10-羟基喜树碱对RAW264.7细胞分化为破骨细胞的影响
发布时间：2019-01-22 15:51
【摘要】：背景及目的类风湿关节炎(Rheumatoid arthritis,RA)是一种以对称性、多关节炎为主要表现的慢性全身性自身免疫性疾病,关节的慢性炎症导致软骨和骨质的破坏,进而可导致关节畸形。关节骨质破坏是关节畸形的重要因素,而破骨细胞是引起骨破坏的关键细胞之一。10-羟基喜树碱(10-hydroxycamptothecin,10-HCPT)源之于植物珙桐科喜树提取物,最先用于肿瘤治疗,研究证实10-HCPT的肿瘤治疗作用与DNA拓扑异构酶I(Topoisomerase I,Topo-I)关系密切,DNA拓扑异构酶类是广泛存在于生物体内调节DNA空间构型的动态变化,并直接参与和/或影响DNA的复制和转录。10-HCPT通过氢键和分子之间的疏水作用与Topo-I和DNA的化合物结合时,形成相对稳定的三元复合物,由此可以抑制Topo-I的活性,阻断DNA的复制,最后导致细胞凋亡。而RA具有类似于“局部恶性肿瘤”的增生性和破坏性的特点,且我们前期研究证实10-HCPT能够抑制类风湿关节炎患者外周血Th17细胞的功能和成纤维样滑膜细胞的增殖,但目前尚无文献报道10-HCPT是否对致关节畸形的破骨细胞具有抑制作用。RAW264.7细胞是小鼠单核巨噬细胞,与核因子κB配体(Receptor activator for nuclear factor-κB ligand,RANKL)和巨噬细胞集落刺激因子(Macrophage colony stimulating factor,M-CSF)共诱导培养,可分化为破骨细胞,是体外研究破骨细胞的一个经典方法。本实验通过体外培养RAW264.7细胞与RANKL和M-CSF共培养,添加不同溶度的10-HCPT,研究10-HCPT对RAW264.7细胞分化为破骨细胞的影响。方法1、细胞培养:体外培养RAW264.7细胞,与RANKL和M-CSF共诱导培养。2、实验分组:分别用不同浓度的10-HCPT处理细胞,空白对照组仅RAW264.7细胞。3、CCK-8法:设置不同浓度梯度10-HCPT处理细胞,检测细胞活性。4、TRAP染色:根据TRAP染色试剂盒说明书操作,将细胞固定和染色。计数TRAP阳性破骨细胞。5、实时荧光定量PCR:检测不同浓度10-HCPT处理下破骨细胞相关标志基因CTSK、TRAP和MMP-9 mRNA的表达。6.、统计学分析:采用SPSS 20.0软件及GraphPad prism 5进行统计分析和图表生成。结果1、10-HCPT对细胞活性的影响:10-HCPT浓度分别为1ng/ml、2ng/ml和5ng/ml与不加10-HCPT的细胞活性相比无明显差异(P0.05)。2、10-HCPT对RAW246.7细胞形成破骨细胞的影响:添加RANKL和M-CSF的对照组和实验组,可见大的多核破骨细胞。加入不同浓度10-HCPT(1、2、5ng/ml)后随着药物浓度增高,破骨细胞数目明显减少(P0.05)。3、实时定量荧光PCR结果示:10-HCPT能显著降低TRAP、CTSK和MMP-9基因的表达,与对照组相比差异有统计学意义(P0.05)。且随着浓度的增加,这种抑制效果更加明显。结论10-HCPT可以减少破骨细胞标志性基因的表达及破骨细胞的形成。
[Abstract]:Background and objective Rheumatoid arthritis (Rheumatoid arthritis,RA) is a chronic systemic autoimmune disease characterized by symmetry and polyarthritis. Chronic inflammation of joints leads to the destruction of cartilage and bone which can lead to joint deformities. Bone destruction is an important factor of joint deformity, and osteoclast is one of the key cells to cause bone destruction. 10-hydroxycamptothecin 10-HCPT was first used in tumor therapy for Camptotheca involucrata. It has been confirmed that the tumor therapy of 10-HCPT is closely related to the DNA topoisomerase I (Topoisomerase I Topo-I, and that DNA topoisomerase is widely present in organisms to regulate the dynamic changes of DNA spatial configuration. And directly participate in and / or affect the replication and transcription of DNA. When 10-HCPT binds to the compounds of Topo-I and DNA through hydrogen bonding and intermolecular hydrophobic interaction, it forms a relatively stable ternary complex, which can inhibit the activity of Topo-I. Blocking the replication of DNA leads to apoptosis. However, RA has the characteristics of proliferative and destructive similar to "local malignant tumor", and our previous studies have demonstrated that 10-HCPT can inhibit the function of Th17 cells and the proliferation of fibroblast synoviocytes in patients with rheumatoid arthritis. However, it has not been reported that 10-HCPT has inhibitory effect on osteoclasts causing joint malformation. RAW264.7 cells are mouse mononuclear macrophages and nuclear factor- 魏 B ligand (Receptor activator for nuclear factor- 魏 B ligand,. RANKL) and macrophage colony stimulating factor (Macrophage colony stimulating factor,M-CSF) co-culture can differentiate into osteoclasts, which is a classical method to study osteoclasts in vitro. The effect of 10-HCPT on the differentiation of RAW264.7 cells into osteoclasts was studied by co-culture of RAW264.7 cells with RANKL and M-CSF in vitro and adding 10-HCPTwith different solubility. Methods 1. Cell culture: RAW264.7 cells were cultured in vitro and co-induced with RANKL and M-CSF. The cells were treated with different concentrations of 10-HCPT, and only RAW264.7 cells were treated in blank control group. CCK-8 method: the cells were treated with different concentration gradient 10-HCPT, and the cell activity was detected. 4The cells were fixed and stained according to the instructions of TRAP staining kit. TRAP positive osteoclasts were counted. 5. Real-time quantitative PCR: was used to detect the expression of osteoclast related marker genes CTSK,TRAP and MMP-9 mRNA under different concentrations of 10-HCPT. Statistical analysis: SPSS 20.0 software and GraphPad prism 5 were used for statistical analysis and chart generation. Results 1the effect of 10-HCPT on cell activity: the concentration of 10-HCPT was 1ng / ml, There was no significant difference in the activity of 2ng/ml and 5ng/ml compared with those without 10-HCPT (P0.05). 2The effect of 10-HCPT on the formation of osteoclasts in RAW246.7 cells: in the control group and experimental group with RANKL and M-CSF, large polynuclear osteoclasts were found. With the increase of drug concentration, the number of osteoclasts decreased significantly (P0.05). The results of real-time quantitative fluorescence PCR showed that 10-HCPT could significantly decrease the expression of TRAP,CTSK and MMP-9 genes. Compared with the control group, the difference was statistically significant (P0.05). And with the increase of concentration, the inhibition effect is more obvious. Conclusion 10-HCPT can reduce the expression of osteoclasts and the formation of osteoclasts.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22
[Abstract]:Background and objective Rheumatoid arthritis (Rheumatoid arthritis,RA) is a chronic systemic autoimmune disease characterized by symmetry and polyarthritis. Chronic inflammation of joints leads to the destruction of cartilage and bone which can lead to joint deformities. Bone destruction is an important factor of joint deformity, and osteoclast is one of the key cells to cause bone destruction. 10-hydroxycamptothecin 10-HCPT was first used in tumor therapy for Camptotheca involucrata. It has been confirmed that the tumor therapy of 10-HCPT is closely related to the DNA topoisomerase I (Topoisomerase I Topo-I, and that DNA topoisomerase is widely present in organisms to regulate the dynamic changes of DNA spatial configuration. And directly participate in and / or affect the replication and transcription of DNA. When 10-HCPT binds to the compounds of Topo-I and DNA through hydrogen bonding and intermolecular hydrophobic interaction, it forms a relatively stable ternary complex, which can inhibit the activity of Topo-I. Blocking the replication of DNA leads to apoptosis. However, RA has the characteristics of proliferative and destructive similar to "local malignant tumor", and our previous studies have demonstrated that 10-HCPT can inhibit the function of Th17 cells and the proliferation of fibroblast synoviocytes in patients with rheumatoid arthritis. However, it has not been reported that 10-HCPT has inhibitory effect on osteoclasts causing joint malformation. RAW264.7 cells are mouse mononuclear macrophages and nuclear factor- 魏 B ligand (Receptor activator for nuclear factor- 魏 B ligand,. RANKL) and macrophage colony stimulating factor (Macrophage colony stimulating factor,M-CSF) co-culture can differentiate into osteoclasts, which is a classical method to study osteoclasts in vitro. The effect of 10-HCPT on the differentiation of RAW264.7 cells into osteoclasts was studied by co-culture of RAW264.7 cells with RANKL and M-CSF in vitro and adding 10-HCPTwith different solubility. Methods 1. Cell culture: RAW264.7 cells were cultured in vitro and co-induced with RANKL and M-CSF. The cells were treated with different concentrations of 10-HCPT, and only RAW264.7 cells were treated in blank control group. CCK-8 method: the cells were treated with different concentration gradient 10-HCPT, and the cell activity was detected. 4The cells were fixed and stained according to the instructions of TRAP staining kit. TRAP positive osteoclasts were counted. 5. Real-time quantitative PCR: was used to detect the expression of osteoclast related marker genes CTSK,TRAP and MMP-9 mRNA under different concentrations of 10-HCPT. Statistical analysis: SPSS 20.0 software and GraphPad prism 5 were used for statistical analysis and chart generation. Results 1the effect of 10-HCPT on cell activity: the concentration of 10-HCPT was 1ng / ml, There was no significant difference in the activity of 2ng/ml and 5ng/ml compared with those without 10-HCPT (P0.05). 2The effect of 10-HCPT on the formation of osteoclasts in RAW246.7 cells: in the control group and experimental group with RANKL and M-CSF, large polynuclear osteoclasts were found. With the increase of drug concentration, the number of osteoclasts decreased significantly (P0.05). The results of real-time quantitative fluorescence PCR showed that 10-HCPT could significantly decrease the expression of TRAP,CTSK and MMP-9 genes. Compared with the control group, the difference was statistically significant (P0.05). And with the increase of concentration, the inhibition effect is more obvious. Conclusion 10-HCPT can reduce the expression of osteoclasts and the formation of osteoclasts.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22
【参考文献】
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1 任丽丽;鲁晓娟;寿记新;王燕;孙晓麟;张丽娟;张o，
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