【作者】 毛碧峰；

【导师】 刘元禄；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 研究背景:自然铜是我国中医骨伤科接骨要药,为我国历版药典所收载。中药自然铜具有散瘀止痛,接骨续筋的功效,临床用于治疗跌打损伤,筋断骨折,血瘀疼痛等。自然铜是接骨续骨最常用的药物,其在骨科方剂中的地位不可替代。临床上自然铜的使用绝大多数是与其他诸位中药配伍使用,并且多以口服方剂为主,有少部分使用浸泡疗法和膏剂案例。骨折发生大多为局部病灶,面对自然铜这样一个对于骨折疗效显著的药物,如果单独使用应选择一种直接在病灶处发挥作用的方式,因此自然铜的局部给药可能是一种理想方式。局部给药使局部组织的药物浓度高,药物主要在局部发挥作用,进入全身血循环的药物较少,对全身的影响较小。皮下注射是其中方法之一,但是局部注射有疼痛、需要多点、多部位注射和注射层次掌握有一定难度,药物分布不均匀,药物在缺血组织中渗透差等不足。低频超声介导下的经皮给药是近年来兴起的局部给药方法之一,是继注射和口服给药后又一种新的给药方式。在一定能量的低频超声介导下,蛋白质等大分子药物可以经局部皮肤被吸收,达到经皮给药的目的,而且这种给药方式不受局部血循环的影响,对缺血组织也同样能够实现局部经皮给药,药物分布均匀。这样局部小剂量的经皮给药,药物就可以在局部形成一个相对的高浓度,发挥其重要的效应。同时由于经皮给药后,药物具有缓释作用,药物进入全身血循环的剂量很小,用药安全,能减少全身的副作用。研究目的:通过应用低频超声透入中药自然铜,观察给药后有效成分组织浓度,自然铜对骨折愈合中BMP-2基因表达的影响以及低频超声透入自然铜对兔肢体骨折愈合的影响。研究方法:1.按随机分组的原则将75只新西兰大耳家兔分成五组,每组15只。治疗方法:治疗1组:浓度为0.5g/ml自然铜煎出液,治疗2组:浓度为1.0g/ml自然铜煎出液,治疗3组:浓度为2.0g/ml自然铜煎出液。治疗1-3组均采用低频超声透入给药,频率为20KHz,强度为100mW/cm²,1次/日,每次20min。对照组:单纯采用超声照射,频率为20KHz,强度为100mW/cm²,1次/日,每次20min。模型组:生理盐水20ml灌胃,1次/日。2.自然铜制备生品自然铜,严格挑选出杂质,将矿石捣碎,用火锻醋淬法:将净自然铜锻至暗红,醋淬至表面呈黑褐色,光泽消失并酥松（如不酥,可反复锻淬至酥）,每100kg自然铜用醋30kg（国家药典委员会.中华人民共和国药典2005年版（一部）:95）。3.X光透视4.骨痂干燥5.骨痂中Fe、Cu、Mn、Zn的测定6.杂交7.洗片8.血清Ca²⁺、P离子浓度、ALP活性检测9.结果统计研究结果:自然铜能够明显提高家兔骨痂中微量元素Fe、Cu的含量（P＜0.05）,从而增加骨痂的生长,表现在X光透视时,试验组家兔骨痂量显著多于对照组。从本次试验所测定的4种微量元素来看,自然铜治疗3组中铁高达1186μg/ml,家兔骨痂铁的含量达（172.68±14.32μg/g）之多,骨痂铜亦明显高于对照组。在BMP-2表达方面,自然铜各治疗组均为棕色,BMP-2表达阳性。对照组及模型组片所有组织均未显色。模型组及对照组所有标本的外骨膜均增厚,治疗组1,2组绝大多数标本骨折断端成纤维细胞和纤维组织及类骨质增多,纤维排列较规则;所有标本外骨膜成骨细胞显著增生,增生细胞融合成一片,骨基质沉着,形成骨样组织、软骨及骨小梁,并向骨折间隙延伸,出现不完全的软骨与骨小梁骨痂相连,在成纤维细胞、成软骨细胞、软骨细胞、成骨细胞、骨细胞等表达阳性。治疗3组编织骨形成,骨髓腔内的骨小梁变细,骨髓腔再通。实验表明自然铜低频超声透入给药能促进血Ca、P、ALP含量增加兔肢体骨折愈合能力,能明显提高家兔骨痂中微量元素的含量（p＜0.05）,增加骨痂生长量,从而促进骨修复过程。结论:1.低频（20KHz）超声药物导入是一种安全、有效的经皮局部给药的方式。2.在低频超声介导下,煅自然铜可以经皮给药,在组织局部发挥其生物效应。3.在低频超声介导下,随给药浓度提高,可以增加局部组织的药物浓度,且单纯的低频超声具有一定的促进愈合功效。4.自然铜在超声介导下可以经皮给药能够诱导BMP-2合成加速骨折愈合;提高血Ca、P、ALP含量增加兔肢体骨折愈合能力;自然铜能明显提高家兔骨痂中微量元素的含量（p＜0.05）,增加骨痂生长量,从而促进骨修复过程。更多还原

【Abstract】 Background:Pyritum is important Chinese medicine for bone fracture and recorded in all ages in our national pharmacopoeia codex.It can help improving blood circulation and relieving bruises and pains and muscle and ligament reunion of bone.It is clinically used for injuries from falls,muscle and bone broken,gore and pains etc.Pyritum is the most common used medicine for bone-knitting and symplectic bone which there is no other orthopaedics prescription to replace.Pyritum is always used matching with other Chinese medicine clinically and in multi-oral prescription.A few of them are used in marinate and electuary ointment.Bone broken always occurs locally.As Pyritum has such an effection in treatment of fracture,it must he a ideal method to administrate locally with Pyritum which can induce a high local drug concentration and drug plays a role locally that there is little drug concentration in body plasma.Hyodermic is one method of it.However it is painful,multi-point needed,difficult to master the level,not well-distrbuted.The disadvantage is low drug concentration in less plasma position.Through using a certain level low frequency ultrasound, giant molecule such as protein can be obtained across the skin arriving on target which will not be affected by local blood circle.It can realize the acquisition of local body insufficient of plasma and drug well-distribute.So local small dose across skin can make a relatively high concentration and has an important effects;meanwhile it has a sustained release effective.There is a little dosage in plasma and it is safe.Also it reduces secondary reaction.Object:After administrating Chinese medicine Pyritum with low frequency ultrasound,we observed local effective agent concentration and the effects of BMp-2’s expression on fracture restitution when using Pyritum and low frequency ultrasound’s administration effects on rabbit’s bone restitution.Method:1.Put 75 New Zealand white big ears rabbits into 5 groups randomly,15 each.Method:one treatment group:0.5g/ml Pyritum decoction;two treatment groups:1.0g/ml Pyritum decoction;three treatment groups: 2.0g/ml Pyritum decoction.Group 1-3 with low frequency ultrasound, once per day.Control group:ultrasound with frequency of 20KHz, intensity of 100mw/cm²,low frequency in vitro irradiation,once per day,20min once.Model group:normal saline 20ml lavage,once per day.2.Pyritum preparation:Pyritum pick up impurity strictly,smash the ore, heat it in fire and dip it into vinegar:heat it to dark red,dip it into vinegar to pitchy,luster disappears and loose（if not loose over and over again till loose ）vinegar 30kg per 100kg Pyritum（NF. Pharmacopoeia of PRC2005 edition（the lst）:95）3.X-ray transmission4.poroma dry5.determination of Fe,Cu,Mn,Zn in poroma6.hybrid7.develop a film8.determination of Ca²⁺,P ion concentration and Alp activity in plasma 9.statistics resultsResultsPyritum can obviously increase Fe,Cu contents in poroma of rabbits.（P＜0.05）,and then increase poroma’s growth.We can observe that poroma in experimental group is more than it in control group when using X-ray.Observed from 4 trace elements from the testing,among 3 Pyritum experimental groups Fe concentration reaches 1186μg/ml,while it reaches（172.68±14.32μg/ml）in rabbit poroma.Cu in poroma in experimental group is higher than it in control group.All Pyritum experimental groups are b（?）n,BMP-2 to express masculine. There is no color development in all control groups and model groups. All samples outside periosteum of model and control group grew thicker. Most samples of fracture mechanocyte,nerve structure and osteoid increased and fiber arrayed regularly in experimental group 1,2.All samples outside periosteum osteoplast got hyperplasia.The hyperplasia got together,bone matrix adipopexised and informed osteoid tissue,cartilage and bone trabecula,and expanded towards fracture clearance.Lame cartilage and bone trabecula’s poroma connected and expressed masculine in mechanocyte,chondroblast cartilage,osteoplast and osteocyte.In 3 experimental groups woven bone got grow and medullary space bone trabecula got thinner and medullary space got open.It improved Ca,P,ALP concentration that union got increased.Pyritum can increase microelement in rabbit poroma obviously（p＜0.05）,increase poroma growth and then improve bone repairing.Conclusion:1.Low frequency（20KHz）ultrasound administration is a safe,effective local percutaneous administration method.2.With low frequency ultrasound,heated Pyritum can be administrated across skin and has bioeffects on local body. 3.With low frequency ultrasound local concentration will increase as dosage increase and single low frequency has effects on wound healing.4.With low frequency ultrasound Pyritum can induce BMP-2 to make broken healing and improve Ca,P,ALP concentration to heal rabbit broken bone.Pyritum can increase trace element in rabbit poroma and then improve course of repair.更多还原