骨折三期治则对家兔骨折愈合过程中FGF-2mRNA，TGF-β_1 mRNA表达调控的研究
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摘要
1研究背景
随着交通运输业的发展,骨折成为临床上的常见病和多发病之一。该病的特点为“年轻化、复杂化、高度致残化”。骨折已严重的影响着人类的生活质量、威胁着人类的健康,因而骨折的修复与愈合一直是骨科界研究的热点问题。在促进骨折愈合治疗上,中医药治疗历史悠久,疗效确切。中医学理论的精髓在于辩证论治,因此中医治疗根据骨折后症候学特点将骨折分为三期:早期活血化瘀、行气止痛;中期接骨续筋、舒筋活络;后期补益肝肾、强筋壮骨。该治则即为中医治疗骨折的指导原则。中医骨折三期治则对于骨折的治疗乃至中医骨科的发展起到了不可估量的作用。
目前,中医骨折三期治则已成为现代中医药治疗骨折的指导原则,然而对于骨折三期治则的分子生物学机制研究则很少。中医骨折三期治则仅是基于对前人经验的总结,尚缺乏实验等方面的科学依据,随着现代分子生物学技术发展,研究骨折三期治则对骨折愈合的分子生物学机制,对指导骨折治疗,丰富骨伤科学内治法,具有重要意义。先期实验已经应用大鼠骨折模型进行蛋白质水平研究,并且初步证实了骨折分期治疗的必要性。从基因水平揭示骨折三期治则的作用机制未见报道。
本次实验已经对骨生长因子VEGF和BMP-2,从X线评分、免疫组化、原位杂交等方面证实了骨折三期治疗效果优于其他治疗组。为进一步揭示中医骨折三期治则的分子生物学机制,应用原位杂交技术检测骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA基因转录水平。研究其分布及表达规律及时间相,从现代医学的角度揭示中医骨折三期治则的科学性,指导骨折的临床治疗,推动中医骨科治疗的快速发展。
2材料与方法
2.1实验动物与骨折模型
选用新西兰家兔,健康,雄性,体重2±0.5 kg;在桡骨中下段1/3处,造成3mm完全横断骨折。
2.2随机分组
将140只家兔随机分为四组,每组35只,分别为三期组(中药三期治疗组)、一期组(中药一期治疗组)、模型组(模型对照组)和阳性药组(阳性药物对照组)。三期组、一期组与阳性药组三组文中统称中药组。每个小组再随机分为7个小组,每组5只。
2.3干预方法
将中药制成含生药5g·ml~(-1)的浓缩口服液,灭菌后装瓶冷藏备用。分早晚2次,灌胃。根据量效反应确定实验最佳用药剂量为2倍等效剂量。根据骨折愈合分期方法与家兔骨折局部组织学变化,将家兔骨折愈合过程分为三期:早期即术后1-7天;中期即术后8-28天;后期即术后29-56天。
三期组根据“中医骨折三期治疗”原则,分三期治疗:早期活血化瘀、行气止痛,选用加减肢伤一方;中期接骨续筋、舒筋活络,选用加减肢伤二方;后期补益肝肾、强筋壮骨,选用加减肢伤三方。一期组将骨折的早期、中期与后期合并为一期,活血化瘀、行气止痛、接骨续筋、补益肝肾、强筋壮骨,选用肢伤一方、二方与三方加减方。阳性药组全程给骨折挫伤胶囊水溶液。模型组给予和实验组等量的蒸馏水灌胃,每日2次。
2.4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,将标本固定、脱钙。
2.5原位杂交
检测骨折标本FGF-2mRNA,TGF-β_1mRNA。
2.6图像及数据分析
对骨折端骨干细胞、骨小梁、内骨膜、外骨膜、纤维骨痂组织各取两个高倍镜视野,对阳性表达细胞进行MOD值测定。用SPSS13.0统计分析软件进行统计分析。
3结果
3.1模型组、一期组、阳性药组与三期组骨折愈合过程中FGF-2mRNA表达MOD值变化
3.1.1各组骨干细胞FGF-2mRNA表达
各组间MOD值的比较显示:术后14天:阳性药组显著高于模型组,p<0.05。术后42天:一期组显著高于模型组,p<0.05;三期组极显著高于模型组,p<0.01。术后56天:组间无差异。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01。
三期组、一期组与阳性药组表达呈三峰曲线。中药组同时出现高峰,三期组峰值最高。模型组高峰出现晚峰值低。
3.1.2各组骨小梁FGF-2mRNA表达
各组间MOD值的比较显示:术后28天:阳性药组显著高于模型组,p<0.05;三期组极显著高于模型组,p<0.01。术后42天:一期组显著高于模型组,p<0.05;阳性药组极显著高于模型组,p<0.01;三期组极显著高于一期组和阳性药组,p<0.01。术后56天:一期组显著高于模型组,p<0.05;三期组极显著高于模型组与阳性药组,p<0.01。各组平均值比较显示:一期组显著高于模型组,p<0.05;阳性药组显著高于模型组,p<0.05;三期组显著高于模型组、一期组、阳性药组,p<0.05。三期组高峰出现晚但峰值高。
3.1.3各组内骨膜FGF-2mRNA表达
各组间MOD值的比较显示:术后3天:三期组显著高于阳性药组,p<0.05。术后9天:一期组显著高于阳性药组,p<0.05;三期组极显著高于阳性药组,p<0.01。术后14天:阳性药组显著高于模型,p<0.05;三期组显著高于模型组,p<0.05。术后28天:阳性药组显著高于模型组与三期组,p<0.05。各组平均值比较显示:一期组极显著高于模型组,p<0.01;三期组极显著高于模型组、一期组与阳性药组,p<0.01。
三期组、一期组在术后9天出现高峰。阳性药组成双峰曲线,在术后28天出现高峰。
3.1.4各组外骨膜FGF-2mRNA表达
各组间MOD值的比较显示:术后9天:三期组有高于模型组、一期组、阳性药组的趋势,p>0.05。术后56天:阳性药组极显著高于模型组,p<0.01;阳性药组显著高于三期组,p<0.05。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01;三期组极显著高于阳性药组,p<0.01。
三期组呈单峰曲线,术后9天出现最高峰。阳性药组呈三峰曲线,术后9天出现最高峰。一期组呈三峰曲线,术后28天出现最高峰。
3.1.5各组纤维骨痂FGF-2mRNA表达
各组间MOD值的比较显示:术后14天:一期组显著高于模型组,p<0.05;阳性药组极显著高于一期组,p<0.01;三期组极显著高于模型组,p<0.01。术后28天:三期组极显著高于模型组,p<0.01。各组平均值比较显示:一期组显著高于阳性药组,p<0.05;一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01;三期组极显著高于阳性药组,p<0.01。
3.2模型组、一期组、阳性药组与三期组骨折愈合过程中TGF-β_1mRNA表达MOD值变化
3.2.1各组骨干细胞TGF-β_1mRNA表达
各组间MOD值的比较显示:术后3天:阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01。术后6天:一期组显著高于模型组,p<0.05;三期组极显著高于模型组,p<0.01;三期组极显著高于阳性药组,p<0.01。术后9天:三期组极显著高于模型组,p<0.01。术后14天:三期组极显著高于模型组,p<0.01。术后28天:一期组极显著高于模型组与阳性药组,p<0.01;三期组显著高于模型组,p<0.05。术后42天:三期组显著高于阳性药组,p<0.05。术后56天:组间无差异。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组、一期组、阳性药组,p<0.01。
三期组呈双峰曲线,三期组出现最高峰早,且峰值高。
3.2.2各组骨小梁TGF-β_1mRNA表达
各组间MOD值的比较显示:术后6天:组间无差异。术后9天:三期组显著高于模型组,p<0.05;阳性药组显著高于一期组,p<0.05;三期组极显著高于一期组,p<0.01。术后14天:一期组极显著高于模型组,p<0.01;一期组显著高于阳性药组,p<0.05;一期组、阳性药组、三期组均显著高于模型组,p<0.05。各组平均值比较显示:各组均显著高于模型组。三期组在9天出现高峰,峰值高。在术后14天,一期组出现高峰,三期组出现高峰早,持续时间长。
3.2.3各组内骨膜TGF-β_1mRNA表达
各组间MOD值的比较显示:术后9天:三期组显著高于模型组,p<0.05。术后14天:三期组极显著高于模型组,p<0.01。术后42天:阳性药组显著高于模型组、一期组,p<0.05;三期组显著高于模型组、一期组,p<0.05。
3.2.4各组外骨膜TGF-β_1mRNA表达
各组间MOD值的比较显示:术后3-14天:三期组有高于模型组、一期组、阳性药组的趋势,p>0.05。各组平均值比较显示:三期组有高于模型组、一期组、阳性药组的趋势,p>0.05。
三期组有高于模型组的趋势,p>0.05。术后6-28天:三期组有高于模型组的趋势,p>0.05。
3.2.5各组纤维骨痂TGF-β_1mRNA表达
各组间MOD值的比较显示:术后14天:阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01;阳性药组显著高于一期组,p<0.05;三期组显著高于一期组,p<0.05。术后28天:一期组显著高于模型组,p<0.05;阳性药组极显著高于模型组,p<0.01;三期组极显著高于一期组,p<0.01。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01。
中药组显著高于模型组,p<0.05。术后14天:三期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01。术后28天:三期组、阳性药组、一期组极显著高于模型组,p<0.01。
4结论
(1)中药能促进骨折愈合过程中不同组织FGF-2mRNA,TGF-β_1mRNA表达,说明中药干预有促进骨折愈合的作用。
(2)骨折三期治疗在不同组织表达都有促进FGF-2mRNA,TGF-β_1mRNA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。
(3)中药对骨折愈合不同阶段各种组织的FGF-2mRNA,TGF-β_1mRNA表达有不同的作用,根据骨折愈合不同阶段施以相应治则的分期治疗较不分期治疗对于FGF-2mRNA,TGF-β_1mRNA表达的促进作用更明显,显示中医骨折分期治疗是必要的。
(4)中医骨折三期治则分别针对骨折愈合不同时期的病理变化,采用相应的中药可以促进相应部位的FGF-2mRNA,TGF-β_1mRNA表达,是比较科学的骨折分期治疗方法。
5创新点
(1)发现骨折愈合过程中不同组织中FGF-2mRNA,TGF-β_1mRNA表达特征
目前对于骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA表达的研究,均是研究骨痂中FGF-2mRNA,TGF-β_1mRNA的定量变化。揭示不同组织FGF-2mRNA,TGF-β_1mRNA表达特征的研究,未见报道。
(2)阐明骨折三期治疗对骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA表达的作用
目前仅对VEGFmRNA和BMP-2mRNA在骨折愈合过程中有过研究,通过研究骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA表达变化情况,揭示骨折三期治则促进骨折愈合的作用机制,未见报道。
Treat fracture into three periods with TCM during fracture healing in rabbit FGF-2mRNA,TGF-β_1mRNA Study of expression regulation
1 Research backgroud
With the development of the transport industry,Bone fractures as a clinical one common and frequently-occurring disease.The characteristics of the disease as "younger,complicated and of a high degree of disability." Bone fracture has been seriously affected the quality of life of mankind,pose a threat to human health,Thus Bone fracture repair and bone healing has been a hot issue in the study.Treatment in the promotion of Bone fracture healing,the treatment of Chinese medicine has a long history,the exact effect.Chinese medicine is the essence of the theory of dialectical theory of governance,Therefore,after treatment with Chinese medicine based on fracture characteristics of the fracture syndrome is divided into three phases:nonage:promoting blomod flow and dissolving congestion、promoting qi circulation to relieve pain;metaphase:reunion of bone、relax and activate the tendons; anaphase:tonifing liver and kidney、strengthening bone and musculature.Chinese medicine is the rule while the guiding principles for the treatment of bone fractures.Governance of Chinese medicine are three fracture treatment for the bone fracture as well as the development of Chinese medicine orthopedic inestimable role to play.
At present,TCM three bone fractures in the governance of modern medicine has become the guiding principles for the treatment of bone fractures,However,for fractures of the three governance mechanisms of the molecular biology research,they are very rarely.TCM three bone fractures in the governance is only based on previous experience,experiments,such as lack of scientific basis,With the development of modern molecular biology techniques,Study the fracture of three governance On the molecular biology of fracture healing mechanism,In guiding the treatment of bone fractures,Rich in bone-setting treatment of science,Of great significance.Pre-experimental rat fracture model has been applied to study the protein level,
Confirmed the fracture and the initial phases of the need for treatment.Gene revealed the bone fracture from the level of the three mechanisms of governance are not reported.
The experiment has been on bone growth factor VEGF and BMP-2,From the X-ray score, immunohistochemistry,in situ hybridization confirmed the fracture treatment is better than the other three treatment groups,Chinese medicine in order to further reveal the fracture of the three governance mechanisms of molecular biology,Application of in situ hybridization detection of fracture healing process of FGF-2,TGF-β_1 gene transcription level.Study the distribution and expression of the law and time.From the perspective of modern medicine revealed three fractures Chinese medicine is the scientific nature of governance,Guide the clinical treatment of fractures,Traditional Chinese Medicine to promote the rapid development of orthopedic treatment.
2 Materials and metheods
2.1 Experimental Animal and Model of the bone fracture
Selecting New Zealand rabbit,healthy,male,body weight 2±0.5 kg;In the lower segment of radial bone1/3 resulted in complete transverse bone fracture of 3mm.
2.2 Randomization
140 rabbits were divided into four groups by computer at random,35 rabbits per group, three-phases group,chinese medicines,one-phase group,the model group,and the positive group.We call the The three-phases group,the one-phase group and the positive group to the traditional Chinese medicine treatment group.Every group were dividied into 7 groups again randomly,5 rabbits in every group.
2.3 Intervention methmods
Selecting the concentrated solution contained crude drugs 5g·ml~(-1),sterilized amd iced storage in the refrigerator,for reserved;Intragastric administrated twice a day on mane et vespere,According to the quantity and effect reaction to decide the best optimization agentia dose was double equivalent dose.Dividing the rabbit bone fracture agglutination into three stages according to the agglutination of fracture staging methmods and bone fracture topographic histology changs:nonage,after bone fracure one～seven days;metaphase,after eight～twenty-eight days;anaphase,after twenty-eight days.
Three-phases group according toThree-phases therapy priciple was adopted according to the bone fracture three station treatment:nonage:promoting blomod flow and dissolving congestion、promoting qi circulation to relieve pain,adopting "jia jian zhi shang one fang"; metaphase:reunion of bone、relax and activate the tendons,adopting "jia jian zhi shang two fang";anaphase:tonifing liver and kidney、strengthening bone and musculature,adopting"jia jian zhi shang three fang".One-phase group put fractured nonage,metaphase and anaphase into together,promoting blomod flow and dissolving congestion、promoting qi circulation to relieve pain、congestion、promoting qi circulation to relieve pain、tonifing liver and kidney、strengthening bone and musculature,adopting"jia jian zhi shang one、two and three fang" "one -phase fang".Positive group deturing bone fracture contund powder solution whole range without stage.Model group administrate tales doses of experimentation group distilled waten intragastric administration,twice a day.
2.4 Specimen management
Every subgroup rabbits were put to death by the aeroembolism methmod after operation from 3,6,9,14,28,42,56 days groups randomly,sinistro-radial bone sample were taken as observed object,fixed,Decalcification.
2.5 Ybridization in situ
Checking up bone fracture collection adopting FGF-2mRNA,TGF-pimRNA biotin labeling hybridization in situ.
2.6 Image and Data analysis
Recipe two high power campus visualis to determine the MOD value of the positive expression cells in diaphysis cell,bone trabecula,bone tunica intima,the periosteum,the fibrillar callus.Statistical analysis use spss13.0 software.
3 Consequence
3.1 The MOD value variation of the FGF-2mRNA of the expression in the three-phases group,the positive group,the one-phase group and the model control group,in the agglutination of fracture
3.1.1 The MOD value variation of the FGF-2mRNA of the expression in bone stem cells in every group
MOD value of inter-group comparison showed:the fourteenth day after operation:the positive group is notably better than the model group,p<0.05.On the fortyth day after operation:the one-phase group is notably better than the model group,p<0.05;the three -phases group is most notably better than the model group,p<0.01;the fifty-sixth day after operation:No difference of inter-group.The average of each group showed:the one -phases group is most notably better than the model group,p<0.01;the positive group is most notably better than the model group,p<0.01;the three -phases group is most notably better than the model group,p<0.01.
The three-phases group,the one-phase group and the positive group present bimodal curve. The peak of the three-phases group,the one-phase group and the positive group achieve at the same time.The three-phases group increase the level of the peak.The peak of model group increase finally and lowly.
3.1.2 The MOD value variation of the FGF-2mRNA of the expression in bone trabecula in every group
MOD value of inter-group comparison showed:the twenty-eighth day after operation: the positive group is notably better than the model group,p<0.05;the three-phases group is most notably better than the model group,p<0.01;On the fortyth day after operation:the one-phase group is notably better than the model group,p<0.05;the positive group is most notably better than the model group,p<0.01;the three-phases group is most notably better than the one-phase group and the positive group,p<0.0\;the fifty-sixth day after operation: the one-phase group is notably better than the model group,p<0.05;the three-phases group is most notably better than the model group and the positive group,p<0.01;The average of each group showed:the one-phase group is notably better than the model group,p<0.05;the positive group is notably better than the model group,p<0.05;the three-phases group is notably better than the model group,the one-phase group and the positive group,p<0.05. Although the peak of the three-phases group is late,the peak of the three-phases group is the highest.
3.1.3 The MOD value variation of the FGF-2mRNA of the expression in the periosteum in every group
MOD value of inter-group comparison showed:the third day after operation:the three-phases group is notably better than the positive group,p<0.05.the ninth day after operation:the one-phases group is notably better than the positive group,p<0.05.the three-phases group is most notably better than the positive group,p<0.01.the fourteenth day after operation:the positive group is notably better than the model group,p<0.05;the three-phases group is most notably better than the model group,p<0.01.the twenty-eighth day after operation:the positive group is notably better than the model group and the three-phases group,p<0.05.The average of each group showed:the one-phases group is most notably better than the positive group,p<0.01;the one-phases group is most notably better than the model group the one-phases group and the positive group,p<0.01.
The peak of the three-phases group and he one-phases group increase in the ninth day after operation.the positive group presents bimodal curve.and the peak of the positive group increase in the twenty-eighth day after operation.
3.1.4 The MOD value variation of the FGF-2mRNA of the expression in bone tunica intima in every group
MOD value of inter-group comparison showed:the ninth day after operation:the three-phases group have the tendency to be better than the model group,the one-phase group and the positive group,p>0.05.the fifty-sixth day after operation:the positive group is most notably better than the model group,p<0.01.the positive group is notably better than the model group,p<0.05.The average of each group showed:the one-phase group is most notably better than the model group,p<0.01.the three-phase group is most notably better than the model group,p<0.01.the three-phase group is most notably better than the positive group, p<0.01.
The three-phases group presents Single-peak curve.and the peak of the three-phases group increase in the ninth day after operation.the positive group presents Sambong urve.and the peak of the positive group increase in the ninth day after operation.The one-phases group presents Sambong curve.and the peak of the one-phases group increase in the twentieth-eighth day after operation.
3.1.5 The MOD value variation of the FGF-2mRNA of the expression in the fibrillar callus.in every group
MOD value of inter-group comparison showed:the ninth day after operation:the one-phase group is notably better than the model group,p<0.05;the positive group is most notably better than the one-phase group,p<0.01;the three-phase group is most notably better than the model group,p<0.01;the twenty-eighth day after operation:the three-phase group is most notably better than the model group,p<0.01.The average of each group showed:the one-phase group is notably better than the positive group,p<0.05;the one-phase group is most notably better than the model group,p<0.01;the positive group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the positive group,p<0.01.
3.2 The MOD value variation of the TGF-β_1mRNA of the expression in the three-phases group,the positive group,the one-phase group and the model control group,in the agglutination of fracture
3.2.1 The MOD value variation of the TGF-β_1mRNA of the expression in bone stem cells in every group
MOD value of inter-group comparison showed:the third day after operation:the positive group is most notably better than the model group,p<0.01;the three-phase group is most
notably better than the model group,p<0.01.the sixth day after operation:the one-phase group is notably better than the model group,p<0.05;the three-phase group is most notably better than the model group,p<0.01.the three-phase group is most notably better than the positive group,p<0.01.the ninth day after operation:the three-phase group is most notably better than the model group,p<0.01.the fourteenth day after operation:the three-phase group is most notably better than the model group,p<0.01.the twentieth-eighth day after operation: the one-phase group is most notably better than the model group and the positive group, p<0.01;the three-phase group is notably better than the model group,p<0.05;the twenty-eighth day after operation:On the fortyth day after operation:the three-phase group is notably better than the model group,p<0.05;the fifty-sixth day after operation:No difference of inter-group.The average of each group showed:the one-phase group is most notably better than the model group,p <0.01;the positive group is most notably better than the model group, p<0.01;the three-phase group is most notably better than the model group,one-phase group and the positive group p<0.01.
The three-phases group presents Bimodal curve,And the peak of the three-phases group achieve the earliest and highest.
3.2.2 The MOD value variation of the TGF-β_1mRNA of the expression in bone trabecula in every group
MOD value of inter-group comparison showed:the sixth day after operation:No difference of inter-group.the ninth day after operation:the three-phase group is notably better than the model group,p<0.05;the positive group is notably better than the three-phase group, p<0.05;the three-phase group is most notably better than the model group,p<0.01.the fourteenth day after operation:the one-phase group is most notably better than the model group,p<0.01;the one-phase group,the positive group and the three-phase group are notably better than the model group,p<0.05.The average of each group showed:the one-phase group, the positive group and the three-phase group are notably better than the model group,p<0.05.
The peak of the three-phases group achieve the highest.and higher than Other groups in the sixth day after operation,the sixth day after operation:The peak of the one-phases group achieve high, The peak of the the three-phase control group achieve the first and prolonge the time of the peak.
3.2.3 The MOD value variation of the TGF-β_1mRNA of the expression in the periosteum in every group
MOD value of inter-group comparison showed:the ninth day after operation:the three-phase group is notably better than the model group,p<0.05.the fourteenth day after operation:the three-phase group is most notably better than the model group,p<0.01.On the fortyth day after operation:,the positive group is notably better than the model group and the one-phase group,p<0.05;the three-phase group is notably better than the model group and the one-phase group,p<0.05.
3.2.4 The MOD value variation of the TGF-β_1mRNA of the expression in bone tunica intima in every group
MOD value of inter-group comparison showed:the ninth day after operation:the third to the fourteenth day after operation:the three-phases group have the tendency to be better than the model group,the one-phase group and the positive group,p>0.05.The average of each group showed:the three-phases group have the tendency to be better than the model group,the one-phase group and the positive group,p>0.05.
3.2.5 The MOD value variation of the TGF-β_1mRNA of the expression in the fibrillar callus.in every group
MOD value of inter-group comparison showed:The fourteenth day after operation:the positive group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the model group,p<0.01;the positive group is notably better than the one-phase group,p<0.05;the three-phase group is notably better than the one-phase group, p<0.05;the twenty-eighth day after operation:the one -phase group is notably better than the model group,p<0.05;the positive group is most notably better than the model group,p<0.01; the three-phase group is most notably better than the one-phase group,p<0.01.The average of each group showed:the one-phase group is most notably better than the model group, p<0.01;the positive group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the model group,p<0.01.
The one-phase group,the positive group and the three-phase group are notably better than the model group,p<0.05.the fourteenth day after operation:the three-phase group is most notably better than the model group,p<0.0l;the positive group is most notably better than the model group,p<0.01.the twenty-eighth day after operation:the three-phase group,the positive group and the one-phase group are most notably better than the model group,p<0.0l.
4 Conclusions
(1)TCM can enhance the expression of FGF-2mRNA,TGF-β_1mRNA in different tissues in the agglutination of fracture,which show that TCM have the effect to enhance the union of fracture.
(2) The three-phases treatment enhance the expression of FGF-2mRNA, TGF-β_1mRNA in different tissues in the agglutination of fracture.It is may be one of the mechanism that the three-phases treatment enhance the union of fracture.
(3) TCM have different effection to the expression of FGF-2mRNA,TGF-β_1mRNA in different tissues in the agglutination of fracture.The effection of treatment by stages is more manifest than treatment no stage.It shows that treatment by stages is necessary.
(4) The three-phase treatment therapeutical principle aim directly at different pathological change of different stags of the agglutination of fracture.Using corresponding burg can enhance the expression of FGF-2mRNA,TGF-β_1mRNA of the corresponding tissue.It is scientific treatment method by stags.
5 Innovative points
(1) Founding that fracture healing process in different organizations FGF-2mRNA, TGF-β_1mRNA expression
At present,The existing research of the expression of FGF-2mRNA,TGF-β_1mRNA in the agglutination of fracture is quantitative study of FGF-2mRNA,TGF-β_1mRNA in callus, which can not response the change character of every tissues in the agglutination of fracture. It is no report to reveal the the expression character of FGF-2mRNA,TGF-β_1mRNA in different tissues.
(2) To clarify the treatment of fractures of the three fracture healing FGF-2mRNA, TGF-β_1mRNA expression
At present,only VEGFmRNA and BMP-2mRNA in fracture healing research process,by studying the fracture healing process of FGF-2mRNA,TGF-β_1mRNA expression changes in governance were revealed three fractures to promote the role of fracture healing mechanisms, not reported.
随着交通运输业的发展,骨折成为临床上的常见病和多发病之一。该病的特点为“年轻化、复杂化、高度致残化”。骨折已严重的影响着人类的生活质量、威胁着人类的健康,因而骨折的修复与愈合一直是骨科界研究的热点问题。在促进骨折愈合治疗上,中医药治疗历史悠久,疗效确切。中医学理论的精髓在于辩证论治,因此中医治疗根据骨折后症候学特点将骨折分为三期:早期活血化瘀、行气止痛;中期接骨续筋、舒筋活络;后期补益肝肾、强筋壮骨。该治则即为中医治疗骨折的指导原则。中医骨折三期治则对于骨折的治疗乃至中医骨科的发展起到了不可估量的作用。
目前,中医骨折三期治则已成为现代中医药治疗骨折的指导原则,然而对于骨折三期治则的分子生物学机制研究则很少。中医骨折三期治则仅是基于对前人经验的总结,尚缺乏实验等方面的科学依据,随着现代分子生物学技术发展,研究骨折三期治则对骨折愈合的分子生物学机制,对指导骨折治疗,丰富骨伤科学内治法,具有重要意义。先期实验已经应用大鼠骨折模型进行蛋白质水平研究,并且初步证实了骨折分期治疗的必要性。从基因水平揭示骨折三期治则的作用机制未见报道。
本次实验已经对骨生长因子VEGF和BMP-2,从X线评分、免疫组化、原位杂交等方面证实了骨折三期治疗效果优于其他治疗组。为进一步揭示中医骨折三期治则的分子生物学机制,应用原位杂交技术检测骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA基因转录水平。研究其分布及表达规律及时间相,从现代医学的角度揭示中医骨折三期治则的科学性,指导骨折的临床治疗,推动中医骨科治疗的快速发展。
2材料与方法
2.1实验动物与骨折模型
选用新西兰家兔,健康,雄性,体重2±0.5 kg;在桡骨中下段1/3处,造成3mm完全横断骨折。
2.2随机分组
将140只家兔随机分为四组,每组35只,分别为三期组(中药三期治疗组)、一期组(中药一期治疗组)、模型组(模型对照组)和阳性药组(阳性药物对照组)。三期组、一期组与阳性药组三组文中统称中药组。每个小组再随机分为7个小组,每组5只。
2.3干预方法
将中药制成含生药5g·ml~(-1)的浓缩口服液,灭菌后装瓶冷藏备用。分早晚2次,灌胃。根据量效反应确定实验最佳用药剂量为2倍等效剂量。根据骨折愈合分期方法与家兔骨折局部组织学变化,将家兔骨折愈合过程分为三期:早期即术后1-7天;中期即术后8-28天;后期即术后29-56天。
三期组根据“中医骨折三期治疗”原则,分三期治疗:早期活血化瘀、行气止痛,选用加减肢伤一方;中期接骨续筋、舒筋活络,选用加减肢伤二方;后期补益肝肾、强筋壮骨,选用加减肢伤三方。一期组将骨折的早期、中期与后期合并为一期,活血化瘀、行气止痛、接骨续筋、补益肝肾、强筋壮骨,选用肢伤一方、二方与三方加减方。阳性药组全程给骨折挫伤胶囊水溶液。模型组给予和实验组等量的蒸馏水灌胃,每日2次。
2.4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,将标本固定、脱钙。
2.5原位杂交
检测骨折标本FGF-2mRNA,TGF-β_1mRNA。
2.6图像及数据分析
对骨折端骨干细胞、骨小梁、内骨膜、外骨膜、纤维骨痂组织各取两个高倍镜视野,对阳性表达细胞进行MOD值测定。用SPSS13.0统计分析软件进行统计分析。
3结果
3.1模型组、一期组、阳性药组与三期组骨折愈合过程中FGF-2mRNA表达MOD值变化
3.1.1各组骨干细胞FGF-2mRNA表达
各组间MOD值的比较显示:术后14天:阳性药组显著高于模型组,p<0.05。术后42天:一期组显著高于模型组,p<0.05;三期组极显著高于模型组,p<0.01。术后56天:组间无差异。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01。
三期组、一期组与阳性药组表达呈三峰曲线。中药组同时出现高峰,三期组峰值最高。模型组高峰出现晚峰值低。
3.1.2各组骨小梁FGF-2mRNA表达
各组间MOD值的比较显示:术后28天:阳性药组显著高于模型组,p<0.05;三期组极显著高于模型组,p<0.01。术后42天:一期组显著高于模型组,p<0.05;阳性药组极显著高于模型组,p<0.01;三期组极显著高于一期组和阳性药组,p<0.01。术后56天:一期组显著高于模型组,p<0.05;三期组极显著高于模型组与阳性药组,p<0.01。各组平均值比较显示:一期组显著高于模型组,p<0.05;阳性药组显著高于模型组,p<0.05;三期组显著高于模型组、一期组、阳性药组,p<0.05。三期组高峰出现晚但峰值高。
3.1.3各组内骨膜FGF-2mRNA表达
各组间MOD值的比较显示:术后3天:三期组显著高于阳性药组,p<0.05。术后9天:一期组显著高于阳性药组,p<0.05;三期组极显著高于阳性药组,p<0.01。术后14天:阳性药组显著高于模型,p<0.05;三期组显著高于模型组,p<0.05。术后28天:阳性药组显著高于模型组与三期组,p<0.05。各组平均值比较显示:一期组极显著高于模型组,p<0.01;三期组极显著高于模型组、一期组与阳性药组,p<0.01。
三期组、一期组在术后9天出现高峰。阳性药组成双峰曲线,在术后28天出现高峰。
3.1.4各组外骨膜FGF-2mRNA表达
各组间MOD值的比较显示:术后9天:三期组有高于模型组、一期组、阳性药组的趋势,p>0.05。术后56天:阳性药组极显著高于模型组,p<0.01;阳性药组显著高于三期组,p<0.05。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01;三期组极显著高于阳性药组,p<0.01。
三期组呈单峰曲线,术后9天出现最高峰。阳性药组呈三峰曲线,术后9天出现最高峰。一期组呈三峰曲线,术后28天出现最高峰。
3.1.5各组纤维骨痂FGF-2mRNA表达
各组间MOD值的比较显示:术后14天:一期组显著高于模型组,p<0.05;阳性药组极显著高于一期组,p<0.01;三期组极显著高于模型组,p<0.01。术后28天:三期组极显著高于模型组,p<0.01。各组平均值比较显示:一期组显著高于阳性药组,p<0.05;一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01;三期组极显著高于阳性药组,p<0.01。
3.2模型组、一期组、阳性药组与三期组骨折愈合过程中TGF-β_1mRNA表达MOD值变化
3.2.1各组骨干细胞TGF-β_1mRNA表达
各组间MOD值的比较显示:术后3天:阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01。术后6天:一期组显著高于模型组,p<0.05;三期组极显著高于模型组,p<0.01;三期组极显著高于阳性药组,p<0.01。术后9天:三期组极显著高于模型组,p<0.01。术后14天:三期组极显著高于模型组,p<0.01。术后28天:一期组极显著高于模型组与阳性药组,p<0.01;三期组显著高于模型组,p<0.05。术后42天:三期组显著高于阳性药组,p<0.05。术后56天:组间无差异。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组、一期组、阳性药组,p<0.01。
三期组呈双峰曲线,三期组出现最高峰早,且峰值高。
3.2.2各组骨小梁TGF-β_1mRNA表达
各组间MOD值的比较显示:术后6天:组间无差异。术后9天:三期组显著高于模型组,p<0.05;阳性药组显著高于一期组,p<0.05;三期组极显著高于一期组,p<0.01。术后14天:一期组极显著高于模型组,p<0.01;一期组显著高于阳性药组,p<0.05;一期组、阳性药组、三期组均显著高于模型组,p<0.05。各组平均值比较显示:各组均显著高于模型组。三期组在9天出现高峰,峰值高。在术后14天,一期组出现高峰,三期组出现高峰早,持续时间长。
3.2.3各组内骨膜TGF-β_1mRNA表达
各组间MOD值的比较显示:术后9天:三期组显著高于模型组,p<0.05。术后14天:三期组极显著高于模型组,p<0.01。术后42天:阳性药组显著高于模型组、一期组,p<0.05;三期组显著高于模型组、一期组,p<0.05。
3.2.4各组外骨膜TGF-β_1mRNA表达
各组间MOD值的比较显示:术后3-14天:三期组有高于模型组、一期组、阳性药组的趋势,p>0.05。各组平均值比较显示:三期组有高于模型组、一期组、阳性药组的趋势,p>0.05。
三期组有高于模型组的趋势,p>0.05。术后6-28天:三期组有高于模型组的趋势,p>0.05。
3.2.5各组纤维骨痂TGF-β_1mRNA表达
各组间MOD值的比较显示:术后14天:阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01;阳性药组显著高于一期组,p<0.05;三期组显著高于一期组,p<0.05。术后28天:一期组显著高于模型组,p<0.05;阳性药组极显著高于模型组,p<0.01;三期组极显著高于一期组,p<0.01。各组平均值比较显示:一期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01;三期组极显著高于模型组,p<0.01。
中药组显著高于模型组,p<0.05。术后14天:三期组极显著高于模型组,p<0.01;阳性药组极显著高于模型组,p<0.01。术后28天:三期组、阳性药组、一期组极显著高于模型组,p<0.01。
4结论
(1)中药能促进骨折愈合过程中不同组织FGF-2mRNA,TGF-β_1mRNA表达,说明中药干预有促进骨折愈合的作用。
(2)骨折三期治疗在不同组织表达都有促进FGF-2mRNA,TGF-β_1mRNA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。
(3)中药对骨折愈合不同阶段各种组织的FGF-2mRNA,TGF-β_1mRNA表达有不同的作用,根据骨折愈合不同阶段施以相应治则的分期治疗较不分期治疗对于FGF-2mRNA,TGF-β_1mRNA表达的促进作用更明显,显示中医骨折分期治疗是必要的。
(4)中医骨折三期治则分别针对骨折愈合不同时期的病理变化,采用相应的中药可以促进相应部位的FGF-2mRNA,TGF-β_1mRNA表达,是比较科学的骨折分期治疗方法。
5创新点
(1)发现骨折愈合过程中不同组织中FGF-2mRNA,TGF-β_1mRNA表达特征
目前对于骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA表达的研究,均是研究骨痂中FGF-2mRNA,TGF-β_1mRNA的定量变化。揭示不同组织FGF-2mRNA,TGF-β_1mRNA表达特征的研究,未见报道。
(2)阐明骨折三期治疗对骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA表达的作用
目前仅对VEGFmRNA和BMP-2mRNA在骨折愈合过程中有过研究,通过研究骨折愈合过程中FGF-2mRNA,TGF-β_1mRNA表达变化情况,揭示骨折三期治则促进骨折愈合的作用机制,未见报道。
Treat fracture into three periods with TCM during fracture healing in rabbit FGF-2mRNA,TGF-β_1mRNA Study of expression regulation
1 Research backgroud
With the development of the transport industry,Bone fractures as a clinical one common and frequently-occurring disease.The characteristics of the disease as "younger,complicated and of a high degree of disability." Bone fracture has been seriously affected the quality of life of mankind,pose a threat to human health,Thus Bone fracture repair and bone healing has been a hot issue in the study.Treatment in the promotion of Bone fracture healing,the treatment of Chinese medicine has a long history,the exact effect.Chinese medicine is the essence of the theory of dialectical theory of governance,Therefore,after treatment with Chinese medicine based on fracture characteristics of the fracture syndrome is divided into three phases:nonage:promoting blomod flow and dissolving congestion、promoting qi circulation to relieve pain;metaphase:reunion of bone、relax and activate the tendons; anaphase:tonifing liver and kidney、strengthening bone and musculature.Chinese medicine is the rule while the guiding principles for the treatment of bone fractures.Governance of Chinese medicine are three fracture treatment for the bone fracture as well as the development of Chinese medicine orthopedic inestimable role to play.
At present,TCM three bone fractures in the governance of modern medicine has become the guiding principles for the treatment of bone fractures,However,for fractures of the three governance mechanisms of the molecular biology research,they are very rarely.TCM three bone fractures in the governance is only based on previous experience,experiments,such as lack of scientific basis,With the development of modern molecular biology techniques,Study the fracture of three governance On the molecular biology of fracture healing mechanism,In guiding the treatment of bone fractures,Rich in bone-setting treatment of science,Of great significance.Pre-experimental rat fracture model has been applied to study the protein level,
Confirmed the fracture and the initial phases of the need for treatment.Gene revealed the bone fracture from the level of the three mechanisms of governance are not reported.
The experiment has been on bone growth factor VEGF and BMP-2,From the X-ray score, immunohistochemistry,in situ hybridization confirmed the fracture treatment is better than the other three treatment groups,Chinese medicine in order to further reveal the fracture of the three governance mechanisms of molecular biology,Application of in situ hybridization detection of fracture healing process of FGF-2,TGF-β_1 gene transcription level.Study the distribution and expression of the law and time.From the perspective of modern medicine revealed three fractures Chinese medicine is the scientific nature of governance,Guide the clinical treatment of fractures,Traditional Chinese Medicine to promote the rapid development of orthopedic treatment.
2 Materials and metheods
2.1 Experimental Animal and Model of the bone fracture
Selecting New Zealand rabbit,healthy,male,body weight 2±0.5 kg;In the lower segment of radial bone1/3 resulted in complete transverse bone fracture of 3mm.
2.2 Randomization
140 rabbits were divided into four groups by computer at random,35 rabbits per group, three-phases group,chinese medicines,one-phase group,the model group,and the positive group.We call the The three-phases group,the one-phase group and the positive group to the traditional Chinese medicine treatment group.Every group were dividied into 7 groups again randomly,5 rabbits in every group.
2.3 Intervention methmods
Selecting the concentrated solution contained crude drugs 5g·ml~(-1),sterilized amd iced storage in the refrigerator,for reserved;Intragastric administrated twice a day on mane et vespere,According to the quantity and effect reaction to decide the best optimization agentia dose was double equivalent dose.Dividing the rabbit bone fracture agglutination into three stages according to the agglutination of fracture staging methmods and bone fracture topographic histology changs:nonage,after bone fracure one～seven days;metaphase,after eight～twenty-eight days;anaphase,after twenty-eight days.
Three-phases group according toThree-phases therapy priciple was adopted according to the bone fracture three station treatment:nonage:promoting blomod flow and dissolving congestion、promoting qi circulation to relieve pain,adopting "jia jian zhi shang one fang"; metaphase:reunion of bone、relax and activate the tendons,adopting "jia jian zhi shang two fang";anaphase:tonifing liver and kidney、strengthening bone and musculature,adopting"jia jian zhi shang three fang".One-phase group put fractured nonage,metaphase and anaphase into together,promoting blomod flow and dissolving congestion、promoting qi circulation to relieve pain、congestion、promoting qi circulation to relieve pain、tonifing liver and kidney、strengthening bone and musculature,adopting"jia jian zhi shang one、two and three fang" "one -phase fang".Positive group deturing bone fracture contund powder solution whole range without stage.Model group administrate tales doses of experimentation group distilled waten intragastric administration,twice a day.
2.4 Specimen management
Every subgroup rabbits were put to death by the aeroembolism methmod after operation from 3,6,9,14,28,42,56 days groups randomly,sinistro-radial bone sample were taken as observed object,fixed,Decalcification.
2.5 Ybridization in situ
Checking up bone fracture collection adopting FGF-2mRNA,TGF-pimRNA biotin labeling hybridization in situ.
2.6 Image and Data analysis
Recipe two high power campus visualis to determine the MOD value of the positive expression cells in diaphysis cell,bone trabecula,bone tunica intima,the periosteum,the fibrillar callus.Statistical analysis use spss13.0 software.
3 Consequence
3.1 The MOD value variation of the FGF-2mRNA of the expression in the three-phases group,the positive group,the one-phase group and the model control group,in the agglutination of fracture
3.1.1 The MOD value variation of the FGF-2mRNA of the expression in bone stem cells in every group
MOD value of inter-group comparison showed:the fourteenth day after operation:the positive group is notably better than the model group,p<0.05.On the fortyth day after operation:the one-phase group is notably better than the model group,p<0.05;the three -phases group is most notably better than the model group,p<0.01;the fifty-sixth day after operation:No difference of inter-group.The average of each group showed:the one -phases group is most notably better than the model group,p<0.01;the positive group is most notably better than the model group,p<0.01;the three -phases group is most notably better than the model group,p<0.01.
The three-phases group,the one-phase group and the positive group present bimodal curve. The peak of the three-phases group,the one-phase group and the positive group achieve at the same time.The three-phases group increase the level of the peak.The peak of model group increase finally and lowly.
3.1.2 The MOD value variation of the FGF-2mRNA of the expression in bone trabecula in every group
MOD value of inter-group comparison showed:the twenty-eighth day after operation: the positive group is notably better than the model group,p<0.05;the three-phases group is most notably better than the model group,p<0.01;On the fortyth day after operation:the one-phase group is notably better than the model group,p<0.05;the positive group is most notably better than the model group,p<0.01;the three-phases group is most notably better than the one-phase group and the positive group,p<0.0\;the fifty-sixth day after operation: the one-phase group is notably better than the model group,p<0.05;the three-phases group is most notably better than the model group and the positive group,p<0.01;The average of each group showed:the one-phase group is notably better than the model group,p<0.05;the positive group is notably better than the model group,p<0.05;the three-phases group is notably better than the model group,the one-phase group and the positive group,p<0.05. Although the peak of the three-phases group is late,the peak of the three-phases group is the highest.
3.1.3 The MOD value variation of the FGF-2mRNA of the expression in the periosteum in every group
MOD value of inter-group comparison showed:the third day after operation:the three-phases group is notably better than the positive group,p<0.05.the ninth day after operation:the one-phases group is notably better than the positive group,p<0.05.the three-phases group is most notably better than the positive group,p<0.01.the fourteenth day after operation:the positive group is notably better than the model group,p<0.05;the three-phases group is most notably better than the model group,p<0.01.the twenty-eighth day after operation:the positive group is notably better than the model group and the three-phases group,p<0.05.The average of each group showed:the one-phases group is most notably better than the positive group,p<0.01;the one-phases group is most notably better than the model group the one-phases group and the positive group,p<0.01.
The peak of the three-phases group and he one-phases group increase in the ninth day after operation.the positive group presents bimodal curve.and the peak of the positive group increase in the twenty-eighth day after operation.
3.1.4 The MOD value variation of the FGF-2mRNA of the expression in bone tunica intima in every group
MOD value of inter-group comparison showed:the ninth day after operation:the three-phases group have the tendency to be better than the model group,the one-phase group and the positive group,p>0.05.the fifty-sixth day after operation:the positive group is most notably better than the model group,p<0.01.the positive group is notably better than the model group,p<0.05.The average of each group showed:the one-phase group is most notably better than the model group,p<0.01.the three-phase group is most notably better than the model group,p<0.01.the three-phase group is most notably better than the positive group, p<0.01.
The three-phases group presents Single-peak curve.and the peak of the three-phases group increase in the ninth day after operation.the positive group presents Sambong urve.and the peak of the positive group increase in the ninth day after operation.The one-phases group presents Sambong curve.and the peak of the one-phases group increase in the twentieth-eighth day after operation.
3.1.5 The MOD value variation of the FGF-2mRNA of the expression in the fibrillar callus.in every group
MOD value of inter-group comparison showed:the ninth day after operation:the one-phase group is notably better than the model group,p<0.05;the positive group is most notably better than the one-phase group,p<0.01;the three-phase group is most notably better than the model group,p<0.01;the twenty-eighth day after operation:the three-phase group is most notably better than the model group,p<0.01.The average of each group showed:the one-phase group is notably better than the positive group,p<0.05;the one-phase group is most notably better than the model group,p<0.01;the positive group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the positive group,p<0.01.
3.2 The MOD value variation of the TGF-β_1mRNA of the expression in the three-phases group,the positive group,the one-phase group and the model control group,in the agglutination of fracture
3.2.1 The MOD value variation of the TGF-β_1mRNA of the expression in bone stem cells in every group
MOD value of inter-group comparison showed:the third day after operation:the positive group is most notably better than the model group,p<0.01;the three-phase group is most
notably better than the model group,p<0.01.the sixth day after operation:the one-phase group is notably better than the model group,p<0.05;the three-phase group is most notably better than the model group,p<0.01.the three-phase group is most notably better than the positive group,p<0.01.the ninth day after operation:the three-phase group is most notably better than the model group,p<0.01.the fourteenth day after operation:the three-phase group is most notably better than the model group,p<0.01.the twentieth-eighth day after operation: the one-phase group is most notably better than the model group and the positive group, p<0.01;the three-phase group is notably better than the model group,p<0.05;the twenty-eighth day after operation:On the fortyth day after operation:the three-phase group is notably better than the model group,p<0.05;the fifty-sixth day after operation:No difference of inter-group.The average of each group showed:the one-phase group is most notably better than the model group,p <0.01;the positive group is most notably better than the model group, p<0.01;the three-phase group is most notably better than the model group,one-phase group and the positive group p<0.01.
The three-phases group presents Bimodal curve,And the peak of the three-phases group achieve the earliest and highest.
3.2.2 The MOD value variation of the TGF-β_1mRNA of the expression in bone trabecula in every group
MOD value of inter-group comparison showed:the sixth day after operation:No difference of inter-group.the ninth day after operation:the three-phase group is notably better than the model group,p<0.05;the positive group is notably better than the three-phase group, p<0.05;the three-phase group is most notably better than the model group,p<0.01.the fourteenth day after operation:the one-phase group is most notably better than the model group,p<0.01;the one-phase group,the positive group and the three-phase group are notably better than the model group,p<0.05.The average of each group showed:the one-phase group, the positive group and the three-phase group are notably better than the model group,p<0.05.
The peak of the three-phases group achieve the highest.and higher than Other groups in the sixth day after operation,the sixth day after operation:The peak of the one-phases group achieve high, The peak of the the three-phase control group achieve the first and prolonge the time of the peak.
3.2.3 The MOD value variation of the TGF-β_1mRNA of the expression in the periosteum in every group
MOD value of inter-group comparison showed:the ninth day after operation:the three-phase group is notably better than the model group,p<0.05.the fourteenth day after operation:the three-phase group is most notably better than the model group,p<0.01.On the fortyth day after operation:,the positive group is notably better than the model group and the one-phase group,p<0.05;the three-phase group is notably better than the model group and the one-phase group,p<0.05.
3.2.4 The MOD value variation of the TGF-β_1mRNA of the expression in bone tunica intima in every group
MOD value of inter-group comparison showed:the ninth day after operation:the third to the fourteenth day after operation:the three-phases group have the tendency to be better than the model group,the one-phase group and the positive group,p>0.05.The average of each group showed:the three-phases group have the tendency to be better than the model group,the one-phase group and the positive group,p>0.05.
3.2.5 The MOD value variation of the TGF-β_1mRNA of the expression in the fibrillar callus.in every group
MOD value of inter-group comparison showed:The fourteenth day after operation:the positive group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the model group,p<0.01;the positive group is notably better than the one-phase group,p<0.05;the three-phase group is notably better than the one-phase group, p<0.05;the twenty-eighth day after operation:the one -phase group is notably better than the model group,p<0.05;the positive group is most notably better than the model group,p<0.01; the three-phase group is most notably better than the one-phase group,p<0.01.The average of each group showed:the one-phase group is most notably better than the model group, p<0.01;the positive group is most notably better than the model group,p<0.01;the three-phase group is most notably better than the model group,p<0.01.
The one-phase group,the positive group and the three-phase group are notably better than the model group,p<0.05.the fourteenth day after operation:the three-phase group is most notably better than the model group,p<0.0l;the positive group is most notably better than the model group,p<0.01.the twenty-eighth day after operation:the three-phase group,the positive group and the one-phase group are most notably better than the model group,p<0.0l.
4 Conclusions
(1)TCM can enhance the expression of FGF-2mRNA,TGF-β_1mRNA in different tissues in the agglutination of fracture,which show that TCM have the effect to enhance the union of fracture.
(2) The three-phases treatment enhance the expression of FGF-2mRNA, TGF-β_1mRNA in different tissues in the agglutination of fracture.It is may be one of the mechanism that the three-phases treatment enhance the union of fracture.
(3) TCM have different effection to the expression of FGF-2mRNA,TGF-β_1mRNA in different tissues in the agglutination of fracture.The effection of treatment by stages is more manifest than treatment no stage.It shows that treatment by stages is necessary.
(4) The three-phase treatment therapeutical principle aim directly at different pathological change of different stags of the agglutination of fracture.Using corresponding burg can enhance the expression of FGF-2mRNA,TGF-β_1mRNA of the corresponding tissue.It is scientific treatment method by stags.
5 Innovative points
(1) Founding that fracture healing process in different organizations FGF-2mRNA, TGF-β_1mRNA expression
At present,The existing research of the expression of FGF-2mRNA,TGF-β_1mRNA in the agglutination of fracture is quantitative study of FGF-2mRNA,TGF-β_1mRNA in callus, which can not response the change character of every tissues in the agglutination of fracture. It is no report to reveal the the expression character of FGF-2mRNA,TGF-β_1mRNA in different tissues.
(2) To clarify the treatment of fractures of the three fracture healing FGF-2mRNA, TGF-β_1mRNA expression
At present,only VEGFmRNA and BMP-2mRNA in fracture healing research process,by studying the fracture healing process of FGF-2mRNA,TGF-β_1mRNA expression changes in governance were revealed three fractures to promote the role of fracture healing mechanisms, not reported.
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44 何虹,黄剑奇,盛列平.海藻酸钙膜引导下颌骨缺损再生机理的实验研究.口腔医学,2001,21(4):185-187.
45 郑军,董福慧,程伟.水蛭对骨折愈合相关基因表达的影响.中国骨伤,2003,16(9):321-324.
46 熊辉,左亚杰.桃红四物汤对实验性骨折愈合过程中VEGFmRNA表达的影响.中医正骨,2004,16(4):8-9.
1 刘柏龄,邓福树,主编.中医骨伤科各家学说.第二版.北京:人民卫生出版社,2000:15-16.
2 韦以宗编著.中国骨科技术史.上海:上海科学技术文献出版社,1983:125-207.
3 董福慧,朱云龙,主编.中医正骨学.北京:人民卫生出版社,1999,35-41.
4 施杞,王和鸣,主编.骨伤科学-中医药学高级丛书.北京:人民卫生出版社,2001,159-171.
5 危立军,李展振,应文世,等.接骨系列方对促进骨折愈合的疗效观察.舟山医学,1999,27(5):1-4.
6 张海燕,刘定安,黄树明,等.骨折三期用药的临床研究.中国骨伤,1999,12(3):46-47.
7 刘定安,张海燕,黄树明,等.骨折三期用药的实验研究.中国中医药科技,1999,6(6):374-376.
8 韩立军,王俊红,孙建国,等.中药治疗骨折的两阶段学说及临床研究.河可北中医药学报,2000,15(1):12-14.
9 郑军.中药对骨愈合相关基因表达的影响.中国中医研究院博士研究生学位论文,2001:41.
10 苏佳灿,许硕贵,张春才.生长因子与骨重建研究进展.临床骨科杂志,2001,4(2)155.
11 苏佳灿,王家林,张春才.骨折愈合中转化生长因子-b的作用机制.第二军医大学学报,2002,23(2):99-101.
12 孙玉鹏,胡蕴玉.β转化生长因子研究进展.中华骨科杂志,1994,14(8):505.
13 孙玉鹏,陆裕补,郁蕴玉,等.TGF-β提取、纯化及其诱导小鼠股骨成骨作用.中华骨科杂志,1995,15(9):610.
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16 孙玉鹏,陆裕朴,胡蕴玉,等.TGF-β诱导体内成骨效应.第四军医大学学报,1997,18(2):30-33.
17 Randy N,Rosier MD,Ragis J,etal.The potential role of Trans forming growth factor beta in fracrure healing.Clin Orthop and Res,1998,355:294-297.
18 Joyce ME,Terek RM,Jingushi S et al.Role of transfor-ming growth factor-beta in fracture healing/Ann N YAcad Sci,1990,593:107-123.
19 Sandberg MM,Am HT,Vuorio EL,et al.Gene expres-sion during fracture repair.Clin Orthop,1993,289:292-312.
20 汤耿民,沈霖,涂意辉,等.补肾活血方对成骨细胞生长因子TGF-β_1mRNA表达的影响.中国中医骨伤科杂志,1999,7(5):5.
21 沈霖,杜靖远,曾辉,等.补肾方对成骨细胞生长因子TGF-β_1mRNA表达的影响.中医正骨,2001,13(5):3.
22 王华松,许申明.骨碎补对骨折愈合中TGF-β_1表达的影响.中国中医骨伤科杂志,2001,9(4):10-13.
23 徐卫东,吴岳嵩,赵定麟,等.成纤维细胞生长因子对兔关节软骨损伤修复作用的初步探讨.中华风湿病学杂志,1998,12(3):150-151.
24 付勤,肖逸鹏,王志为.生长因子促成年兔关节软骨细胞增殖的研究.中国修复重建外科杂志,2002,16(4):219-222.
25 时光达,安又贤,沈冯君,等.皮质骨在实验性骨折愈合过程中的形念学观察.中华骨科杂志,1982,2(5):292-293.
26 季鸣,顾云伍,刘杰文.骨外膜剥离对实验性骨折愈合的影响.超微结构研究.中华骨科杂志,1994,1 4(8):498-499.
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28 孙永生,温建民,吴林生,等.骨折三期治疗对骨生长因子FGF-2表达影响的实验研究.中国骨伤,2004,17(4):222-223.
29 Solheim E.Growth factors in bone.Int Orthop,1998,22(6):410-416.
30 舒旭,刘贾坤,张长春.中药贯穿疗法在骨折愈合中的应用.中医正骨,2005,17(9):7-9.
31 张俐,叶俊材.活血化瘀汤对骨折早期血管内皮细胞因子活性的影响.中国临床康复,2004,8(23):4798-4799.
32 张俐,叶俊材,陈伯仪,等.活血化瘀汤对骨折早期缺氧诱导因子-1α及血管内皮生长因子影响的实验研究.中国骨伤,2005,18(4):216-218.
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10 苏佳灿,王家林,张春才.骨折愈合中转化生长因子-b的作用机制.第二军医大学学报,2002,23(2):99-101.
11 孙玉鹏,胡蕴玉.p转化生长因子研究进展.中华骨科杂志,1994,14(8):505.
12 孙玉鹏,陆裕补,郁蕴玉,等.TGF-β提取、纯化及其诱导小鼠股骨成骨作用.华骨科杂志,1995,15(9):610.
13 Centrella M,Thomas M,Canalis W.Skeletal tissue andtransforming growth factor & FASEBI,1998,2(10):3066-3073.
14 Hill DJ,Logan A,M c Carry M,et ak Control of protein and matxrix molecule synthesis in isolaled ovine fetalgrowth factors I and n,inurlin and transforming growth factors beta Endocrinol 1992,133(3):363-367.
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16 Randy N,Rosier MD,Ragis J,etal.The potential role of Trans forming growth factor beta in fracrure healing.Clin Orthop and Res,1998,355:294-297.
17 Joyce ME,Terek RM,Jingushi Setal.Role of transfor-ming growth factor-beta in fracture healing/Ann N YAcad Sci,1990,593:107-123.
18 Sandberg MM,Am HT,Vuorio EL,et al.Gene expres-sion during fracture repair.Clin Orthop,1993,289:292-312.
19 汤耿民,沈霖,涂意辉,等.补肾活血方对成骨细胞生长因子TGF-β_1mRNA表达的影响.中国中医骨伤科杂志,1999,7(5):5.
20 沈霖,杜靖远,曾辉,等.补肾方对成骨细胞生长因子TGF-β_1mRNA表达的影响.中医正骨,2001,13(5):3.
21 王华松,许申明.骨碎补对骨折愈合中TGF-β_1表达的影响.中国中医骨伤科杂志,2001,9(4):10.
22 史炜镔,杜宁,等.丹参有效部位对骨折愈合中胶原基因表达的影响.中国中西医结合杂志,2000,20(4):269.
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25 刘道德,王凡.淫羊藿对家兔骨折愈合影响的实验研究.四川解剖学杂志,2004,12(2):101-105.
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33 徐卫东,吴岳嵩,赵定麟,等.成纤维细胞生长因子对兔关节软骨损伤修复作用的初步探讨.中华风湿病学杂志,1998,12(3):150-151.
34 陈纪宁,屠重棋,龚全,等.酸性成纤维细胞生长因子促进引导性骨再生的实验研究中国修复重建外科杂志,1999,13(5):309-314.
35 梁晓萍.骨质疏松大鼠血清IGF-1水平和TGF-β_1骨表达的实验研究.中国老年学杂志,2002,22:47-49.
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37 朱晓峰,张荣华,彭柯萍.益骨胶囊促进成骨细胞增殖和泌IGF-2的作用研究.安徽中医学院学报,2003,22(2):45-47.
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41 初同伟,王正国,朱佩芳,等.骨折愈合过程中血管内皮生长因子及其受体的表达.中华创伤杂志,2001,17(6):344-346.
42 武永刚.血管内皮细胞生长因子在骨折愈合过程中的表达.西安医科大学学报,2001,22(1):51-52.
43 马信龙,谢军,王沛,等.vegf,tgf-b1在骨缺损不愈合中表达的实验研究.中华骨科杂志,2002,22(9):561-566.
44 何虹,黄剑奇,盛列平.海藻酸钙膜引导下颌骨缺损再生机理的实验研究.口腔医学,2001,21(4):185-187.
45 郑军,董福慧,程伟.水蛭对骨折愈合相关基因表达的影响.中国骨伤,2003,16(9):321-324.
46 熊辉,左亚杰.桃红四物汤对实验性骨折愈合过程中VEGFmRNA表达的影响.中医正骨,2004,16(4):8-9.
1 刘柏龄,邓福树,主编.中医骨伤科各家学说.第二版.北京:人民卫生出版社,2000:15-16.
2 韦以宗编著.中国骨科技术史.上海:上海科学技术文献出版社,1983:125-207.
3 董福慧,朱云龙,主编.中医正骨学.北京:人民卫生出版社,1999,35-41.
4 施杞,王和鸣,主编.骨伤科学-中医药学高级丛书.北京:人民卫生出版社,2001,159-171.
5 危立军,李展振,应文世,等.接骨系列方对促进骨折愈合的疗效观察.舟山医学,1999,27(5):1-4.
6 张海燕,刘定安,黄树明,等.骨折三期用药的临床研究.中国骨伤,1999,12(3):46-47.
7 刘定安,张海燕,黄树明,等.骨折三期用药的实验研究.中国中医药科技,1999,6(6):374-376.
8 韩立军,王俊红,孙建国,等.中药治疗骨折的两阶段学说及临床研究.河可北中医药学报,2000,15(1):12-14.
9 郑军.中药对骨愈合相关基因表达的影响.中国中医研究院博士研究生学位论文,2001:41.
10 苏佳灿,许硕贵,张春才.生长因子与骨重建研究进展.临床骨科杂志,2001,4(2)155.
11 苏佳灿,王家林,张春才.骨折愈合中转化生长因子-b的作用机制.第二军医大学学报,2002,23(2):99-101.
12 孙玉鹏,胡蕴玉.β转化生长因子研究进展.中华骨科杂志,1994,14(8):505.
13 孙玉鹏,陆裕补,郁蕴玉,等.TGF-β提取、纯化及其诱导小鼠股骨成骨作用.中华骨科杂志,1995,15(9):610.
14 Centrella M,Thomas M,Canalis W.Skeletal tissue andtrans forming growth factor&FASEBI,1998,2(10):3066-3073.
15 Hill DJ,Logan A,M c Carry M,et ak Control of protein and matxrix molecule synthesis in isolaled ovine fetal growt h factors I and n,inurlin and trans forming growth factors beta I.J Endocrinol.l992,133(3):363-367.
16 孙玉鹏,陆裕朴,胡蕴玉,等.TGF-β诱导体内成骨效应.第四军医大学学报,1997,18(2):30-33.
17 Randy N,Rosier MD,Ragis J,etal.The potential role of Trans forming growth factor beta in fracrure healing.Clin Orthop and Res,1998,355:294-297.
18 Joyce ME,Terek RM,Jingushi S et al.Role of transfor-ming growth factor-beta in fracture healing/Ann N YAcad Sci,1990,593:107-123.
19 Sandberg MM,Am HT,Vuorio EL,et al.Gene expres-sion during fracture repair.Clin Orthop,1993,289:292-312.
20 汤耿民,沈霖,涂意辉,等.补肾活血方对成骨细胞生长因子TGF-β_1mRNA表达的影响.中国中医骨伤科杂志,1999,7(5):5.
21 沈霖,杜靖远,曾辉,等.补肾方对成骨细胞生长因子TGF-β_1mRNA表达的影响.中医正骨,2001,13(5):3.
22 王华松,许申明.骨碎补对骨折愈合中TGF-β_1表达的影响.中国中医骨伤科杂志,2001,9(4):10-13.
23 徐卫东,吴岳嵩,赵定麟,等.成纤维细胞生长因子对兔关节软骨损伤修复作用的初步探讨.中华风湿病学杂志,1998,12(3):150-151.
24 付勤,肖逸鹏,王志为.生长因子促成年兔关节软骨细胞增殖的研究.中国修复重建外科杂志,2002,16(4):219-222.
25 时光达,安又贤,沈冯君,等.皮质骨在实验性骨折愈合过程中的形念学观察.中华骨科杂志,1982,2(5):292-293.
26 季鸣,顾云伍,刘杰文.骨外膜剥离对实验性骨折愈合的影响.超微结构研究.中华骨科杂志,1994,1 4(8):498-499.
27 尚天裕医学文集.第1版.北京:中国科学技术出版社,1991-102.
28 孙永生,温建民,吴林生,等.骨折三期治疗对骨生长因子FGF-2表达影响的实验研究.中国骨伤,2004,17(4):222-223.
29 Solheim E.Growth factors in bone.Int Orthop,1998,22(6):410-416.
30 舒旭,刘贾坤,张长春.中药贯穿疗法在骨折愈合中的应用.中医正骨,2005,17(9):7-9.
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