| 1 Research backgroudBone fracture has been the common disease and frequently encountered disease in clinicalong with the society develops, the communication and the carrying trade becomeprosperous. How to encourage the bone fracture concrescent has always been the research hotspot in the orthopedics occasion because of the bone fracture caused by various kinds ofreasons is late healed and disunion, which harms people life quality severely. Applying TCMto cure bone fracture has a long history and certain curative effect, and because of itseconomics and convenient, has received more and more reconstruction, to be one of the mainmethmods to cure bone fracture. Differentiation of symptoms and signs and discussing curingis the theory cores and essence in TCM, by which the bone fracture can be divided into threestages-nonage, metaphase and anaphase based on symptoms and histopathology changing.Based above all of which, the bone fracture three-stage treatment has been proposed, whichhas been the guidance principle in the modern TCM curing the catagma. But the therapeuticalprinciple of the bone fracture three-stage treatment based on the experience of the predecessor,the scientific experiment evidence is deficiency, and the mechanism of action of drugs staystill in the macroscopic and vague level. How to keep and develop the features anddominances of the department of fracture and wounds in TCM and promote TCMmodernization to the world must not avoidant.Currently there is a tremendous dispute aboutthe effect of the therapeutical principle of the bone fracture three-stage treatment and a severedissension about the methmods of TCM curing the catagma. The clinical guidance principleof the bone fracture three-stage treatment has to be faced with draconic challenge, whichrestrict the development of TCM curing the catagma severely. But the former research aboutthe therapeutical principle of the bone fracture three-stage treatment was very poor,furthermore, most of which were clinical rough initial observation and the comparison to themodel groupor the officinal drug. The area is vacant than revealing the function and the mechanisms system of the therapeutical principle of the bone fracture three station treatmentfrom the gene level.This research planed to observe the treatment of the three-phases of the fracture, one-phase curing, the influence of the bone growth factor express and distribute disciplinarian inthe "gu zhe cuo shang san " curing the bone fracture, by the way of immunity class bromodand hybridization in situ technique to detect the bone growth factor BMP, BMP in thelocation of the fracture. Illuminating the effect and the mechanism of the fracture in the TCMthree-phases about the fracture from the molecular and gene level, approaching the beststaging methmods, supporting the more scientific theory evidence for TCM to cure thefracture, guiding the treatment of the bone fracture.2 Materials and metheods2．1 Experimental Animal and Model of the bone fractureNew Zealand rabbits. A 3mm deossification was made in radius at distal end of mulslespronator teres as fracture healing model. After operation, 140 rabbits were divided into fourgroups randomly by SPSS, which are three-phases group, one-phase group, the model groupand the positive group, 35 rabbits per group.2．2 Intervention methmods2．2．1 Three-phases groupThree-phases therapy priciple was adopted according to the bone fracture three stationtreatment: nonage, after bone fracure one～seven days: promoting blomod flow anddissolving congestion,promoting qi circulation to relieve pain, adopting "jia jian zhi shangone fang"; metaphase, after eight～twenty-eight days: reunion of bone,relax and activate thetendons, adopting "jia jian zhi shang two fang"; anaphase, after twenty-eight days: tonifingliver and kidney,strengthening bone and musculature, adopting"jia jian zhi shang threefang".2．2．2 One-phase group Put fractured nonage, metaphase and anaphase into together, promoting blomod flow anddissolving congestion,promoting qi circulation to relieve pain,congestion,promoting qicirculation to relieve pain,tonifing liver and kidney,strengthening bone and musculature,adopting"jia jian zhi shang one,two and three fang" "one-phase fang".2．2．3 Positive groupDeturing bone fracture contund powder solution whole range without stage.2．2．4 Medication doseAccording to the quantity and effect reaction to decide the best optimization agentia dosewas double equivalent dose, Intragastric administrated twice a day.2．2．5 Model groupAdministrate tales doses of experimentation group distilled water,intragastricadministration, twice a day.2．3 Specimen managementA subgroup rabbits of every group were selected randomly and put to death by theaeroembolism methmod after operation at day 3, 5, 7,14, 28,42, 56．Their radials was takenout and taken CR films at same condition. The status of bone fracture healing was observedand scored by the borderline of broken ends, the quantity of bony callus, bony callus densities,bony callus borderline. Then the left radius were used to check the BMP-2 and mRNAexpression with Immunity class chemical coloratura ( S-P methmod ) and Hybridization in situ.Recipe two high power campus visualis to determine the MOD value of the positiveexpression cells in diaphysis cell, the periosteum, bone tunica intima, hematoma, the fibrillarcallus, the chondral callus, bone trabecula, intertrabecular tissue and cavitas medullaris,2．4 Data processingStatistical analysis use spss13．0 software.3 Consequence3．1 The X-ray scores of the influence to the agglutination of fracture in the modelcontrol group, the one-phase group, the three-phases group and the positive group The comparison in every group evids that the three-phases group and the one-phase group is most notably better than the model control group and the positive group, p<0．01, On the sixth day after operation: the three-phases group and the one-phase group is most notably better than the model control group, p<0．01, the three-phases group is most notably better than the positive group, p<0．01, On the ninth day after operation: the three-phases group and the one-phase group is most notably better than the model control group and the positive group, p<0．01．3．2 The MOD value variation of the BMP-2 of the expression in the three-phases group, the one-phase group, the positive group and the model control group, in the agglutination of fracture3．2．1 The MOD value variation of the BMP-2 of the expression in bone stem cells in every groupThe comparison in every group evids that the three-phases group and the one-phase 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 three-phases group is notably better than the one-phase group, p<0．05, and the three-phases group is most notably better than the positive group, p<0．01．From the ninth day to the forty-twice day, after operation: the three-phases group is most notably better than the model group, p<0．01; the three-phases group is notably better than the positive group, p<0．05．The three-phases group, the one-phase group and the positive group presents three-peak curve. The peak of the the model control group achieve the first, but the lowest. The three-phases group, the one-phase group and the positive group present the peak at the same time, and the peak of the three-phases group is the highest.3．2．2 The MOD value variation of the BMP-2 of the expression in hematoma, in every group The comparison in every group evids that the one-phase group is better than the model group, p<0．05; the three-phases group is notably better than the model group, p<0．01 On the sixth day after operation: the three-phases group is most notably better than the model group, p<0．01, the one-phases group is notably better than the model group; On the nighth to the forteenth day after operation, there is no statistical significance in every group.The positive group present bimodal curve. The peak of the three-phases group and the one-phase group achieve there days earlier than that of the positive group. The peak of the the model control group achieve the first, but the lowest. The peak of the three-phases group is the highest. The three-phases group increase the level of the peak.3．2．3 The MOD value variation of the BMP-2 of the expression in periosteum in every groupThe comparison in every group evids that the three-phases group and the one-phase group is most notably better than the model group, p<0．01, the one-phase treatment 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, and the positive group have the tendency to be better than the model group.On the ninth day after operation: the three-phases group is notably better than the model control group, p<0．05, On the fourteenth day after operation: the three-phases group is most notably better than the model group, p<0．01, the three-phases group , the one-phase treatment, the positive group is notably better than the model control group, p<0．05, the three-phases treatment notably better than the positive group, P<0．05．The three-phases group, the one-phase group and the positive group present bimodal curve. The peak of the the one-phase group achieve the first, but the lowest. The three-phases group, present the peak on the forteenth day after operation and the peak of the three-phases group is notably better than the model group.3．2．4 The MOD value variation of the BMP-2 of the expression in bone tunica intima in every group The comparison in every group evids that the three-phases group is most notably better than the model group, p<0．01, and the one-phase group have the tendency to be better than the model group, P>0．05．On the fourteenth day after operation: the three-phases group is most notably better than the model group, p<0．01, the positive group have the tendency to be better than the model group.p>0．05．On the fortyth day after operation: the positive group have the tendency to be better than the model control group, and the three-phases group have the tendency to be better than the model group, p>0．05．The three-phases group, the one-phase group and the positive group present bimodal curve. The peak of the the one-phase group achieve the first, but the lowest. The three-phases group and the positive group present the peak at the same time. The peak of the three-phases group is notably better than the other group.3．2．5 The MOD value variation of the BMP-2 of the expression in cavum ossis in every groupThe comparison in every group evids that the three-phases group and the one-phase group are most notably better than the model group, p<0．01, the positive group is notably better than the model group, p<0．05, the three-phases group is notably better than the positive group, p<0．05, and the three-phases treatment grou have the tendency to be better than the one-phase group, P>0．05．On the nighth day after operation: the three-phases group is notably better than the model group, p<0．05, the one-phase group have the tendency to be better than the one-phase group; the positive group have the tendency to be better than the one-phase group. On the fourteenth day after operation: the three-phases group is notably better than the model control group, p<0．05．The three-phases group and the positive group present three-peak curve. The one-phase group and the model grouppresent bimodal curve. The peak of the three-phases group, the one-phase group and the positive group achieve there days earlier than that of the model group.From the nighth to the fortyth day after operation, the peak of the three-phases group last the higher level, which show that the three-phases group enhance the level of the peak and prolonge the time of the peak.3．2．6 The MOD value variation of the BMP-2 of the expression in cartilaginous callus in every groupThe comparison in every group evids that the one-phase group is most notably better than the model group, p=0．01, the three-phases group is most notably better than the positive group, p<0．01．On the ninth day after operation: the three-phases group is most notably better than the model group, p<0．01, the three-phases group is notably better than the one-phase group, p<0．05．the three-phases group have the tendency to be better than the one-phase treatment, p>0．05．On the fourteenth day after operation: the three-phases group is most notably better than the model group, p<0．01, On the forty-twice day, the three-phases group is notably better than the model control group, p<0．05．Every group presents bimodal curve. The three-phases group, the one-phase group and the model group achieve the peak at the same time. The positive group have no peak. The peak of the three-phases group is notably better than the model group. And the three-phases group enhance the level of the peak.3．3 The MOD value variation of the BMP-2 mRNA of the expression in the three-phases group, the one-phase group, the positive group and the model control group, in the agglutination of fracture3．3．1 The MOD value variation of the BMP-2 mRNA of the expression in bone stem cells in every groupThe comparison in every group evids that the three-phases group has the tendency to be better than the positive group and the one-phase treatment, P>0．05, and is notably better than the model group, P<0．01． On the ninth day after operation: the three-phases group is notably better than the model group, p<0．05．On the fourteenth to the twenty-eighth day after operation: the three-phases group is notably better than the model group, p<0．05．The positive group presents bimodal curve.The three-phases group and the one-phase group treatment presents three-peak curve. The three-phases group, the one-phase group and the positive group achieve the peak at the same time,and five days earlier than that of the model group. The three-phases group enhance the level of the peak, The three-phases group increase the level of the peak.3．3．2 The MOD value variation of the BMP-2 mRNA of the expression in hematoma of broken ends of fractured bone, in every groupThe comparison in every group evids that the three-phases group and the one-phase group is most notably better than the model group, p<0．01．The positive groupis notably better than the model group, p<0．05; the three-phases group is most notably better than the positive group, p<0．05．On the sixth day after operation: the three-phases group have the tendency to be better than the model group, p>0．05．On the nighth day after operation, the three-phases grou and the one-phase group is most notably better than the model group, p<0．01．The three-phases grou and the one-phase group present bimodal curve. The three-phases group and the one-phase group achieve the peak at the same time. The peak of the three-phases group is the highest.The other group has no peak.3．3．3 The MOD value variation of the BMP-2 mRNA of the expression in periosteum in every groupThe comparison in every group evids that the three-phases group and the one-phase group is most notably better than the model group, p<0．01, the three-phases group have the tendency to be better than the one-phase group, and the three-phases group is most notably better than the positive group, p<0．05．On the ninth day after operation: the three-phases group is notably better than the positive group, p<0．05．On the fourteenth day after operation: the three-phases group is most notably better than the model group, p<0．01,the three-phases group , the one-phase treatment, the positive group is notably better than the model group, p<0．05, the three-phases treatment notably better than the positive group,P<0．05．The three-phases group presents three-peak curve. The one-phase group and the positive group present bimodal curve. The three-phases group and the positive group achieve the peak at the same time, and five days earlier than that of the one-phases group. The peak of the three-phases group is the higher than the model group .3．3．4 The MOD value variation of the BMP-2 mRNA of the expression in bone tunica intima in every groupThe comparison in every group evids that the three-phases group have the tendency to be better than the one-phase group, P>0．05; the three-phases group and the one-phase group is most notably better than the model group, p<0．01, the three-phases group is most notably better than the positive group, p<0．01, ans the one-phase group is notably better than the positive group, p<0．05．On the ninth day after operation: the three-phases group is notably better than the model group, p<0．05, have the tendency to be better than the positive group, p>0．05．On the fourteenth day after operation: the three-phases group is notably better than the model group, p<0．05．the one-phase group is notably better than the model group, p<0．05．The three-phases group and the one-phase group present bimodal curve. The peak of three-phases group achieve three days earlier than that of the one-phases group. The peak of the three-phases group have the tendency to be better than the model group, The peak of the three-phases group last the higher level ,which achieve the earliest and last longer.3．3．5 The MOD value variation of the BMP-2 mRNA of the expression in cavum ossis in every groupThe comparison in every group evids that the three-phases group and the one-phase group are most notably better than the model group, p<0．01, the three-phases group is most notably better than the positive group, p<0．01, and the one-phase group is notably better than the positive group, P<0．05．From the ninth day to the fourteenth day after operation: the three-phases group is most notably better than the model group, p<0．01, the one-phase group is notably better than the model group, p<0．05．the three-phases group is notably better than the positive group, p<0．05．The three-phases group, the one-phase group and the positive group present trith-peak curve. The peak of the three-phases group and the one-phases group achieve five days earlier than that of the positive group. The peak of the three-phases group is notably higher than the model group.3．3．6 The MOD value variation of the BMP-2 RNA of the expression in cartilaginous callus in every groupThe comparison in every group evids that the three-phases group,the one-phase group and the positive group are most notably better than the model group, p<0．01, the three-phases group is most notably better than the positive group, p<0．05．From the ninth day to the forty-twice day after operation: the three-phases group is most notably better than the model group, p<0．01, the one-phase group and the positive group are notably better than the model group, p<0．05．The one-phase group presents bimodal curve. The three-phases group and the one-phases group achieve the peak at the same time,and forteenth day ealier than the model group and the positive group. The peak of the three-phases group and the one-phases group is most notably higher than that of the model group.The peak of the positive group is notably higher than that of the model group. The peak of the three-phases group last the higher level,and enhance the level of the peak.3．3．7 The MOD value variation of the BMP-2 mRNA of the expression in intertrabecular space in every group The comparison in every group evids that the three-phases group and the one-phase group is most notably better than the model control group, p<0．01, the three-phases group have the tendency to be better than the positive group.On the fourteenth day after operation: the three-phases group is most notably better than the model group, p=0．01．the one-phase group is notably better than the model control group, p<0．05．The peak of every group achieve the peak at the same time, and all present bimodal curve.The three-phases group enhance the level of the peak.4 Conclusions4．1 TCM can enhance the expression of BMP-2,BMP-2 mRNA in different tissues in theagglutination of fracture, which show that TCM have the effect to enhance the union of fracture.4．2 The three-phases treatment enhance the expression of BMP-2,BMP-2 mRNA 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.4．3 TCM have different effection to the expression of BMP-2,BMP-2 mRNA 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,4．4 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 BMP-2,BMP-2 mRNA of the corresponding tissue. It is scientific treatment method by stags.5 Innovative points5．1 Reveal the expression rule of BMP-2,BMP-2 mRNA in every born tissues in the agglutination of fracture, on the protein-level and the gene-levelThe agglutination of fracture is a complicated process. Many tissues participate and intercommunicate. The existing research of the expression of BMP in the agglutination of fracture is quantitative study of BMP 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 BMP in different tissues.5．2 Reveal the partial mechanism of the three-phases treatment to fracrure on the genetic level, the bone induction aspectAt present, the research of TCM enhance the union of fracture is mainly about the single burg or the single recipe. The research of the three-phases therapeutical principle is few. It is no report to research the expression of BMP-2．BMP-2 mRNA in the agglutination of fracture or the mechanism of the three-phases treatment enhance the union of fracture. |
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