| Articular cartilage is mainly composited by cartilage cells and matrix, and its function is conducting and distributing loading, maintaining and bearing the contact stress of articular surface. Articular cartilage is the organization without vessels, lymphatics and nerve, so its repair capacity is very limited, minor injury may cause progressive damage. The emergence of tissue engineering methods provides an ideal way which is the possibe one to solve the problem eventually. The tissue-engineered cartilage built in the past which selected mainly chondrocytes as seed cells, there are a lot of disadvantages: For example, the function of providing areas is disabled; the amount of cells obtained is too little; the cells are easy to differentiate in the process of vitro amplification, and allogeneic chondrocytes exist immunogenicity and the possibility of carrying virus potentially. But BMSCs (Bone Marrow Stromal Cells) as prerequisite cells for chondrocytes,that its source is acquired easyly, it is geted conveniently, proliferation ability is strong, and can be amplified in vitro without the loss of much large-scale differentiation potential, and rejection is small at the time of allograft. It is a class of stem cells that have differentiated potential of multi-directional, some of which have the capacity of self-replicating, high degree of proliferation and multilineage differentiation. In specific culture conditions,they can transformate to mesenchymal tissue cells, such as bone cells, cartilage cells, glial cells, myocardium cells, skeletal muscle cells, fat cells, vascular endothelial cells etc,so these cells also were called mesenchymal stem cells.The way of BMSCs that were induced and differentiated to the cartilage cells was very maturated, and BMSCs were considered the most valuable seed cells of cartilage tissue engineering. In vivo and vitro the process of BMSCs which were induced and differentiated to the cartilage cells, BMSCs required the role of certain factors, such as TGF-β(transforming growth factor-β), IGF (insulin-like growth factor), BMP (bone morphogenetic protein), FGF (fibroblast growth factor). They played a role at all stages which were induced and differentiated to the cartilage cells, through the way of autocrine and paracrine.IGF is a kind of peptides which have Proinsulin-like structure, from bone, cartilage and synthesis of a variety of other organizations, play a role of mitogen on a variety of tissues and cells. IGF can promote chondrocytes'proliferation, enhance cells'function,promote proteoglycan and type II collagen's synthesis. Madry's study showed that insulin-like growth factor 1(IGF-1) promoted the process of BMSCs differentiated to the cartilage cells, and inhibited extracellular accumulation of pyrophosphate induced by TGF-β1 and prevent the formation of calcium phosphate crystallization and further formated bone, it is a essential cofactor of TGF-β1. Clinical observation showed that adults serum leptin levels was correlated with the cycle of IGF-1 significantly positively.Leptin was a low molecular weight polypeptide protein which was an obese (ob) gene encodes, produced by white adipose tissue, participated the regulation of energy metabolism, loss of appetite, growth and development. In recent years, both at home and abroad, study of leptin involved a number of areas of research and found that leptin were closely related to a variety of diseases including obesity, diabetes, hypertension, renal failure, osteoporosis, breast cancer. Cornish etc confirmed that there were leptin receptor on human articular chonodrocytes, leptin could promote the proliferation of the sheep and dog cartilage cells, and increase the thickness of epiphyseal growth plate when adult mouse was applicated leptin. After leptin was injected to the knee joint of rats, it could stimulate the two types of cartilage growth factors'(IGF-1 and TGF-β1) expression, and then increase the proteoglycan's synthesis. Maor et al considered that leptin could act directly on the receptor of growth plate chondrocytes, to promote the proliferation of chondrocytes'precursor cells and increase mandibular condylar cartilage cells'activity of proliferation and differentiation in vitro, IGF-1 pathway may play an important role in the mediation. Therefore, we could speculate that leptin may affect the cartilage process of BMSCs by IGF-1, to promote the synthesis of extracellular matrix.Now, the reports about the leptin's effects on BMSCs were also less, the present study used leptin to interfere the rat BMSCs in vitro, observed the differentiation and proliferation of BMSCs, the expression of IGF-1 and the synthesis of extracellular matrix, to learn about leptin's effects and mechanisms on rat BMSCs, and provided a theoretical basis for it that leptin could involve in repairing cartilage injuries in clinic.ObjectiveThe present study was undertaken to explore:1. Effects of leptin on rat BMSCs in the early differentiation (<72h) in cultured rat BMSCs to chondrocytes;2. Determination effects of different concentrations of leptin on expression of IGF-1 in BMSCs to chondrocytes during differentiation at gene level;3. The optimum effect of the concentration Leptin in vitro cultured rat BMSCs to chondrocytes during differentiation.Through these results, explore the effect and mechanism of leptin on the process of BMSCs induced and differentiated to chondrocytes, so as to better guide the clinical treatment of osteoarthritis and to promote the development of cartilage tissue engineering.motheds1. With the method of cell cultivation in vitro, bone marrow stromal stem cells of 4 weeks SD rat were choosed as experimental research material.2. Rat bone marrow stromal stem cells cultured were morphologically observed.3. Cultured the BMSCs to the third generation, randomly divided them into 6 groups, the first group was the blank group, added induction DMEM (DMEM, 5% fetal bovine serum, recombinant human transforming growth factor-β1 (TGF-β1) 10 ug / L, dexamethasone 10-7 mol / L, vitamin C 50μg / L), the rest were the experimental groups, separately added 0.1 ng/ml,1 ng/ml,10 ng/ml,100 ng/ml,1000 ng/ml leptin in induction DMEM.4. The proliferations of BMSCs cultured in vitro with the stimulus of different concentration leptin were detected with the method of MTT.5. The ecretions of type II collagen by BMSCs inductively cultured in vitro with the stimulus of different concentration leptin were detected with immunocytochemical method.6. The expressions of IGF-I by BMSCs inductively cultured in vitro with the stimulus of different concentration leptin were detected with the method of PCR.7. The results were analyzed statistically with the software SPSS15.0.Results1. BMSCs were cultured successfully with the method of cell cultivation in vitro, and confirmed by morphological observation. Immunocytochem- ical method was used to prove the transformed cells to be chondrocytes.2. At the first 24h, the cells'proliferation of the experimental group were higher than the control group(P<0.05); At the 48h - 72h, the OD values of experimental group that added 10 ng/ml leptin was significantly higher , and were significantly different from other groups3. The expressions of IGF-1 had increase vary degrees, in experimental groups after leptin stimulated, the biggest effect concentration is 10 ng/ml, but the promotive effect of high concentration's leptin is not so obvious.Conclusions1. Different concentrations of leptin can promote the proliferation of BMSCs cultured vary degrees in vitro, and increase the expression of IGF-1.2. Leptin can promote cell proliferation at the early period of BMSCs differentiated into chondrocytes, and can play a promoting role on directed differentiation of BMSCs to cartilage cells through increasing the expression of IGF-1 of BMSCs.
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