国新牌骨痛膏促进家兔骨愈合的实验研究

【作者】 杨济洲；

【导师】 徐林；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 研究背景骨折是骨科临床的常见病和多发病,随着社会经济的发展,交通运输高度发达,而且中国逐渐迈入老龄化社会,骨折成高发态势。据统计约有5%-10%的骨折可因各种原因发生骨折迟缓性愈合及不愈合。目前,国外对于药物促进骨折愈合的研究较少,国内的研究也多侧重于中药内服,相对中药内服促进骨愈合而言,外用中药促进骨愈合的研究水平不高。中医药治疗骨折已有漫长的历史,经验丰富。1931年出土的《居延汉简》中就记录了汉代军医以膏药为主治疗各种创伤的方药和方法,可见早在秦汉时代应用敷贴治疗骨伤就已经很普遍了。因此利用现代分子生物学及细胞生物学深入研究并揭示外用中药治疗骨折的疗效及机制有重要的现实意义和社会意义。国新牌骨痛膏源自吉林省民间验方,为促进骨愈合的一种外用膏剂,在临床应用多年,疗效确切,治愈大量骨折、骨不连、骨坏死病人,但是作用机制仍不清楚,本研究拟通过检测血生化指标、X线评分、组织切片观察、胶原分型、骨形态发生蛋白(BMP-2)免疫组化、骨形态发生蛋白(BMP-2)原位杂交等方法验证其促进促进骨愈合的效果,从现代医学的角度探讨其促进骨愈合的的作用机制,进而从现代医学角度为外用中药促进骨愈合提供更为科学的依据。研究目的探讨国新牌骨痛膏促骨愈合效果,揭示其促进骨愈合的可能机理。研究方法将30只日本大耳白兔均以手术造成双桡骨中段宽约3mm、深达髓腔的骨缺损模型,术后观察日本大白兔的一般情况。再将造模成功的30只日本大耳白兔随机分成国新牌骨痛膏外用药组和空白对照组,再将每组15只随机分成三个亚组,即第1周取标本组(A组)、第2周取标本组(B组)、第4周取标本组(C组),空白对照组以石膏固定,给药组外敷国新牌骨痛膏后以石膏固定,给药组每2天换一次药,1周处死A组,2周处死B组,4周处死C组。每组处死前均予耳缘静脉取血,检测血中ALP、钙、磷、镁浓度,两组行t检验；处死后拍摄双侧桡骨侧位X光片肉眼观察评分,A、B、C各组行t检验比较；病理学HE、Masson染色观察；Ⅰ、Ⅲ型胶原苦味天狼星染色后偏正光显微镜观察分析；免疫组化法检测桡骨缺损处骨形态发生蛋白(Bone Morphogenetic Protein, BMP-2)的表达,测MOD值后A、B、C各组行t检验；原位杂交法检测桡骨缺损处骨形态发生蛋白(Bone Morphogenetic Protein, BMP-2)的表达,测MOD值后A、B、C各组行t检验。研究结果术后各组实验兔的一般情况良好；术后1、2、4周时,生化指标检查：给药组与对照组血中ALP、钙、磷、镁及钙磷乘积值均无统计学差异；放射学检查：术后第1周给药组即有骨痂形成,对照组未见骨痂生长。术后第2周两组均可见骨痂形成,给药组骨折缺损区有较完全的骨痂填满并见大量外骨痂形成,对照组骨折缺损区可见软性骨痂填充,大部分无外骨痂。术后第4周给药组缺损区骨皮质重建,骨干外形恢复,髓腔再通,对照组缺损区骨痂基本填满,仍有连接性骨痂阴影,髓腔未通。各组X线片肉眼观察评分结果给药组的得分均高于空白对照组,统计学差异显著；病理学HE、Masson染色观察发现给药组第1周即出现骨痂,第2周外骨痂明显,第4周缺损部位骨痂中胶原明显减少,骨缺损修复质量好。对照组第1周时未见骨痂形成,第2周大部分无外骨痂,第4周缺损部位骨痂仍含有大量胶原,修复质量较给药组差；Ⅱ、Ⅲ型胶原苦味天狼星染色结果,给药组的Ⅰ型胶原出现较对照组早,且Ⅰ型胶原含量高；BMP-2免疫组化及原位杂交结果给药组BMP-2mRNA的表达出现时间早于对照组,表达程度明显优于对照组,统计学差异显著。结论(1)国新牌骨痛膏对血ALP、钙、磷、镁及钙磷乘积没有影响。(2)国新牌骨痛膏可以促进Ⅰ型胶原的表达。(3)国新牌骨痛膏可以调节BMP-2的合成、分泌并促进BMP-2的表达(4)国新牌骨痛膏可以使骨痂提前出现,提高骨愈合质量,缩短愈合时间,促进骨愈合更多还原

【Abstract】 Study backgroundFracture is the common disease in the clinical practice. Along with social economy’s development, the transportation is highly developed, moreover China enters into the aging society gradually, the fracture becomes more frequently. Statistically, there is approximate 5%-10% fractures may lead to the slow cicatrization and disunion by each kind of reason. At present, there are few research about promoting healing by medicine overseas. The domestic research also stresses on the oral administration of traditional Chinese medicine, the level of research about traditional Chinese medicine external using promoting healing is not high compared to the oral administration. The history is long in Fracture treated with the Chinese medicine, and also has the rich experience. "Occupied Extends Chinese Jan" unearthed in 1931 had recorded the medicine external use which could treat wound by military doctor in Han Dynasty. Obviously it is very common as early as in the Qin and Han Dynasty. Therefore, curative effect and the mechanism of the external use of traditional Chinese medicine through modern molecular biology and cytobiology has the vital practical significance and the social meaning."Guoxin ostalgia ointment" is a folk recipe and comes from Jilin Province. It is a kind of external use medicinal and can promote fracture healing. It has been many years in clinical practice and has the excellent curative effect.“Guoxin ostalgia ointment”had cured massive patient with bone fractures, the bone do not link and the osteonecrosis. But the mechanism is not clear yet, through testing biochemical indicator, X ray grading, tissue slice analysis, the collagen typing, immunohistochemistry test of the bone morphogenesis protein (BMP-2) and hybridization in situ test for bone morphogenesis protein (BMP-2), author hopes to reveal the mechanism of promoting fracture healing and also hopes to provide more scientific evidence for external use of traditional Chinese medicine from the view of modern medicine.Study objectiveTo study the therapy effect of "Guoxin ostalgia ointment" and to reveal the possible mechanism of promoting fracture healing.Study methodsFracture models were made by operation.3mm bone defect was made on the middle shafts of both Japanese rabbits in each bone. Observe the post-operation general conditions of the Japanese rabbits. Thirty rabbits were randomly assigned to receive "Guoxin ostalgia ointment" and blank control groups.15 rabbits in each of the two groups. Then each group were assigned to 3 sub-groups. With specimen collection from rabbits postoperative 1 week (A),2 week (B) and 4 week(C).All of the experimental rabbits were made to the bone defect model with 3mm wide to the pulp chamber located on the midpiece of bi-radial. Rabbits in blank control group were received by gypsum exclusively. Rabbits in experimental group were received by"Guoxin ostalgia ointment" external use add gypsum fixation. Experimental group changed addressing per two days. Execute the rabbits in A sub-group after first week. Execute the rabbits in B sub-group after second week. Execute the rabbits in C sub-group after fourth week. Test ALP, Calcium, Phosphorus and Magnesium from blood of ear vein. Shot the bi-radial X ray film. T-test was used to statistic the three group(A,B,C). Pathology test by the method of HE, Masson dyeing. light microscope observation ofⅠ,Ⅲcollagen bitter taste sirius dyeing. BMP-2 was tested by the method of immunohistochemistry and hybridization in situ. T test was applied to the A,B,C each group after testing the value of MOD.Study resultsEach group of rabbit’s general conditions is good. Biochemical indicator of 1,2,4 week after operation showed:There is no difference between experimental group and blank control group on the effect of blood density of ALP, Calcium, Phosphorus, Magnesium and calcium-phosphorus product. The result of postoperative 1st week showed:"Guoxin ostalgia ointment" can enhance the BMP-2 expression and bony callus formulation; Reduce healing time. Callus formation was occurred by the radiological examination and there is no bony callus in the blank control group. The result of postoperative 2nd week showed:There exist bony callus in both two group. Bony callus stuffed the bone defect area and massive external callus formation appeared in the experimental group. And there is no external callus but soft callus stuff in the blank group. The result of postoperative 4th week showed:medicine group’s bone cerebral cortex reconstruction was appeared in the damage area, the backbone contour restored, the medulla cavity passed again. And callus filled up basically in the control group damage area, connective callus shadow still existed, the medulla cavity had not pass. Through macroscopic observation of X ray, the store of medicine group is higher than the control group and there is obvious difference in statistics. Through the HE and Masson dyeing, in the 1st week, there appeared bony callus in the medication group and there existed obvious bony callus in the 2nd week. In the 4th week, the collagen reduction was obvious in the defect area and the quality was good; In the control group, callus formed in the 1st week, there did not have the external callus in the 2nd week, there existed massive collagens in the 4th week in the damage area and the requirement quality was worse than the experimental group.Ⅰ,Ⅲcollagen bitter taste sirius dyeing result:experimental group appeared earlier than the control group and had more I collagen; BMP-2 was tested by the method of immunohistochemistry and hybridization and the result indicated that the experimental group appeared earlier than the control group, the expressive degree was obvious outweigh the control group and there was obvious difference in statistic.Conclusion(1) "Guoxin ostalgia ointment" doesn’t affect the ALP, Calcium, Phosphorus, Magnesium and calcium-phosphorus product.(2) "Guoxin ostalgia ointment" can enhance the expression of I type collagen.(3)“Guoxin ostalgia ointment”can regulate the formulation、externalization and promote the BMP-2 expression.(4) "Guoxin ostalgia ointment" can make the bony callus appears earlier, improve the bone healing quality, reduce the healing time and promote the healing progress.更多还原