【摘要】：背景及目的類(lèi)風(fēng)濕關(guān)節(jié)炎(Rheumatoid arthritis,RA)是一種以對(duì)稱(chēng)性、多關(guān)節(jié)炎為主要表現(xiàn)的慢性全身性自身免疫性疾病,關(guān)節(jié)的慢性炎癥導(dǎo)致軟骨和骨質(zhì)的破壞,進(jìn)而可導(dǎo)致關(guān)節(jié)畸形。關(guān)節(jié)骨質(zhì)破壞是關(guān)節(jié)畸形的重要因素,而破骨細(xì)胞是引起骨破壞的關(guān)鍵細(xì)胞之一。10-羥基喜樹(shù)堿(10-hydroxycamptothecin,10-HCPT)源之于植物珙桐科喜樹(shù)提取物,最先用于腫瘤治療,研究證實(shí)10-HCPT的腫瘤治療作用與DNA拓?fù)洚悩?gòu)酶I(Topoisomerase I,Topo-I)關(guān)系密切,DNA拓?fù)洚悩?gòu)酶類(lèi)是廣泛存在于生物體內(nèi)調(diào)節(jié)DNA空間構(gòu)型的動(dòng)態(tài)變化,并直接參與和/或影響DNA的復(fù)制和轉(zhuǎn)錄。10-HCPT通過(guò)氫鍵和分子之間的疏水作用與Topo-I和DNA的化合物結(jié)合時(shí),形成相對(duì)穩(wěn)定的三元復(fù)合物,由此可以抑制Topo-I的活性,阻斷DNA的復(fù)制,最后導(dǎo)致細(xì)胞凋亡。而RA具有類(lèi)似于“局部惡性腫瘤”的增生性和破壞性的特點(diǎn),且我們前期研究證實(shí)10-HCPT能夠抑制類(lèi)風(fēng)濕關(guān)節(jié)炎患者外周血Th17細(xì)胞的功能和成纖維樣滑膜細(xì)胞的增殖,但目前尚無(wú)文獻(xiàn)報(bào)道10-HCPT是否對(duì)致關(guān)節(jié)畸形的破骨細(xì)胞具有抑制作用。RAW264.7細(xì)胞是小鼠單核巨噬細(xì)胞,與核因子κB配體(Receptor activator for nuclear factor-κB ligand,RANKL)和巨噬細(xì)胞集落刺激因子(Macrophage colony stimulating factor,M-CSF)共誘導(dǎo)培養(yǎng),可分化為破骨細(xì)胞,是體外研究破骨細(xì)胞的一個(gè)經(jīng)典方法。本實(shí)驗(yàn)通過(guò)體外培養(yǎng)RAW264.7細(xì)胞與RANKL和M-CSF共培養(yǎng),添加不同溶度的10-HCPT,研究10-HCPT對(duì)RAW264.7細(xì)胞分化為破骨細(xì)胞的影響。方法1、細(xì)胞培養(yǎng):體外培養(yǎng)RAW264.7細(xì)胞,與RANKL和M-CSF共誘導(dǎo)培養(yǎng)。2、實(shí)驗(yàn)分組:分別用不同濃度的10-HCPT處理細(xì)胞,空白對(duì)照組僅RAW264.7細(xì)胞。3、CCK-8法:設(shè)置不同濃度梯度10-HCPT處理細(xì)胞,檢測(cè)細(xì)胞活性。4、TRAP染色:根據(jù)TRAP染色試劑盒說(shuō)明書(shū)操作,將細(xì)胞固定和染色。計(jì)數(shù)TRAP陽(yáng)性破骨細(xì)胞。5、實(shí)時(shí)熒光定量PCR:檢測(cè)不同濃度10-HCPT處理下破骨細(xì)胞相關(guān)標(biāo)志基因CTSK、TRAP和MMP-9 mRNA的表達(dá)。6.、統(tǒng)計(jì)學(xué)分析:采用SPSS 20.0軟件及GraphPad prism 5進(jìn)行統(tǒng)計(jì)分析和圖表生成。結(jié)果1、10-HCPT對(duì)細(xì)胞活性的影響:10-HCPT濃度分別為1ng/ml、2ng/ml和5ng/ml與不加10-HCPT的細(xì)胞活性相比無(wú)明顯差異(P0.05)。2、10-HCPT對(duì)RAW246.7細(xì)胞形成破骨細(xì)胞的影響:添加RANKL和M-CSF的對(duì)照組和實(shí)驗(yàn)組,可見(jiàn)大的多核破骨細(xì)胞。加入不同濃度10-HCPT(1、2、5ng/ml)后隨著藥物濃度增高,破骨細(xì)胞數(shù)目明顯減少(P0.05)。3、實(shí)時(shí)定量熒光PCR結(jié)果示:10-HCPT能顯著降低TRAP、CTSK和MMP-9基因的表達(dá),與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。且隨著濃度的增加,這種抑制效果更加明顯。結(jié)論10-HCPT可以減少破骨細(xì)胞標(biāo)志性基因的表達(dá)及破骨細(xì)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R593.22

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