| I. Research design, at random, blind method, contrast, cell cultureII. Research backgroundWith the development of society, the flourishing transportation industry, the frequently launched activities, Various injuries and fractures have become common clinical diseases and frequently-occurring diseases, which seriously threaten the health of human being as well as people's working ability. How to speed up the recovery of fractures is a key research subject in the field of Orthopaedics. Treating fracture with Chinese herbal medicine has a long history and has excellent clinical effects and is one of the main methods to treat fracture at present. The Science of Traditional Chinese Medicine has a Special View in cognition and methodology, which causes it to remain at a macroscopic and vague level when it comes to explaining the functioning mechanism of herbal medicine. How to keep up and develop the fracture-treating features and advantages of Chinese Medical Orthopaedics as well as to modernize traditional Chinese medicine has become an unavoidable topic.Fracture recovery is a complicated biological restoringprocess. For years, Scholars at home and abroad carry out a lot of experiments and observation on fracture by means of histology, histological chemistry, Ultramicroscopic structure and biomechanics and also conduct extensive discussion on fracture recovery mechanism. They use various methods to promote the recovery of fracture clinically, but the fracture delays recovery and the problem of unrecovery is still not completely solved. For recent years, because of the development and application of gene technology, people' s cognition about fracture has been deepened from the level of cell biology to the level of molecule biology. With the development of molecule biology, they have realized that fracture recovery is connected with many kinds of biological factors. In addition to the internal system and the metabolic factors, there are many growth factors and the part of fracture, which have important regulating effects on fracture recovery.The research of the effects of Chinese herbal medicine on fracture recovery genes has made preliminary progress. The research subject led by my instructor, professor DongFuHui: "The research of the regulating effects of Chinese herbal medicine on the relevant cell signal conducting system in the process of fracture recovery" explores the mechanism of Chinese herbal medicine promoting fracture recovery in terms of molecule biology and has finished the research of the effects of some traditional Chinese medicine such asdonkey-hide gelatin, rhizome davallia, cuttlebone and leech on fracture recovery gene expression in the process of fracture recovery and reveals that different traditional Chinese medicine has different effect on gene expression, which provides theoretical basis for traditional Chinese medicine promoting fracture recovery, At present, the research of the effects of traditional Chinese medicine on the relevant gene expression in the process of fracture recovery is still at the preliminary stage and the focus is on the effects of traditional Chinese medicine on animal models, The research of traditional Chinese medicine and bone cell culture out side the body focuses on the reproducing effects of traditional Chinese medicine on bone, cells, while the research of the effect of traditional Chinese medicine rhizome of davallia on the relevant gene expression by means of bone-cell-culture outside-the-body technology in the process of fracture recovery has not been reported yet. III. Research aim.Observe the effects of rhizome of davallia on the reproducing function of the bone cells cultured outside the mouse body, as well as the effects of rhizome of davallia on the bone cell TGF- P i , TGF- P RII, VEGF , bFGFgene expression, explore the mechanism and the effects of rhizome of davallia on fracture recovery, provide the theoretical basis for traditional Chinese medicinepromoting fracture recovery and thus guide the clinical treatment of fracture. IV. Materials and methods.Experiment materials: Prepared rhizome of davallia original medicinal liquid, Original bone cells separated from wistar Milk Mouse' s skull. Dosage of medicine used in experiment: The rhizome davallia original medicinal liquid is divided into 5mg/ml, lmg/ml and 0.lmg/ml groups according to the preparatory experiment the serum containing the medicine is made according to the mouse body size in proportion to the human body size, seven times that of clinical adult dosage is the low dosage group, twice that of the low dosage group is the middle dosage group and four times that of the middle dosage group is the high dosage group. The mice are given the medicine twice a day and three days after they are given the medicine, the mice are killed to take the serum, Groupings blank serum control group, cell model control group, rhizome of davallia original medicine low dosage group(0. lmg/ml) , rhizome of davallia original medicine middle dosage group(lmg/ml), rhizome of davallia original medicine high dosage group(5mg/ml), rhizome of davallia medicine-containing serum low dosage group, rhizome of davallia medicine-containing serum middle dosage group, rhizome of davallia medicine-containing serum high dosage group. Experiment interference:Apply the rhizome davallia original medicinalliquid and the medicine-containing serum to the second and the third generation bone cell sample I) process: (immunity organized) Take a 24-hole board, put a piece of processed glass (IX lcm) into it. Then plant the bone cell of mice (in the density of 4 X 103) into the 24-hole board and culture the bone cell with DMEM culture medium containing 10% cattle serum for 24 hours. Take the medium away and apply the clear liquid. Then add all-conditioned liquid containg 10% serum, culture it for 48 hours, then take it away and apply the clear liquid, wash it with PBS(0. 1u,PH7.4) three time. Fis it with 10% formaldehyde liquid for 30 minutes and prepare it for use, II.) (RT-PCR). Plant the bone cell of mice (in the density of 4 X 103) into a 12-hole board, culture it with DMEM culture medium containing 10% cattle serum for 24 hours. Then take the medium away and apply the clear liquid, add all conditioned liquid containing 10% serum and culture it for 48 hours, take the medium in the holes away and wash it with cold PBS (0. 01u , PH7. 4) twice, add TRI Z01, each hole 0.5ml and blow it repeadedly for three minutes. Take away the TRI Z01 and put it into a 1.5ml Eppendorf tube, make it static at the temperature of 22 °C for 5 minutes. Put it into the refrigerator at the temperature of -70°C and prepare it for use. Test: Use the li TT method to test the cell' s growth rate;use the RT-PCR method to test TGF- 0 imRNA, TGF- 0 RII mRNA, VEGFmRNA, bFGFmRNA;Use the immunity organized method to lest TGF- 0 ,TGF- |3 RII, VEGF, b FGF;Use computer pattern analyzing system to analyze it and conduct the average grey level test on the bone growth factors expression positive cells. V.ResultThe effects of the rhizome of davallia on the reproducing function of the bone cells cultured out side the mouse body.The result of this experiment: Compared with the blank serum control group, the OD value in the cell model control group, the rhizome of davallia original medicinal liquid low dosage group, the rhizome of davallia original medicinal liquid middle dosage group, the rhizome of davallia original medicinal liquid high dosage group and the rhizome of davallia medicine-containing serum high dosage group decreases obviously(P<0.01), while the OD value in the rhizome of davallia medicine-containing serum low dosage group and the rhizome of davallia medicine-containing serum middle dosage group doesn' t show obvious change(P>0. 05), Compared with the cell model control group, the OD value in the rhizome of davallia medicine-containing serum low dosage group and the rhizome of davallia medicine-containing serum middle dosage group increases obviously (P<0.01) while the OD value in the rhizome of davallia original medicinal liquid low dosage group, the rhizome of davallia original medicinal liquid middle dosage group and the rhizome of davallia medicine-containing serumhigh dosage group show no clear changes(P>0.05), On the whole, compared with the rhizome of davallia original medicinal liquid group, the OD value in the rhizome of davallia medicine-containing serum group increases(P<0. 05).The effects of the rhizome of davallia on the bone cell culturedoutside themouse body TGF- ￡ i, TGF- 3 RII.VEGF and bFGF gene expression.I. The effects of the rhizome of davallia on the bone cell cultured outside the mouse body TGF- ￡ i, and gene expression.The result of this experiment: Compared with the blank serum control group, the three dosage groups of the rhizome of davallia original medicinal liquid and the low dasage and the middle dosage groups of the rhizome of davallia medicine-containing serum show no decrease in TGF- 13 imRNA, expression (P<0.05) and the rhizome of davallia medicine-containing high dosage group shows no clear changes(P>0. 05) compared with the cell model control group, the blank serum control group shows increase in expression (P<0.05) and the other groups show no changes (P>0. 05) The result of TGF-P i, albumen expression: Compared with the blank serum control group, the low dosage and the middle dosage groups of the rhizome of davallia original medicinal liquid as well as the middle dosage group of the rhizome of davallia medicine-con taining serum show decrease in expression (P<0.05). Compared with the cell model control group, thelow dosage group of the rhizome of davallia original medicinal liquid and the middle dosage group of the rhizome of davallia medicine-containing serum show decrease in expression while the high dosage group of the rhizome of davallia medicine-containing serum shows increase in expression (P<0.01).III. The effects of the rhizome of davallia on the bone cell cultured outside the mouse body VEGF andgene expression.The result of this experiment;Compared with the blank serum control group, the cell model control group shows no obvious changes in expression(P>0.05), the three dosage groups of the rhizome of davallia original medicinal liquid and the low dosage group of the rhizome of davallia medicine-containing serum show. Decrease in VEGFmRNA expression (P<0. 01)and the middle dosage and the high dosage groups of the rhizome of davallia medicine-containing serum, show obvious increase in VEGFmRNA expression (P<0.01). Compared with the cell model control group, the blank serum group shows no obvious changes in VEGF mRNA expression (P>0.05), the three dosage groups of the rhizome of davallia original medicinal liquid and the low dosage group of the rhizome of davallia medicine-containing serum show decrease in VEGFmRNA expression(P<0. 01), the middle dosage group of the rhizome of davallia medicine-containing serum shows a slight increase in VEGFmRNA expression but this increase has no sense(P>0.05). The result in VEGF albumen expression: Compared with the blank serum control group, the cell model group as well as the low dosage and the high dosage group of the rhizome of davalliaoriginal medicinal liquid show no changes in expression (P>0.05), the middle dosage group of the rhizome of davallia original medicinal liquid shows obvious decrease in expression (P<0. 01), the low dosage group of the rhizome of davallia medicine containing serum shows decrease in expression (P<0.05) the middle dosage group of the rhizome of davallia medicine-containing serum shows increase in expression (P<0. 05) , while the high dosage group of the rhizome of davallia medicine-containing serum shows obvious increase in expression (P<0.01). Compared with the cell model control group, the cell control group, the tow dosage and the high dosage groups of the rhizome of davallia original medicinal liquid show no changes in expression (P>0.05), the middle dosage group of the rhizome of davallia original medicinal liquid shows obvious decrease in expression (P<0.01), the low dosage group of the rhizome of davallia medicine- containing serum shows decrease in expression (P<0. 05), the middle dosage group of the rhizome of davallia (P<0.05), the high dosage group of the rhizome of davallia medicine- containing serum shows obvious increase in expression (P<0.01).IV. The effects of the rhizome of davallia on the bone cell cultured outside the mouse body bFGFand gene expression.The result of this experiment: Compared with the blank serum control group, the cell model control group, the low dosage and the middle dosage groups of the rhizome of davallia original medicinal liquid show decrease in bFGFmRNA expression (P<0.01), the high dosage group of the rhizome of davallia original medicinal liquid shows no changes in bFGFmRNA expression (P>0.05), the three dosage groups ofthe rhizome of davallia medicine-containing serum show obvious increase in bFGFmRNA expression (P<0.01). Compared with the cell model control group, the blank serum control group,the high dosage group of the rhizome of davallia original medicinal liquid and the three dosage groups of the rhizome of davallia medicine- containins serum show obvious increase in bFGFmRNA expression (P<0.01). Besides, the three dosage groups of the rhizome of davallia medicine-containins- serum show more obvious increase than the three dosage groups of the rhizome of davallia original medicinal liquid in 6FGFmRNA expression (P<0.01). Compared with the blank serum control group, the cell model control group, the low dosage and the high dosage groups of the rhizome of davallia original medicinal liquid and the low dosage group of the rhizome of davallia medicine containing group show no change in albumen expression (P>0.05), the middle dosage group of the rhizome of davallia original medicinal liquid shows decrease in albumen expression (P>0.05), the middle dosage group of the rhizome of davallia original medicinal liquid shows decrease in albumen expression (P<0. 05), the high dosage group of the rhizome of davallia original medicinal liquid as the three dosage groups of the rhizome of davallia medicine- containing serum show obvious increase in albumen expression(P<0.01), Compared with the cell model control group, the high dosage group of the rhizome of davallia original medicinal liquid and the three dosage groups of the rhizome of davallia medicine-containing serum show obvious increase in albumen expression (P<0.01), the other groups show no obvious changes (P>0.05).Conclusion:I. The rhizome of davallia has no obvious promoting effects on the reproduction of bone cells, what' s more, when the concentration of the rhizome of davallia reaches a certain level, it has checking effects of the bone cell' s reproduction or growth. II. The rhizome of davallia has no obvious promoting effects on the TGF- j3 imRNA, and TGF- $ i, expression of the bone cell cultured outside the mouse body;however, the rhizome of davallia has a certain regulating effect on the TGF- 3 imRNA, expression of the bone cell cultured outside the mouse body.ill. The rhizome of davallia has obvious promoting effects on the VEGFand gene expression of the bone cell cultured outside the mouse body and has a certain amount-effect relationship.IV. The rhizome of davallia has promoting effects on the bFGF and geneexpression of the bone cell cultured outside the mouse body and has a certain amount-effect relationship.V. The rhizome of davallia has promoting effects on the TGF- 3 RII, mRNA, VEGF, bFGF andgene expression of the bone cell cultured outside the louse body. It is likely that the rhizome of davallia has the effect of promoting the fracture recovery.VI. The medicine-containing serum of the rhizome of davallia has more obvious promoting effects on the. TGF-P RII mRNA, VEGF andgene, bFGF and gene expression than the original medicinal liquid of the rhizome of davallia.
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