中医骨折分期治疗对骨折愈合bFGF、TGF-β、VEGF、BMP-2基因表达影响的实验研究
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摘要
1、研究背景
骨折分期的辨证施治方法是中医治疗骨折的一大特色,是前人经过不断总结发展而来的。中医骨伤学家总结了骨折三期辨证治疗,骨折三期治疗原则是中医药治疗骨折的指导原则。中医骨折三期治则临床疗效已证实,但实验证据不完善。虽然临床疗效已得到充分的验证,但对其研究往往局限于临床疗效观察。目前对中医骨折三期治则的作用存在诸多争议,无论在是否分期还是在分期的方法上都有明显的分歧,出现了不分期的治疗,以及分期的三期治疗和二期治疗的不同情况,使得骨折三期治疗做为中医药治疗骨折的指导原则的地位受到严峻的挑战,导致了中医药在治疗骨折的发展受到严重的制约。但是既往研究较多为临床疗效的初步观察,而且均是中药汤剂组与空白对照组、单味中药与空白对照组或某种中成药与空白对照组之间的相互做比较,所以认为不分期的一期治疗或二期治疗能够促进骨折愈合,但并均不能充分说明一期治疗与二期治疗疗效强于三期治疗。而关于中医骨折三期治疗原则的作用及其机制系统的实验研究,尚未见文献报道。
本课题组前期研究发现在SD大鼠骨痴愈合过程中,二期治疗不但可以明显提高其骨折愈合过程中bFGF (Basic fibroblast growth factor碱性成纤维细胞生长因子)的表达量,二期治疗对骨折愈合过程中bFGF表达的促进作用显著强于期治疗和一期治疗。并在前期的家兔骨折愈合过程的实验研究中,发现在骨折愈合时间及质量上分期的治疗组明显强于不分期治疗组,骨折三期治疗组无论在外骨膜、骨干细胞、骨小梁组织、骨痂质量及骨髓组织等骨折端组织的VEGF (Vascular endothelial growth factor血管内皮生长因子)和VEGF mRNA表达的促进作用明显优于一期治疗组。
bFGF有促血管形成、诱导胚胎发育、促进神经生长等功能。另外TGF-p (Transforming growth factor-β转化生长因子β),可促进骨膜间充质细胞增殖和分化,促进成骨细胞增殖、刺激Ⅱ型胶原、骨连接素和骨桥蛋白的合成,TGF-p在骨组织代谢过程中起重要的作用。而VEGF是目前公认最强的促血管形成因子,不仅能够促进血管重建,而且还能够增强软骨细胞活性,其他促血管生成因子对于血管重建时全部或部分通过VEGF的作用得以实现。bFGF与VEGF两因子都是对血管重建较为重要的因子,而骨的愈合过程中,骨形成的最关键因子的莫过于BMPs (Bone morphogenetic proteins骨形成蛋白),其具有诱导成骨作用,尤其是间充质细胞发生化学趋向、聚集、分化形成软骨和骨,最终形成骨髓,因此,被认为是最强的骨诱导分化因子,而实验研究方面对于在骨折愈合过程中较为重要的BMPS多进行其家族的BMP-2研究。我们为较全面的从分子生物水平更进一步探讨中医骨折分期治疗的作用及其机制,研究骨折治疗的合理分期方法,因此,本实验对中医分期治疗将从bFGF、TGF-β、VEGF及BMP-2四个生长因子同时进行全方位的分析。
2、研究目的
本研究拟通过对家兔双侧桡骨中下1/3旋前圆肌远端3mm横断骨折的bFGF、TGF-β、VEGF、BMP-2及其bFGFmRNA、TGF-PmRNA、VEGFmRNA、 BMP-2mRNA的基因表达、细胞因子的表达及其分布规律的影响情况,从分子水平层次对家兔桡骨下段骨缺损的愈合进行分期,进而从器官、细胞与分子水平系统地阐明中医骨折分期治疗对骨折愈合的作用及其机制,确定最佳的分期治疗方案,阐明中医骨折分期治疗的原则,从现代医学的角度为中医药分期治疗骨折提供更为科学的理论根据,指导骨折的治疗。
3、材料与方法
3.1研究对象及建立骨折模型
本实验研究对象为实验用雄性新西兰家兔174只,于双侧桡骨中下1/3处在旋前圆肌肌止远端用锯造成3mm横断骨折,不作任何固定。用计算机产生随机数字的方法将174只家兔先随机取出6只作为对照组,其余168只家兔随机分为四组,每组42只。分别为中药三期治疗组、中药二期治疗组、中药一期治疗组、模型对照组。每个小组再随机分为7个小组,每组6只。分别为3、6、9、14、28、42、56天组。
3.2干预方法
3.2.1给药方法：将家兔桡骨骨折愈合过程分为三期：早期,即血肿机化演进期,骨折后1-7天；中期,即原始骨痂形成期,骨折后8-28天；后期,即改建塑形期,骨折后28天以后。
本研究所选用的肢伤一方、二方及三方选自高等中医药院校统编教材《中医正骨学》。
1组：中药三期治疗组将骨折愈合过程分为三期,早期,活血化瘀、行气止痛,给予肢伤一方,中期,接骨续筋、舒筋活络,给予肢伤二方：末期,补益肝肾、强筋壮骨,给予肢伤三方。
2组：中药二期治疗组将骨折愈合过程分为两期,早期,给予肢伤一方加减方；将中医骨折三期治疗的中期和末期合并为后期,给予肢伤二方与三方的综合方(后期方)。
3组：中药一期治疗组将骨折愈合过程不分期,给予肢伤一方、二方与三方的综合方(一期方)。
4组：模型对照组给予等量蒸馏水,灌胃,每日2次。
5组：空白对照组给予等量蒸馏水,灌胃,每日2次。3.3标本处理
术后第3、6、9、14、28、42、56天每组随机抽取一小组家兔用空气栓塞法处死,取左侧桡骨标本为观察对象,经固定,脱钙及HE染色切片制作等程序,采用免疫组化及原位杂交的方法观察骨折不同时间点bFGF、TGF-β、VEGF、BMP-2及其bFG|mRNA、TGF-βmRNA、VEGFmRNA、BMP-2mRNA的表达情况,在骨折中心以及两断端附近相同位置取9个高倍镜视野,采用美国3Y彩色图像分析系统与Pixelpro Ver4.0图像分析软件对阳性表达细胞进行光密度测定。
3.4统计分析
采用SPSS20.0统计软件对实验数据进行统计分析,采用一般线性模型的重复测量过程对骨折愈合过程中每种组织MOD值组间差异进行比较,并应用LSD法对每个时间点组间差异进行比较,结果采用均数±标准差(χ±s),表示检验水准α=0.05。
4结果
4.1bFGF及bFGFmRNA表达的结果
4.1.1外骨膜bFGF表达结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01。术后第9天,三组治疗组均达到高峰,但明显分期组明显最高,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于一期组,三期组高于模型对照组,均有显著性差异,P<0.05。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.1.2外骨膜bFGFmRNA表达结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组达到高峰,二期组高于一期组,有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.1.3内骨膜bFGF表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于一期组、模型对照组,差异非常显著,P<0.01；三期组高于模型对照组,有显著性差异,P<0.05。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.1.4内骨膜bFGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组和三期组,差异非常显著,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于一期组、模型对照组,差异非常显著,P<0.01；也高于三期组,有显著性差异,P<0.05。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.1.5骨髓腔bFGF表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,三期组高于一期组,均有显著性差异,P<0.05。术后第14天,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于三期组,有显著性差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.1.6骨髓腔bFGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,三期组高于一期组,均有显著性差异,P<0.05。术后第14天,三期组高于模型对照组,差异非常显著,P<0.01；二期组高于模型对照组,有显著性差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
4.2TGF-β及TGF-β mRNA表达的结果
4.2.1外骨膜TGF-β表达结果
均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,三期组高于一期组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、三期组、模型对照组,三期组高于一期组,均差异非常显著,P<0.01。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.2.2外骨膜TGF-β mRNA表达结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,有显著性差异,P<0.05。术后第42天,二期组高于一期组、模型对照组,均差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,均有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.2.3内骨膜TGF-β表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、三期组、模型对照组,均差异非常显著,P<0.01；三期组高于一期组、模型对照组,均有显著性差异,P<0.05。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.2.4内骨膜TGF-β mRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、模型对照组,三期组高于一期组,均差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组、模型对照组,均有显著性差异,P<0.05。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.2.5骨干细胞TGF-β表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、模型对照组,均差异非常显著,P<0.01；二期组高于三期组,三期组高于模型对照组,均有显著性差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.2.6骨干细胞TGF-βmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于模型对照组、一期组；三期组高于模型对照组,均差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,均有显著性差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
4.3VEGF及VEGFmRNA表达的结果
4.3.1外骨膜VEGF表达结果
组间两两比较,三组中药治疗组MOD值显著强于模型对照组,P<0.01。其中二期组的平均MOD值最高,二期组高于三期组,有显著性差异,P<0.05,三期组高于一期组,有显著性差异,P<0.05,一期组高于模型对照组,差异非常显著,P<0.01。术后第6天,二期组最高,与一期组及模型对照组相比,差异非常显著,P<0.01；三期组高于一期组和模型对照组,有显著性差异,P<0.05。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.3.2外骨膜VEGFmRNA表达结果
组间两两比较,二期、三期治疗组MOD值显著强于模型对照组,P<0.01,一期治疗组MOD值显著高于模型对照组,P<0.05。其中二期组的平均MOD值最高,二期组高于三期组,有显著性差异,P<0.05,三期组高于一期组,有显著性差异,P<0.05,一期组高于模型对照组,有显著性差异,P<0.05。术后第6天,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于一期组,有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.3.3内骨膜VEGF表达的结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05；三期组高于一期组,有显著性差异,P<0.05。术后第6天,二期组高于模型对照组,有显著性差异,P<0.05；二期组高于一期组,差异非常显著,P<0.01。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.3.4内骨膜VEGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组强于模型对照组,差异具有统计学意义,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01：三期组高于一期组,有显著性差异,P<0.05。术后第6天和术后第9天,二期组均高于模型对照组,差异非常显著,P<0.01；二期组均高于一期组,有显著差异,P<0.05；三期组均高于模型对照组,有显著差异,P<0.05。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.3.5骨髓腔VEGF表达的结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于模型对照组,有显著性差异,P<0.05；二期组均高于期组,差异非常显著,P<0.01；三期组均高于一期组,有显著差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.3.6骨髓腔VEGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组强于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01：三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于模型对照组,有显著性差异,P<0.05；二期组均高于一期组,差异非常显著,P<0.01；三期组均高于一期组,有显著差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
4.4BMP-2及BMP-2mRNA表达的结果
4.4.1外骨膜BMP-2表达结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第42天,二期组、三期组均高于模型对照组,差异非常显著,P<0.01；一期组强于模型对照组,有显著性差异,P<0.05。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.4.2外骨膜BMP-2mRNA表达结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,差异非常显著,P<0.01。术后第42天,二期组、三期组均高于模型对照组,差异非常显著,P<0.01；一期组强于模型对照组,有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.4.3骨干细胞BMP-2表达的结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第28天,二期组强于模型对照组,差异非常显著,P<0.01；二期组、三期组均高于一期组,有显著性差异,P<0.05；二期组高于三期组,有显著性差异,P<0.05。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.4.4骨干细胞BMP-2mRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第14天,二期组高于模型对照组、一期组和三期组,差异非常显著,P<0.01。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.4.5骨髓腔BMP-2表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第42天,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于一期组,三期组高于模型对照组,有显著性差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.4.6骨髓腔BMP-2mRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于
三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第42天,二期组、三期组均高于模型对照组,有显著性差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
5、结论
1)家兔骨折愈合过程中,各部位各时间点的bFGF、TGF-β、VEGF、BMP-2表达的无论从免疫组化还是原位杂交检查中,中药都能够促进骨折愈合过程中不同组织的bFGF、TGF-β、VEGF、BMP-2的表达,可能是中药促进骨折愈合的机制之一。
2)骨折三期治疗在不同组织表达都有促进bFGF、TGF-β、VEGF、BMP-2及bFGFmRNA、TGF-βmRNA、VEGFmRNA、BMP-2mRNA的表达作用,这是骨折三期治疗促进骨折愈合的作用机制之一,更显示出骨折分期治疗的必要性。
3)骨折愈合过程中的二期组能够更好的发挥作用,使bFGF、TGF-β、VEGF、 BMP-2及bFGFmRNA、TGF-βmRNA、VEGFmRNA、BMP-2mRNA的表达无论峰值水平,还是峰值时间都得到充分的提高,因此,二期治疗是骨折分期的治疗的最合理的分期。
6、创新点
1)多因子的全面分析骨折愈合过程
分析了骨折愈合过程中的促进作用的研究往往为单个因子或者双因子的表达,对各组织当中的bFGF、TGF-β、VEGF、BMP-2表达定量全面分析研究未见报道。
2)多基因的表达分析骨折分期治疗的愈合机制
通过免疫组化和原位杂交技术研究了骨折分期治疗促进骨折愈合中的bFGF、TGF-β、VEGF、BMP-2表达的影响,探求了骨折分期治疗的愈合机制,揭示了骨折分期治疗的合理分期,尚未报道。
1.Background
Three period treatment(TPT) is characteristic of Traditional Chinese Medine (TCM) for fractures. During the Sui and Tang dynasties, Lin DaoRen inherit and develop treatment for fracture in the experience of previous,and proposed Lin's seven-step traditional method for fracture injury, creating the first fracture differential period treatment of TCM. Orthopedics scientist summed up the three period treatment for fractures.Three period treatment principle is the guiding principle of TCM of bone fractures.
TPT of TCM for cure bone fracture has a long history and certain curative effect,but lack of scientific experimental evidence.Although clinical practice has confirmed its efficacy, but very few studies for it and only limited to the initial observation of clinical efficacy. In recent years, some scholars in the clinical work, they use one period treatment which is combine mid-period and post-period of fractures, achieved a certain effect, thus proposed TPT of TCM fracture point of view. Some scholars consider not to the staging (one period treatment) of the treatment of fractures in clinical and experimental research, also achieved a certain effect. Thus, the three period treatment has much controversy in fracture of TCM, so there are serious chaos appear three period treatment,two period treatment(WPT) and one period treatment(OPT), therefore the fracture of three period treatment faces serious challenges in the status of the guiding principles with fracture of TCM,which severely restricts the development of Chinese medicine treatment of fractures. But previous studies mostly preliminary observation of clinical efficacy, and are comparison between the control group and blank, so only prompted WPT and OPT also has a role to promote fracture healing, and can not explain the WPT and OPT are better than the TPT. Experimental study has not been reported in the literature about TPT of TCM for fracture to explain the effect and mechanism of the system.
We have study the callus healing process with SD rats in previous studies, and found WPT not only can significantly improve the expression of Basic fibroblast growth factor (bFGF) in fracture healing process,WPT on the bFGF expression was significantly stronger than the TPT and OPT in the process of fracture healing.Stage treatment group was significantly better than the treatment group with time and quality of callus formation in rabbits callus healing process, and expression of Vascular endothelial growth factors (VEGF) in TPT groups was significantly superior to OPT with outside the periosteum,the periosteum and bone marrow tissue of the fracture, and expression of VEGFmRNA have same results.
The function of bFGF promote angiogenesis, induct of embryonic development, promote nerve growth and so on. And VEGF is recognized as the strongest pro-angiogenic formation factor, it can promote the formation of new blood vessels, maintain the cartilage cell survival, accelerate bone turnover, and pro-angiogenic of other factors can be achieved in whole or part through the VEGF. Two growth factors are the more important factor for vascular reconstruction.And another important bone factor is Bone morphogenetic proteins (BMPs) during the bone healing process, which has induced osteogenesis, first mesenchymal quality cell chemotaxis, aggregation, differentiate into cartilage and bone, and ultimately become the bone marrow, it is recognized as the strongest bone induced differentiation factor, has an extremely important role in regulating fracture healing, more experimental research on BMP-2. In addition, Transforming growth factor-β (TGF-β) can promote periosteal mesenchymal cell proliferation and differentiation, promote the proliferation of osteoblasts, stimulate collagen type II, bone connected to the synthesis of the hormone and osteopontin, TGF-β in the play an important role in the metabolic process of the bone tissue. Therefore, in order to further explore the molecular biological level mechanism of TCM treatment for fracture, a reasonable way to study fracture stage, the experimental period treatment of Chinese medicine on bFGF,TGF-β,VEGF and BMP-2growth factor analyzed simultaneously.
2.Objective
We study the gene expression, cytokine expression and its distribution pattern on bFGF, TGF-p, VEGF and BMP-2,as well as bFGF mRNA,TGF-p mRNA, VEGFmRNA and BMP-2mRNA on the rabbit fracture which in the bilateral radial bone in the lower1/3a pronator teres remote3mm transverse fracture,and staging from the molecular level hierarchy in healing rabbit radial bone defects, and clarify TCM fractures treatment effect and mechanism in fracture healing from the organ, cell and molecular level system, to determine the best staging and clarify the stage treatment principles of Chinese medicine fracture, to provide more scientific and theoretical basis guiding treatment for TCM fractures treatment from the perspective of modern medicine. Promote Chinese medicine treatment of fractures to achieve towards standardization and modernization,and to create conditions for Chinese medicine treatment of fractures into the world.
3Materials and methods:
3.1the object of study and the establish a fracture model174male and healthy rabbits from New Zealand were made3mm transverse fracture with a saw at the lower1/3of pronation ended circular muscle, there was not for any fixation.Those rabbits were remove6as a control group randomly by computer.Then the remaining168rabbits were seperated into four groups randomly,42rabbits in each group.They were TPT group(TTG), WPT group(WTG), OPT group (OTG), and model control group(MCG).Each group was seperated into seven subgroups randomly,6rabbits per subgroup.There was3th,6th,9th,14th,28th,42th,56th day group which at after operation.
3.2Intervention methods
3.2.1Medicine given Method:The process of rabbit radial fracture healing is divided into three periods:The first period is from1st day to7th day after operation, it is hematoma evolving period.The second is8th to28th day after the operation, it is the original callus formating.The day after28th day is the third period, and it si shaping alterations of bone.
The prescription of medine of our study were No.1ZhiShang prescription,No.2ZhiShang prescription,and No.3ZhiShang prescription,that were both selected from the uniform textbooks "The bonesetting medicine of TCM".
TTG:TTG divided into three phases in the process of fracture healing,rabbits of TTG were feed No.1ZhiShang prescription at first period, and in second period of fracture healing,No.2ZhiShang prescription was given.The third period of fracture healing,use No.3ZhiShang prescription.
WTG:WTG divided into two phase in the process of fracture healing,rabbits of WTG were feed No.1ZhiShang prescription at first period, and combinated prescription of No.2ZhiShang prescription and No.3ZhiShang prescription in second and third period.
OTG:OTG not to stage,and rabbits of OTG were feed combinated prescription of
No.1ZhiShang prescription,No.2ZhiShang prescription and No.3ZhiShang prescription in whole fracture healing process.
MCG:MCG were not given any prescription or drug but distilled water as same dose as other group.and twice one day. Control Group:Control Group were given water as same dose as other group,and twice one day.
3.3Collection and handling of specimens
At day of3,6,9,14,28,42,56,one subgroup rabbits were selected from every groups and killed by air embolism.Their left radius were taken out and make the specimens by tchique process like stabilized and decalcificated,then all specimens were made into pathological sections,and then were observed by HE stain. Immunohistochemistry and in situ hybridization were performed to examinate the bFGF、TGF-β、VEGF、BMP-2expression with the fracture center and near the two ends of the same position, and analysis positive cells taken nine high-power field use USA3Y color image analysis system and Pixelpro Ver4.0image analysis software.
3.4Statistical analysis
Experimental data statistical analysis by SPSS20.0statistical software, comparing the difference between the fracture healing process in each organization MOD value group by the general linear model repeated measurement process and apply LSD method of differences between each time point group.The results wirte as mean±standard deviation (χ±s),test level a=0.05.
4results
4.1MOD vlue of bFGF and bFGFmRNA expression in bone marrow of groups at different time points during fracture healing
4.1.1The bFGF expression of outer periosteum results
Pairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than the MCG (P<0.01); OTG is higher than that in the MCG (P<0.05); including the highest group was WTG, higher than TTG (P<0.05), also higher thanOTG(P<0.01). At9th day after operation, TTG reached the peak, but the obvious staging group was significantly highest, WTG was higher than MCG(P<0.01); WTG was higher than OTG(P<0.05),and TTG was higher than MCG(P<0.05). Overall, the staging group maintained a high level, and significantly higher than MCG and the non-staging group.
4.1.2The outer periosteum bFGFmRNA expression results
Pairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than the MCG(P<0.01); OTG was higher than MCG(P<0.05), including WTG was highest, and higher than MCG(P<0.05);and higher than TTG(P<0.05), also higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At9th day after surgery, the WTG reached a peak,and higher than OTG(P<0.05). Overall, the staging group regardless of the peak of the peak average higher than not staging group and MCG.
4.1.3The bFGF expression of periosteum results
Pairwise comparisons between the average MOD value of groups,WTG and TTG were significantly higher than MCG(P<0.01); including OTG was higher than that MCG(P<0.05);WTG was the highest value, higher than TTG(P<0.05), also higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At9th day after operation, WTG was higher than OTG and MCG(P<0.01); TTG was higher than MCG (P<0.05). Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.1.4The bFGFmRNA Expression of periosteum results
Pairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than MCG, and WTG was higher than TTG and OTG (P<0.01);OTG was higher than MCG(P<0.05);TTG was higher than OTG(P<0.05). After9th day, WTG was higher than OTG and MCG(P<0.01); also higher than TTG(P<0.05). Overall, the peak level and the peak of the staging group was higher than one pertiod group and model control group.
4.1.5The bFGF expression of marrow cavity results
Pairwise comparisons between the average MOD value of groups, three groups of MOD values were significantly stronger than the model control group, two group than in a group, the difference was significant (P<0.01); two groups above three groupsa group is higher than that in the untreated control group, the three group than a group, there were significant differences (P<0.05). After14days, two groups higher than that in the untreated control group, the difference was very significant (P<0.01); two groups above the three groups, there is a significant difference (P<0.05). Overall, the peak level and the peak period of the staging group were stronger than a group staging.
4.1.6The bFGFmRNA expression of marrow cavity results
The average MOD value pairwise comparised between two groups, WTG and TTG of MOD values were significantly higher than MCG, WTG was higher than OTG(P<0.01);WTG was higher than TTG, OTG was higher than MCG, there were significant differences (P<0.05).After14th day. TTG was higher than MCG (P<0.01); WTG was higher than MCG(P<0.05). Overall, the peak level and the peak of staging group was higher than not staging group at most of the time points.
4.2MOD vlue of TGF-β and TGF-β mRNA expression in bone marrow of groups at different time points during fracture healing
4.2.1The TGF-P expression of outer perichondrium results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG (P<0.01);WTG was higher than OTG (P<0.01); WTG above TTG and OTG was higher than MCG, TTG was higher than OTG, there were significant differences(P<0.05). After42nd day,WTG was higher than OTG, TTG, MCG;TTG more than OTG, the differences were very significant (P<0.01). Overall, the staging group maintained a high level, and significantly higher than the model control group and the non-staging group.
4.2.2The TGF-βmRNA expression of outer periosteum results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, WTG was higher than OTG,the difference was significant(P<0.01); WTH was higher than TTG(P<0.05). After42nd day, WTG was higher than OTG and MCG, the differences were very significant(P<0.01);WTG was higher than TTG, TTG was higher than OTG, there were significant difference(P<0.05).Overall,the staging group regardless of the peak of the peak average higher than non-staging group and the model control group.
4.2.3The TGF-P expression of endosteal results
The average MOD value pairwise comparised between two groups, TTG and WTG of MOD values were significantly higher than the MCG,WTG was higher than OTG, the difference were significant(P<0.01); WTG was higher than TTG, TTG was higher than MCG, there were significant differences(P<0.05). The42nd day after operation, WTG was higher than OTQTTG and MCG(P<0.01);TTG was higher than OTG and MCG(P<0.05). Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.2.4The TGF-βmRNA expression of the periosteum results
The average MOD value pairwise comparised between two groups, TTG and WTG were significantly higher than MCG,WTG was higher than OTG(P<0.01); WTG was higher than TTQTTG was higher than OTG,OTG was higher than MCG, there were significant differences(P<0.05).At42nd day after operation, WTG was higher than OTG, MCG and TTG was higher than OTG, the differences were very significant(P<0.01);WTG was higher than TTG,TTG was higher than OTG and MCG(P<0.05). Overall, the peak level and the peak of the staging group is higher than not staging group and model control group.
4.2.5TGF-β expression of backbone cells results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, WTG was higher than OTG(P<0.01);WTG was higher than TTG,TTG was higher than OTG,OTG was higher than MCG, there were significant differences(P<0.05).At42nd day after operation, WTG was more than OTG and MCG(P<0.01);WTG was higher than TTG,TTG was higher than MCG(P <0.05). Overall, the peak level and the peak period of the staging group were higher than one staging group.
4.2.6TGF-PmRNA expression of backbone Cell results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, and WTG was higher than OTG(P <0.01);WTG was higher than TTQand TTG was higher than OTG, and OTG was higher than MCG,there were significant differences(P<0.05).At42nd day after operation,WTG was higher than MCG and MCG,and TTG was higher than MCG, the differences were very significant(P<0.01);WTG was higher than TTG,and OTG was higher than MCG(P<0.05). Overall, the staging group of peak level and the peak time were higher than one staging group at most of the time points.
4.3MOD vlue of VEGF and VEGFmRNA expression in bone marrow of groups at different time points during fracture healing
4.3.1The VEGF expression of outer perichondrium results
The average MOD value pairwise comparised between two groups, the MOD value of three Chinese medicine treatment groups was significantly higher than MCG(P<0.01). The average MOD of WTG was highest, and WTG was higher than TTG (P<0.05),TTG was higher than OTG(P<0.05),OTG was higher than MCG (P<0.01). At6th day after operation,WTG was highest, compared with OTG and MCG,the difference was very significant(P<0.01);TTG was higher than OTG and MCG(P<0.05). Overall, the staging group maintained a high level and significantly higher than the model control group and the non-staging group.
4.3.2The VEGFmRNA expression of outside periosteum results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher thanMCG(P<0.01),OTG was significantly higher than MCG(P<0.05). The average MOD of WTG was highest,WTG was higher than TTG(P<0.05),TTG was higher than OTG(P<0.05),OTG was higher than MCG(P <0.05). At6th day after operation,WTG was higher than MCG (P<0.01);WTG was higher than OTG (P<0.05). Overall, the staging group regardless of the peak of time and the peak higher than not staging group and the model control group.
4.3.3The VEGF expression of periosteum results
The average MOD value pairwise comparised between two groups,OTG,WTG and TTG was significantly higher than MCG(P<0.01); WTG was highest one, higher than TTG(P<0.05);TTG was higher than OTG(P<0.05). At6th day after operation,WTG was higher than MCG(P<0.05);WTG was higher than OTG (P<0.01).Overall,the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.3.4The VEGFmRNA expression of periosteum results
The average MOD value pairwise comparised between two groups,WTG and TTG were significantly higher than MCG(P<0.01);and OTG was higher than MCG(P<0.05); and WTG was the highest one,was higher than TTG(P<0.05),and higher than OTG(P<0.01); TTG was higher than OTG(P<0.05). At6th day and9th after operation, WTG was higher than MCG(P<0.01);WTG was higher than OTG (P<0.05);TTG was higher than MCG(P<0.05). Overall, the peak level and the peak of time for staging group were higher than not staging group and model control group.
4.3.5The VEGF expression of marrow cavity results
The average MOD value pairwise comparised between two groups,OTG,WTG and TTG were significantly higher than MCG(P<0.01); highest value was WTG,was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At9th day after operation,WTG was higher than MCG(P<0.05);WTG was higher than OTG,the difference was very significant(P<0.01);TTG was higher than OTG(P<0.05).Overall, the peak level and the peak time of the staging group was higher than not staging group.
4.3.6The VEGFmRNA expression of marrow cavity results
The average MOD value pairwise comparised between two groups,WTG and TTG were significantly higher than MCG(P<0.01);OTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG(P<0.05), also higher than OTG(P<0.01);TTG was higher than OTG(P<0.05). At9th day after operation, WTG was higher than MCG(P<0.05);WTG was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).Overall, the staging group peak level and the peak time were higher than not staging group at most of the time points.
4.4MOD vlue of BMP-2and BMP-2mRNA expression in bone marrow of groups at different time points during fracture healing
4.4.1The BMP-2expression of the outer periosteum results
The average MOD value pairwise comparised between two groups, MOD value of OTG, WTG and TTG were significantly higher than MCG(P<0.01); and WTG was the highest one,and was higher than TTG(P<0.05),was higher than OTG(P<0.01); TTG was higher thanOTG(P<0.05).At42nd day after operation,WTG and TTG were higher than MCG(P<0.01); OTG was higher than MCG(P<0.05). Overall, the staging group maintained a high level,and significantly higher than the model control group and the non-staging group.
4.4.2The BMP-2mRNA express of outer perichondrium results
The average MOD value pairwise comparised between two groups, MOD value of OTG, WTG and TTG were significantly higher than MCG(P<0.01); the highest was WTG, and was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.01).At42nd day after operation,WTG and TTG were higher than MCG(P<0.01); OTG was higher than MCG(P<0.05). Overall, the staging group regardless of the peak time and the average peak were higher than not staging a group and the model control group.
4.4.3The BMP-2expression of backbone cell results
The average MOD value pairwise comparised between two groups, MOD value of OTG,WTG and TTG were significantly higher than MCG(P<0.01); and was highest one,was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At28th day after surgery,WTG was higher than MCG(P<0.01);WTG and TTG were higher than OTG(P<0.05); WTG was higher than TTG(P<0.05).Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.4.4The BMP-2mRNA expression of bone cell results
The average MOD value pairwise comparised between two groups, the MOD value of the WTG and TTG were significantly higher than the MCG(P<0.01); including WTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG(P<0.05), also higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At14th day after operation, WTG was higher than MCG, OTG and TTG (P<0.01). Overall, the peak level and the peak time of staging group were higher than not staging group and model control group.
4.4.5marrow cavity BMP-2expression results
The average MOD value pairwise comparised between two groups, the MOD value of WTG and TTG were significantly higher than MCG(P<0.01); including WTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG (P<0.05), also was higher than OTG(P<0.01); TTG was higher than OTG (P<0.05).At42nd day after operation,WTG was higher than MCG(P<0.01);WTG was higher than OTG, and TTG was higher thanMCG(P<0.05).Overall, the peak level and the peak time of the staging group were higher than not staging group.
4.4.6The BMP-2mRNA expression of marrow cavity results
The average MOD value pairwise comparised between two groups,the MOD value of WTG and TTG were significantly higher than MCG(P<0.01);WTG was the highest one,and was higher than TTG(P<0.05);and was higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At42nd day after operation, WTG and TTG were higher than theMCG(P<0.05). Overall, the peak level and the peak time of the staging group were higher than not staging group at most of the time points.
5Conclusion
1) TCM can promote the expression of bFGF, TGF-β, VEGF, BMP-2with immunohistochemistry or situ hybridization examination with different organizations at the various parts of each time point in rabbits fracture healing process, it may be one of the mechanisms of TCM to promote fracture healing.
2) TPT treatment have to promote the expression of bFGF, TGF-p, VEGF, BMP-2and bFGFmRNA, TGF-p mRNA, VEGFmRNA, BMP-2mRNA in different tissues, and this is not only one of the mechanism of action the three pertiod treatment of fractures to promote fracture healing, but also shows the need for treatment of fractures in stage.
3) The WTG of bFGF, TGF-p, VEGF, BMP-2and of bFGFmRNA, TGF-β mRNA, VEGF mRNA, BMP-2mRNA expression are best than the other of groups, were able to play a role in the process of fracture healing, so regardless of the peak level, or peak timehave been fully improved, while two pertiod is the treatment of best method of reasonable fractures staging.
6New creation
1) Multi-factor comprehensive analysis of the fracture healing process
Study in fracture healing process often is the expression of a single factor or two-factor, but studies analysis the organizations of bFGF, TGF-β,VEGF, BMP-2expression quantitative comprehensive have not been reported.
2) Multi-gene expression analysis the mechanism of fracture healing staging treatment
By immunohistochemistry and in situ hybridization studies treatment of fractures installments to promote fracture healing of bFGF, TGF-β, VEGF, BMP-2expression, and explore the mechanisms of fracture healing stage treatment, revealed the fracture installmentstreatment of reasonable installments, not yet reported.
骨折分期的辨证施治方法是中医治疗骨折的一大特色,是前人经过不断总结发展而来的。中医骨伤学家总结了骨折三期辨证治疗,骨折三期治疗原则是中医药治疗骨折的指导原则。中医骨折三期治则临床疗效已证实,但实验证据不完善。虽然临床疗效已得到充分的验证,但对其研究往往局限于临床疗效观察。目前对中医骨折三期治则的作用存在诸多争议,无论在是否分期还是在分期的方法上都有明显的分歧,出现了不分期的治疗,以及分期的三期治疗和二期治疗的不同情况,使得骨折三期治疗做为中医药治疗骨折的指导原则的地位受到严峻的挑战,导致了中医药在治疗骨折的发展受到严重的制约。但是既往研究较多为临床疗效的初步观察,而且均是中药汤剂组与空白对照组、单味中药与空白对照组或某种中成药与空白对照组之间的相互做比较,所以认为不分期的一期治疗或二期治疗能够促进骨折愈合,但并均不能充分说明一期治疗与二期治疗疗效强于三期治疗。而关于中医骨折三期治疗原则的作用及其机制系统的实验研究,尚未见文献报道。
本课题组前期研究发现在SD大鼠骨痴愈合过程中,二期治疗不但可以明显提高其骨折愈合过程中bFGF (Basic fibroblast growth factor碱性成纤维细胞生长因子)的表达量,二期治疗对骨折愈合过程中bFGF表达的促进作用显著强于期治疗和一期治疗。并在前期的家兔骨折愈合过程的实验研究中,发现在骨折愈合时间及质量上分期的治疗组明显强于不分期治疗组,骨折三期治疗组无论在外骨膜、骨干细胞、骨小梁组织、骨痂质量及骨髓组织等骨折端组织的VEGF (Vascular endothelial growth factor血管内皮生长因子)和VEGF mRNA表达的促进作用明显优于一期治疗组。
bFGF有促血管形成、诱导胚胎发育、促进神经生长等功能。另外TGF-p (Transforming growth factor-β转化生长因子β),可促进骨膜间充质细胞增殖和分化,促进成骨细胞增殖、刺激Ⅱ型胶原、骨连接素和骨桥蛋白的合成,TGF-p在骨组织代谢过程中起重要的作用。而VEGF是目前公认最强的促血管形成因子,不仅能够促进血管重建,而且还能够增强软骨细胞活性,其他促血管生成因子对于血管重建时全部或部分通过VEGF的作用得以实现。bFGF与VEGF两因子都是对血管重建较为重要的因子,而骨的愈合过程中,骨形成的最关键因子的莫过于BMPs (Bone morphogenetic proteins骨形成蛋白),其具有诱导成骨作用,尤其是间充质细胞发生化学趋向、聚集、分化形成软骨和骨,最终形成骨髓,因此,被认为是最强的骨诱导分化因子,而实验研究方面对于在骨折愈合过程中较为重要的BMPS多进行其家族的BMP-2研究。我们为较全面的从分子生物水平更进一步探讨中医骨折分期治疗的作用及其机制,研究骨折治疗的合理分期方法,因此,本实验对中医分期治疗将从bFGF、TGF-β、VEGF及BMP-2四个生长因子同时进行全方位的分析。
2、研究目的
本研究拟通过对家兔双侧桡骨中下1/3旋前圆肌远端3mm横断骨折的bFGF、TGF-β、VEGF、BMP-2及其bFGFmRNA、TGF-PmRNA、VEGFmRNA、 BMP-2mRNA的基因表达、细胞因子的表达及其分布规律的影响情况,从分子水平层次对家兔桡骨下段骨缺损的愈合进行分期,进而从器官、细胞与分子水平系统地阐明中医骨折分期治疗对骨折愈合的作用及其机制,确定最佳的分期治疗方案,阐明中医骨折分期治疗的原则,从现代医学的角度为中医药分期治疗骨折提供更为科学的理论根据,指导骨折的治疗。
3、材料与方法
3.1研究对象及建立骨折模型
本实验研究对象为实验用雄性新西兰家兔174只,于双侧桡骨中下1/3处在旋前圆肌肌止远端用锯造成3mm横断骨折,不作任何固定。用计算机产生随机数字的方法将174只家兔先随机取出6只作为对照组,其余168只家兔随机分为四组,每组42只。分别为中药三期治疗组、中药二期治疗组、中药一期治疗组、模型对照组。每个小组再随机分为7个小组,每组6只。分别为3、6、9、14、28、42、56天组。
3.2干预方法
3.2.1给药方法：将家兔桡骨骨折愈合过程分为三期：早期,即血肿机化演进期,骨折后1-7天；中期,即原始骨痂形成期,骨折后8-28天；后期,即改建塑形期,骨折后28天以后。
本研究所选用的肢伤一方、二方及三方选自高等中医药院校统编教材《中医正骨学》。
1组：中药三期治疗组将骨折愈合过程分为三期,早期,活血化瘀、行气止痛,给予肢伤一方,中期,接骨续筋、舒筋活络,给予肢伤二方：末期,补益肝肾、强筋壮骨,给予肢伤三方。
2组：中药二期治疗组将骨折愈合过程分为两期,早期,给予肢伤一方加减方；将中医骨折三期治疗的中期和末期合并为后期,给予肢伤二方与三方的综合方(后期方)。
3组：中药一期治疗组将骨折愈合过程不分期,给予肢伤一方、二方与三方的综合方(一期方)。
4组：模型对照组给予等量蒸馏水,灌胃,每日2次。
5组：空白对照组给予等量蒸馏水,灌胃,每日2次。3.3标本处理
术后第3、6、9、14、28、42、56天每组随机抽取一小组家兔用空气栓塞法处死,取左侧桡骨标本为观察对象,经固定,脱钙及HE染色切片制作等程序,采用免疫组化及原位杂交的方法观察骨折不同时间点bFGF、TGF-β、VEGF、BMP-2及其bFG|mRNA、TGF-βmRNA、VEGFmRNA、BMP-2mRNA的表达情况,在骨折中心以及两断端附近相同位置取9个高倍镜视野,采用美国3Y彩色图像分析系统与Pixelpro Ver4.0图像分析软件对阳性表达细胞进行光密度测定。
3.4统计分析
采用SPSS20.0统计软件对实验数据进行统计分析,采用一般线性模型的重复测量过程对骨折愈合过程中每种组织MOD值组间差异进行比较,并应用LSD法对每个时间点组间差异进行比较,结果采用均数±标准差(χ±s),表示检验水准α=0.05。
4结果
4.1bFGF及bFGFmRNA表达的结果
4.1.1外骨膜bFGF表达结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01。术后第9天,三组治疗组均达到高峰,但明显分期组明显最高,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于一期组,三期组高于模型对照组,均有显著性差异,P<0.05。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.1.2外骨膜bFGFmRNA表达结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组达到高峰,二期组高于一期组,有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.1.3内骨膜bFGF表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于一期组、模型对照组,差异非常显著,P<0.01；三期组高于模型对照组,有显著性差异,P<0.05。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.1.4内骨膜bFGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组和三期组,差异非常显著,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于一期组、模型对照组,差异非常显著,P<0.01；也高于三期组,有显著性差异,P<0.05。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.1.5骨髓腔bFGF表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,三期组高于一期组,均有显著性差异,P<0.05。术后第14天,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于三期组,有显著性差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.1.6骨髓腔bFGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,三期组高于一期组,均有显著性差异,P<0.05。术后第14天,三期组高于模型对照组,差异非常显著,P<0.01；二期组高于模型对照组,有显著性差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
4.2TGF-β及TGF-β mRNA表达的结果
4.2.1外骨膜TGF-β表达结果
均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,三期组高于一期组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、三期组、模型对照组,三期组高于一期组,均差异非常显著,P<0.01。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.2.2外骨膜TGF-β mRNA表达结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,有显著性差异,P<0.05。术后第42天,二期组高于一期组、模型对照组,均差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,均有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.2.3内骨膜TGF-β表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、三期组、模型对照组,均差异非常显著,P<0.01；三期组高于一期组、模型对照组,均有显著性差异,P<0.05。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.2.4内骨膜TGF-β mRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、模型对照组,三期组高于一期组,均差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组、模型对照组,均有显著性差异,P<0.05。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.2.5骨干细胞TGF-β表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于一期组、模型对照组,均差异非常显著,P<0.01；二期组高于三期组,三期组高于模型对照组,均有显著性差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.2.6骨干细胞TGF-βmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,二期组高于一期组,差异非常显著,P<0.01；二期组高于三期组,三期组高于一期组,一期组高于模型对照组,均有显著性差异,P<0.05。术后第42天,二期组高于模型对照组、一期组；三期组高于模型对照组,均差异非常显著,P<0.01；二期组高于三期组,一期组高于模型对照组,均有显著性差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
4.3VEGF及VEGFmRNA表达的结果
4.3.1外骨膜VEGF表达结果
组间两两比较,三组中药治疗组MOD值显著强于模型对照组,P<0.01。其中二期组的平均MOD值最高,二期组高于三期组,有显著性差异,P<0.05,三期组高于一期组,有显著性差异,P<0.05,一期组高于模型对照组,差异非常显著,P<0.01。术后第6天,二期组最高,与一期组及模型对照组相比,差异非常显著,P<0.01；三期组高于一期组和模型对照组,有显著性差异,P<0.05。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.3.2外骨膜VEGFmRNA表达结果
组间两两比较,二期、三期治疗组MOD值显著强于模型对照组,P<0.01,一期治疗组MOD值显著高于模型对照组,P<0.05。其中二期组的平均MOD值最高,二期组高于三期组,有显著性差异,P<0.05,三期组高于一期组,有显著性差异,P<0.05,一期组高于模型对照组,有显著性差异,P<0.05。术后第6天,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于一期组,有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.3.3内骨膜VEGF表达的结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05；三期组高于一期组,有显著性差异,P<0.05。术后第6天,二期组高于模型对照组,有显著性差异,P<0.05；二期组高于一期组,差异非常显著,P<0.01。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.3.4内骨膜VEGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组强于模型对照组,差异具有统计学意义,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01：三期组高于一期组,有显著性差异,P<0.05。术后第6天和术后第9天,二期组均高于模型对照组,差异非常显著,P<0.01；二期组均高于一期组,有显著差异,P<0.05；三期组均高于模型对照组,有显著差异,P<0.05。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.3.5骨髓腔VEGF表达的结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于模型对照组,有显著性差异,P<0.05；二期组均高于期组,差异非常显著,P<0.01；三期组均高于一期组,有显著差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.3.6骨髓腔VEGFmRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组强于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01：三期组高于一期组,有显著性差异,P<0.05。术后第9天,二期组高于模型对照组,有显著性差异,P<0.05；二期组均高于一期组,差异非常显著,P<0.01；三期组均高于一期组,有显著差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
4.4BMP-2及BMP-2mRNA表达的结果
4.4.1外骨膜BMP-2表达结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第42天,二期组、三期组均高于模型对照组,差异非常显著,P<0.01；一期组强于模型对照组,有显著性差异,P<0.05。总体而言,分期组均保持较高的水平,且明显强于模型对照组以及不分期的一期组。
4.4.2外骨膜BMP-2mRNA表达结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,差异非常显著,P<0.01。术后第42天,二期组、三期组均高于模型对照组,差异非常显著,P<0.01；一期组强于模型对照组,有显著性差异,P<0.05。从总体而言,分期组无论高峰期还是峰值水平均强于不分期的一期组和模型对照组。
4.4.3骨干细胞BMP-2表达的结果
平均值组间两两比较,一期组、二期组、三期组的MOD值显著强于模型对照组,P<0.01；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第28天,二期组强于模型对照组,差异非常显著,P<0.01；二期组、三期组均高于一期组,有显著性差异,P<0.05；二期组高于三期组,有显著性差异,P<0.05。从总体而言,分期治疗组峰值水平仍高于不分期组及模型对照组。
4.4.4骨干细胞BMP-2mRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第14天,二期组高于模型对照组、一期组和三期组,差异非常显著,P<0.01。总体而言,分期组峰值水平以及高峰期均强于一期组及模型对照组。
4.4.5骨髓腔BMP-2表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第42天,二期组高于模型对照组,差异非常显著,P<0.01；二期组高于一期组,三期组高于模型对照组,有显著性差异,P<0.05。从总体而言,分期组的峰值水平及高峰期均强于不分期的一期组。
4.4.6骨髓腔BMP-2mRNA表达的结果
平均值组间两两比较,二期组、三期组的MOD值显著强于模型对照组,P<0.01；一期组高于模型对照组,有显著性差异,P<0.05；其中二期组最高,高于
三期组,有显著性差异,P<0.05,也高于一期组,差异非常显著,P<0.01；三期组高于一期组,有显著性差异,P<0.05。术后第42天,二期组、三期组均高于模型对照组,有显著性差异,P<0.05。从总体而言,分期组大部分时间点的峰值水平及高峰期均强于不分期的一期组。
5、结论
1)家兔骨折愈合过程中,各部位各时间点的bFGF、TGF-β、VEGF、BMP-2表达的无论从免疫组化还是原位杂交检查中,中药都能够促进骨折愈合过程中不同组织的bFGF、TGF-β、VEGF、BMP-2的表达,可能是中药促进骨折愈合的机制之一。
2)骨折三期治疗在不同组织表达都有促进bFGF、TGF-β、VEGF、BMP-2及bFGFmRNA、TGF-βmRNA、VEGFmRNA、BMP-2mRNA的表达作用,这是骨折三期治疗促进骨折愈合的作用机制之一,更显示出骨折分期治疗的必要性。
3)骨折愈合过程中的二期组能够更好的发挥作用,使bFGF、TGF-β、VEGF、 BMP-2及bFGFmRNA、TGF-βmRNA、VEGFmRNA、BMP-2mRNA的表达无论峰值水平,还是峰值时间都得到充分的提高,因此,二期治疗是骨折分期的治疗的最合理的分期。
6、创新点
1)多因子的全面分析骨折愈合过程
分析了骨折愈合过程中的促进作用的研究往往为单个因子或者双因子的表达,对各组织当中的bFGF、TGF-β、VEGF、BMP-2表达定量全面分析研究未见报道。
2)多基因的表达分析骨折分期治疗的愈合机制
通过免疫组化和原位杂交技术研究了骨折分期治疗促进骨折愈合中的bFGF、TGF-β、VEGF、BMP-2表达的影响,探求了骨折分期治疗的愈合机制,揭示了骨折分期治疗的合理分期,尚未报道。
1.Background
Three period treatment(TPT) is characteristic of Traditional Chinese Medine (TCM) for fractures. During the Sui and Tang dynasties, Lin DaoRen inherit and develop treatment for fracture in the experience of previous,and proposed Lin's seven-step traditional method for fracture injury, creating the first fracture differential period treatment of TCM. Orthopedics scientist summed up the three period treatment for fractures.Three period treatment principle is the guiding principle of TCM of bone fractures.
TPT of TCM for cure bone fracture has a long history and certain curative effect,but lack of scientific experimental evidence.Although clinical practice has confirmed its efficacy, but very few studies for it and only limited to the initial observation of clinical efficacy. In recent years, some scholars in the clinical work, they use one period treatment which is combine mid-period and post-period of fractures, achieved a certain effect, thus proposed TPT of TCM fracture point of view. Some scholars consider not to the staging (one period treatment) of the treatment of fractures in clinical and experimental research, also achieved a certain effect. Thus, the three period treatment has much controversy in fracture of TCM, so there are serious chaos appear three period treatment,two period treatment(WPT) and one period treatment(OPT), therefore the fracture of three period treatment faces serious challenges in the status of the guiding principles with fracture of TCM,which severely restricts the development of Chinese medicine treatment of fractures. But previous studies mostly preliminary observation of clinical efficacy, and are comparison between the control group and blank, so only prompted WPT and OPT also has a role to promote fracture healing, and can not explain the WPT and OPT are better than the TPT. Experimental study has not been reported in the literature about TPT of TCM for fracture to explain the effect and mechanism of the system.
We have study the callus healing process with SD rats in previous studies, and found WPT not only can significantly improve the expression of Basic fibroblast growth factor (bFGF) in fracture healing process,WPT on the bFGF expression was significantly stronger than the TPT and OPT in the process of fracture healing.Stage treatment group was significantly better than the treatment group with time and quality of callus formation in rabbits callus healing process, and expression of Vascular endothelial growth factors (VEGF) in TPT groups was significantly superior to OPT with outside the periosteum,the periosteum and bone marrow tissue of the fracture, and expression of VEGFmRNA have same results.
The function of bFGF promote angiogenesis, induct of embryonic development, promote nerve growth and so on. And VEGF is recognized as the strongest pro-angiogenic formation factor, it can promote the formation of new blood vessels, maintain the cartilage cell survival, accelerate bone turnover, and pro-angiogenic of other factors can be achieved in whole or part through the VEGF. Two growth factors are the more important factor for vascular reconstruction.And another important bone factor is Bone morphogenetic proteins (BMPs) during the bone healing process, which has induced osteogenesis, first mesenchymal quality cell chemotaxis, aggregation, differentiate into cartilage and bone, and ultimately become the bone marrow, it is recognized as the strongest bone induced differentiation factor, has an extremely important role in regulating fracture healing, more experimental research on BMP-2. In addition, Transforming growth factor-β (TGF-β) can promote periosteal mesenchymal cell proliferation and differentiation, promote the proliferation of osteoblasts, stimulate collagen type II, bone connected to the synthesis of the hormone and osteopontin, TGF-β in the play an important role in the metabolic process of the bone tissue. Therefore, in order to further explore the molecular biological level mechanism of TCM treatment for fracture, a reasonable way to study fracture stage, the experimental period treatment of Chinese medicine on bFGF,TGF-β,VEGF and BMP-2growth factor analyzed simultaneously.
2.Objective
We study the gene expression, cytokine expression and its distribution pattern on bFGF, TGF-p, VEGF and BMP-2,as well as bFGF mRNA,TGF-p mRNA, VEGFmRNA and BMP-2mRNA on the rabbit fracture which in the bilateral radial bone in the lower1/3a pronator teres remote3mm transverse fracture,and staging from the molecular level hierarchy in healing rabbit radial bone defects, and clarify TCM fractures treatment effect and mechanism in fracture healing from the organ, cell and molecular level system, to determine the best staging and clarify the stage treatment principles of Chinese medicine fracture, to provide more scientific and theoretical basis guiding treatment for TCM fractures treatment from the perspective of modern medicine. Promote Chinese medicine treatment of fractures to achieve towards standardization and modernization,and to create conditions for Chinese medicine treatment of fractures into the world.
3Materials and methods:
3.1the object of study and the establish a fracture model174male and healthy rabbits from New Zealand were made3mm transverse fracture with a saw at the lower1/3of pronation ended circular muscle, there was not for any fixation.Those rabbits were remove6as a control group randomly by computer.Then the remaining168rabbits were seperated into four groups randomly,42rabbits in each group.They were TPT group(TTG), WPT group(WTG), OPT group (OTG), and model control group(MCG).Each group was seperated into seven subgroups randomly,6rabbits per subgroup.There was3th,6th,9th,14th,28th,42th,56th day group which at after operation.
3.2Intervention methods
3.2.1Medicine given Method:The process of rabbit radial fracture healing is divided into three periods:The first period is from1st day to7th day after operation, it is hematoma evolving period.The second is8th to28th day after the operation, it is the original callus formating.The day after28th day is the third period, and it si shaping alterations of bone.
The prescription of medine of our study were No.1ZhiShang prescription,No.2ZhiShang prescription,and No.3ZhiShang prescription,that were both selected from the uniform textbooks "The bonesetting medicine of TCM".
TTG:TTG divided into three phases in the process of fracture healing,rabbits of TTG were feed No.1ZhiShang prescription at first period, and in second period of fracture healing,No.2ZhiShang prescription was given.The third period of fracture healing,use No.3ZhiShang prescription.
WTG:WTG divided into two phase in the process of fracture healing,rabbits of WTG were feed No.1ZhiShang prescription at first period, and combinated prescription of No.2ZhiShang prescription and No.3ZhiShang prescription in second and third period.
OTG:OTG not to stage,and rabbits of OTG were feed combinated prescription of
No.1ZhiShang prescription,No.2ZhiShang prescription and No.3ZhiShang prescription in whole fracture healing process.
MCG:MCG were not given any prescription or drug but distilled water as same dose as other group.and twice one day. Control Group:Control Group were given water as same dose as other group,and twice one day.
3.3Collection and handling of specimens
At day of3,6,9,14,28,42,56,one subgroup rabbits were selected from every groups and killed by air embolism.Their left radius were taken out and make the specimens by tchique process like stabilized and decalcificated,then all specimens were made into pathological sections,and then were observed by HE stain. Immunohistochemistry and in situ hybridization were performed to examinate the bFGF、TGF-β、VEGF、BMP-2expression with the fracture center and near the two ends of the same position, and analysis positive cells taken nine high-power field use USA3Y color image analysis system and Pixelpro Ver4.0image analysis software.
3.4Statistical analysis
Experimental data statistical analysis by SPSS20.0statistical software, comparing the difference between the fracture healing process in each organization MOD value group by the general linear model repeated measurement process and apply LSD method of differences between each time point group.The results wirte as mean±standard deviation (χ±s),test level a=0.05.
4results
4.1MOD vlue of bFGF and bFGFmRNA expression in bone marrow of groups at different time points during fracture healing
4.1.1The bFGF expression of outer periosteum results
Pairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than the MCG (P<0.01); OTG is higher than that in the MCG (P<0.05); including the highest group was WTG, higher than TTG (P<0.05), also higher thanOTG(P<0.01). At9th day after operation, TTG reached the peak, but the obvious staging group was significantly highest, WTG was higher than MCG(P<0.01); WTG was higher than OTG(P<0.05),and TTG was higher than MCG(P<0.05). Overall, the staging group maintained a high level, and significantly higher than MCG and the non-staging group.
4.1.2The outer periosteum bFGFmRNA expression results
Pairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than the MCG(P<0.01); OTG was higher than MCG(P<0.05), including WTG was highest, and higher than MCG(P<0.05);and higher than TTG(P<0.05), also higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At9th day after surgery, the WTG reached a peak,and higher than OTG(P<0.05). Overall, the staging group regardless of the peak of the peak average higher than not staging group and MCG.
4.1.3The bFGF expression of periosteum results
Pairwise comparisons between the average MOD value of groups,WTG and TTG were significantly higher than MCG(P<0.01); including OTG was higher than that MCG(P<0.05);WTG was the highest value, higher than TTG(P<0.05), also higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At9th day after operation, WTG was higher than OTG and MCG(P<0.01); TTG was higher than MCG (P<0.05). Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.1.4The bFGFmRNA Expression of periosteum results
Pairwise comparisons between the average MOD value of groups, WTG and TTG were significantly higher than MCG, and WTG was higher than TTG and OTG (P<0.01);OTG was higher than MCG(P<0.05);TTG was higher than OTG(P<0.05). After9th day, WTG was higher than OTG and MCG(P<0.01); also higher than TTG(P<0.05). Overall, the peak level and the peak of the staging group was higher than one pertiod group and model control group.
4.1.5The bFGF expression of marrow cavity results
Pairwise comparisons between the average MOD value of groups, three groups of MOD values were significantly stronger than the model control group, two group than in a group, the difference was significant (P<0.01); two groups above three groupsa group is higher than that in the untreated control group, the three group than a group, there were significant differences (P<0.05). After14days, two groups higher than that in the untreated control group, the difference was very significant (P<0.01); two groups above the three groups, there is a significant difference (P<0.05). Overall, the peak level and the peak period of the staging group were stronger than a group staging.
4.1.6The bFGFmRNA expression of marrow cavity results
The average MOD value pairwise comparised between two groups, WTG and TTG of MOD values were significantly higher than MCG, WTG was higher than OTG(P<0.01);WTG was higher than TTG, OTG was higher than MCG, there were significant differences (P<0.05).After14th day. TTG was higher than MCG (P<0.01); WTG was higher than MCG(P<0.05). Overall, the peak level and the peak of staging group was higher than not staging group at most of the time points.
4.2MOD vlue of TGF-β and TGF-β mRNA expression in bone marrow of groups at different time points during fracture healing
4.2.1The TGF-P expression of outer perichondrium results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG (P<0.01);WTG was higher than OTG (P<0.01); WTG above TTG and OTG was higher than MCG, TTG was higher than OTG, there were significant differences(P<0.05). After42nd day,WTG was higher than OTG, TTG, MCG;TTG more than OTG, the differences were very significant (P<0.01). Overall, the staging group maintained a high level, and significantly higher than the model control group and the non-staging group.
4.2.2The TGF-βmRNA expression of outer periosteum results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, WTG was higher than OTG,the difference was significant(P<0.01); WTH was higher than TTG(P<0.05). After42nd day, WTG was higher than OTG and MCG, the differences were very significant(P<0.01);WTG was higher than TTG, TTG was higher than OTG, there were significant difference(P<0.05).Overall,the staging group regardless of the peak of the peak average higher than non-staging group and the model control group.
4.2.3The TGF-P expression of endosteal results
The average MOD value pairwise comparised between two groups, TTG and WTG of MOD values were significantly higher than the MCG,WTG was higher than OTG, the difference were significant(P<0.01); WTG was higher than TTG, TTG was higher than MCG, there were significant differences(P<0.05). The42nd day after operation, WTG was higher than OTQTTG and MCG(P<0.01);TTG was higher than OTG and MCG(P<0.05). Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.2.4The TGF-βmRNA expression of the periosteum results
The average MOD value pairwise comparised between two groups, TTG and WTG were significantly higher than MCG,WTG was higher than OTG(P<0.01); WTG was higher than TTQTTG was higher than OTG,OTG was higher than MCG, there were significant differences(P<0.05).At42nd day after operation, WTG was higher than OTG, MCG and TTG was higher than OTG, the differences were very significant(P<0.01);WTG was higher than TTG,TTG was higher than OTG and MCG(P<0.05). Overall, the peak level and the peak of the staging group is higher than not staging group and model control group.
4.2.5TGF-β expression of backbone cells results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, WTG was higher than OTG(P<0.01);WTG was higher than TTG,TTG was higher than OTG,OTG was higher than MCG, there were significant differences(P<0.05).At42nd day after operation, WTG was more than OTG and MCG(P<0.01);WTG was higher than TTG,TTG was higher than MCG(P <0.05). Overall, the peak level and the peak period of the staging group were higher than one staging group.
4.2.6TGF-PmRNA expression of backbone Cell results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher than MCG, and WTG was higher than OTG(P <0.01);WTG was higher than TTQand TTG was higher than OTG, and OTG was higher than MCG,there were significant differences(P<0.05).At42nd day after operation,WTG was higher than MCG and MCG,and TTG was higher than MCG, the differences were very significant(P<0.01);WTG was higher than TTG,and OTG was higher than MCG(P<0.05). Overall, the staging group of peak level and the peak time were higher than one staging group at most of the time points.
4.3MOD vlue of VEGF and VEGFmRNA expression in bone marrow of groups at different time points during fracture healing
4.3.1The VEGF expression of outer perichondrium results
The average MOD value pairwise comparised between two groups, the MOD value of three Chinese medicine treatment groups was significantly higher than MCG(P<0.01). The average MOD of WTG was highest, and WTG was higher than TTG (P<0.05),TTG was higher than OTG(P<0.05),OTG was higher than MCG (P<0.01). At6th day after operation,WTG was highest, compared with OTG and MCG,the difference was very significant(P<0.01);TTG was higher than OTG and MCG(P<0.05). Overall, the staging group maintained a high level and significantly higher than the model control group and the non-staging group.
4.3.2The VEGFmRNA expression of outside periosteum results
The average MOD value pairwise comparised between two groups, WTG and TTG were significantly higher thanMCG(P<0.01),OTG was significantly higher than MCG(P<0.05). The average MOD of WTG was highest,WTG was higher than TTG(P<0.05),TTG was higher than OTG(P<0.05),OTG was higher than MCG(P <0.05). At6th day after operation,WTG was higher than MCG (P<0.01);WTG was higher than OTG (P<0.05). Overall, the staging group regardless of the peak of time and the peak higher than not staging group and the model control group.
4.3.3The VEGF expression of periosteum results
The average MOD value pairwise comparised between two groups,OTG,WTG and TTG was significantly higher than MCG(P<0.01); WTG was highest one, higher than TTG(P<0.05);TTG was higher than OTG(P<0.05). At6th day after operation,WTG was higher than MCG(P<0.05);WTG was higher than OTG (P<0.01).Overall,the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.3.4The VEGFmRNA expression of periosteum results
The average MOD value pairwise comparised between two groups,WTG and TTG were significantly higher than MCG(P<0.01);and OTG was higher than MCG(P<0.05); and WTG was the highest one,was higher than TTG(P<0.05),and higher than OTG(P<0.01); TTG was higher than OTG(P<0.05). At6th day and9th after operation, WTG was higher than MCG(P<0.01);WTG was higher than OTG (P<0.05);TTG was higher than MCG(P<0.05). Overall, the peak level and the peak of time for staging group were higher than not staging group and model control group.
4.3.5The VEGF expression of marrow cavity results
The average MOD value pairwise comparised between two groups,OTG,WTG and TTG were significantly higher than MCG(P<0.01); highest value was WTG,was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At9th day after operation,WTG was higher than MCG(P<0.05);WTG was higher than OTG,the difference was very significant(P<0.01);TTG was higher than OTG(P<0.05).Overall, the peak level and the peak time of the staging group was higher than not staging group.
4.3.6The VEGFmRNA expression of marrow cavity results
The average MOD value pairwise comparised between two groups,WTG and TTG were significantly higher than MCG(P<0.01);OTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG(P<0.05), also higher than OTG(P<0.01);TTG was higher than OTG(P<0.05). At9th day after operation, WTG was higher than MCG(P<0.05);WTG was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).Overall, the staging group peak level and the peak time were higher than not staging group at most of the time points.
4.4MOD vlue of BMP-2and BMP-2mRNA expression in bone marrow of groups at different time points during fracture healing
4.4.1The BMP-2expression of the outer periosteum results
The average MOD value pairwise comparised between two groups, MOD value of OTG, WTG and TTG were significantly higher than MCG(P<0.01); and WTG was the highest one,and was higher than TTG(P<0.05),was higher than OTG(P<0.01); TTG was higher thanOTG(P<0.05).At42nd day after operation,WTG and TTG were higher than MCG(P<0.01); OTG was higher than MCG(P<0.05). Overall, the staging group maintained a high level,and significantly higher than the model control group and the non-staging group.
4.4.2The BMP-2mRNA express of outer perichondrium results
The average MOD value pairwise comparised between two groups, MOD value of OTG, WTG and TTG were significantly higher than MCG(P<0.01); the highest was WTG, and was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.01).At42nd day after operation,WTG and TTG were higher than MCG(P<0.01); OTG was higher than MCG(P<0.05). Overall, the staging group regardless of the peak time and the average peak were higher than not staging a group and the model control group.
4.4.3The BMP-2expression of backbone cell results
The average MOD value pairwise comparised between two groups, MOD value of OTG,WTG and TTG were significantly higher than MCG(P<0.01); and was highest one,was higher than TTG(P<0.05),and was higher than OTG(P<0.01);TTG was higher than OTG(P<0.05).At28th day after surgery,WTG was higher than MCG(P<0.01);WTG and TTG were higher than OTG(P<0.05); WTG was higher than TTG(P<0.05).Overall, the peak level of the staging treatment group was still higher than the non-staging group and model control group.
4.4.4The BMP-2mRNA expression of bone cell results
The average MOD value pairwise comparised between two groups, the MOD value of the WTG and TTG were significantly higher than the MCG(P<0.01); including WTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG(P<0.05), also higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At14th day after operation, WTG was higher than MCG, OTG and TTG (P<0.01). Overall, the peak level and the peak time of staging group were higher than not staging group and model control group.
4.4.5marrow cavity BMP-2expression results
The average MOD value pairwise comparised between two groups, the MOD value of WTG and TTG were significantly higher than MCG(P<0.01); including WTG was higher than MCG(P<0.05);WTG was the highest one, was higher than TTG (P<0.05), also was higher than OTG(P<0.01); TTG was higher than OTG (P<0.05).At42nd day after operation,WTG was higher than MCG(P<0.01);WTG was higher than OTG, and TTG was higher thanMCG(P<0.05).Overall, the peak level and the peak time of the staging group were higher than not staging group.
4.4.6The BMP-2mRNA expression of marrow cavity results
The average MOD value pairwise comparised between two groups,the MOD value of WTG and TTG were significantly higher than MCG(P<0.01);WTG was the highest one,and was higher than TTG(P<0.05);and was higher than OTG(P<0.01); TTG was higher than OTG(P<0.05).At42nd day after operation, WTG and TTG were higher than theMCG(P<0.05). Overall, the peak level and the peak time of the staging group were higher than not staging group at most of the time points.
5Conclusion
1) TCM can promote the expression of bFGF, TGF-β, VEGF, BMP-2with immunohistochemistry or situ hybridization examination with different organizations at the various parts of each time point in rabbits fracture healing process, it may be one of the mechanisms of TCM to promote fracture healing.
2) TPT treatment have to promote the expression of bFGF, TGF-p, VEGF, BMP-2and bFGFmRNA, TGF-p mRNA, VEGFmRNA, BMP-2mRNA in different tissues, and this is not only one of the mechanism of action the three pertiod treatment of fractures to promote fracture healing, but also shows the need for treatment of fractures in stage.
3) The WTG of bFGF, TGF-p, VEGF, BMP-2and of bFGFmRNA, TGF-β mRNA, VEGF mRNA, BMP-2mRNA expression are best than the other of groups, were able to play a role in the process of fracture healing, so regardless of the peak level, or peak timehave been fully improved, while two pertiod is the treatment of best method of reasonable fractures staging.
6New creation
1) Multi-factor comprehensive analysis of the fracture healing process
Study in fracture healing process often is the expression of a single factor or two-factor, but studies analysis the organizations of bFGF, TGF-β,VEGF, BMP-2expression quantitative comprehensive have not been reported.
2) Multi-gene expression analysis the mechanism of fracture healing staging treatment
By immunohistochemistry and in situ hybridization studies treatment of fractures installments to promote fracture healing of bFGF, TGF-β, VEGF, BMP-2expression, and explore the mechanisms of fracture healing stage treatment, revealed the fracture installmentstreatment of reasonable installments, not yet reported.
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