Effects of exogenous recombinant human basic fibroblast growth factor on the healing of muscles in rats after deep tissue injury of pressure ulcers
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摘要: 目的 探讨外源性重组人bEGF(rhbFGF)对大鼠压疮深部组织损伤(DTI)后肌肉修复的作用。 方法 将48只SD大鼠按随机数字表法分为正常对照组、损伤对照组、伤后4 d组、伤后7 d组、伤后14 d组、伤后21 d组,每组8只。正常对照组大鼠不做任何处理,后5组大鼠在两侧后肢股薄肌处建立压疮DTI模型。损伤对照组大鼠伤后不做任何处理;另4组大鼠在伤后即刻分别于左侧后肢股薄肌处皮下注射100 μg/mL的rhbFGF 0.1 mL,右侧后肢股薄肌处注射等体积生理盐水(NS),隔天注射1次。损伤对照组大鼠于伤后即刻、正常对照组大鼠于相同时相点,另4组大鼠分别于伤后第4、7、14、21天切取两侧后肢股薄肌处肌肉组织。HE染色观察股薄肌组织形态,免疫荧光法检测成肌素表达,蛋白质印迹法检测肌肉结构蛋白肌球蛋白重链(MyHC)、磷酸化蛋白激酶B(Akt)、磷酸化哺乳动物雷帕霉素靶蛋白(mTOR)表达。对数据行单因素方差分析、LSD检验。 结果 (1)正常对照组大鼠股薄肌细胞胞核大小均匀、排列紧密、结构清晰,无明显的炎性细胞浸润;损伤对照组大鼠股薄肌出现肌细胞固缩、溶解断裂,结构紊乱的病理退化现象;伤后4 d组、伤后7 d组、伤后14 d组、伤后21 d组大鼠经rhbFGF处理的股薄肌细胞肿胀、炎性浸润、结构紊乱等病理损伤现象均较组内经NS处理的股薄肌减轻,肌细胞增殖数目增多。(2)正常对照组、损伤对照组大鼠股薄肌成肌素表达阳性细胞数分别为(28±17)、(42±28)个;伤后4 d组、伤后7 d组、伤后14 d组大鼠经NS处理的股薄肌成肌素表达阳性细胞数分别为(100±50)、(196±87)、(460±110)个,经rhbFGF处理的股薄肌成肌素表达阳性细胞数分别为(174±34)、(717±182)、(613±122)个,各组大鼠经rhbFGF处理的股薄肌成肌素表达阳性细胞数明显多于组内经NS处理的股薄肌(
P <0.05或P <0.01);伤后21 d组大鼠经rhbFGF处理的股薄肌成肌素表达阳性细胞数为(109±34)个,明显低于组内经NS处理的股薄肌[(218±71)个,P <0.05]。(3)正常对照组大鼠股薄肌MyHC表达较高,损伤对照组大鼠股薄肌MyHC表达较正常对照组大鼠有所降低;伤后4 d组、伤后7 d组、伤后14 d组、伤后21 d组大鼠经NS处理的和经rhbFGF处理的股薄肌MyHC表达均呈逐渐增高趋势,各组大鼠经rhbFGF处理的股薄肌MyHC表达明显高于组内经NS处理的股薄肌(P <0.05或P <0.01);伤后21 d组大鼠经rhbFGF处理的股薄肌MyHC表达较高,与正常对照组大鼠相近(P >0.05)。正常对照组大鼠股薄肌磷酸化Akt、磷酸化mTOR表达较低,损伤对照组大鼠股薄肌磷酸化Akt表达较正常对照组大鼠有少量增高,磷酸化mTOR表达有少量降低;伤后4 d组、伤后7 d组、伤后14 d组、伤后21 d组大鼠经rhbFGF处理的股薄肌磷酸化Akt、磷酸化mTOR表达呈先升高后降低的趋势;伤后4 d组大鼠经rhbFGF处理的股薄肌磷酸化Akt、磷酸化mTOR表达明显低于组内经NS处理的股薄肌(P <0.05或P <0.01);伤后7 d组、伤后14 d组、伤后21 d组大鼠经rhbFGF处理的股薄肌磷酸化Akt、磷酸化mTOR表达明显高于组内经NS处理的股薄肌(P <0.05或P <0.01)。 结论 外源性rhbFGF能有效促进大鼠压疮DTI肌肉修复,其作用机制可能与提高成肌素表达,增强肌肉生长相关信号通路的蛋白表达有关。-
关键词:
- 肌肉发育 /
- 成纤维细胞生长因子2 /
- 肌球蛋白重链 /
- 压疮 /
- 深部组织损伤 /
- 蛋白激酶B /
- 哺乳动物雷帕霉素靶蛋白
Abstract: Objective To investigate the effect of exogenous recombinant human basic fibroblast growth factor (rhbFGF) on the healing of muscles in rats after deep tissue injury of pressure ulcers. Methods Forty-eight SD rats were randomly divided into normal control group, injury control group, post injury day (PID) 4 group, PID 7 group, PID 14 group, PID 21 group according to the random number table, with 8 rats in each group. The rats in normal control group did not receive any treatment, whereas the rats in the latter 5 groups were established the deep tissue injury of pressure ulcer model on both sides of the gracilis muscle on the hind limb. The rats in injury control group did not receive any treatment after injury, while the rats in the latter 4 groups were given subcutaneous injection of 0.1 mL rhbFGF to the left gracilis in a dosage of 100 μg/mL immediately after injury, and an equal volume of normal saline (NS) was injected to right gracilis, once every other day. The rats in injury control group were sacrificed immediately after injury, and the rats in normal control group were sacrificed at the same time point. The rats in the other 4 groups were sacrificed on PID 4, 7, 14, 21, and the gracilis muscles on both sides were harvested respectively. The morphology of the gracilis muscle was examined after HE staining. The expression of myogenin in the tissues was detected by immunofluorescence method. The levels of muscle structural proteins myosin heavy chain (MyHC), phosphorylated protein kinase B (Akt), and phosphorylated mammalian target of rapamycin (mTOR) were determined by Western blotting. Data were processed with one-way analysis of variance and LSD test. Results (1) In normal control group, the nuclei of graciles cells were in uniform size, and they were closely arranged with clear structure, and there were no significant infiltration of inflammatory cells. In injury control group, the nuclei of graciles cells showed signs of pyknosis, dissolution, fracture and structural disorder. Swelling of muscle cells, inflammation infiltration, structural disorder and other pathological signs of injury phenomena in graciles of PID 4 group, PID 7 group, PID 14 group, PID 21 group after rhbFGF treatment were milder compared with those after NS treatment. In addition, the numbers of regenerated myocytes in graciles of PID 4 group, PID 7 group, PID 14 group, PID 21 group after rhbFGF treatment were higher than those after NS treatment. (2) The numbers of graciles myogenin positive cells in normal control group and injury control group were respectively 28±17 and 42±28. The numbers of graciles myogenin positive cells in PID 4 group, PID 7 group, PID 14 group after NS treatment were 100±50, 196±87, 460±110 respectively, while the numbers of graciles myogenin positive cells in PID 4 group, PID 7 group, PID 14 group after rhbFGF treatment were 174±34, 717±182, 613±122 respectively, and the numbers of graciles myogenin positive cells after rhbFGF treatment were significantly higher than those after NS treatment in each group(P <0.05 orP <0.01). The number of graciles myogenin positive cells in PID 21 group after rhbFGF treatment was 109±34, which was significantly lower than that after NS treatment (218±71,P <0.05). (3) The expression of MyHC in graciles in normal control group was high, which was decreased in injury control group. Both the expressions of MyHC in graciles in PID 4 group, PID 7 group, PID 14 group, PID 21 group after treatment of NS and rhbFGF showed a trend of gradual elevation, while the expressions of MyHC in graciles after rhbFGF treatment were significantly higher than those after NS treatment (P <0.05 orP <0.01). The expression of MyHC in graciles in PID 21 group showed a high level, and it was similar to that of the normal control group (P >0.05). The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of normal control group were low, and the expression of phosphorylated Akt in graciles increased in injury control group, while the expression of phosphorylated mTOR in graciles decreased in injury control group. The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of PID 4 group, PID 7 group, PID 14 group, PID 21 group after treatment with rhbFGF showed a trend of elevation in the beginning but declined afterwards. The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of PID 4 group after rhbFGF treatment were significantly lower than those after NS treatment (P <0.05 orP <0.01). The expressions of phosphorylated Akt and phosphorylated mTOR in graciles of PID 7 group, PID 14 group, PID 21 group after rhbFGF treatment were significantly higher than those after NS treatment (P <0.05 orP <0.01). Conclusions Exogenous rhbFGF may effectively facilitate the healing of muscle structure and accelerate the regeneration of muscles in rats after deep tissue injury of pressure ulcers, and its mechanism may be related to the improvement of the expression of myogenin and enhancement of the expression of protein of muscle growth-related signaling pathways.
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