| The skeleton is built the embryonic form of the skeleton according to genetic regulation and control, and according to the need that bear loadedly to reconstruct and reinvent constantly while being one's own among growth of bone, in order to attempt to realize that reaches the largest intensity of bone with the minimum bone amount, this is function adaptability of bone. The experiment has already proved , it is the basic reason that a skeleton is structured and reinvented that the machinery stress meets an emergency. The suitable stress environment can impel the bone supersession to develop towards the best level, can cause the bad consequence on the contrary. In recent years, the research about the skeleton function adaptability mechanism has some new discoveries in cell's biological field. Research verifies , some cells in the skeleton can perceive the biological physics signal (such as mechanics signal, electric signal ) , process and combine these signals. And turn it into corresponding skeleton structure change. This function of the cell is known as the mechanics signal transfering to and leading the function. This indicates that in the function adaptability mechanism of the skeleton, the construction , reinventing of mechanics phenomenon and skeleton changes through the functionality of the cell but directly connected.Stress growth , absorption and building up regulating action again of the bone. The result of study of a large number of experiments indicates that the state of weightlessness will cause the bone to absorb and increase, the increase that the opposite skeleton is loaded can increase the boneand is formed and reduces the bone to absorb , these cause change , bone of quantity through osteoblast that department produce mainly until environmental stress change. No matter the oral cavity is treating and passing the function of exerting strength on the tooth lopsidedly on clinic, make the alveolar bone reconstruct the living beings, lead one and become bone in bone skill and organize and become function that osteoblasts receive the traction force in the skill, it is all results changed into the biological effect finally after mechanical signals act on the organism to make the bone organize and get and draw waiting a moment. How to conduct the signal in the afterbirth after regarding as the osteoblast and receiving the mechanical signal of the external world, how to regulate its physiological function to adapt to the external mechanics environment accordingly, the study on these questions will contribute to exploring further that organizes the response to the external mechanics environment with bone, and treat and offer the new theoretical foundation to the bone organizational project clinically.This subject is divided into four respects and carried on research:Section 1 The study oil primery culture and biological characteristics of osteoblastIn order to establish a new model and method of osteoblast culture in vitro, Sprague Dawley rats for one day of new life, in aseptic to fetch cranium cover bone , utilize 0.25% trypsin and Model 0.1% II collagen enzyme a lot of enzyme digest law go on osteoblast take the place of and train originally, go down to posterity repeatedly again. The ones that trained became the osteoblast and carried on the biological characteristic to appraise, including examing by fhase-contrast microscope, light microscope, HE staining, ALP staining, calcium tubercle staining (Von-Kossa stain )etc. Come to verify into the characteristics , such as the form of the osteoblast, division function ,etc.The result shows, the cell that inoculates is trained by 24 h and can stick to the wall, the cell triangular in shape, spinning many kinds of forms , such as Chui's shape and polygon ,etc, the afterbirth thick liquid is spread outwards, and stretch out protrudingly. Cell's quantity increases 5 days later, the volume increases, the cell presents half a state of merging. The cell merges in flakes 8 days later, the cell presents the index and grows. Afterbirth body expand , border difficult todistinguish the fact cell have , and gather into group's phenomena after the one day, demonstrate that collects and leaves one kind of trends of growing. With collect and leave expanding constantly, each collecting the peripheral cell's form while falling has a lot of arrow protruding forms for polygon cells , such as short shuttleless loom shape , triangle , polygon ,etc, the nuclear ovum occupies one side roundly, cell's tubercle takes shape, the host is piled up . The calcification tubercle takes shape 18 days later. After the cell merges, dyes ALP, most cells present the positive reaction, it is obvious that the particle presents positive reaction in the cell membrane and afterbirth thick liquid. After the cell forms the calcification tubercle , dyes plain and red the madder, it is obvious that the tubercle is red.The above-mentioned results show , the method that this research adopts a lot of collagen enzymes and digests, cell isolated to make have in large quantity, purity high, 24 h can stick to wall, form intact to grow the vigorous , high advantage of survival rate to observe cell under the mirror, set up better method of training the big mouse to the osteoblasf s experimental model outside body. What experiment this obtain osteoblast have body become many kinds of biological characteristics , bone of cell, study in order to observe into the cell's external growth law of bone and biological characteristic, and then in order to study various kinds of regulation factors to the function becoming the osteoblast further, have laid a good foundation.Section 2A biomechanical system that can apply fluid sheer stress to osteoblast cells in vitroAn important subject studied with cell mechanics that the impact on cell's biological behavior of mechanical force is cell biology at present. Cell mechanics is an important respect in the organizational project field, cell mechanics experimental technique whether cell basic plateform that mechanics study flow use extensively one cell mechanics experiment method cell mechanics experiment, it is mainly the function of seizing among research cell and cell, among the cell and basis material . Because the internal environment is complicated, the size of the human cell is while getting a few dozen micron in more than ten, it is unable to use that routine macroscopical mechanics loads the method and experimental technique. So it is cell mechanics to train cell'smechanical thrust augmentation device to study outside the body and study one of the foundations. We method is it make one utilize gravity is it is it flow to plateform go on system flowed to irritate and improve to this system to flow to irritate to make by oneself according to Frangos (1988), and to irritating flowing and carrying on corresponding mechanics analysis systematically, a of shear strength and fluid are irritated and flowed and is the high relational expression the ware liquid is collected in the bottle to get and irritate and flow systematic flow, plateform and flow.r = 0.63(V3.68 + fc -1.91) (pa)The above-mentioned results show : Fluid that research set up this shear strength load system can for study stick to the wall grow become cell's response to fluid shearing stress of bone, glue the size of the relay , the intensity of sticking of the cell and host material between the cells, the cell seizes influence that the factor seizes to the cell ,etc. The easy analysis of origin cause of formation of the experimental result, the experimental result one is better than sex, repeatability. System this can after the division those can use go down to posterity and easy a large number of cell of hyperplasia also, research of broken the osteoblast, bone marrow host cell, becoming the fibre cell, becoming endothelial cells of the skin cell, blood vessel and Hie previous body cell of some of cells ,etc. Through perfect body train cell mechanical thrust augmentation device study, simulation better become internal environment, bone of cell, realize accurately that osteoblast mechanics is loaded , thus in order to understand more deeply into mechanics of the osteoblast transfers to the mechanism of leading and establishes the foundation.Section 3AP-1 members in response to changes in wall-shear stress in osteoblastic cellsAP-l exists of various kinds of animals cell extensively, involve cell's signal and transmit each level of the system: Including the signal, such as growth factor, receptor (such as the growth factor receptor ) , membrane G protein (ras family ) , membrane and protein phosphoric acidkinase and nuclear interior nucleoprotein of afterbirth quality outside the cell Domestic and international research has indicated in recent years , c-fos gene and becoming osteoblasts in the early gene family is in close relations at once . Numerous research indicate , osteoblasts when led tension , shorn strength , strength overweight , little gravity, c-Fos is strengthened in the expression of mRNA and protein level, and may be with activating having something to do of the thorough fare of MAPK. In addition some other experiments verify the function while the bone happens of other members besides c-Fos of AP-1 family, for example: Fra-1 passes and expresses and causes the osteosclerosis, whether it is that the members of AP-1 families may all participate in into the signal that the osteoblast changes to the external stress and conduct the thorough fare to point out us. So on the foundation of the fluid shearing stress system trained to be the osteoblast outside becoming the osteoblast to take the place of and train and set up and used in the body originally in this research, the strength scheme is to use the level with year and shear: Load level shear strength including: 0.8 Pa , 1.2 Pa , 1.4 Pa and 1.6 Pa The loading time that every group shears the strength level is 10 min, 15 min, 30 min, 60 min. Measure into c-Fos in the osteoblast, FosB , c-Jun, JunB, Fra-1, the expressions of Fra-2 and JunD mRNA, aim at responding different all kinds of physiology of shearing strength of fluid through studying AP-1 family, explore into a osteoblast receives the early response after strength.The result shows that a result of the test indicates that acts on and can be stimulated into c-Fos in the osteoblast effectively most when become the fluid on osteoblast surface and shear the size of strength , FosB, c-Jun, the expressions of JunB and JunD mRNA. After the fluid shears strength to stimulate a osteoblast into to begin for 10 minutes, c-Fos, FosB, c-Jun, JunB and JunD mRNA express competence is it increase to begin promptly , is it can reach the highest peak after the 15 minutes such as thrust augmentation to begin. With the extension of time after this, it expressed competence to drop to the level for about 10 minutes gradually, indicate into the cell's extension to the thing that the response of shearing strength of fluid up to timed of bone, its sensitiveness is reducing too. Find too from studying , all increase after Fra-1 and Fra-2 shear strength and stimulate in the fluid at the same time , but there is not a remarkable difference (P> 0.05 ) between the group for each time , disappear after the expression of Fra-2 mRNA is being shorn strength by the fluid and stimulated for 60 minutes at the same time .The result shows c-Fos in AP-1 family, FosB, Fra-1 , Fra-2, c-Jun, JunB and JunD,etc. haveparticipated in mechanics and stimulated the course causing cells to respond. AP-1 osteoblast after not stimulating by external mechanics , transmit external information that signal stimulus cause grades of antithetical couplet play uniting function occasionally important in reacting, serve as nuclear inside the third courier and gene and duplicate the molecular switch adjusted and controlled.Section 4Nitric Oxide Regulates c-fos Expression in osteoblastic cells Induced by wall-shearIt is a free radical of a kind of height disperse that one oxidizes the nitrogen (nitric oxide , NO ), is the second important courier and neurotransmitter in organism. NO has extensive function in the respects, such as neurotransmitter releasing, blood pressure regulating, immunity repelling , inflammation reacting , cell's function and information transmission ,etc.. In recent years, NO research in the field of dept. of orthopedics makes some achievements too, includes the respects , such as emergence , bone reconstructing , bone supersession , bone arthritis that organize with bone ,etc.. On the foundation of the fluid shearing stress system trained to be the osteoblast outside becoming the osteoblast to take the place of and train and set up and used in the body originally in this research, become the expression situation of NOS of the osteoblasts after determining the competence of loading and shearing strength , after then use inhibitor L-NMMA of NO and other source NO to be for body SNP function to the osteoblast 24h separatelying, become NOS , the expression situations of c-fos mRNA and C-FOS protein in the osteoblast after determining the competence of loading and shearing strength , become influence that the osteoblast C-FOS expresses under shearing strength function on the fluid in order to the change which observes NO level, explore NO is it lead mechanical stress to stimulus that bone organize, promote bone function that have in forming to transfer to. So on the foundation of the fluid shearing stress system trained to be the osteoblast outside becoming the osteoblast to take the place of and train and set up and used in the body originally in this research, become the expression situation of NOS of the osteoblasts after determining the competence of loading andshearing strength , after then use inhibitor L-NMMA of NO and activator SNP of NO to treated with the osteoblast 24h separatelying, become NOS , the expression situations of c-fos mRNA and C-FOS protein in the osteoblast after determining the competence of loading and shearing strength , become influence that the osteoblast C-FOS expresses under shearing strength function on the fluid in order to the change which observes NO level, explore NO is it lead mechanical stress to stimulus that bone organize, promote bone function that have in forming to transfer to.The result shows that the mechanical stress can be stimulated into the activation of NOS of the osteoblast and broken osteoblast, the activation of NOS probably has something to do with the response that the bone is amazing to the mechanical stress, the osteoblast and is shorn total NOS and iNOS after strength function lOmin by 1.2Pa fluid and expressed and dropped notably, compared with the situation that contrast the group there is a remarkable difference (PO.05 ). At the 15min, total NOS and iNOS is it drop further to express, have remarkable difference compared with other groups (PO.05). At the 30min, total NOS and iNOS express to some extent going up, total NOS and iNOS express and go up further at the 60min, express closely when but with 30min. At the same time cNOS is presented and expressed low-levelly continuously all the time. After treated with 0.6mmmol/L L-NMMA 24h, the osteoblast cells were load 1.2Pa fluid shear strength lOmin , total NOS and iNOS is it yet drop notably to express at the 15min, at the 30min, total NOS and iNOS express to some extent going up, total NOS and iNOS express and go up further at the 60min, express closely when but with 30min. At the same time cNOS is presented and expressed low-levelly continuously all the time. The expression level of c-fos mRNA has not used function group L-NMMA to reduce obviously than, there is significance difference (P<0.05 ) among the groups. Is it is it can see from cell immune fluorescence thrust augmentation 30min C-FOS weak to express while being positive occasionally to pursue to dye, it is expressed that there is no masculine gender in thrust augmentation 60min hour C-FOS. After other source NO of O.lmmmol/L density supports body SNP function 24h, load 1.2Pa fluid shear strength lOmin , total NOS and iNOS is it yet drop notably to express at the 15min and then, at the 30min, total NOS and iNOS express to some extent going up, total NOS and iNOS express and go up further at the 60min, express closely when but with 30min. At the same time cNOS is loading 1. 2Pa fluid shear strength after the lOmin, is it rise notably to express, 30min, and cNOS express than contrast group rise obvious at the 60min, compare remarkable difference (PO.05 ) while contrasting thegroup and group of group L-NMMA with the blank at the same time . The expression level of c-fos mRNA has not used group SNP and used function group L-NMMA to increase notably than, there is significance difference (PO.05 ) among the groups. Is it is it can see from cell immune fluorescence thrust augmentation 30min C-FOS strong to express while being positive occasionally to pursue to dye, it is expressed that there is masculine gender in thrust augmentation 60min hour C-FOS.The above-mentioned results show: Expressing c-fos mRNA and C-FOS protein after culture with the NO inhibitor L-NMMA is stimulated by stress of machinery to the osteoblast has obvious inhibition. Expressing c-fos mRNA and C-FOS protein after culture with the activator SNP is stimulated by stress of machinery to the osteoblast has obvious facilitation. The mechanical stress stimulates NO produced to be may be the bone and organizes the activating and causing of eNOS in cells. NO right to osteoblast c-fos in short-term up regulating action, may organize cell is it is it lead biochemical signal, people of cell to transfer mechanical stress to to irritate bone NO have under the function, key signal molecule promoting the bone to form. |
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