Effects of basic fibroblast growth factor on healing of Mycobacterium tuberculosis infective wound in New Zealand rabbit after debridement
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摘要: 目的 探讨外用碱性成纤维细胞生长因子(bFGF)对新西兰兔结核性创面清创后愈合的影响。 方法 取32只3~4个月龄雌雄不拘新西兰兔,均于臀部皮内注射弗氏完全佐剂0.1 mL致敏,6周后于兔背部两侧注射浓度5×107集落形成单位/mL的牛型结核分枝杆菌减毒株菌液各0.5 mL,建立新西兰兔结核性创面模型。建模成功后,将32只兔按随机数字表法分为生长因子组、抗结核药物组、联合治疗组和空白对照组,每组8只。对创面进行彻底清创后,生长因子组兔创面均匀涂抹重组牛bFGF凝胶(300 IU/cm2,每个创面约涂0.45 g);抗结核药物组兔创面采用6 mL异烟肼注射液和0.15 g利福平粉针剂混匀浸透纱布后覆盖;联合治疗组兔创面同前用重组牛bFGF凝胶均匀涂抹后,用异烟肼和利福平浸透纱布覆盖;空白对照组兔创面仅用无菌纱布覆盖。各组隔日换药1次,至创面完全愈合。于术后即刻、7、14、21、28 d,肉眼观察创面大体情况,拍照并计算术后7、14、21、28 d的创面愈合率并记录创面完全愈合时间。术后7、14、21、28 d,取创缘组织行苏木精-伊红染色、Masson染色观察组织形态学情况。于术后21 d,取创缘组织采用免疫组织化学法行微血管计数。于术后7、14、21、28 d,采用酶联免疫吸附测定法检测创缘羟脯氨酸含量。以上实验样本数均为8。对数据行重复测量方差分析、析因设计方差分析、单因素方差分析、LSD检验及Bonferroni校正。 结果 (1)4组兔均存活至实验完成。术后即刻,4组兔创面基底水肿明显。术后7 d,4组兔创面较前缩小,创面覆盖痂皮,水肿减轻,其中生长因子组和联合治疗组兔创面组织更加红润。术后14 d,4组兔创面缩小明显,抗结核药物组和联合治疗组兔创面均无明显渗出;生长因子组兔1个创面和空白对照组兔2个创面红肿,有脓性分泌物。术后21 d,生长因子组、联合治疗组兔创面基本愈合,空白对照组兔有2个创面仍有脓性分泌物,愈合迟缓。术后28 d,4组兔创面基本愈合,空白对照组兔有2个创面红肿,难以愈合。(2)术后7~28 d,生长因子组、抗结核药物组及联合治疗组兔创面愈合率均明显高于空白对照组(
P <0.05)。术后14、21 d,生长因子组和联合治疗组兔创面愈合率明显高于抗结核药物组(P <0.05)。术后7~28 d,生长因子组和联合治疗组兔创面愈合率相近(P >0.05)。(3)生长因子组、抗结核药物组及联合治疗组兔创面完全愈合时间明显短于空白对照组(P <0.05),生长因子组和联合治疗组兔创面完全愈合时间明显短于抗结核药物组(P <0.05),生长因子组和联合治疗组兔创面完全愈合时间相近(P >0.05)。(4)术后7 d,4组兔创面组织均可见大量炎性细胞浸润,生长因子组、抗结核药物组和联合治疗组兔创面组织可见少量上皮细胞生长。术后14 d,抗结核药物组和联合治疗组兔创面组织炎性细胞明显减少,生长因子组和联合治疗组兔创面组织上皮细胞增加明显。术后21 d,生长因子组和联合治疗组兔创面组织结构完整,层次较为清晰;抗结核药物组和空白对照组兔创面组织新生上皮较薄,且空白对照组有炎性细胞浸润。术后28 d,4组兔创面组织皮肤组织完整,其中生长因子组、抗结核药物组、联合治疗组兔创面组织新生上皮较空白对照组厚。(5)术后7、14 d,生长因子组和联合治疗组兔创面组织胶原纤维数量多于其余2组。术后21 d,生长因子组和联合治疗组兔创面组织中成纤维细胞(Fb)排列密集,胶原纤维排列规则;抗结核药物组Fb密度尚可,胶原纤维排列杂乱;空白对照组兔创面组织Fb和胶原纤维稀少。术后28 d,4组兔创面组织胶原纤维数量接近正常皮肤组织,其中生长因子组和联合治疗组胶原纤维排列更加规则、有序。(6)术后21 d,生长因子组[(31.6±1.2)个]、抗结核药物组[(27.5±1.3)个]及联合治疗组[(32.8±1.6)个]兔每200倍视野下创缘微血管数明显多于空白对照组[(22.3±1.7)个,P <0.05],生长因子组和联合治疗组兔创缘微血管数明显多于抗结核药物组(P <0.05),生长因子组和联合治疗组兔创缘微血管数相近(P >0.05)。(7)术后7、28 d,4组兔创缘羟脯氨酸含量相近(F =0.916、1.752,P >0.05)。术后14、21 d,生长因子组、抗结核药物组及联合治疗组兔创缘羟脯氨酸含量明显高于空白对照组(P <0.05),生长因子组和联合治疗组兔创缘羟脯氨酸含量明显高于抗结核药物组(P <0.05),生长因子组和联合治疗组兔创缘羟脯氨酸含量相近(P >0.05)。 结论 彻底清创后局部单独应用bFGF或联合抗结核药物均可促进新西兰兔结核性创面的愈合,优于单用抗结核药物。Abstract: Objective To investigate the effects of basic fibroblast growth factor (bFGF) on healing ofMycobacterium tuberculosis infective wound in New Zealand rabbit after debridement. Methods Thirty-two New Zealand rabbits (3 to 4 months old, no matter male or female) were intradermally injected with 0.1 mL of complete Freund′s adjuvant on the buttocks. Six weeks later, each rabbit was injected with 0.5 mL 5×107 colony forming unit/mLBacillus Calmette -Guerin on both sides of the back to reproduce the model ofMycobacterium tuberculosis infective wound in New Zealand rabbit. After successful modeling, the 32 rabbits were divided into growth factor (GF) group, antituberculosis drug (AD) group, combined treatment (CT) group, and blank control (BC) group according to the random number table, with 8 rabbits in each group. After a complete debridement, the wounds of rabbits in group GF were treated with recombinant bovine bFGF gel (300 IU/cm2, about 0.45 g for each wound), the wounds of rabbits in group AD were covered with gauze which was impregnated with 6 mL isoniazid injection and 0.15 g rifampicin powder-injection, the wounds of rabbits in group CT were covered with gauze which was impregnated with isoniazid injection and rifampicin powder-injection after being treated with recombinant bovine bFGF gel as before, the wounds of rabbits in group BC were covered with sterile gauze, with dressing change of once every two days until the wounds were completely healed. Immediately after surgery and on post surgery day (PSD) 7, 14, 21, and 28, the wounds of rabbits in each group were observed with naked eyes and photos. On PSD 7, 14, 21, and 28, the wound healing rate was calculated and the complete healing time of wound was recorded. On PSD 7, 14, 21, and 28, the tissue samples of wound edge were collected for histomorphological observation with hematoxylin and eosin staining and Masson staining. On PSD 21, the number of microvessels was counted with immunohistochemical method. On PSD 7, 14, 21, and 28, the content of hydroxyproline in wound edge was determined by enzyme-linked immunosorbent assay. The numbers of samples of above-mentioned experiments were all 8. Data were processed with analysis of variance for repeated measurement, analysis of variance of factorial design, one-way analysis of variance, least significant difference test and Bonferroni correction. Results (1) The rabbits in four groups all survived to the end of experiment. Immediately after surgery, edema was observed in basal wounds of rabbits in the four groups. On PSD 7, the wounds of rabbits in the 4 groups were contracted with scabs and less edema. The wounds of rabbits in groups GF and CT became redder. On PSD 14, the wounds of rabbits in the 4 groups contracted obviously. There were no obvious exudates in wounds of rabbits in groups AD and CT, while 1 wound of rabbit in group GF and 2 wounds of rabbits in group BC became red and swelling with purulent exudates. On PSD 21, wounds of rabbits in groups GF and CT were basically healed, while 2 wounds of rabbits in group BC healed slowly with purulent secretion. On PSD 28, wounds of rabbits in the 4 groups were basically healed, while 2 wounds of rabbits in group BC hardly healed with redness and swelling. (2) From PSD 7 to 28, the wound healing rates of rabbits in groups GF, AD, and CT were significantly higher than those in group BC (P <0.05). On PSD 14 and 21, the wound healing rates of rabbits in groups GF and CT were significantly higher than those in group AD (P <0.05). From PSD 7 to 28, the wound healing rates of rabbits in group GF were close to those in group CT (P >0.05). (3) The complete healing time of wounds of rabbits in groups GF, AD, and CT was significantly shorter than that in group BC (P <0.05). The complete healing time of wounds of rabbits in groups GF and CT was significantly shorter than that in group AD (P <0.05). The complete healing time of wounds of rabbits in group GF was close to that in group CT (P >0.05). (4) On PSD 7, a large number of inflammatory cells infiltration were observed in wound tissue of rabbits in the 4 groups and a few epithelial cells were observed in wound tissue of rabbits in groups GF, AD, and CT. On PSD 14, more epithelial cells were observed in wound tissue of rabbits in groups GF and CT, and an obvious reduction of inflammatory cells infiltration was observed in wound tissue of rabbits in groups AD and CT. On PSD 21, there was a complete wound tissue structure and distinctive nuance of dyeing in wound tissue of rabbits in groups GF and CT while thinner new epithelium in wound tissue of rabbits in groups AD and BC, and inflammatory cell infiltration was observed in wound tissue of rabbits in group BC. On PSD 28, there was a complete wound tissue structure in wound tissue of rabbits in the 4 groups, the new epithelium in wound tissue of rabbits in groups GF, AD, and CT was thicker than that in group BC. (5) On PSD 7 and 14, the quantity of collagen fibers in wound tissue of rabbits in groups GF and CT was larger than that in the other two groups. On PSD 21, a large quantity of fibroblasts and well reorganized collagen fibers were observed in wound tissue of rabbits in groups GF and CT, a moderate quantity of fibroblasts and collagen fibers in a random arrangement were observed in wound tissue of rabbits in group AD, and a little quantity of fibroblasts and collagen fibers were observed in wound tissue of rabbits in group BC. On PSD 28, the quantity of collagen fibers in wound tissue of rabbits in the 4 groups was close to that of normal skin tissue, and the collagen fibers performed more well reorganized in wound tissue of rabbits in groups GF and CT. (6) On PSD 21, the numbers of microvessels per 200-time visual field in wound edge of rabbits in groups GF (31.6±1.2), AD (27.5±1.3), and CT (32.8±1.6) were significantly higher than the number in group BC (22.3±1.7,P <0.05). The numbers of microvessels in wound edge of rabbits in groups GF and CT were significantly higher than the number in group AD (P <0.05). The number of microvessels in wound edge of rabbits in group GF was close to that in group CT (P >0.05). (7) On PSD 7 and 28, there were no statistically significant differences in content of hydroxyproline in wound edge of rabbits in the 4 groups (F =0.916, 1.752,P >0.05). On PSD 14 and 21, the content of hydroxyproline in wound edge of rabbits in groups GF, AD, and CT was significantly higher than that in group BC (P <0.05). The content of hydroxyproline in the wound edge of rabbits in groups GF and CT was significantly higher than that in group AD (P <0.05). The content of hydroxyproline in the wound edge of rabbits in group GF was close to that in group CT (P >0.05). Conclusions bFGF can be used solely or combined with AD to promoteMycobacterium tuberculosis infective wound healing in New Zealand rabbit after complete debridement of wound, which is better than single use of AD.-
Key words:
- Mycobacterium tuberculosis /
- Fibroblast growth factors /
- Wound healing /
- Models, animal
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