乳酸调控“骨—软骨”组织成分细胞的效应及其机制研究

发布时间：2019-05-15 17:02

【摘要】：骨和软骨损伤是常见的运动系统疾患,组织工程技术构建人工骨和软骨,以及再生医学技术促进骨和软骨再生是其有效治疗方法。p oly(lactic-co-glycolic acid) (PLGA)和poly-L-lactate acid (PLLA)等乳酸基质材料是骨-软骨损伤修复与再生常用的支架材料和药物载体,乳酸是其合成的基质材料也是其体内降解的主要产物。有研究显示PLGA和PLLA的降解产物可影响“骨-软骨”组织功能,但是乳酸在“骨-软骨”组织中的功能尚不清楚。本课题将研究乳酸对软骨细胞(骨关节炎(OA)及正常软骨细胞)以及骨组织细胞(骨髓间充质干细胞,MSC)的作用。研究共分2部分：1乳酸影响软骨细胞稳态的研究以生物材料为支架的组织工程再生技术和自体软骨细胞移植技术是治疗软骨缺损的有效方法,但这两种技术都存在缺陷：PLGA和PLLA等常用生物支架降解后影响软骨功能；软骨细胞体外培养易发生去表型丢失。本实验研究PLGA和PLLA的主要降解产物乳酸对OA软骨细胞的调控作用及其机制,为后期优化用于软骨再生的PLGA和PLLA支架材料设计提供理论支持；以乳酸作用于体外培养的正常软骨细胞,检测其对软骨细胞表型维持的作用,为软骨细胞体外培养表型维持提供新方法。1.1适宜乳酸作用抑制骨关节炎软骨细胞的病理进程我们检测了不同分子量的PLGA和PLLA降解液中乳酸的浓度,并以降解液作用于人软骨细胞,检测其对软骨细胞增殖和基质蛋白基因表达的作用；以不同浓度和时间的乳酸作用于人正常及OA软骨细胞,检测细胞的功能变化；将乳酸分别以脉冲式和一次性模式作用于软骨细胞,优化其最佳作用效果；采用基因芯片筛选并基因敲降方法,验证乳酸调控细胞功能的可能机制。具体研究内容如下：第一部分、PLGA和PLLA降解液对人软骨细胞的作用。主要发现：1)不同分子量PLGA和PLLA降解液对软骨细胞增殖和COL2A1和ACAN的表达作用不同。2)不同分子量P LGA和PLLA降解液中乳酸浓度和pH值不同,分子量越小,乳酸浓度越高,pH值越低。第二部分、乳酸对人软骨细胞的作用。主要发现：乳酸对人软骨细胞的作用呈时间和浓度依赖性,短时高浓度乳酸可促进COL2A1和ACAN的表达；并且短时高浓度乳酸还可抑制ADAMTS5的表达。第三部分、乳酸不同作用模式对软骨细胞的效果。结果发现3天中每天1次,每次8小时的乳酸脉冲式作用模式,相比3天仅给予1次8小时乳酸的一次性作用模式,可显著上调COL2A1的表达。第四部分、乳酸对软骨细胞的作用机制。主要发现：1)通过基因芯片筛查乳酸作用后软骨细胞的基因表达和生物过程变化,发现低氧诱导因子1A (hypoxia inducible factor 1A, HIF1A)下游调控基因差异显著,提示HIF1A参与调控乳酸对软骨细胞的作用。2)通过敲降HIF1A及其家族成员低氧诱导因子2A (hypoxia inducible factor 2A, HIF2A)方法,发现乳酸的作用主要由HIF1A介导而非HIF2A;且发现乳酸调控的HIF1A蛋白表达增强主要是由于上调其基因表达,而非抑制其蛋白降解。上述结果表明,适宜的乳酸作用可通过上调COL2A1和抑制ADAMTS5的表达,促进软骨细胞的稳态；设计用于软骨再生的PLGA和PLLA材料时需考虑降解产物乳酸的影响。1.2适宜乳酸作用促进体外培养的正常软骨细胞的表型维持我们以1.1中筛选的乳酸最佳作用浓度和时间作用于人软骨细胞,检测其对软骨细胞表型维持的作用；从乳酸调控软骨细胞糖酵解代谢角度,阐释乳酸促进表型维持的机制。具体研究内容如下：第一部分、乳酸对软骨细胞表型维持的作用。主要发现：原代软骨细胞传代过程中COL2A1、AcAN和SOX9的表达持续下降,乳酸作用可上调COL2A1、ACAN和SOX9的表达,维持软骨细胞表型。第二部分、乳酸维持软骨细胞表型的机制。主要发现：1)乳酸可上调软骨细胞糖酵解相关基因表达。2)软骨细胞表型与糖酵解相关：低氧和TGF-β3作用可上调糖酵解,传代过程中糖酵解下调。3)抑制糖酵解后,乳酸调控软骨细胞表型的功能下降。上述结果表明,乳酸可通过上调糖酵解促进软骨细胞体外培养过程中的表型维持。2乳酸调控骨髓间充质干细胞的自我更新能力的研究骨髓间充质干细胞是骨组织的主要前体细胞。间充质干细胞的一个显著特征是具有自我更新能力,研究显示细胞代谢可通过不同的代谢中间产物调控干细胞自我更新能力。乳酸是PLGA、PLLA的降解产物,也是细胞无氧酵解代谢的主要终产物,其对干细胞的自我更新能力的调控尚不清楚。本实验研究乳酸对于细胞自我更新能力的作用,并从KDM6B调控糖酵解角度探讨乳酸调控干细胞的自我更新能力的机制,以增加乳酸调控干细胞功能的新认识。具体研究内容如下：第一部分、乳酸对hMSC的增殖与自我更新能力的作用。主要发现：1)乳酸抑制hMSC的增殖。2)低浓度乳酸促进hMSC的自我更新能力。3)乳酸促进hMSC干性基因表达。第二部分、乳酸调控]MSC自我更新能力的机制(乳酸对糖酵解的调控)。主要发现：1)乳酸可上调hMSC糖酵解。2)调控糖酵解可影响hMSC的自我更新能力：上调糖酵解可促进hMSC的自我更新能力,下调糖酵解可抑制hMSC的自我更新能力。3)抑制糖酵解后,乳酸调控hMSC的自我更新能力下降。第三部分、乳酸对糖酵解的调控机制(乳酸对KDM6B的调控)。主要发现：1)乳酸可上调KDM6B的表达。2)KDM6B调控1MSC糖酵解和自我更新能力：KDM6B敲降可下调hMSC糖酵解,KDM6B敲降可抑制hMSC自我更新能力。3)敲降KDM6B后,乳酸调控hMSC的自我更新能力下降。上述结果表明,乳酸可通过KDM6B介导的糖酵解调控hMSC的自我更新能力。
[Abstract]:Bone and cartilage damage is a common system disorder, tissue engineering techniques to construct artificial bone and cartilage, and regenerative medicine to promote bone and cartilage regeneration is an effective method of treatment. The lactic acid matrix material such as p-toly (lactic-co-glycolic acid) (PLGA) and poly-L-lacate acid (PLLA) is a commonly used scaffold material and drug carrier for the repair and regeneration of bone-cartilage damage. Studies have shown that the degradation products of PLGA and PLLA may affect the "bone-cartilage" tissue function, but the function of lactic acid in the "bone-cartilage" tissue is not clear. This subject will study the effect of lactic acid on chondrocytes (OA) and normal cartilage cells) and bone tissue cells (bone marrow mesenchymal stem cells, MSC). The research is divided into two parts:1 lactic acid influences the steady state of the cartilage cell, and the tissue engineering regeneration technology and the autocartilage cell transplantation technology of the biological material as the support are effective methods for treating the cartilage defect, but the two technologies have the defects that: The cartilage function is affected by the degradation of the common biological scaffold, such as PLGA and PLLA, and the in vitro culture of the chondrocytes is susceptible to the loss of the phenotype. The effect and mechanism of lactic acid on OA chondrocytes in PLGA and PLLA were studied in this experiment. The theoretical support was provided for the design of PLGA and PLLA scaffolds for cartilage regeneration in the later stage, and the normal chondrocytes cultured in vitro by lactic acid. it is suitable for lactic acid to inhibit the pathological process of the osteoarthritis cartilage cell, and the lactic acid concentration in the PLGA and the PLLA degradation liquid with different molecular weights is detected, and the degradation liquid acts on the human cartilage cell to detect the effect of the degradation liquid on the proliferation of the chondrocyte and the expression of the basic protein gene, and the lactic acid acting on the human normal and the OA cartilage cells at different concentrations and times is used for detecting the function change of the cells; The lactic acid is applied to the chondrocytes in a pulse mode and a one-time mode respectively, and the optimal action effect thereof is optimized; a gene chip is adopted to screen and the gene knock-down method, and the possible mechanism of the function of the lactic acid regulation and control cell is verified. The specific contents of the study are as follows: The first part, PLGA and PLLA degradation liquid have effect on human chondrocytes. The results were as follows:1) The different molecular weight of PLGA and PLLA degradation solution to the proliferation of chondrocytes and the expression of COL2A1 and ACAN.2) The concentration of lactic acid and the pH value in the different molecular weight of P-LGA and PLLA-degrading liquid were different, the smaller the molecular weight, the higher the lactic acid concentration, and the lower the pH value. The second part, the effect of lactic acid on the human chondrocytes. It is found that the effect of lactic acid on the human chondrocytes is time and concentration dependent, and the short-term high-concentration lactic acid can promote the expression of COL2 A1 and ACAN, and the short-term high-concentration lactic acid can also inhibit the expression of the ADAMTS5. The third part, the effect of the different modes of lactic acid on the chondrocytes. The results show that the expression of COL2A1 can be up-regulated by one-time mode of one-time action of 8-hour lactic acid only once every 8 hours in 3 days. The fourth part is the mechanism of the action of lactic acid on the chondrocytes. It was found that:1) The gene expression and the biological process of the chondrocytes after the lactic acid action were screened by the gene chip, and the difference of the regulatory genes downstream of the hypoxia-inducible factor 1A (HIF1A) was found to be significant. HIF1A was suggested to be involved in the regulation of the effect of lactic acid on the chondrocyte.2) The effect of lactic acid was found to be mainly mediated by HIF1A and not HIF2A by the method of HIF1A and its family member hypoxia inducible factor 2A (HIF2A). And the expression of the HIF1A protein regulated by lactic acid is found to be mainly due to the up-regulation of the gene expression and the non-inhibition of the protein degradation. The results show that the appropriate lactic acid action can promote the steady state of the chondrocytes by up-regulating the expression of COL2 A1 and inhibiting the expression of the ADAMTS5. The effect of the degradation product lactic acid needs to be taken into account when designing the PLGA and PLLA materials for cartilage regeneration. The mechanism of lactic acid to promote the maintenance of the phenotype was explained. The specific contents of the study are as follows: The first part, the effect of lactic acid on the phenotype of the chondrocyte. It was found that the expression of COL2A1, AAN and SOX9 decreased continuously during the passage of primary chondrocytes, and the expression of COL2A1, ACAN and SOX9 could be up-regulated by lactic acid, and the phenotype of chondrocyte was maintained. The second part, the mechanism of the lactic acid maintenance of the chondrocyte phenotype. It is found that 1) lactic acid can increase the expression of the related gene of the glycolysis of the chondrocyte.2) The phenotype of the chondrocyte is related to the glycolysis: the action of hypoxia and TGF-3 can increase the glycolysis, and the glycolysis in the course of the passage is down-regulated.3) After the glycolysis is inhibited, The function of lactic acid to regulate the phenotype of the chondrocyte decreased. The results show that lactic acid can promote the phenotype of the in vitro culture of the chondrocytes by up-regulation of glycolysis. The study of the self-renewal ability of the lactic acid to regulate the self-renewal of the bone marrow-derived mesenchymal stem cells is the main precursor of the bone tissue. A significant feature of the mesenchymal stem cells is the ability to self-update, and the study shows that cell metabolism can regulate the self-renewal capacity of stem cells through different metabolic intermediates. The lactic acid is the degradation product of PLGA and PLLA, and it is the main end product of the anaerobic glycolysis of the cell. The regulation of the self-renewal ability of the stem cells is not clear. In this experiment, the role of lactic acid on the self-renewal ability of the cells was studied, and the mechanism of the self-renewal capacity of the lactic acid-regulating stem cells was discussed from the viewpoint of the control of the glycolysis of the KDM6B, so as to increase the new recognition of the function of the lactic acid to regulate the stem cells. The specific content of the study is as follows: The first part, the effect of lactic acid on the proliferation and self-renewal capacity of hMSCs. 1) The lactic acid inhibits the proliferation of hMSCs.2) The low concentration of lactic acid promotes the self-renewal capacity of hMSCs.3) The lactic acid promotes the expression of hMSCs. The second part, the mechanism of the self-renewal capacity of the MSC (the regulation of the glycolysis of the lactic acid). it is found that 1) lactic acid can increase the glycolysis of hMSCs.2) The regulation of glycolysis can affect the self-renewal ability of hMSCs: up-regulation of glycolysis can promote the self-renewal capacity of hMSCs, and the down-regulation of glycolysis can inhibit the self-renewal capacity of hMSCs.3) After the glycolysis is inhibited, The self-renewal ability of the hMSCs was decreased by lactic acid. The third part, the control mechanism of the lactic acid to glycolysis (the regulation of the lactic acid to the KDM6B). It is found that 1) lactic acid can increase the expression of KDM6B.2) KDM6B regulates the glycolysis and self-renewal capacity of the 1MSC: the KDM6B knock-down can reduce the glycolysis of hMSCs, and the KDM6B knock-down can inhibit the self-renewal capacity of hMSCs.3) After the KDM6B is knocked down, the self-renewal ability of the hMSCs is reduced. The results show that lactic acid can regulate the self-renewal capacity of hMSC through the glycolysis mediated by KDM6B.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R68;R318.08

【相似文献】