【作者】 王济训；

【导师】 姜杰；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2007， 硕士

【摘要】 目的：探讨兔关节软骨细胞分离培养的方法和生物学特性，观察中药当归注射液对体外培养兔关节软骨细胞的增殖以及对肿瘤坏死因子-α诱导的软骨细胞损伤的保护作用，探讨其治疗骨关节炎的机制。方法：体外培养从3周龄新西兰兔关节分离培养的软骨细胞，倒置显微镜下观察原代及传代培养的细胞形态学变化，用组织化学甲苯胺蓝染色法鉴定软骨细胞。将传一代的软骨细胞接种于96孔培养板，细胞贴壁后，将其分成当归组A—F组与空白对照组G组，分别用含10％，5％，2.5％，1.25％，0.625％，0.312％和0％的当归注射液的含10％血清DMEM培养基进行培养，H组未接种细胞，只加无血清DMEM培养基做为调零组。培养48h后用MTT比色法测定各组软骨细胞的增殖情况。取第二代软骨细胞接种到24孔培养板中，培养5d后将细胞随机分为8组，分别换用不同的培养液。当归A—F组分别以含10％，5％，2.5％，1.25％，0.625％，0.312％当归注射液的含10％血清DMEM培养基培养，并在每孔加入100U肿瘤坏死因子-α诱导细胞损伤。实验对照组不加当归注射液，仅以含10％血清DMEM培养基培养细胞，并在每孔加入100U肿瘤坏死因子-α。空白对照组只以不含当归注射液的含10％血清DMEM培养基培养。培养72h后收集细胞上清液，用化学比色法测定SOD及NOS含量，观察当归注射液对软骨细胞的保护作用。结果：成功建立体外培养兔关节软骨细胞的实验方法。原代培养的软骨细胞呈多角形，传代3次后出现去分化。形态学、组织化学染色显示细胞培养3代以内可以保持表型的稳定。5％，2.5％，1.25％，0.625％，0.312％的当归注射液对软骨细胞的增殖有明显的促进作用，与对照组相比有非常显著性的差异。其中0.625％当归组作用最强，10％当归组对软骨细胞的增殖无促进作用，与对照组相比无显著性差异。肿瘤坏死因子-α可显著升高软骨细胞中NOS活性，降低SOD活性，当归注射液组能对抗上述结果并呈浓度依赖性。结论：本实验建立的体外培养关节软骨细胞的方法简单可行。细胞具有良好的生物学活性，细胞表型可保持3代稳定。药物实验显示中药当归注射液可有效的促进软骨细胞的增殖，并可有效的防止和保护肿瘤坏死因子-α诱导的细胞损伤作用，调节软骨细胞代谢，减缓骨关节炎关节软骨的退变。从而证实当归注射液可有效的防治骨关节炎。更多还原

【Abstract】 Objective:To investigate the method of isolation and culture of rabbit articular chondrocytes for biological characteristic studies, To observe the effect of DangGui injection on proliferation of rabbit articular chondrocyte and the protective effect of DangGui injection on chondrocyte lesion induced by TNF-α, and discuss the mechanism of DangGui injection for treating Osteoarthritis.Methods:Articular chondrocytes were isolated from the cartilages of 3 week-old New Zealand rabbits were cultured in vitro. The morphological changes and growth feature of primary culture and subculture of chondrocytes were observed under the inverted microscope each day. Toluidine blue staining was used to indentify the chondrocytes .The first subculture chondrocytes were inoculated into 96- hole culture plate and divided into the experimental groups DangGui A - F group and the G group, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25 %, 0.625%, 0.312%and 0% of DangGui injection, respectively. G Group is the blank control group were cultured in 10% serum DMEM medium, H Group were cultured in serum-free DMEM medium and no chondrocyte. After cultured for 48 hours,chondrocytes proliferation was assessed by MTT chromometry. The second subculture chondrocytes were inoculated into two 24-hole culture plates. After incubation for five days, chondrocytes were divided into 8 groups randomly with different medium. DangGui A-F groups, which were cultured in the 10% serum DMEM media containing 10%, 5%, 2.5%, 1.25%, 0.625%, 0.312% of Dang Gui injection, with100U/ml TNF-α, respectively. Experimental group were cultured in 10% serum medium with 100U/ml TNF-α, Blank control group were cultured in 10% serum DMEM only, after incubation for 72 hours, culture supernatants were collected for SOD and NOS measurement.Results:The primarily cultured chondrocytes was in polygonal shape, and became dedifferentiation after 3 passage. The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages . Compared with the control, 5%, 2.5%, 1.25%, 0.625%, 0.312% of DangGui injection could promote the proliferation of chondrocytes with significant difference. 0.625% DangGui injection had the greatest function in the six doses with a significant difference from other groups. 10% DanShen injection had no significant promotion effect difference. TNF-αcould promote the NOS activity and depress SOD activity in chondrocytes. DangGui injection could altered the above mentioned results in a concentration dependent manner.Conclusions:The method used in this work for isolation and culture of chondrocytes is simple and feasible. The chondrocytes cultured in vitro maintained the specific chondrocytes phenotype in the first 3 passages. The following experiment suggested that DangGui injection can effectively promote the proliferation of chondrocytes and prevent and protect against chondrocytes lesion induced by TNF-α, and regulate anabolism of chondrocytes, thus it can defer the degeneration of osteoarthritis cartilage. It demonstrated that DangGui injection was effective to prevent and cure osteoarthritis.更多还原