| ObjectiveOsteoarthritis (OA) is a chronic progressive joint disease which the degradation and synthesis of chondrocyte, extracellular matrix and groundsubstance under cartilage induced by mechanics and biology factors. With the life expectancy extension and the alteration of the population structure, OA has been a serious global public healthy problem. Nowadays It's a international research hot spot how to prevent and cure OA. The pathogenesy of OA is still unknown now, which severely hold up the new indeed effective therapy methods and pharmacal developments that prevent the advancement of OA. Articular cartilage damage is a distinctive pathology alteration of OA, which pathogenesy has close correlation with the change of articular cartilage. So it is significant to the clinical diagnosis, treatment and prognostic evaluation by probing the molecular and biology mechanism pathology of articular cartilage damage.Articular cartilage is special connective tissue which composed by cartilage matrix and cartilage cell. The main feature of OA is osteoclasia of cartilage. The demolish begin with the focal damage in cartilaginous surface, and metabolism change happened in the cartilage matrix several days or several weeks later, then bigger range osteoclasia will happen. now days the research of the pathomechanism of the articular cartilage destructive mainly concentrate the function of all kinds of cytokine, inflammatory factor and protease. so we say that the wide of its research is narrow and the index is too single and replicate and breakthrough is so much few. It is a very difficult responsibility to how to study the osteoarthritis pathomechanism based on the more administrative level and more lucubratise .many study have supported powerfully that OA is a multiple gene and multifactorial inheritance disease.So aimed at the pathology change, annotated the relation of multiple gene each others hunted the key gene and its function, it has not only great significance but practicable for investigate morbidity mechanism and the foundation of the key pathological changes. And will widen our research field.Human genome project (HGP) that has been fulfilled wholly gene sequencing of many species give our great help to study systematically the gene function and hereditary disease. Now we have accumulated much structure and function information of individual gene. But it is a important topic how to utilize so much information which could reveal the rule of diagnose and treatment human's disease. Gene chip has great characteristic for simultaneous treatments high-fluxsinicromation and automation. And could high density detect polygene of the disease simultaneously. And it offer a new moment to illuminating polygene's function and effect of OA.OA means rheumatism involving bone in traditional Chinese medicine (TCM). The internal cause is the deficiency of the vital-qi, the external cause is wind cold dampness evil factors invaded. The internal and external cause combined, so the disease happened. There exist abundant experience in TCM for OA. Professor Jizhang Chen, who is my tutor, according to the TCM theory, for example, balance theory, concept of wholism, diagnosis and treatment on the basis of an overall analysis of the illness and the patient's condition and the kidney being in charge of the bone, bring forward that invigorate the kidney to treat OA. A series of experimental and clinical research gone suggested that invigorate the kidney can accelerate the proliferation of cartilage cell, regulate the apoptosis of cartilage cell, delay the pathological change of articular cartilage obviously, especially the pathological change of chondrocyte. The prescription of the invigorate the kidney has no side effects, is an ideal medicament for prevention and cure of OA. With the development of the modernization of the TCM, investigate it's material foundation and mechanism of action , which had been became a focal point which the herbs prevention and cure OA. But mechanism of action of the herds is too complex. We should draw assistance from the new view and technology for developing and consummating the theory system of the TCM. So bonding the research outcome of the modern medicine, educing the ascendancy of traditional medicine, which differentiation of symptoms and signs and differential diagnosis of diseases are conjunct, contacting tightly pathomechanism of OA, all of those whichwill cue the mechanism of action of the complex prescription prevention and cure OA.Guided the methodology of the modern system scientific, we plan to establish the model which is the unpregnancy SD female rat aged 4 mouths which the ovaries be eccoped and the cruciate ligaments of the knee be cut off. We named this model is knee ostarthritis, which syndrome and illness are conjunct. we will lucubrate the relation between the rule of gene express and the mutilated cartilage . then the key gene in which specificity index is intimate interrelated the degradation of the articular cartilage will be found, those gene and protein should be detected using the method RT-PCR and Western-blotting, at the same time, We will elucidate initially the function of the target molecule which be involved the pathomechanism of the mutilated cartilage . From the point of the molecular biology we have something in mind the alteration pathology and understand widely the change the profile of the gene of the ostarthritis articular cartilage cataplasia in this study. And some key point gene which induced OA will be preliminary screened. The effective mechanism which the complex prescription treat OA will be probed from the point of which the complex prescription regulate the express of the key gene of the ostarthritis articular cartilage cataplasia. MethodsClean-class grade of 150 healthy female big mice of SD, age of 4 mouths, weight 220 ±20g ( offered by the centre of animal, the traditional Chinese medicine university of Guangzhou, number of the certificate of quality (Guangdong supervises 2003-0001 of number 2005A031SCXK of the card (Guangdong )). The uneven law divides the animals into 6 groups at random adopting the random software, group A is 15 normal mice, group B is 30 mice spayed simply, group C, includes 31 mice which were excised the PCL ,ACL and medial collateral ligament of the right knee simply, group D includes 31 mice which not only were excised the PCL , ACL and medial collateral ligament of the right knee simply but also spayed, group E includes 31 mice which not only were excised the PCL ,ACL and medial collateral ligament of the right knee simply but also were spayed, and were drenched traditional Chinese medicine, group F includes 12 mice acted as preliminary test in order to test in advance. All SD rat are feeding in thermostatic, wet clean environment, the environment temperature is (25±1) °C, it is in humidity 70%, daily 12h illumination /darkness, the mouse arbitrary eating standard mouse feed and water the raiser give.The method of the castration mould: peritoneal injection according to 0. 35ml/100g with 10% of the chloral hydrate solution, after 5minutes, the big mouse of SD totally anaesthetizes, the castration group adopts the dorso-approach, such as Pernille etc. to excise one pair of ovary. The SD mouse adopt prostrate location, below the arch of ribs, the third lumbar vertebrae place, unhairing^ degermating^ cutting the leather, the notch is generally lcm. blunt separation is caused the muscle layer, the left side 0. 5cm blunt separation muscle of lumbar vertebrae, cut off the peritonaeum carefully, deepen the notch with tissue clamp , lift abdominal cavity's fat carefully, open the fat lightly, finding the plum blossom ovary , ligature with 1# asepsis line, cut off and separate the ovary from the oviduct with eye scissors, after oppression arrests blood, put the fat into the abdominal cavity carefully , and sew up muscle aponeurosis The simple ligament cuts off group' s operation and the approach are the same with the above, raise but not excise the ovary after finding it, put back the ovary into the abdominal cavity again after only excising the fat of the ovary equivalent, it is the same to close the notch method, the same method , excise the right side ovary , drip into a small amount of penicillin aqueous solution in the wound, sew up the skin , disinfect the wound.The method of the KOA mould : Anaesthetize it well, the area in which performing the operation and its around region shave with the razor blade and wash clean with the wet gauze, disinfect with the iodine, paves the cloth routinely, cutting the skin and articular cavity close to the in-board, put knee joint in heaviest buckling state, turn over the kneecap to outside, and excise the medial collateral ligament , expose articular cavity, then cut off PCL and ACL operate it carefully so as not to damage the cartilage, washes the joint with physiological saline , sew up articular capsule and skin with the fine rule of 1 # asepsis line, disinfect with the iodine , and stick with injected-pave, wash the joint with penicillin after the operation, at last put rat into the cage to raise, run free as it is. Three days in succession after making the mould, inject penicillin to mouse gluteus lml/lOOg.The simple ligament cuts off group's operation and the approach are the same with the above, raise but not excise the ovary after finding it , putback the ovary into the abdominal cavity again after only excising the fat of the ovary equivalent, it is the same to close the notch method. Medicine preparationChinese medicine herb of invigorate the kidney was constituted with ten kinds of herbs such as old arable soil, Fructus Psoraleae, Salvia miltiorrhiza and milk veteh, etc. The pharmacognostics contents of the extract of this polypharmaceutical was 0. 8377g/ml, according to the body surface area of the human to rat. Therapy methodA month of a half after the operation, the OA model of ovariectomy rat was succeed. We began to cure. E group was drenched one time every day, 6ml/kg every time. Access methodExperiment method: We obtained the materials from F group in the 2^ 4> 6 week and from the A^ Ek C% D groups in the 10> 14n 16 week respectively after operation. Fasting diet and weighting but not prohibiting water before drawing the materials. We extirpated the both eyeballs of rats to take the blood and place the sample in the anticoagulation tube two minutes standing. Abstraction serum for 25 minutes in the hydroextractor of 3500 turn every minute, checked the content of E2.We reject the muscle around the knee to obtain the femur and tibia metaphysis in right side of rats two in F group 2> 4> 6 weeks after operation, one in A group and three in D, fore in B> CU E group 10 weeks after operation, two in A group and three in D group, four in B> C> E group 14 weeks after operation, two in A group and four in B> C\ D^ E group 18 weeks after operation. Preserving the samples in the 4% PFA in order to make HE and saffren-0 staining. We evaluated the pathology change of the cartilaginous tissue with OARSI method. Checking the samples with immunity class influence of MMP-13.We reject the muscle around the knee to obtain the femur and tibia metaphysis in right side of rats two in A> D group 10 weeks after operation, two in A group and four in D group 14 weeks after operation. Putting the samples in the Eppendorf tubes of 1.5 ML which with symbol were put into the LN and maintained in the ice cabinet of -80°C. We got out RNA in accordance with Trizol method and depurated samples with QIAquick PCR Purification Kit method. Checked the A value with spectrophotometer and carried out experiment ofincubation. Making quality examination with AGE method checking 28s^ 18s strip. Preparing the fluorescent probe and quantitated and purified it with QIAGEN RNeasy minikit. Detecting and analyzing gene chip with Oligo chip and relevant kit made from Qiagen CO.We reject the muscle around the knee to obtain the femur and tibia metaphysis in right side of rats two in A group and five in D> B-. C> E group 10 weeks after operation, three in A group and four in C> five in Bn D> E group 14 weeks after operation, two in A group and four in B> C> DN E group 18 weeks after operation. Putting the samples in the Eppendorf tubes of 1.5 ML which with symbol were put into the LN and maintained in the ice cabinet of -80°C. Detecting the DDR2 and the expression of HIF—la RAN with RT-PCR. Making con-RNA, carrying out inverse transcription with Reverse Transcription System kit made from Promega CO. designing primer and running the amplification of PCR with endo-reference of house-keeping gene GAPDH. Checking DDR2 and HIF-la proteinum' s expressing with Western blotting method. Cutting and abrading the bone in microtherm. Adding the extract of protein sample, abrading the sample again and again, then adding the PMSF in microtherm. Subpackaging the sample into the Eppendorf tube of 1. 5 ML and breaking into pieces with hypersound in ice water. Centrifuging sample 30 minutes in 12000r/min. Quantitating protain' s sample with better Bradford method and BCA Protein Assay Reagent Kit. Taking albumen and buffer solution part. aeq. to make 10%SDS2PAGE gum, electrotransfering it to the NC membrane. The samples were sealed with 5% skim milk and incubated 1 hour with anti-FLAG antibody in room temperature. Washing the membrane 10 minutes for 3 times with 50mmol/LTris-HCl/ljTween20 and incubating with HRP. Then the samples were illuminated with Super Signal Femto Substrate kit and exposed on the X-ray. Result 1. Pathologic changes :10 weeks passed after the model operated, the surface of the cartilage is smooth in the A and B group, surface cell of cartilage existed, cartilage in every layer arranged in order, which was the ordinary change in the lens of the cartilage. The surface of the cartilage was lack of smooth. SAF' staining appeared: ecderon worn out company with the base material in the superficial area loss. There were lots of hypertrophic chondrocyte in the 2/3 of the cartilage. The color of the base understained. The pathologic changeof OA account for between 25-50% in the section of the cartilage. The surface of the cartilage in the D group gravanened, the surface cartilage in the articular cartilage thinningzed, asperited and submit to villus in some area. SAF' staining appeared: there were vertical rhagades in the surface of the cartilage. Prismatical CHS disordered. The pathologic change of OA account for 25%—50% in the section of the cartilage. The surface of the cartilage in the E group gravanened. The surface cartilage disappeared. SAF' staining appeared: the surface of the cartilage was worn out and the base disappeared. There were lots of hypertrophic chondrocyte in the 1/3 of the cartilage. The pathologic change of OA account for less than 25% in the section of the cartilage.14 weeks passed after the model operated, the surface of the cartilage existed in the A group, cartilage in every layer arranged in order, which was the ordinary change in the lens of the cartilage. The surface of the cartilage was lack of smooth in B group. The cartilage became hypertrophia and deformed. The surface of the cartilage was lack of smooth in C group, The cartilage in the surface disappeared. Cartilage nucleolus in the reflecting layer shrinked back, the cartilage clustered in the calcification layer. SAF' staining appeared: there were vertical fissuaring in the surface of the cartilage. Prismatical CHS disordered. The pathologic change of OA account for 25%— 50% in the section of the cartilage. The cartilage surface was gravamen in the D group. The cartilage cell in every layer disordered. SAF' staining appeared: there were lots of complex verical fissuaring into the cartilage layer. The surface of cartilage was still smooth, the cartilage in surface disappeared. There were not pannus in the surface. Cartilage was in order. The cluster of cartilage still resisted, but the number is less than that of 8 week' s. SAF' staining appeared: the surface of the cartilage was worn out and the base disappeared. There were lots of hypertrophic chondrocyte in the 1/3 of the cartilage. The pathologic change of OA account for less than 25% in the section of the cartilage. After the 8 week' s treatment with Chinese crude drug, histopathology in E group was more gently than that in C and D group obviously. Patho-change in D group was more obviously than that in C group. There was patho-change of cartilage in B group barely, the base material had no change, which hint the estrogenic decrease resulting in patho-change in cartilage.18 weeks passed after the model operated, the surface of the cartilage existed in the A group, cartilage in every layer arranged in order, which was the ordinary change in the lens of the cartilage. The surface of the cartilage was lack of smooth in B group. Number of the hypertrophia and deformed, cartilage became more. The surface of the cartilage was still smooth in C group, The cartilage in the surface existed but was not integrated. SAF' staining appeared: there were vertical f issuaring in the surface of the cartilage. There was anabrotic change in surface of cartilage in D group. The cartilage in surface disappeared and cell was in disorder. Base in lamina superficialis of part of sample desquamated. There was cymbo-change in intercellular layer. There was cystis degeneration in base of cartilage. In E group, the surface of cartilage was still smooth, the cartilage in surface still existed, there were not pannus in the surface, and cartilage was in order. SAF' staining appeared: the surface of the cartilage was worn out and the base disappeared. There were lots of hypertrophic chondrocyte in the 1/3 of the cartilage. The pathologic change of OA account for less than 25% in the section of the cartilage. After the 12 week' s treatment with Chinese crude drug, patho-change in C and D groups were more and more seriously. Patho-change in D group was more obviously than that in C group. Patho-change of cartilage in B group became more serious, but there was no obvious change in the base. 2. OARSI scoresModified The Osteoarthritis Research Society International(OARSI) catilage histopathologic scores. The OARSI scores between group C and D after 10 weeks post-operation showed no statistic significance difference (P>0. 5), but the scores of group D was higher than group C. The OARSI scores of group B compared with group C, group D or group E showed significant difference (p<0. 05). The OARSI scores of group E compared with group D , with significant difference (p<0. 05). The OARSI scores of group C compared with group D after 14 weeks post-operation ,with no significant difference(P>0. 5), but the scores of group D was also higher than the group C . The OARSI scores of group B compared with group C\ group D or group E showed having significant difference(P<0.05). Between group E and group D, with significant difference(P<0.05). After 18 weeks post-operation :the OARSI scores of group B compared with group C-. group D or group E showed having significant difference(P<0. 01). The group E compared with group C or group D,with significant difference (P<0.05). The OARSI scores of group D compared with group E showed having significant difference (P<0.01).3. Estradiol level 'The change of estradiol level :The group A compared with group BN D E showed having significant difference (P<0. 05). The group C compared with group B n D and group E showed having significant difference (P<0. 05). Among group An group C and group E .without significant difference. The group B compared with group D showed no significant difference. After 14 weeks post-operation , the group A compared with group B, D showed significant difference (P<0.01), Between the group D and group E , there was significant difference (P<0.05). The group C and group E compared with group B or group D showed having significant difference(P<0. 05). Among group A^ group C and group E , there were no significant difference. Between group B and group D , without significant difference. After 8 weeks post-operation , the group A compared with group Bn group D or group E showed significant difference (P<0. 01). group C and group E compared with group B or group D, with significant difference (P<0. 01). Among group A^ group C and group E, without significant difference. Between group B and group D, without significant difference.4. Genetic profile expressionGenetic profile expression of osteoarthritis in kidney deficiency type indicates: there is significant difference in 98 genetic express. Among the total, 58 genetic expression is up-regulated, and 40 genetic expression is down-regulated. Immunity factor related genes, growth factor related genes, growth and development related genes, obesity related genes, metabolic related genes, and signals transduction genes play appropriate roles in the pathogensis of osteoarthritis. The research find out 4 immune factors related genes, 11 growth fators related genes, 9 growth and development related genes, 3 obesity related genes, 8 energetic metabolism related genes, 10 genes related to cell signal transduction molecules and passage, 6 genes related to protein degradation enzymes.5. Genetic profile expression After the contribution of TCM,52 items genetic expression of renal deficiency type OA' s gene profile have significant change, the gene of the proliferation apoptosis of the cartilage cell and could inhibit the gene of the prolease and inflammatory factor and could reinforce the immune function of the body and could rebuildthe steady state of the senile immunization and could remolding the optimum balance of the expression of the gene and could delay the cartilage cataplasia had been involved.6. The effects of the possible key gene involved articular cartilage cataplasia on the invigorate the kidney herbsThe outcome of gene chip shows: DDR2> PPM13 and HIF-la are high expression (the ratio score of three is respectively 16.18 -. 13.15> 10.86). According to some records have been published , we infer that three of them have great action during OA procedur. The outcome of RT-PCR% Western blotting shows: the expression of DDR and HIF-la of the C> D groups have a tendency to increase progressively with straight line , so do HIF-la of the B groups and An B groups is nothing or low after the operation of l(k 14n 18 weeks. The outcome of MMP-13 shows: the cartilage cell of the CUD groups could express brownish-black positive cells, which its color is deep. So do B group. So we guess DDR is key gene of the pathological change of OA .7. The analysis dependability of the MMP-13 and OARSI scoreThe analysis dependability of the MMP-13 and OARSI score shows: they are positive correlation (the coefficient correlation is 0.514,p=0.038<0.05).it also cue that MMP-13 could degraded the articular cartilage matrix and OARSI score is rationality dependability.8. The effects of the DDR2n HIF-la> MMP-13 and articular cartilage lesion on the invigorate the kidney herbsThe outcome of HE> Safranin 0 staining and OARSI score hint that the invigorate the kidney herbs could inhibit apoptosis of the cartilage cell and could prevent the loss of the extra-cellular matrix (EMC). The outcome of RT-PCRn Western blotting and immunohistochemistry hint the invigorate the kidney herbs could down regulation the expression of DDR2> HIF-laN MMP-13. Conclusion:1. The method for the type of renal deficiency osteoarthritis is easy and its cycle time is short . the costs and the request for the sites of breeding is so low and the comparison is better, the newest research outcome of the molecular biology might be studied the osteoarthritis using this model.2. For the scoring system of the OARSI, could rational give expression to the interclass difference in the difference stage of the osteoarthritis3. The expression of the gene profile of the type of the renal deficiencyosteoarthritis. Many gene have great effects on the process of the OA. Such as : the related gene of the immunization factor the related gene of the growth factors the related gene of the growth and developments the related gene of the obesitys the related gene of the energy metabolisms the related gene of the information conductions the related gene of the protein catabolic enzymes and the related gene of the ion channel.4. DDR2 might be the key gene which induce the degradation of the articular cartilage.5. The prescription of the invigorate the kidney could inhibit apoptosis of the cartilage cell and could prevent the loss of the extra-cellular matrix (EMC).6 . The prescription of the invigorate kidney could up-regulation the gene of the proliferations apoptosis of the cartilage cell and could inhibit the gene of the prolease and inflammatory factor and could reinforce the immune function of the body and could rebuild the steady state of the senile immunization and could remolding the optimum balance of the expression of the gene and could delay the cartilage cataplasia.7. The mechanism of action of the traditional Chinese medicine of the invigorate the kidney is a whole process which is multicomponents multisystenK multi-targets multi-channels networking.8. To study and research of the OA using the technique of the gene-chip have good specificity and have high sensitivity and have quickly and reliable result. This technology have a great perspective.
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