| At present, osteoporosis is the mainly disease to be prevented in the world, while people ageing. According to the latest standard, osteoporosis can divided into three kinds; the primary osteoporosis, the secondary osteoporosis, and the idiopathetic osteoporosis. And the morbidity of primary osteoporosis is highest, which is described as osteopenia, degeneration of the bone microstructure (the spongy bone trabecula turn thinner, break, and decrease; the compact bone turn porosity and thinner) , and the increase of the bone fragility and the fracture risk. Fracture is the mainly complication of osteoporosis, which can decrease the osteal quality and intension at the fracture part, and there are all splintered fracture , which is difficultly treated. Recently, we got great improvement on osteoporosis prevention, however, we didnt have the definite methods on the osteoporosis treatment yet.Mundy reported that descend blood lipid drugs of statin may promote the bone formation. But it is unclear of the effect on the fracture union of osteoporosis. In this investigation, we can observe the effect of fluvastatin on the callus of the fracture union process, by studying the histology, x - ray, ultramicrostruc-ture, and the change of the regional vitodynamics. And what is more, by the immunochemistry and hybridization in situ methods, we also observe some important cytokines for consummating the clinical treatment theory, including bone morphogenetic protein - 7 ( BMP - 7 ) , interleukin - 6 (IL - 6 ) , vascular endo-thelial growth factor (VEGF).Experimental materials1. Animals: 60 SD female rats; 6 months; weight 290 -340G, provided by China Medical University laboratory animal center.2. The main agent: Fluvastatin ( Beijing Nuohua Limited Company) ; BMP - 7 detecting kit of immunochemistry and hybridization in situ; IL — 6 detectingkit of immunochemistry and hybridization in situ (Wuhan Boshide biologic product company ) ; VEGF immunochemistry and "hybridization in situ ( Wuhan Boshide biologic product company ).3. The equipment: Olympus microscope(Japan) ; Prodigy bone density apparatus (America LUNAR company) ; XR - d X - ray (American GE company) ; JEM1200EX transmission electron microscope ( Japan) ; AG -5000A electron almighty equipment by computer (Japan) ; MetaMorph/Olympus BX41 mi-crogramme analytical system (Japan) ; surgical instruments ( China).Experiment Method1. Sample of osteoprosis animal60 female SD rats (supported by experimental animal center of China Medical University) , age of 6 months, 290 -340 grams. After introperitoneal injection of ketamine( 100mg/kg) , incise at median abdominal under asepsis. Remove bilateral ovasis totally, and hypodesis ductus Meller and comites, then close the abdominal after douching. Raise separately and standardly after operation with freely action and under sun 12 hours/day, at 25 2℃, room temperature.2. Administration method; After fracture fixation, divide the animals into two groups ( experiment group and control group) randomly, 30 rats in each group. Lavage fluvastatin (Beijing Nuohua Limited Company) 5mg/kg.g, and placebo in control group. This begins from the day after operation, and lasts for 6 weeks.3. 1 Radiation observation; Choose 6 rats randomly in each group at 7th,14th,21th,28th,and 42th day separately. After intraperitoneal anesthesia with ket-amine(100mg/kg) , orthotropia DR(50kv,2. 5ms) with XR/dX( American GE company ).3. 2 The change of BMP -7, IL -6,VEGF in the callus during the healing of fracture: test the expression of BMP - 7,IL - 6 ,VEGF in the callus at the 7th,14th,21st,28th and 42nd day, using SABC method, and analyze with MetaMorp/ Olympus BX41 micro - image analysis system. 3. 3 The change of BMP -7mRNA,IL -6mRNA,VEGFmRNA in the callus during the healing of fracture; test the expression of BMP -7mRNA ,IL -6mRNA,VEGFmRNA in callus at the 7th J4th,21st,28th,and 42nd day with hybridization in situ.3.4 The change of ultramicrostructure of local callus during healing of fracture ; observe the ultrastructure of callus at the 7th ,4th,21st,28th and 42nd day u-sing electron microscope.3.5 The change of vitodynamics of local callus during healing of fracture : test the tri -bending load of femus at 7th, 14th,21st, 28th, and 42nd day using AG- 5000A electro - experiment.Experiment Result1. The change of radiation; during the healing of fracture, the change of radiation is similar. At 7th day, there is little callus around the line of fracture. At 21st day, there is fusiform -like callus around the fracture, but the fracture line is clear. At 42nd day, the density of callus is higher, and the fracture line is vu-gur.2. Histological observation; at 7th day, the two groups are similar. The periocytes start changing into osteoblast, and there is newborn trabecular, and prechondrocyte in the around granulation generate, and there has already been newborn chondrocyte. At 14th day, there is cardilangious callus intead of fibrous callus, and early -cartilage island in fibrous callus. The chondrocytes around the vessels are round immature chondrocyte, as time goes by, the chondrocytes around the callus become large and partly necrosis, and also there is little proto- trabecular. In the experiment group, there is a lot of regular osteoblast on thesurface of trabecular, while less in control group. From the 28th to 42nd days, there is more newborn trabecular around the callus, and in the experiment group the newborn tracebular is regular and there is a lot of osteoblast around, while in control group, is unregular and less. The newborn trabecular gradually becomes necrosis or differentiate into osteocyte, the chondrocyte island becomes smaller. The chondrocyte in the experiment group is less than the control group.3. Observation of immune class and hybridization in situ; The gene expression and location of BMP -7,IL -6,VEGF cell factor and gene is similar. BMP-7 and BMP-7mRNA mainly locates in interstitial cell , phagocyte ,osteoblast and chondrocyte, etc. VEGF and VEGFmRNA mainly locates in interstitial cell,phagocyte,newborn vessel endothelial cell and chondrocyte, etc. IL - 6 and IL -6mRNA mainly locates in osteoblast and chondrocyte, etc. At 7th day after fracture, there is some cells which are weak positive in BMP - 7 and BMP-7mRNA,VEGF and VEGFmRNA, but no IL -6 and IL -6mRNA. At 14th day after fracture, there are expression of BMP 7 and BMP -7mRNA,VEGF and VEGFmRNA in interstitial cell ? osteoblast and chondrocyte in which most strong positive. And there are IL - 6 and IL — 6mRJNfA in osteoblast and chondrocyte. At 21st there is still BMP -7 and BMP -7mRNA,VEGF and VEGFmRNA in osteoblast and chondrocyte, and IL —6 and IL — 6mRNA in chondrocyte and osteoblast, and the positive cell is still much. At 28th day, the positive cell is less. At 42nd day, the positive is little.4. The image analysis of immune class: At 14th day, the positive of BMP — 7 , IL - 6 and VEGF in experiment group is stronger than that in control group, it has statistics sense( p < 0. 05).5. The change of ultramicrostructure of callus: the cell that takes part in the healing during the first 14 days in the experiment group and control group is similar; after 14th day, the osteoblast and osteoclast in experiment are more active than that in control group; after 28th day, the necrosis chondrocyte in experiment is less than control group; after 42 nd day, the osteoblast and osteoclast all disappear.6. The change of vitodynamics in callus; although the index of vitodynamics in experiment group in each period is higher than control group, but it hasnt sta-tistics sense ( p > 0. 05).DiscussionThe pathologic factor of osteoporosis is less bone in local or body, both organic and inorganic. In the early stage the tiabecular becomes thinner,broken or disappear, and the reducing of trabecular makes the load of the rest trabecular larger, and microfracture occurs, the structure of bone brakes. What' s more, the cortical substance of bone gradually changes into spongy bone, thinner and weaker. Both elasticity and hardness decrease, so fracture easily occurs.The treatment of osteoporosis fracture should obey the rules of position , fixation , functional exercise and drug. The first three is similar with the common fracture. Nowadays, there are two kinds of drugs; one inhibit the activity of os-teoclast to inhibit the obsorption of bone, such as diphosphate,estrogen and cal-citonin; the other enhance the activity of osteoblast to irritate the formation of bone, such as fluroide,steroid hormone,small dose parathyrin, but for the location of such drugs, the use of this kind drug is less. So the exploitation of this kind drug has important sense in treatment of osteoporosis fracture.Fluvastatin in this experiment is 2nd tatin - drugs, which is an artificial ra-cemic, has the factor of safe , high effect , tolerence , little adverse effect.. The mechanism is unknown, perhaps its pharmacological action is to inhibit the hydroxymethoglutaryl coenzyme ( HMGCo — A ) reductase, whose metabolic product mevalonat can inhibit the promoter of BMP - 2 gene, thus promote the formation of BMP, and healing of fracture.BMP - 7,, IL - 6 and VEGF are all the important cell factor in healing of fracture, the result of this experiment shows that the early reaction in experiment group and control group is similar, both has the procession of intramembrane ossification and intracartilage ossification, and the location and gene expression of BMP-7,IL -6 and VEGF is similar, though the experiment group is stronger positive, it hasnt statistics sense. So, the action of fluvastatin in the early stage is unobvious. About 2 weeks later, the callus in two groups becomes calcification, the location and gene expression of BMP -7,IL -6 and VEGF are same,...
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