| ObjectiveKnee osteoarthritis is a common disease in the elderly, connects with rheumatism involving bone in Chinese medicine, the main pathogenesis are renal deficiency and blood stasis. Treating knee osteoarthritis with Kidney-Supplementing Blood-Quickening Chinese medicine gained good outcome, clinical studies before showed that Traditional Chinese medicine and celecoxib had similar clinical efficacy, no significant difference. However, with the development of medical model, the efficacy standard only focusing on improving the clinical symptoms can not meet patients'requirements. Damage of physiological function, psychological function, physical role, social role, et al, involve in the quality of life (QOL) caused by illness received more and more attention. This study was to observe the effect of QOL of patients with knee osteoarthritis treated with Kidney-Supplementing Blood-Quickening Chinese medicine(Guan Jie Kang), try to introduce the QOL assessment to clinical trials of Chinese medicine and promote and apply, in order to modify composition of clinical evaluation index in Chinese medicine, and to better verify the efficacy of Chinese medicine.Modern medical research showed that subchondral micro-fractures played an important role in the pathogenesis of knee OA. This study was to confirm that by experimental Kidney-Supplementing medicine might promote the repair of subchondral micro-fracture by promotion of proliferation of BMSCs and induction them to differentiation into osteoblasts, to block the pathological process that the increase of inflammatory components and growth factors, and induce of the inflammatory inhibitors. In order to ecplore the mechanism of treatment KOA by Guan Jie Kang. Chinese medicine considers the kidney generating marrow and dominating bone, which was studied mainly on the bone metabolism itself by modern medical research. Studies also found that features of mesenchymal stem cells (MSCs) had relationship with "the kidney being in charge of bone". Based on the important role of Duzhong(Cortex Eucommiae) in the Chinese medicine invigorating the kidney, the study chose Duzhong (Cortex Eucommiae) which was the Sovereign drug in Guan Jie Kang tables as the research object to observe proliferation of BMSCs induced by serum containing Duzhong (Cortex Eucommiae), and differentiation results of BMSCs into osteoblasts, in order to expound the theory of "the kidney generating marrow" from cytology and to explain the mechanism of Duzhong(Cortex Eucommiae) invigorating the kidney.Usually biological characteristics of stem cells were researched at the level of genome. However, the analysis of biological function by transcription was often not sufficient, because the cells depends on protein, it is protein involved in metabolism and regulation that decided cell survival. This study used proteomics research methods to establish functional protein of BMSCs differentiating into osteoblasts induced by Duzhong (Cortex Eucommiae), and analyze molecular mechanism of BMSCs differentiating into osteoblasts. MethodsAll the cases who attending out-patient first were selected from the department of Orthopaedics of the First Affiliated Hospital of Guangzhou University of Traditional Chinese medicine, there was a total of 55 cases, all the cases were divided into Guan Jie Kang group and the celecoxib group by random table. Before and after treatment all patients completed SF-36 quality of life questionnaire to calculate scores in each group, t test was used to compare before and after treatment within group, two sample t test was used to compare score difference between the two groups.BMSCs were isolated from the two femurs and tibias of SD rats, cultured by the whole bone marrow method in vitro, purification and amplification by trypsin digestion. The quartus generation cells were used as the experimental cells.The experiment was divided into 3 groups named blank group, DXM group and Duzhong (Cortex Eucommiae) group. Inoculated quartus generation MSCs in nine nity-six-holes cell culture plates and two six-holes cell culture plates by 5×106 cells per milliliter, all the holes were divided into three groups, each group had eight holes in nity-six-holes cell culture plates and two holes in six-holes cell culture plates. Two days later, changed medium and add new medium. The medium of DXM group contained 10mmol/Lβ-sodium glycerophosphate, 0.lumol/L DXM,50mg/L vitamin C, that of Duzhong (Cortex Eucommiae) group contained 10% serum containing Duzhong (Cortex Eucommiae). Measured cell number by MTT of nity-six-holes plates from the second day to the eighth day, one of the six-holes plates was colored by ALP staining solution at the tenth day, the last one was colored by alizarin bordeaux at the twentieth day.Extracted total protein of cells induced by serum containing Duzhong (Cortex Eucommiae) for 0day,2days,7days,14days, and runouted them by two-dimensional electrophoresis, searched for different protein spot by PDQuest-7.3.0 software for analyzing gel picture, to establish functional proteome by mass chromatographic analysis and data base querying, to analyze molecular mechanism of BMSCs differentiation into osteoblasts induced by Duzhong (Cortex Eucommiae).ResultsGuan Jie Kang group and celecoxib group got improvement of score of each dimension after treatment, with statistical significance. It showed that Guan Jie Kang and celecoxib could significantly improve the QOL of patients with knee OA. But there were different results between the two groups from different dimensions, the two groups could not be compared in improving role limitations due to physical health and bodily pain, which was consistent with the previous clinical observation results; but Guan Jie Kang group improve physical functioning, general health, energy, social functioning, role limitations due to emotional problems, emotional well being better than celecoxib group.BMSCs could be isolated from the bone marrow of SD rat by whole bone marrow culture, and could be further purified by trypsin digestion and passage.The OD value of Duzhong (Cortex Eucommiae) group rose obviously, blank group and DXM group had no obvious ascension; on the tenth day DXM group and Duzhong(Cortex Eucommiae) group were colored masculine by ALP staining solution; white calcium nodus could be seen at the undersurface of the holes 13days later, which could be colored masculine by alizarin bordeaux twenty days later.Total protein concentration in each group were determined by the bradford method, they were 3.24μg/μL on the Oday,4.10μg/μL on the 2nd day, 4.7μg/μL on the 7th day,7.27μg/μL on the 14th day. Which descripted that serum containing Duzhong (Cortex Eucommiae) could promote cell proliferation.We separated proteins by two-dimensional electrophoresis and gained 4 maps. Analysis of all the maps, the distinction between points was more obvious, the difference of protein expression may be analyzed. We used PDQuest-7.3.0 analysis software to analyze and identified a total of 641 protein spots,75 of witch were selected to receive mass spectrometry analysis and database query. 6 points of them were positive. They were:F18, stromal cell-derived factor (SDF-1), can be used as a sign of detection differentiation degree of BMSCs;G21, superoxide dismutase (SOD), was metal enzyme, was a major antioxidant enzyme, superoxide can eliminate oxygen-derived free radicals, and play an important role in the defense of oxygen toxicity, suppression of senile disease, prevention of senescence and so on;E17, calcium reticulocyte protein (CRT), involved in calcium storage and signal transduction; cell adhesion; gene expression, etc;E4, vimentin, vimentin was a source of intermediate filament protein existing in mesenchymal cells, attached to the nucleus, endoplasmic reticulum and the side or the end of mitochondria, so it was believed that vimentin played an important role in supporting and anchoring organelle in protoplasm, It can also maintain cell shape, integrity of cytoplasm and stability of the cytoskeleton interaction;F6, lamin A, it and vimentin belonged to a kind of intermediate filament proteins, it was a necessary structural protein for structure of all eukaryocyte, mainly involved in constructing nuclear, and playing an important role in the processes of nuclear construction;F3, annexin A6, involved in membrane transport and a series of calmodulin dependent activities at membrane surface in cells, including vesicle transport, membrane fusion of exocytosis, signal transduction and calcium channel formation, control inflammation, cell differentiation, and interaction between cytoskeletal proteins, etc. ConclusionTCM treatment based on determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs, it synthetically considered functional state of each demic system, and adjusted the whole functional state of human from more links, so it had.a greater advantage in the treatment of chronic physical and psychological diseases with complex etiopathogenisis in modern society. Introducing the QOL assessment to clinical trials of Chinese medicine and promoting and applying, could modify composition of clinical evaluation index in Chinese medicine, in order to verify the efficacy of Chinese medicine better.But there were various QOL scale, each had its own scope of application. In this study, SF-36 form was used, this table was a universal QOL scale, although responsed to effect on patients'QOL of disease from different angles, but in terms of knee osteoarthritis, some items can not responsed QOL very well. Therefore, a study of a QOL scale for knee osteoarthritis is our further objective of the study.Guan Jie Kang minht cure KOA by promoting repair of subchondral microfracture which blocked the pathological process that increase of the production of inflammatory component and growth factor and decrease of the production of inflammatory inhibitors.It was more effectively to obtain BMSCs by extraction from bone marrow in vivo, which was benefit for growth, division and proliferation of BMSCs on the parietes. Whole bone marrow culture was simple and effective, cell density affected cell proliferation. Serum containing Duzhong (Cortex Eucommiae) could promote the proliferation of BMSCs and induced differentiation of BMSCs into osteoblasts. In the differentiation process, cell density played an important role. Differentiation of BMSCs into osteoblasts was a complex process, experiments thought that Duzhong (Cortex Eucommiae) invigorating the kidney possibly promoted cell differentiation by increasing vimentin, lamin A expression, reduced the express of calreticulin to release intracellular calcium, which involved in the formation of calcium nodules, and related to the process of bone mineralization in vivo. Superoxide dismutase and annexin A6 had relation with the vitality of the cells, regulated cell division, proliferation and differentiation; but whether they had closely relation with directing differentiation of osteoblasts remained to be further studied. |
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