本文選題：異位骨化 + 類風(fēng)濕性關(guān)節(jié)炎�。� 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：盤(pán)狀結(jié)構(gòu)受體2(DDR2)是一種受體型蛋白酪氨酸激酶,DDR2調(diào)控的相關(guān)信號(hào)在機(jī)體的生長(zhǎng)發(fā)育以及腫瘤、纖維化、動(dòng)脈粥樣硬化、骨性關(guān)節(jié)炎(OA)和類風(fēng)濕性關(guān)節(jié)炎(RA)等許多疾病的發(fā)病過(guò)程中起重要作用,因此DDR2成為了治療相關(guān)疾病潛在的靶分子。在本實(shí)驗(yàn)中,我們運(yùn)用抑制DDR2的策略,分別研究GD856和達(dá)沙替尼兩種DDR2小分子抑制劑對(duì)異位骨化和膠原誘導(dǎo)關(guān)節(jié)炎小鼠動(dòng)物模型的影響。實(shí)驗(yàn)一DDR2缺失對(duì)小鼠骨形成的影響DDR2可以促進(jìn)軟骨細(xì)胞的增殖和分化、成骨細(xì)胞的分化,并能夠抑制破骨細(xì)胞的分化,它在骨形成的作用中發(fā)揮重要作用。雖然在細(xì)胞層面對(duì)DDR2的骨形成作用有較為廣泛的探索,但是在體內(nèi)實(shí)驗(yàn)中仍缺乏對(duì)DDR2骨形成作用較為系統(tǒng)的研究。本實(shí)驗(yàn)通過(guò)比較DDR2缺失小鼠和野生型小鼠的骨骼生長(zhǎng)發(fā)育以及骨折后、異位骨化后的骨形成情況,研究DDR2在體內(nèi)對(duì)骨形成的作用。結(jié)果顯示DDR2缺失小鼠骨骼發(fā)育顯著異常,機(jī)體以及骨骼長(zhǎng)度顯著小于野生型小鼠,通過(guò)microCT重建股骨中部皮質(zhì)和股骨遠(yuǎn)端骨小梁后可以發(fā)現(xiàn),DDR2缺失小鼠骨皮質(zhì)的厚度和骨小梁的相關(guān)骨形成參數(shù)顯著低于野生型小鼠,同時(shí)股骨的三點(diǎn)彎曲實(shí)驗(yàn)表明ddr2缺失小鼠股骨所能承受的最大力和股骨剛度顯著小于野生型小鼠,雙鈣黃綠素?zé)晒鈱?shí)驗(yàn)顯示ddr2缺失小鼠的骨代謝速度顯著低于野生型小鼠。我們通過(guò)手術(shù)建立了小鼠開(kāi)放式骨折模型,骨折后ddr2缺失小鼠的骨痂形成能力顯著降低,骨折愈合能力顯著減弱。我們對(duì)ddr2缺失小鼠和野生型小鼠建立了創(chuàng)傷-燒傷異位骨化模型,建模10周后通過(guò)microct掃描重建發(fā)現(xiàn)ddr2缺失小鼠的異位骨化能力顯著低于野生型小鼠。本實(shí)驗(yàn)結(jié)果表明,ddr2缺失后小鼠的骨骼生長(zhǎng)發(fā)育、機(jī)體的骨代謝、以及骨折和異位骨化后的骨形成能力受到顯著抑制。實(shí)驗(yàn)二ddr2小分子抑制劑gd856對(duì)小鼠異位骨化的影響gd856是一種新研制的ddr2小分子抑制劑,能夠在較低的濃度下對(duì)膠原誘導(dǎo)的ddr2磷酸化有較顯著的抑制作用。我們通過(guò)誘導(dǎo)成骨細(xì)胞系mc-3t3-e1分化以及建立小鼠異位骨化模型,研究gd856對(duì)小鼠異位骨化的影響。我們將成骨細(xì)胞系分為未加藥的對(duì)照組、10μm的低劑量組和50μm的高劑量組,在向成骨細(xì)胞誘導(dǎo)的同時(shí)加gd856藥物刺激,三周后對(duì)細(xì)胞進(jìn)行茜素紅染色、堿性磷酸酶(alp)染色和成骨相關(guān)分子的mrna測(cè)定。結(jié)果表明低劑量組細(xì)胞的茜素紅陽(yáng)性強(qiáng)度略低于對(duì)照組,而高劑量組的陽(yáng)性強(qiáng)度則顯著低于對(duì)照組和低劑量組。低劑量組細(xì)胞的alp陽(yáng)性強(qiáng)度顯著強(qiáng)于對(duì)照組,而高劑量組的alp陽(yáng)性強(qiáng)度則顯著低于對(duì)照組和低劑量組。低濃度組和高劑量組opn、ocn和bmp2mrna的表達(dá)均顯著少于對(duì)照組,高劑量組alp的mrna表達(dá)顯著低于對(duì)照組,而低濃度組alp和colia1以及高劑量組runx2在mrna水平的表達(dá)則顯著高于對(duì)照組。為了在體內(nèi)驗(yàn)證gd856對(duì)小鼠異位骨化的影響,我們建立了創(chuàng)傷-燒傷異位骨化模型,小鼠分為對(duì)照組和給藥組,將給藥組小鼠的飲水為濃度為0.08mg/ml的gd856水溶液。建模10周后我們發(fā)現(xiàn)給藥組小鼠的骨代謝速度顯著減慢,根骨的異位骨化嚴(yán)重程度顯著減輕。本實(shí)驗(yàn)結(jié)果表明gd856可以抑制成骨細(xì)胞的分化,并能夠減緩體內(nèi)的骨代謝速度,抑制異位骨化的發(fā)生。實(shí)驗(yàn)三DDR2小分子抑制劑達(dá)沙替尼對(duì)小鼠膠原誘導(dǎo)關(guān)節(jié)炎的影響達(dá)沙替尼是一種第二代酪氨酸激酶抑制劑,對(duì)DDR2活性有較好的抑制作用。達(dá)沙替尼能夠抑制RA發(fā)病過(guò)程中的多個(gè)關(guān)鍵基因,本實(shí)驗(yàn)通過(guò)建立膠原誘導(dǎo)關(guān)節(jié)炎的小鼠模型,研究達(dá)沙替尼對(duì)膠原誘導(dǎo)關(guān)節(jié)炎(CIA)小鼠的治療作用。我們通過(guò)異種膠原二次免疫的方法建立小鼠的關(guān)節(jié)炎模型,在建模成功后將小鼠分為給藥組和對(duì)照組分別給予達(dá)沙替尼和安慰劑灌胃。結(jié)果表明給藥組小鼠的體重顯著大于對(duì)照組,同時(shí)給藥組小鼠四爪的腫脹程度以及關(guān)節(jié)炎的嚴(yán)重程度都顯著低于對(duì)照組。通過(guò)microCT對(duì)小鼠的關(guān)節(jié)周?chē)墙M織掃面重建我們發(fā)現(xiàn),給藥組小鼠關(guān)節(jié)周?chē)约肮切×旱墓琴|(zhì)破壞程度顯著輕于對(duì)照組。小鼠踝關(guān)節(jié)周?chē)M織的組織學(xué)染色結(jié)果顯示給藥組小鼠關(guān)節(jié)內(nèi)的滑膜細(xì)胞以及炎性細(xì)胞數(shù)量顯著減少,關(guān)節(jié)軟骨和骨質(zhì)的破壞以及血管翳的形成被顯著抑制。本實(shí)驗(yàn)結(jié)果表明達(dá)沙替尼可以減輕CIA發(fā)病的嚴(yán)重程度,緩解CIA的關(guān)節(jié)破壞程度,對(duì)小鼠膠原誘導(dǎo)關(guān)節(jié)炎模型有較好的治療作用。實(shí)驗(yàn)四DDR2小分子抑制劑達(dá)沙替尼對(duì)類風(fēng)濕性關(guān)節(jié)炎滑膜細(xì)胞的作用成纖維樣滑膜細(xì)胞(FLS)在RA的發(fā)病過(guò)程中對(duì)關(guān)節(jié)的破壞起重要作用,也是治療RA的一個(gè)重要靶細(xì)胞。本實(shí)驗(yàn)通過(guò)原代培養(yǎng)RA患者的關(guān)節(jié)FLS,研究達(dá)沙替尼對(duì)RA FLS的作用,探索達(dá)沙替尼治療RA的可能機(jī)制。我們通過(guò)BRDU免疫熒光染色研究達(dá)沙替尼對(duì)RA FLS增殖能力的影響,結(jié)果表明經(jīng)達(dá)沙替尼刺激24小時(shí)后的FLS的BRDU陽(yáng)性細(xì)胞數(shù)量顯著減少,這表明FLS的增殖能力顯著受到抑制。通過(guò)細(xì)胞劃痕實(shí)驗(yàn)和Transwell小室遷移實(shí)驗(yàn)判斷FLS遷移能力的改變,結(jié)果表明達(dá)沙替尼刺激24小時(shí)后FLS的遷移能力被顯著抑制。用流失細(xì)胞儀檢測(cè)FLS的凋亡能力我們發(fā)現(xiàn)在達(dá)沙替尼刺激的第3天和第5天凋亡的細(xì)胞數(shù)量顯著增多。運(yùn)用RT-PCR我們發(fā)現(xiàn)達(dá)沙替尼刺激24小時(shí)后,FLS中VEGF、FGF、MMP13和DKK1在mRNA水平的表達(dá)顯著降低。本實(shí)驗(yàn)結(jié)果表明,達(dá)沙替尼可以抑制RA FLS的增殖和遷移,同時(shí)能夠誘導(dǎo)FLS的凋亡,且可以在mRNA水平抑制VEGF、FGF、MMP13和DKK1的表達(dá)。這可能是達(dá)沙替尼治療Ra的重要機(jī)制,表明FLS可能是達(dá)沙替尼治療RA的重要靶細(xì)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R593.22;R-332

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