长时间周期性拉伸形变对软骨细胞生化环境的影响

发布时间：2018-04-01 14:01

本文选题：软骨细胞　切入点：周期性伸形变　出处：《南方医科大学》2011年硕士论文

【摘要】：[目的] 由于无血液供应软骨及软骨细胞外基质的增殖能力较其他组织明显低下,软骨组织通常在受损的情况下其自身低下的恢复能力,是各类型关节炎的主要原因。不同的模型研究中,应力强度频率及作用的方式是变化的标尺,所以很难比较之前发表的文章中的多种结果。已有文献证明20%拉伸幅度的周期性拉伸应力应用于单层培养的软骨细胞致软骨基质中蛋白成分降低,使MMP-1、MMP-3、MMP-9表达水平调高。持续性周期性拉伸应力导致基质向重新塑造的转变以及潜在的基质成分的改变。并且也发现只有在炎症因子(IL-1p和TNF-α)存在的前提下周期性拉伸(5%)应力能显示其对炎症因子的拮抗作用。本实验的目的是研究软骨细胞在单层细胞培养中受到拉伸机械应变时几种分解代谢酶(金属蛋白酶3、5及带血小板凝集素敏感蛋白样模体的解整联蛋白金属蛋白酶5)及与合成代谢有紧密关系的转化生长因子p等各种细胞因子的基因水平变化,以了解在不同拉伸幅度下长时间拉伸后软骨细胞内外生化环境的变化情况。 [方法] 本研究采用获取经典新西兰兔髋、膝关节透明软骨制成原代软骨细胞,按5×105/孔的密度将细胞混悬液接种于flexercellⅠ型培养板中,显微镜下显示大部分细胞贴壁牢靠并生长成亚融合状态后,利用FLEXERCELL-5000T细胞拉伸系统对单层培养细胞施加拉伸应变。实验采用组别与时间两因素析因分析设计,组别分为空白组和低幅度(5%)和高幅度(15%)拉伸组,拉伸5天,每天作用8个小时,分别取第3天和第5天两个时间点的细胞行(1)甲苯胺蓝染色,倒置显微镜下观察、拍照,及(2)荧光实时定量PCR观察MMP3、MMP13、ADAMTS-5、TGF-β1的基因表达水平。 [结果] MMP3、ADAMTS-5第3天时各拉伸组均显著升高,第5天时较第3天不同程度下降。MMP13第3天时各刺激组均高于空白组,第5天时高幅度组较前继续升高,低幅度组较前无明显改变。组间比较高幅度组高于低幅度组。TGF-β1在第5天时刺激组均高于空白组,低幅度组高于高幅度组。 [结论] 小幅度(5%)拉伸可提高细胞的合成、分解代谢,及对软骨细胞的保护作用。大幅度拉伸对软骨细胞具有促分解作用,并且使软骨细胞产生的不同类型分解代谢酶中在不同时间点提高基因表达水平,其中MMP-13在分解代谢中起到关键作用。长时间大幅度拉伸刺激下TGF-β1在软骨细胞的病理性修复中可能起到的重要作用。
[Abstract]:[Objective]
The blood supply of cartilage and cartilage extracellular matrix proliferation was significantly lower than the other tissues, cartilage tissue is usually in the damaged condition of its own low recovery capability, is the main reason for the various types of arthritis. The research of different models, the stress intensity and the frequency of the mode of action is the change of scale, a variety of results so it is hard to compare the.
It is proven that the cartilage cell cycle of tensile stress amplitude of 20% tension applied to monolayer induced proteins in cartilage matrix decreased, MMP-1, MMP-3, MMP-9 expression level increased. Persistent tensile stress leads to the re shaping of the transformation matrix and the change of matrix potential and also found only. In the inflammatory factors (IL-1p and TNF-) are under the premise of cyclical tensile stress (5%) can show its antagonistic effects on inflammatory cytokines.
The purpose of this experiment is to study the tensile mechanical strain by cartilage cells of several catabolic enzymes in monolayer cell culture (metalloproteinase 3,5 and platelet agglutinin sensitive protein like motif disintegrin metalloproteinase 5) changes in gene level and synthesis and metabolism are closely related to the transforming growth factor P and other cytokines and in order to understand the different amplitude under long time after tensile tensile chondrocytes inside and outside the biochemical environment changes.
[method]
This study was used to obtain the classic New Zealand rabbit hip, knee joint cartilage into the primary chondrocytes by 5 * 105/ hole in the density of the cell suspension were inoculated in flexercell type culture plate, under the microscope showed that most cells adhered firmly and grow into sub fusion state, the use of FLEXERCELL-5000T drawing system of single cell cell culture applied tensile strain. Experimental group and time using two factor factorial design analysis, groups were divided into blank group (5%) and low amplitude and high amplitude (15%) tensile tensile group, 5 days, 8 hours a day, third days and fifth days were taken at two time points (1 cell line) camera observation, toluidine blue staining, inverted microscope, and (2) fluorescence quantitative real-time PCR observation of MMP3, MMP13, ADAMTS-5, gene expression level of TGF- beta 1.
[results]
MMP3 ADAMTS-5, the third day of each stretch group were significantly increased at fifth days compared to third days decreased.MMP13 third days when the stimulation group were higher than that in control group, fifth days of high magnitude group than before to continue to rise, the low amplitude group compared with no alteration. Comparison between groups of high amplitude is higher than low amplitude group.TGF- beta 1 on the fifth day stimulation group were higher than that in control group, low amplitude group was higher than that of high magnitude group.
[Conclusion]
Small amplitude (5%) can improve the metabolism of cell stretch, synthesis, and protective effect on cartilage cells. Substantial tensile promoting effect on the decomposition of cartilage cells, and the cartilage cells produce different types of catabolic enzymes in different time points to improve the level of gene expression, in which MMP-13 catabolism plays a key effect of long time greatly. Under mechanical stretch TGF- beta 1 in chondrocytes in important pathological repair may play a role.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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