绿原酸对缺氧环境下干细胞来源软骨样细胞凋亡的影响

发布时间：2018-05-06 09:34

本文选题：低氧 + 骨髓间充质细胞来源软骨样细胞　；参考：《南昌大学》2011年硕士论文

【摘要】：研究背景和目的：软骨细胞是构成关节软骨的主要基础,但因其高度分化增殖能力低下造成软骨的再生能力差,使得软骨损伤后难以自然修复。干细胞及其来源软骨细胞移植治疗解决了软骨细胞低增殖力问题,已成为临床上治疗软骨损伤的有效方法。大量研究发现体外常氧环境下培养的细胞移植进入类似关节腔内缺氧环境后活性及存活率均下降,Wakitani等研究中认为移植细胞低存活率使得干细胞分化的软骨的厚度、机械强度等均不及正常软骨,所以认为细胞移植的低存活率是制约这一方法应用的因素。绿原酸(chlorogenic acid,CGA)为苯丙素类极性有机酸,具有强抗氧化能力可抑制氧化应急引起的凋亡。绿原酸对缺氧环境下细胞内活性氧改变引起的凋亡的作用,目前还不清楚。本实验拟体外诱导骨髓间充质细胞为软骨样细胞,0.1% O2缺氧环境下模拟关节腔内的缺氧环境模型,观察绿缺氧环境下原酸对软骨样细胞的作用,探讨其对软骨细胞的影响及可能机制。研究内容和方法：采用体外培养大鼠间充质干细胞,以含0.2mg·L-1 BMP-2的DMEM诱导诱导液培养12天使其成为软骨样细胞后进行实验。分为(A)正常对照组；(B)绿原酸组；(C)0.1% O2缺氧环境组；(D)绿原酸+0.1% O2缺氧环境组。进而检测各组Hoechst33258荧光染色检测细胞凋亡、细胞内活性氧水平；RT-PCR检测不同组软骨细胞Caspase-3和Bcl-2基因的表达。结果：与正常对照组比较,0.1%O2缺氧环境12小时能明显造成干细胞来源软骨样细胞的凋亡(P0.05),绿原酸可降低0.1% O2缺氧引起的软骨样细胞凋亡率(P0.05),活性氧水平下降,Bcl-2表达增强,Caspase-3表达减弱。结论：绿原酸可抑制0.1%02缺氧引起的内皮细胞凋亡,其作用机制可能与降低细胞内的活性氧水平,稳定细胞的氧化还原状态来保护线粒体膜电位,促进凋亡抑制基因Bcl-2的表达及抑制Caspase-3的表达有关。
[Abstract]:Background and objectives of the study: Chondrocytes are the main base of articular cartilage, but the regeneration ability of cartilage is poor due to its low differentiation and proliferation, which makes it difficult to repair cartilage naturally after injury. Transplantation of stem cells and chondrocytes from chondrocytes has solved the problem of low proliferation of chondrocytes and has become an effective method for the treatment of cartilage injury. A large number of studies have found that the viability and survival rate of cells cultured in normoxic environment after transplantation into the anoxic environment of articular cavity decreased. Wakitani and other studies suggested that the low survival rate of transplanted cells resulted in the thickness of the cartilage of stem cell differentiation. The mechanical strength was not as good as normal cartilage, so the low survival rate of cell transplantation was considered to be a factor restricting the application of this method. Lv Yuan chlorogenic acid CGA is a polar organic acid of phenylpropanoid, which has strong antioxidant ability to inhibit the apoptosis induced by oxidation emergency. The effect of Lv Yuan on apoptosis induced by changes of reactive oxygen species in anoxic environment is unclear. The aim of this experiment was to induce bone marrow mesenchymal cells to be chondroid cells in 0.1% O2 hypoxia environment, and to observe the effect of protonic acid on chondroid cells in green anoxic environment. Objective: to investigate the effect of chondrocytes on chondrocytes and its possible mechanism. Contents and methods of the study: Rat mesenchymal stem cells (MSCs) were cultured in vitro, and 12 angels were cultured into chondroid cells in DMEM induction medium containing 0.2mg L-1 BMP-2. They were divided into two groups: normal control group (B)) Lv Yuan group with 0.1% O2 anoxic environment group and D acid 0.1% O2 anoxic environment group. Apoptosis was detected by Hoechst33258 fluorescence staining and the expression of Caspase-3 and Bcl-2 genes in different chondrocytes were detected by RT-PCR. Results: Compared with the control group, 0.1 O _ 2 hypoxia environment for 12 hours could significantly induce apoptosis of chondroid cells derived from stem cells. Lv Yuan acid could decrease the apoptosis rate of chondroid cells induced by 0.1% O _ 2 hypoxia and increase the expression of Caspase-3 in the chondroid cells induced by 0.1% O _ 2 hypoxia. Conclusion: Lv Yuan can inhibit the apoptosis of endothelial cells induced by anoxia. The mechanism may be related to reducing the level of reactive oxygen species in the cells and stabilizing the redox state of the cells to protect the mitochondrial membrane potential. Promoting the expression of apoptosis suppressor gene Bcl-2 and inhibiting the expression of Caspase-3 are related.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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