.67±7.47) g〕, Plt;0.05. On day 7 after operation, relative expression of ghrelin mRNA in the fundus of stomach of rats in ghrelin group was 0.08±0.04, which was significantly lower than that in saline group (0.22±0.07), Plt;0.01. Compared with saline group, ghrelin-treated rats displayed significantly higher bursting pressure 〔（155.83±6.62） mm Hg vs. （172.33±10.44） mm Hg, Plt;0.05〕 higher hydroxyproline content 〔 (0.43±0.05) μg/mg wet tissue vs. （0.50±0.29） μg/mg wet tissue, Plt;0.01〕 at the anastomotic stoma. ConclusionGhrelin may effectively promote the early recovery of rats after subtotal gastrectomy.
ObjectiveTo explore the feasibility of medical ozone in treatment of pulmonary fibrosis. MethodsForty Wistar rats were randomly divided into an experimental group and a control group, with 20 rats in each group.All rats were intratreacheally instilled with bleomycin to induce pulmonary fibrosis.Then the rats were intraperitoneally injected with physiological saline every other day in the control group, and with medical ozone every other day in the experimental group.After 28 days, 10 rats in each group were sacrificed after lung function test.Right lung tissues were sampled for pathological examination, and left lung tissues were sampled for measurement of superoxide dismutase (SOD) and hydroxyproline.The remaining 10 rats in each group continued to be normally fed and intraperitoneally injected for observation of the survival time. ResultsThe lung function of the control group significantly decreased compared with the experimental group.The degree of lung fibrosis in the control group was more severe than that in the experimental group (lung fibrosis score: 1.9±0.5 vs.1.2±0.4, P < 0.05). The level of SOD in lung tissue was significantly higher and the level of hydroxyproline was significantly lower in the experimental group compared with the control group [(208.48±29.37)U·mg-1·pro-1 vs.(163.34±21.42) U·mg-1·pro-1, (2.25±0.28) mg/g vs.(2.68±0.37) mg/g, P < 0.05].The rats in the experimental group had longer survival time compared with the control group (79 d vs.59 d, P < 0.05). ConclusionMedical ozone can delay the progress of pulmonary fibrosis in rats.