抑制DDR2策略在异位骨化和类风湿性关节炎小鼠动物模型中的应用

发布时间：2018-05-30 15:43

本文选题：异位骨化 + 类风湿性关节炎　；参考：《第四军医大学》2016年博士论文

【摘要】：盘状结构受体2(DDR2)是一种受体型蛋白酪氨酸激酶,DDR2调控的相关信号在机体的生长发育以及肿瘤、纤维化、动脉粥样硬化、骨性关节炎(OA)和类风湿性关节炎(RA)等许多疾病的发病过程中起重要作用,因此DDR2成为了治疗相关疾病潜在的靶分子。在本实验中,我们运用抑制DDR2的策略,分别研究GD856和达沙替尼两种DDR2小分子抑制剂对异位骨化和胶原诱导关节炎小鼠动物模型的影响。实验一DDR2缺失对小鼠骨形成的影响DDR2可以促进软骨细胞的增殖和分化、成骨细胞的分化,并能够抑制破骨细胞的分化,它在骨形成的作用中发挥重要作用。虽然在细胞层面对DDR2的骨形成作用有较为广泛的探索,但是在体内实验中仍缺乏对DDR2骨形成作用较为系统的研究。本实验通过比较DDR2缺失小鼠和野生型小鼠的骨骼生长发育以及骨折后、异位骨化后的骨形成情况,研究DDR2在体内对骨形成的作用。结果显示DDR2缺失小鼠骨骼发育显著异常,机体以及骨骼长度显著小于野生型小鼠,通过microCT重建股骨中部皮质和股骨远端骨小梁后可以发现,DDR2缺失小鼠骨皮质的厚度和骨小梁的相关骨形成参数显著低于野生型小鼠,同时股骨的三点弯曲实验表明ddr2缺失小鼠股骨所能承受的最大力和股骨刚度显著小于野生型小鼠,双钙黄绿素荧光实验显示ddr2缺失小鼠的骨代谢速度显著低于野生型小鼠。我们通过手术建立了小鼠开放式骨折模型,骨折后ddr2缺失小鼠的骨痂形成能力显著降低,骨折愈合能力显著减弱。我们对ddr2缺失小鼠和野生型小鼠建立了创伤-烧伤异位骨化模型,建模10周后通过microct扫描重建发现ddr2缺失小鼠的异位骨化能力显著低于野生型小鼠。本实验结果表明,ddr2缺失后小鼠的骨骼生长发育、机体的骨代谢、以及骨折和异位骨化后的骨形成能力受到显著抑制。实验二ddr2小分子抑制剂gd856对小鼠异位骨化的影响gd856是一种新研制的ddr2小分子抑制剂,能够在较低的浓度下对胶原诱导的ddr2磷酸化有较显著的抑制作用。我们通过诱导成骨细胞系mc-3t3-e1分化以及建立小鼠异位骨化模型,研究gd856对小鼠异位骨化的影响。我们将成骨细胞系分为未加药的对照组、10μm的低剂量组和50μm的高剂量组,在向成骨细胞诱导的同时加gd856药物刺激,三周后对细胞进行茜素红染色、碱性磷酸酶(alp)染色和成骨相关分子的mrna测定。结果表明低剂量组细胞的茜素红阳性强度略低于对照组,而高剂量组的阳性强度则显著低于对照组和低剂量组。低剂量组细胞的alp阳性强度显著强于对照组,而高剂量组的alp阳性强度则显著低于对照组和低剂量组。低浓度组和高剂量组opn、ocn和bmp2mrna的表达均显著少于对照组,高剂量组alp的mrna表达显著低于对照组,而低浓度组alp和colia1以及高剂量组runx2在mrna水平的表达则显著高于对照组。为了在体内验证gd856对小鼠异位骨化的影响,我们建立了创伤-烧伤异位骨化模型,小鼠分为对照组和给药组,将给药组小鼠的饮水为浓度为0.08mg/ml的gd856水溶液。建模10周后我们发现给药组小鼠的骨代谢速度显著减慢,根骨的异位骨化严重程度显著减轻。本实验结果表明gd856可以抑制成骨细胞的分化,并能够减缓体内的骨代谢速度,抑制异位骨化的发生。实验三DDR2小分子抑制剂达沙替尼对小鼠胶原诱导关节炎的影响达沙替尼是一种第二代酪氨酸激酶抑制剂,对DDR2活性有较好的抑制作用。达沙替尼能够抑制RA发病过程中的多个关键基因,本实验通过建立胶原诱导关节炎的小鼠模型,研究达沙替尼对胶原诱导关节炎(CIA)小鼠的治疗作用。我们通过异种胶原二次免疫的方法建立小鼠的关节炎模型,在建模成功后将小鼠分为给药组和对照组分别给予达沙替尼和安慰剂灌胃。结果表明给药组小鼠的体重显著大于对照组,同时给药组小鼠四爪的肿胀程度以及关节炎的严重程度都显著低于对照组。通过microCT对小鼠的关节周围骨组织扫面重建我们发现,给药组小鼠关节周围以及骨小梁的骨质破坏程度显著轻于对照组。小鼠踝关节周围组织的组织学染色结果显示给药组小鼠关节内的滑膜细胞以及炎性细胞数量显著减少,关节软骨和骨质的破坏以及血管翳的形成被显著抑制。本实验结果表明达沙替尼可以减轻CIA发病的严重程度,缓解CIA的关节破坏程度,对小鼠胶原诱导关节炎模型有较好的治疗作用。实验四DDR2小分子抑制剂达沙替尼对类风湿性关节炎滑膜细胞的作用成纤维样滑膜细胞(FLS)在RA的发病过程中对关节的破坏起重要作用,也是治疗RA的一个重要靶细胞。本实验通过原代培养RA患者的关节FLS,研究达沙替尼对RA FLS的作用,探索达沙替尼治疗RA的可能机制。我们通过BRDU免疫荧光染色研究达沙替尼对RA FLS增殖能力的影响,结果表明经达沙替尼刺激24小时后的FLS的BRDU阳性细胞数量显著减少,这表明FLS的增殖能力显著受到抑制。通过细胞划痕实验和Transwell小室迁移实验判断FLS迁移能力的改变,结果表明达沙替尼刺激24小时后FLS的迁移能力被显著抑制。用流失细胞仪检测FLS的凋亡能力我们发现在达沙替尼刺激的第3天和第5天凋亡的细胞数量显著增多。运用RT-PCR我们发现达沙替尼刺激24小时后,FLS中VEGF、FGF、MMP13和DKK1在mRNA水平的表达显著降低。本实验结果表明,达沙替尼可以抑制RA FLS的增殖和迁移,同时能够诱导FLS的凋亡,且可以在mRNA水平抑制VEGF、FGF、MMP13和DKK1的表达。这可能是达沙替尼治疗Ra的重要机制,表明FLS可能是达沙替尼治疗RA的重要靶细胞。
[Abstract]:Discoid receptor 2 (DDR2) is a type of protein tyrosine kinase, and the related signal regulated by DDR2 plays an important role in the growth and development of the body, as well as in the pathogenesis of many diseases such as tumor, fibrosis, atherosclerosis, osteoarthritis (OA) and rheumatoid arthritis (RA). Therefore, DDR2 has been the potential for the treatment of related diseases. Target molecules. In this experiment, we used the strategy of inhibiting DDR2 to study the effects of two DDR2 small molecule inhibitors of GD856 and dasatinib on heterotopic ossification and collagen induced arthritis in mice. The effect of DDR2 deletion on the formation of bone in mice DDR2 could promote the proliferation and differentiation of bone cells and the differentiation of osteoblasts. It can inhibit the differentiation of osteoclasts and play an important role in the role of bone formation. Although the osteogenesis of DDR2 is widely explored in the cell layer, there is still a lack of systematic study on the osteogenesis of DDR2 in vivo. This experiment compares the skeletal growth of DDR2 deficient mice and wild type mice. The formation of bone after long development and fracture, after ectopic ossification, studies the role of DDR2 in bone formation in the body. The results show that the skeletal development of DDR2 deficient mice is significantly abnormal, and the length of the body and bone is significantly smaller than that of the wild type mice. After the reconstruction of the middle femur cortex and the distal femur trabecula by microCT, the DDR2 deletion mice can be found. The thickness of the cortical bone and the bone formation parameters of the trabecular bone were significantly lower than that of the wild type mice. At the same time, the three point bending test of the femur showed that the maximum strength and the femur stiffness of the femur of the DDR2 deficient mice were significantly lower than that of the wild type mice. The fluorescence test of the DDR2 deficient mice showed that the bone metabolism rate of the lost mice was significantly lower than that of the wild type. Mice. We established the open fracture model of mice by operation. The callus formation ability of DDR2 deficient mice was significantly reduced and the fracture healing ability decreased significantly. We established the model of trauma and burn ectopic ossification in DDR2 deficient mice and wild type mice. After modeling for 10 weeks, the DDR2 deletion mice were found through the microCT scan reconstruction. The ability of heterotopic ossification was significantly lower than that of wild type mice. The results of this experiment showed that the bone growth and development, bone metabolism, and bone formation ability after fracture and ectopic ossification were significantly inhibited after DDR2 deletion. Experiment two DDR2 small molecule inhibitor gd856 on the ectopic ossification of mice gd856 was a newly developed DDR2 Molecular inhibitors can significantly inhibit collagen induced DDR2 phosphorylation at a lower concentration. We study the effect of gd856 on heterotopic ossification in mice by inducing mc-3t3-e1 differentiation in osteoblasts and the model of heterotopic ossification in mice. We divide the osteoblast lines into an unmedicated control group, with a low dose of 10 mu m. Group and 50 m high dose group were stimulated with gd856 drugs at the same time as osteoblasts. After three weeks, the cells were stained with alizarin red, alkaline phosphatase (ALP) staining and mRNA determination of bone related molecules. The results showed that the positive intensity of alizarin red in the low dose group was slightly lower than that of the control group, while the positive intensity of the high dose group was significantly lower. In the control group and the low dose group, the ALP positive intensity of the cells in the low dose group was significantly stronger than the control group, while the ALP positive intensity of the high dose group was significantly lower than the control group and the low dose group. The expression of OPN, OCN and BMP2mRNA in the low concentration group and the high dose group were significantly less than those in the control group. The mRNA expression of ALP in the high dose group was significantly lower than that in the control group, and the low concentration was lower than the control group, but the low concentration group was lower than the control group. In order to verify the effect of gd856 on heterotopic ossification in mice, the expression of ALP and COLIA1 in the degree group and the high dose group Runx2 was significantly higher than that of the control group. In order to verify the effect of gd856 on heterotopic ossification in mice, we established a model of ectopic ossification of trauma and burn. The mice were divided into control and drug groups, and the drinking water in the mice was gd856 solution of 0.08mg/ml concentration of 0.08mg/ml. Modeling 1 0 weeks later, we found that the rate of bone metabolism in the mice was significantly slowed down, and the severity of heterotopic ossification in the root bone was significantly reduced. The results showed that gd856 could inhibit the differentiation of osteoblast, slow down the bone metabolism in the body and inhibit the occurrence of heterotopic ossification. Experiment three DDR2 small molecule inhibitor dasitinib on mouse collagen The effect of dasatinib, a second generation tyrosine kinase inhibitor, has a good inhibitory effect on DDR2 activity. Dasinii can inhibit multiple key genes in the pathogenesis of RA. By establishing a mouse model of collagen induced arthritis, dasatinib was used to study the treatment of dasatinib on collagen induced arthritis (CIA) mice. After the modeling was successful, the mice were divided into two groups: davatinib and placebo. The results showed that the body weight of the mice was significantly greater than that of the control group, and the degree of swelling of the four paws and arthritis in the mice was also given. The severity of the scavenging of the bone around the joints of the mice was significantly lower than that of the control group. We found that the bone destruction around the joints and trabecular bone in the mice was significantly lighter than that of the control group. The histological staining results of the tissue around the ankle in mice showed that the synovial cells in the joint of the mice were given to the synovial cells in the drug group as well as in the microCT mice. The number of inflammatory cells decreased significantly, the destruction of articular cartilage and bone and the formation of pannus were significantly inhibited. The results of this experiment showed that satinib could reduce the severity of CIA, alleviate the joint destruction of CIA, and have a good therapeutic effect on the model of collagen induced arthritis in mice. Experiment four DDR2 small molecule inhibitors The effect of satinib on synovial cells of rheumatoid arthritis (FLS) plays an important role in the destruction of the joint during the pathogenesis of RA. It is also an important target cell for the treatment of RA. In this experiment, the effect of dasatinib on RA FLS was studied by the joint FLS of the primary culture of RA patients and the possibility of dasatinib in the treatment of RA was explored. Mechanism. We studied the effect of dasatinib on the proliferation of RA FLS by BRDU immunofluorescence staining. The results showed that the number of BRDU positive cells in FLS decreased significantly after 24 hours of dasatinib stimulation, indicating that the proliferation ability of FLS was significantly inhibited. The migration ability of FLS through the cell scratch test and the Transwell chamber migration test was used to determine the migration ability of FLS. The results showed that the migration ability of FLS was significantly suppressed after 24 hours of ddacatinib stimulation. The apoptotic capacity of FLS was detected by the flow cytometry. We found that the number of apoptotic cells in the third and fifth days of dasatinib increased significantly. Using RT-PCR we found that after 24 hours of dasatinib stimulation, VEGF, FGF, MMP13 and DKK1 in FLS were in M. The expression of RNA level was significantly reduced. The results of this experiment showed that dasatinib could inhibit the proliferation and migration of RA FLS, and could induce apoptosis of FLS and inhibit the expression of VEGF, FGF, MMP13 and DKK1 at mRNA level. This may be an important mechanism for dasitinib in the treatment of Ra, indicating that FLS may be an important target cell for dasatinib in the treatment of RA.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R593.22;R-332

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