| PrefaceThe injury and destruction of articular cartilage is very common, but its restoring competence is very poor. The development of tissue engineering provides a new method for cartilage tissue cultivation in vitro and its repairing, but the chondrocytes in vitro which growth slowly and easy aging can not fit to the clinical demands. Cytokine is a substance which play an important role in the growth, proliferation, development and metabolism of cells. It can be inoculated into chondrocytes in vitro, as a result, it accelerates cell proliferation and postpones cell aging. HGF was found about 10 years ago. It can promote the tissue repairing and regeneration of hepatocyte, but as a multi -functional cytokine, it can play a part in various tissues. HGF receptor can be expressed by chondrocyte. HGF can regulate the proliferation and differentiation of chondrocyte both in vivo and in vitro. Ecto-genetic HGF can enhance the morphogenesis of bone and cartilage and the repairing capability of chondrocyte. We cultured chondrocyte cells in vitro, to verify its shape of chondrocytes by Giemsa stsining and im-munohistochemistry in the microscope, to certify its capability of secreting type - II collagen that belong to chondrocytes. HGF was inoculated in cultured chondrocytes in vitro. We want to verify its exact role and explore its optimal concentration by immunohistochemisty, MTT technique and flow cy tome try.Materials and MethodsEquipment; carbon dioxide hatch box, enzymoimmune detector,flow cytometer, poly - pore cultural plate, invert microscope, etc. REAGENT: DMEM cultural medium, HGF, IGF - I , etc. ANIMALS; 2 weeks age New Zealand white rabbits. METHODS: Articular cartilages of rabbits was incised under sterile condition, made cell suspension and cultured with DMEM. To observe the shape of chon-drocytes after inoculation and margination by invert microscope. To observe the growth status by Giernsa staining. To detect the expressing condition of type - II collagen by immunohistochemistry. After incubation with cytokine, to comprehend the influence of cytokine on the survival cells number and proliferation of chondrocytes by MTT and flow cytometer respectively.Results1. The cells after inoculation have the characteristics of chondro-cyte, and transfer of culture is fine. Invert microscopic observation, Giemsa staining and immunohistochemistry can confirm it.2. HGF can enhance the proliferation of chondrocyte in different concentrations. Its optimal concentration is lOng/ml, in contrast, the same role of IGF is more obvious, and its optimal concentration is 50ng/ml.3. Both HGF and IGF can stimulate the proliferation of chondrocyte, and they have synergistic action.Discussion1. The articular cartilage defect which caused by trauma or degenerative disease often can be seen clinically. The mitosis of chon-drocyte is limited, so do the experiment in vitro. There are two patterns by which cytokines enhance cell proliferation: ( 1 ) to increase the number of cells. (2) to transit dormant cells into active one. HGF have both of the patterns mentioned above, and it is the only one which have multi - function, so far, even if its role is weaker than IGF.2. The role of HGF have dosage - effect in it. We detect cell proliferation by MTT method. MTT is a kind of dye which can accept hydrogen atom. Ectogenetic MTT can reduce into purple crystal by succinate dehydrogenase in mitochondrion in live cell. So, to certain extent, the dose of MTT crystal is parallel to the live cell number.3. With the development of growth factors, it is confirmed that there are many growth factors in chondrocyte, and they have interaction with each other. In our study, IGF and HGF have synergistic action, but as for other growth factors, it needs further investigation.Conclusion1. Cultured cells are active chondrocytes.2. HGF plays an important role in enhancing the proliferation of chondrocytes.3. IGF and HGF have synergistic action in it. |
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