| Objective: Osteoporosis is a systemic metabolic bone dis- ease,at persent,it has become one of diseases with the largest health-care casts and highest mortality.According to statistics, there are about 200 million people suffering from osteoporosis all over the world. The incidence of osteoporosis often"silent"and"unconsciously", if the signs be discoveried, it always was late.Therefore,this disorder is causing more and more attention. On the cytological study,the pathogenesis of osteoporosis and evaluation of treatable efficacy has become a basic and clinical studied focus. Osteoblasts and osteoclasts have played an important role in the maintenance of dynamic balance between the normal bone structure and metabolism. The uncoupling of bone resorption and bone formation,if the former than the latter,it will result to osteoporosis. Many studies have proved that inhibited osteoblast proliferation and reduced the degree of differentiation is an important cause of osteoporosis.Traditional Chinese Medicine(TCM) believes that the close relationship of bone and kidney, which in the prevention and treatment of osteoporosis and fracture there is a long history. There are a lot of names of osteoporosis-like have writed down in Huang Di Nei Jing.Throughout history,there are still many treatments of osteoporosis prescriptions. Nourishing Kidney Herbs of our experiment composed of the Prepared Radix Rehmanniae, Epimedii, Yam, Salvia, Drynariae and Duhuo. Effective parts of the monomer compounds composed of the Catalpol,Icariin,Diosgenin,TanshinonⅡA,Naringin and Hymecr -omone.Cell culture technology plays an indispensable role in the whole bio-engineering techniques. We take multiple enzyme digestion-paste ways to culture primary osteoblasts of SD rat in vitro,then obtaining sufficient osteoblasts. First, we observe the morphological changes in the growth process,then, select osteoblasts of 5~6generations as the research object. At different concentrations, different times and different combinations of circumstances, we use different Monomer Drug Compatibility, Crude herbs Compatibility and Icariin monomer to intervent osteoblasts culture.We adopted MTT and pNPP to measure and observe osteoblasts proliferation and differentiation. Comparing with the results of different combinations of Nourishing Kidney Herbs, we determine the effective prescriptions of Bushen,com- ponent compatibility and concentration range preliminarily.Methods: 1 we selected newborn (<24h) SD rat calvaria bone for the test material, used multiple enzyme digestion-paste to culture rat primary osteoblasts. Adopting inverted microscope to observe morphological changes of primary osteoblasts; 2 Nourishing Kidney Herbs compatibility grouping:Accord -ing to TCM theory and the basic rules of prescription,we divide six herbs into two categories and name TMed and CMed."TMed"inculdes Icariin+Yellow+yam and"CMed"inculdes Drynariae + Salvia + Duhuo.Tonic Medicine (TMed) and Cathartic Medicine (CMed) is divided into Crude herbs (TC extract ingredients directly) and monomer (effective chemical composition) are known as Crude Tonics herbs Compatibility Group(cTHCG) and Crude Cathartic herbs Compatibility Group (cCHCG) respectively with CT and CC said,Monomer Tonics Drug Compatibility Group (mTDCG) and Monomer Cathartic Drug Compatibility Group (mCDCG) separately with MT and MC express. Our experiment is divided into 8 groups,①CT>CC,②CT=CC,③CTMCGroup,⑥MT = MC Group,⑦MT Cathartic Medicine (CMed): Dihuang: Icariin: yam: Drynariae: Salvia: Duhuo (2:2:2:1:1:1); Tonic Medicine (TMed) = Cathartic Medicine (CMed): Dihuang: Icariin: yam: Drynariae: Salvia: Duhuo (1:1:1:1:1:1); Tonic Medicine (TMed) < Cathartic Medicine (CMed): Dihuang: Icariin: yam: Drynariae: Salvia: Duhuo (1:1:1:2:2:2);4 Osteoblast proliferation; Add the concentration of each 10ng/ml, 20ng/ml, 30ng/ml of the crude herbs and monomer; continuing to cultivate 24h, 28h, 72h, then adding MTT 20μl. Then using microplate reader to measure OD value.5 Osteoblast differentiation; According to the group above, replacing the intervental herbs or monomers, Crude Herbs and monomer, the concentration in 10ng/ml,20ng/ml,30ng/ml;contin -uing to cultivate 24h, 28h, 72h, abandoning culture medium, then add 0.25ml 0.1% Triton solution, according to Kit descrip- tion determination per gram of protein in ALP International units (U/g protein) express.Results:1 Rat primary osteoblasts in vitro Morphological observation1.1 Inverted microscope observation revealed that the original generation of osteoblasts climbing from rat calvaria bone, adherenting,extending,showing triangular polygon; just subcult- ured osteoblasts spherical. During 24h, living cells has been the settlement adherent, completely start triangle, spindle, nuclear significantly increased; subculture 5 days, cell morphology can be seen diversity; cultivate 8~15 days, they can be seen polygonal, cord-shaped, growth overlap;about 15 days later, we can observe the accumulation of collagen and calcium deposition.1.2 Alkaline phosphatase staining: we can observe the activity of osteoblasts site showing granular purple.2 Rat osteoblast cell proliferation2.1 Concentration of 10ng/ml, compared with the control group ,measured at 24h are MT>MCgroup is slightly higher (P<0.05); at 48h measured CT>CC group,CT=CCgroup, MT> MC group have proliferation significantly (P<0.05); at 72h , MT> MC group had significant proliferation (P<0.05);2.2 Concentration of 20ng/ml, compared with the control group,measured at 24h,CT>CC and MT>MC have proliferation (P<0.05); at 48h measured results show that CT>CC group was significantly (P<0.05); at 72h, CT>CC and MT>MC group enhance proliferation (P <0.05);2.3 Concentration of 30ng/ml, compared with the control group, measured at 24h are CT>CC group has obvious prolifera -tion (P<0.05); at 48h measured CT>CC group, MT>MC Group, MTCC group strong proliferation (P<0.05);3 Rat osteoblast differentiation3.1 Concentration of 10ng/ml, compared with the control group, at 24h, osteoblast ALP of MT>MC group are higher(P< 0.05); at 48h, measured MT>MC Group has ALP increased significantly (P<0.05);at 72h, measured results show osteoblast ALP of MT>MC Group, MT=MC group were significantly increased, (P <0.05);3.2 Concentration of 20ng/ml, compared with the control group, at 24h CT>CC group, CT=CC group, MT>MC and MT=MCgroup have higher ALP (P <0.05); at 48h and 72h,the results show that CT>CC group has promoted ALP obviously (P<0.05) ;3.3 Concentration of 30ng/ml, compared with the control group, at 24h , CT>CC group, MT>MC group has increased ALP ,CT>CC group of the more obvious (P <0.05); at 48h and 72h, measured results demonstrated that CT>CC group has promoted ALP obviously (P<0.05);Conclusions:1. At 10ng/ml concentration and in 72h, MT>MC group has osteoblast proliferation and differentiation ;2. MT>MC Group has the capacity-effect relationship: with the concentration (dose) increasing ,the rat osteoblast proliferati -on and differentiation are dampening; Low concentration (10ng/ml) , MT>MC group is also time-effect relationship.3. At 30ng/ml, CT>CC have obvious function of osteoblast proliferation and differentiation, With the concentrations (dose) and times increasing ,the osteoblast proliferation and differentiation are enhancement, this is capacity-effect and time-effect relationship of CT>CC group;4. TMed>CMed may have a better treatable effect on clinical osteoporosis; In terms of our prescription ,the effective parts may be Catalpol, Icariin, diosgenin and tanshinoneⅡA, naringin, Hymecromone;However,the results of CT>CC showed that the CT and CC may be more effective than MT and MC, it is possible to include other unknown ingredients;5. The complexity of traditional Chinese medicine derived from traditional Chinese medicine combined in the process of emerging, or contain other unknown ingredients. This thesis was carried out beneficial exploration in the research of prescription; On basic and clinical experiment,it also provided an idea and method for TCM-WM.
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