内质网应激介导棕榈酸诱导的成骨细胞凋亡

发布时间：2018-06-27 05:40

本文选题：成骨细胞 + 凋亡　；参考：《郑州大学》2015年硕士论文

【摘要】：背景:骨质疏松(Osteoporosis,OP)及与其相关的骨折的发病率和死亡率在世界范围内不断上升,已经成为威胁公众健康的重大问题。越来越多的研究发现,肥胖与骨质疏松的发生发展密切相关,可能是导致骨质疏松的另一危险因素。肥胖患者血清游离脂肪酸水平普遍增高,并且可以对包括成骨细胞在内的多种细胞产生脂毒性作用。成骨细胞是骨代谢的基础,其工作状态和相对数量的改变都可能引起骨代谢异常并最终进展为骨质疏松。因此,体内高脂环境引起的成骨细胞凋亡增多可能是肥胖患者发生骨质疏松的重要机制。棕榈酸为一种动植体内常见的高级饱和脂肪酸,已有研究发现,棕榈酸可以通过其脂毒性诱导成骨细胞凋亡,然而,其具体机制目前尚不明确。内质网应激(endoplasmic reticulum stress,ERS)可以通过CHOP途径、Caspase-12途径和JNK途径参与细胞凋亡进程,这些通路的激活可以通过下游一系列反应最终导致细胞凋亡的发生。因此,我们推测高脂环境,即棕榈酸的干预,可能通过内质网应激途径诱导成骨细胞凋亡从而导致骨质疏松的发生。为证实这一设想,我们拟建立棕榈酸诱导成骨细胞凋亡的模型,同时应用内质网应激抑制剂4-苯基丁酸(4-phenylbutyric acid,4-PBA)预处理细胞,进而探讨内质网应激在此进程中的作用。目的:建立棕榈酸诱导成骨细胞凋亡的模型,探讨内质网应激在棕榈酸诱导成骨细胞凋亡中的作用,为骨质疏松的治疗提供新的思路。方法:用含不同浓度棕榈酸(0,100,250,500μmol/L)的完全培养基分别孵育成骨细胞24h;选择棕榈酸敏感浓度500μmol/L孵育成骨细胞不同时间(0,6,12,24 h);用含500μmol/L棕榈酸的完全培养基干预成骨细胞24 h并应用内质网应激抑制剂4-PBA进一步验证棕榈酸对成骨细胞增殖和凋亡的影响。CCK-8法检测细胞增殖活力;Annexin V/PI双染色法流式细胞术检测细胞凋亡率;Real-time quantitative PCR检测各组细胞内质网应激相关基因GRP78、CHOP、Caspase-12m RNA的表达。结果:1、与对照组相比,100μmol/L棕榈酸干预成骨细胞24 h对其增殖率无明显影响(P0.05),250μmol/L(P0.05)和500μmol/L(P0.01)浓度组成骨细胞增殖率均下降,且呈剂量依赖性。用500μmol/L的棕榈酸干预6,12,24 h后,细细胞增殖率明显降低且呈时间依赖性(P0.01)。与500μmol/L棕榈酸组相比,用4-PBA预处理细胞后再加入500μmol/L的棕榈酸干预细胞24 h可在一定程度上提高细胞的增殖活性,差异有统计学意义(P0.05)。2、与对照组相比,100μmol/L棕榈酸干预组细胞凋亡率无明显差异(P0.05),而250μmol/L和500μmol/L棕榈酸干预成骨细胞24 h可明显增加细胞凋亡率(P0.01)且呈剂量依赖性。用500μmol/L的棕榈酸干预成骨细胞6 h(P0.05),12 h(P0.01)和24 h(P0.01)可显著升高细胞凋亡率,且呈时间依赖性。与500μmol/L棕榈酸组相比,用4-PBA预处理细胞后再加入500μmol/L的棕榈酸干预细胞24 h可在一定程度上降低细胞的凋亡率,差异有统计学意义(P0.01)。3、与对照组相比,用250μmol/L和500μmol/L棕榈酸干预成骨细胞24 h后,CHOP和GRP78 m RNA的表达水平呈剂量依赖性增高(P0.01),而Caspase-12 m RNA的表达水平则有所降低(P0.05),100μmol/L浓度组各基因表达量无明显差异(P0.05)。500μmol/L棕榈酸分别处理细胞6,12,24 h后,CHOP的表达水平呈时间依赖性增高(P0.01),GRP78 m RNA的表达水平在12 h开始升高(P0.05),24 h达到峰值(P0.01),Caspase-12 m RNA的表达水平则在12 h达到峰值(P0.05),24 h后开始下降(P0.01)。用4-PBA预处理细胞则可在一定程度上逆转棕榈酸的以上作用结果,差异有统计学意义(P0.01)。结论:1、棕榈酸可能通过内质网应激途径诱导成骨细胞凋亡。2、4-PBA可以通过抑制内质网应激在一定程度上减少棕榈酸诱导的成骨细胞凋亡。
[Abstract]:Background: the incidence and mortality of Osteoporosis (OP) and its associated fractures are rising worldwide and have become a major threat to public health. More and more studies have found that obesity is closely related to the development of osteoporosis and can be another risk factor for osteoporosis. Obese patients. The levels of serum free fatty acids are generally higher and can produce lipid toxic effects on a variety of cells, including osteoblasts. Osteoblasts are the basis of bone metabolism. Their working state and relative changes may cause abnormal bone metabolism and eventually progress to osteoporosis. Therefore, the osteoblasts caused by high fat environment in the body are withered. The increase in death may be an important mechanism for the occurrence of osteoporosis in obese patients. Palmitic acid is a common high saturated fatty acid in the plant. It has been found that palmitic acid can induce apoptosis of osteoblasts through its lipid toxicity. However, its specific mechanism is not yet clear. Endoplasmic reticulum stress (ERS) can be a specific mechanism. Through the CHOP pathway, the Caspase-12 pathway and the JNK pathway participate in the apoptosis process. The activation of these pathways may eventually lead to apoptosis through a series of downstream reactions. Therefore, we speculate that the high fat environment, that is, palmitic acid, may induce osteoblast apoptosis through endoplasmic reticulum stress pathway and lead to osteoporosis. To confirm this idea, we intend to establish a model of palmitic acid induced osteoblast apoptosis, and the use of endoplasmic reticulum stress inhibitor 4- phenylene butyric acid (4-phenylbutyric acid, 4-PBA) preconditioning cells, and then explore the role of endoplasmic reticulum stress in this process. The role of net stress in the apoptosis of osteoblast induced by palmitic acid provides a new way of thinking for the treatment of osteoporosis. Methods: the osteocyte 24h was incubated with the complete medium containing different concentrations of palmitic acid (0100250500 mol/L); the osteocytes were incubated for different time (0,6,12,24 h) with the sensitive concentration of palmitic acid (0,6,12,24 h); with 500 micron mol/ The total culture of L palmitic acid was 24 h and the endoplasmic reticulum stress inhibitor 4-PBA was used to further verify the effect of palmitic acid on the proliferation and apoptosis of osteoblasts by.CCK-8 method; Annexin V/PI double staining flow cytometry was used to detect the apoptosis rate; Real-time quantitative PCR detected the cell endoplasmic reticulum in each group. The expression of stress related genes GRP78, CHOP, and Caspase-12m RNA. Results: 1, compared with the control group, the proliferation rate of osteoblasts was not significantly affected by the intervention of 100 mu mol/L palmitic acid in osteoblasts (P0.05), 250 mu mol/L (P0.05) and 500 micron mol/L (P0.01) concentration were decreased in a dose-dependent manner. 500 micron mol/L palmitic acid was used to intervene. After h, the proliferation rate of fine cells decreased significantly and was time dependent (P0.01). Compared with 500 mol/L palmitic acid group, the proliferation activity of cells was improved to a certain extent by adding 500 mu of palmitic acid to 24 h with 4-PBA pretreated cells, and the difference was statistically significant (P0.05).2, compared with the control group, the 100 - mol/L palmitic acid intervention group There was no significant difference in apoptosis rate (P0.05), while the intervention of 250 mu mol/L and 500 mol/L palmitic acid in osteoblasts with 24 h significantly increased the apoptosis rate (P0.01) and was dose-dependent. 500 mu of palmitic acid was used to interfere with osteoblasts 6 h (P0.05), 12 h (P0.01) and 24 h (P0.01) could significantly increase the cell apoptosis rate, and it was time dependent. And 500 micron. Compared with the palmitic acid group, the cell apoptosis rate was reduced to a certain extent by adding 500 mol/L palmitic acid to the cells after 4-PBA pretreatment of the cells, and the difference was statistically significant (P0.01).3. Compared with the control group, the expression level of CHOP and GRP78 m RNA was in a dose dependent manner after the intervention of 250 mu mol/L and 500 mu mol/L palmitate in the osteoblast 24 h. The expression level of Caspase-12 m RNA decreased (P0.05), and there was no significant difference in gene expression in 100 mol/L concentration group (P0.05).500 u mol/L palmitic acid treated cells 6,12,24 h, the expression level of CHOP was increased in time dependent, and the level of expression of CHOP was increased at the beginning of 12. 24 At the peak (P0.01), the expression level of Caspase-12 m RNA reached a peak (P0.05) at 12 h (P0.05) and decreased after 24 h (P0.01). The results were reversed to a certain extent by using 4-PBA pretreated cells. The difference was statistically significant (P0.01). Conclusion: 1, palmitic acid may induce osteoblast apoptosis.2 by endoplasmic reticulum stress pathway. 4-PBA can reduce the apoptosis of palmitic acid induced osteoblasts to some extent by inhibiting endoplasmic reticulum stress.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580

【相似文献】