基于破骨细胞功能探讨腺苷激酶敲除小鼠和贞术调脂胶囊对骨代谢的影响
发布时间：2018-09-13 21:35
【摘要】：【目的】我们在构建髓性单核细胞敲除腺苷激酶(Adenosine Kinase,ADK)的小鼠过程中,与对照组小鼠相比,显示体重明显下降。因此,我们将利用髓性单核细胞系特异性敲除ADK小鼠进行破骨细胞(osteoclast,OC)分化和骨吸收功能的研究,探讨髓性单核细胞敲除ADK对骨代谢的影响。同时在建立的破骨细胞体外培养体系下,评价贞术调脂胶囊(FTZ)对破骨细胞分化和骨吸收功能的影响。【方法】1.髓性单核细胞敲除ADK小鼠的鉴定方法选取杂交繁殖后的子代鼠尾进行PCR基因型分析鉴定,筛选出ADKf/f、ADKf/flysmcre/cre、ADKf/flysmcre/+三种基因敲除小鼠。采用Western-blot技术检测不同基因型小鼠原代巨噬细胞ADK蛋白的表达量。2.体外破骨细胞分化及骨吸收功能检测方法利用ADKf/f、ADKf/flysmcre/cre、ADKf/flysmcre/+三种基因敲除小鼠,体外获取分离小鼠的原代骨髓单核细胞,在M-CSF和RANKL的刺激下诱导培养破骨细胞,同时收集野生型BL/6J小鼠原代骨髓单核细胞并给予ADK抑制剂(ABT-702)干预,进行抗酒石酸酸性磷酸酶特异性Trap染色、骨吸收实验、骨代谢功能相关TRAP、NFATc1、c-Fos、MMP-9、Cts K、c-Src、CTR基因RT-PCR检测,评价髓性单核细胞敲除ADK对体外破骨细胞分化及骨吸收功能的作用。3.基因型小鼠骨形态计量学检测方法比较4月龄ADKf/f、ADKf/flysmcre/cre、ADKf/flysmcre/+雄性小鼠的体重、股骨湿重;收集胫骨上、中段组织,采用HE染色观察小鼠骨组织病理形态学改变,通过骨组织静态学分析系统分析小鼠体内骨量的变化。4.FTZ对体外破骨细胞分化和骨吸收的影响利用已建立的破骨细胞体外培养体系,收集野生型BL/6J小鼠原代骨髓单核细胞,并添加大鼠含药血清干预,进行抗酒石酸酸性磷酸酶特异性TRAP染色、骨吸收实验、骨代谢功能相关TRAP、NFATc1、c-Fos、MMP-9、Cts K、c-Src、CTR基因RT-PCR检测,评价FTZ对破骨细胞分化和吸收功能的作用。【结果】1.ADKf/flysmcre/cre小鼠ADK的敲除效率比ADKf/fLysmcre/+小鼠、ADKf/f小鼠高。(1)PCR检测鼠尾基因型鉴定结果:ADK纯合子条带为360bp;ADKf/f小鼠在350bp处有1条清晰条带,ADKf/flysmcre/cre小鼠在700bp处有一条清晰条带,ADKf/flysmcre/+小鼠在350bp、700bp处有两条条带。(2)Western blot检测髓性单核细胞ADK蛋白水平结果:与ADKf/f组相比较,ADKf/fLysmcre/cre组ADK蛋白表达水平降低69.77%(P0.01),ADKf/fLysmcre/+组ADK蛋白表达水平降低36.83%(P0.05)。2.髓性单核细胞敲除ADK小鼠体外破骨细胞分化增多,骨吸收功能增强。(1)髓性单核细胞敲除ADK小鼠增加了破骨细胞分化:不同基因型小鼠体外破骨细胞培养结果发现,与ADKf/f组相比,ADKf/fLysmcre/cre组的破骨细胞数量较多,具有显著性差异(P0.01);但ADKf/f Lysmcre/+组的破骨细胞数量无明显变化(P0.05);与ADKf/fLysmcre/cre组相比,ADKf/fLysmcre/+组的破骨细胞数量有显著性差异(P0.01)。ABT-702干预破骨细胞培养结果发现,分别与control组、5%Et OH组相比,100n M组的破骨细胞数量较多,有显著性差异(P0.01)。破骨细胞分化相关基因RT-PCR检测结果,相比于ADKf/f组,ADKf/fLysmcre/cre组的TRAP(P0.01)、NFATc1(P0.05)基因表达量增加;c-Fos基因表达量无差异(P0.05),但ADKf/f Lysmcre/+组上述基因无明显变化(P0.05);与ADKf/f Lysmcre/cre组相比,ADKf/fLysmcre/+组的TRAP和NFATc1基因表达量有统计学差异(P0.05),c-Fos基因表达量无差异(P0.05)。ABT-702干预破骨细胞分化相关基因RT-PCR结果显示,相比于5%Et OH组,100n M组的TRAP、NFATc1的m RNA基因表达量增多,具有统计学差异(P0.05);c-Fos基因表达量无显著性差异(P0.05)。(2)髓性单核细胞敲除ADK增强了破骨细胞吸收功能:不同基因型小鼠体外破骨细胞骨吸收实验结果发现,与ADKf/f组相比,ADKf/fLysmcre/cre组培养的破骨细胞的骨陷窝面积显著增大(P0.01)和数量增多(P0.05),ADKf/f Lysmcre/+组的破骨细胞骨陷窝面积和数量比较,无明显变化(P0.05);与ADKf/f Lysmcre/cre组相比,ADKf/f Lysmcre/+组的破骨细胞骨陷窝面积和数量均具有显著性差异(P0.01)。ABT-702干预破骨细胞骨吸收实验结果发现,与5%Et OH组相比,100n M骨陷窝面积增大和数量增多,有显著性差异(P0.01)。破骨细胞吸收功能相关基因RT-PCR检测结果,相比于ADKf/f组,ADKf/fLysmcre/cre组的MMP-9、Cts K、CTR基因表达量增多,具有显著性差异(P0.01),c-Src基因表达量无显著性差异(P0.05),ADKf/f Lysmcre/+组的Cts K基因表达量有统计学差异(P0.05);与ADKf/f Lysmcre/cre组相比,ADKf/f Lysmcre/+组的MMP-9、Cts K、CTR基因表达量具有统计学差异(P0.05)。ABT-702干预破骨细胞骨吸收相关基因结果显示,相比于5%Et OH组,100n M组的MMP-9(P0.01)、Cts K(P0.05)基因表达量增多,但c-Src、CTR基因表达量无明显变化(P0.05)。3.髓性单核细胞敲除ADK小鼠体内骨量的变化相比于ADKf/f小鼠,ADKf/flysmcre/cre小鼠体重和股骨湿重最轻,具有显著性差异(P0.01),ADKf/flysmcre/+小鼠无明显变化(P0.05);与ADKf/f Lysmcre/cre小鼠相比,ADKf/fLysmcre/+小鼠体重和股骨重量均有显著性差异(P0.01)。ADKf/f组松质骨部分骨小梁丰富呈浅紫粉红色,结构致密粗实,连续性好呈网状,ADKf/fLysmcre/cre组骨小梁明显减少,排列稀疏,断裂状,ADKf/fLysmcre/+组骨小梁结构介于两组间,骨小梁细长,部分断裂。骨组织静态参数结果显示,ADKf/flysmcre/cre小鼠分别与ADKf/f小鼠、ADKf/flysmcre/+小鼠相比,%Tb.Ar、Tb.Th、Tb.N、%Ob.S/BS、Ob.N/BS、Tb.Sp、%Oc.S/BS、Oc.N/BS均有统计学差异(P0.01;P0.05)。同样,胫骨中段分析,ADKf/f Lysmcre/cre组与其余两组计较,%Ct.Ar、%Ma.Ar参数变化明显(P0.01)。4.FTZ抑制了破骨细胞分化和吸收功能不同浓度剂量对破骨细胞有不一样的抑制作用,与control组相比,Alendronate组、FTZ-H组和FTZ-M组的破骨细胞数量(P0.01)、骨陷窝面积(P0.05)和骨陷窝数量均减少(P0.01),比FTZ-L组的破骨细胞数量少(P0.05)和骨陷窝数量少(FTZ-H组P0.01;FTZ-M组P0.05);与Alendronate组相比,FTZ-L组的骨陷窝数量具有显著性差异(P0.01)。破骨细胞分化相关基因RT-PCR结果显示,相比于control组,FTZ-H组的TRAP、NFATc1基因表达量明显降低(P0.05),c-Fos基因表达量无统计学意义(P0.05)。骨吸收相关基因表达检测结果显示,与control组相比,FTZ-H组MMP-9基因表达量下降,具有统计学差异(P0.01),Cts K、c-Src、CTR基因表达量无明显变化(P0.05)。【结论】1、髓性单核细胞敲除ADK小鼠增强了破骨细胞分化功能。2、髓性单核细胞敲除ADK小鼠增强了破骨细胞吸收功能。3、髓性单核细胞细胞敲除ADK小鼠显示骨量下降,骨结构异常。4、FTZ改善骨质疏松通过抑制破骨细胞的分化和吸收功能。
[Abstract]:[Objective] Compared with the control group, the weight of ADK mice was significantly decreased during the construction of ADK knockout mice. Therefore, the osteoclast (OC) differentiation and bone resorption function of ADK mice were studied by using myelomonocyte line specific knockout mice. The effect of sexual monocyte knockout ADK on bone metabolism was evaluated in vitro. The effects of Zhenzhu Tiaozhi capsule (FTZ) on osteoclast differentiation and bone resorption were evaluated under the established osteoclast culture system. ADK f/f, ADK f/flysmcre/cre, ADK f/flysmcre/+ three gene knockout mice were identified and screened. The expression of ADK protein in primary macrophages of different genotypes of mice was detected by Western blot. 2. Osteoclast differentiation and bone resorption function in vitro were detected by ADK f/f, ADK f/flysmcre/cre, ADK f/flysmcre/+ three gene knockout mice. Osteoclasts were induced by M-CSF and RANKL stimulation. Primary bone marrow mononuclear cells of wild type BL/6J mice were collected and interfered with ADK inhibitor (ABT-702), tartrate-resistant acid phosphatase specific Trap staining, bone resorption test, TRAP, NFATc1 related to bone metabolism were performed. The effects of myelomonocyte knockout ADK on osteoclast differentiation and bone resorption in vitro were evaluated by RT-PCR. 3. Bone morphometry of 4-month-old ADK f/f, ADK f/flysmcre/cre, ADK f/flysmcre/+ male mice was compared with that of 4-month-old ADK f/f, ADK f/flysmcre/+ male mice. HE staining was used to observe the pathomorphological changes of bone tissue in mice. The changes of bone mass in mice were analyzed by bone histostatic analysis system. 4. The effect of FTZ on osteoclast differentiation and bone resorption in vitro. The primary bone marrow mononuclear cells of wild type BL/6J mice were collected and added into the culture system of osteoclasts in vitro. Drug serum intervention, tartrate-resistant acid phosphatase-specific TRAP staining, bone resorption test, Bone Metabolism-Related TRAP, NFATc1, c-Fos, MMP-9, Cts K, c-Src, CTR gene RT-PCR assay were used to evaluate the effect of FTZ on osteoclast differentiation and absorption function. [Results] 1. The knockout efficiency of ADKf/smcre/cre/ADflyK was higher than that of ADKf/fLysms/+mice. ADK f / flysmcre / CRE mice had a clear band at 350 bp. ADK f / flysmcre / CRE mice had a clear band at 700 bp. ADK f / flysmcre / + mice had two bands at 350 BP and 700 bp. (2) Western blot was used to detect ADK protein level in myeloid monocytes. Compared with ADK f/f group, ADK protein expression in ADK f/fLysmcre/cre group was decreased by 69.77% (P 0.01), and ADK protein expression in ADK f/fLysmcre/+ group was decreased by 36.83% (P 0.05). 2. Osteoclast differentiation and bone resorption were increased in ADK-knockout mice. (1) Osteoclast differentiation was increased in ADK-knockout mice: no The results of osteoclasts culture in vitro showed that the number of osteoclasts in ADKf/fLysmcre/cre group was significantly higher than that in ADKf/fLysmcre/cre group (P 0.01), but there was no significant change in the number of osteoclasts in ADKf/fLysmcre/+ group (P 0.05). The results showed that the number of osteoclasts in 100nM group was higher than that in control group and 5% Et OH group (P 0.01). The expression of NFATc1 (P 0.05) gene in ADKf / fLysmcre / CRE group was higher than that in ADKf / F group by RT-PCR. There was no significant difference in the expression of OS gene (P 0.05), but there was no significant change in the above genes in ADKf / fLysmcre /+ group (P 0.05). Compared with ADKf / fLysmcre / cre group, the expression of TRAP and NFATc1 gene in ADKf / fLysmcre /+ group was significantly different (P 0.05), and there was no difference in the expression of c-Fos gene (P 0.05). The results of RT-PCR showed that ABT-702 intervened osteoclast differentiation-related genes. Compared with 5% Et OH group, the expression of TRAP and NFATc1 m RNA in 100N M group increased, with statistical difference (P 0.05); the expression of c-Fos gene had no significant difference (P 0.05). (2) Myelogenic monocyte knockout ADK enhanced osteoclast resorption function: The results of osteoclast bone resorption in vitro in different genotypes of mice showed that ADK f was significantly higher than that in ADK f/f group. Osteoclast lacunae area and number of osteoclasts cultured in / fLysmcre / CRE group increased significantly (P 0.01) and increased significantly (P 0.05). There was no significant difference in lacuna area and number of osteoclasts cultured in ADKf / fLysmcre /+ group compared with ADKf / fLysmcre / CRE group (P 0.05). ABT-702 intervention osteoclasts bone resorption experiment results showed that compared with 5% Et OH group, 100nM bone lacuna area increased and the number increased, there was a significant difference (P 0.01). Osteoclasts absorption function related gene RT-PCR detection results, compared with ADKf/fLysmcre/cre group, ADKf/fLysmcre/cre group MMP-9, Cts K, CTR gene expression increased, with a significant difference. There was no significant difference in the expression of c-Src gene (P 0.05) between ADKf/f Lysmcre/+ group and ADKf/f Lysmcre/cre group (P 0.05). Compared with ADKf/f Lysmcre/cre group, the expression of MMP-9, Cts-K and CTR gene in ADKf/f Lysmcre/+ group was significantly different (P 0.05). The results of ABT-702 intervention osteoclast bone resorption related gene showed that the expression of ADKf/f Lysmcre/f Lysmcre/cre group was significantly different from that of ADKf Compared with 5% Et OH group, the expression of MMP-9 (P 0.01) and Cts K (P 0.05) increased in 100N M group, but the expression of c-Src and CTR genes did not change significantly (P 0.05). 3. Bone mass of ADK mice with myelomonocyte knockout was the lightest in body weight and femoral wet weight in ADK f/smcre/cre mice, with significant difference (P 0.01). Compared with ADKf/fLysmcre/cre mice, the body weight and femoral weight of ADKf/fLysmcre/+ mice were significantly different (P In ADKf / fLysmcre /+ group, the trabecular bone structure was between the two groups, and the trabecular bone was slender and partially broken. Static parameters of bone tissue showed that ADKf / flysmcre / CRE mice were significantly different from ADKf / F mice, ADKf / flysmcre /+ mice,% Tb. Ar, Tb. Th, Tb. N,% Ob. S / BS, Ob. N / BS, Tb. Sp,% Oc. N / BS (P 0.05), respectively. Segment analysis showed that compared with the other two groups,% Ct. Ar,% Ma. Ar parameters changed significantly (P 0.01). 4. FTZ inhibited the osteoclast differentiation and absorption function at different doses. Compared with the control group, the number of osteoclasts (P 0.01), the area of bone lacunae (P 0.01) and the number of osteoclasts (P 0.01) in the Alendronate group, FTZ-H group and FTZ-M group were inhibited. The number of osteoclasts and lacunae in FTZ-L group was significantly lower than that in FTZ-L group (P 0.05), and the number of osteoclasts and lacunae in FTZ-H group (P 0.01) and FTZ-M group (P 0.05). The expression of FATc1 gene was significantly decreased (P 0.05), but the expression of c-Fos gene was not statistically significant (P 0.05). The expression of MMP-9 gene in FTZ-H group was significantly lower than that in control group (P 0.01). The expression of Cts K, c-Src and CTR gene was not significantly changed in FTZ-H group (P 0.05). [Conclusion] Cell knockout ADK mice enhanced osteoclast differentiation. 2, myelomonocyte knockout ADK mice enhanced osteoclast absorption. 3, myelomonocyte knockout ADK mice showed decreased bone mass and abnormal bone structure. 4, FTZ improved osteoporosis by inhibiting osteoclast differentiation and absorption.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580
[Abstract]:[Objective] Compared with the control group, the weight of ADK mice was significantly decreased during the construction of ADK knockout mice. Therefore, the osteoclast (OC) differentiation and bone resorption function of ADK mice were studied by using myelomonocyte line specific knockout mice. The effect of sexual monocyte knockout ADK on bone metabolism was evaluated in vitro. The effects of Zhenzhu Tiaozhi capsule (FTZ) on osteoclast differentiation and bone resorption were evaluated under the established osteoclast culture system. ADK f/f, ADK f/flysmcre/cre, ADK f/flysmcre/+ three gene knockout mice were identified and screened. The expression of ADK protein in primary macrophages of different genotypes of mice was detected by Western blot. 2. Osteoclast differentiation and bone resorption function in vitro were detected by ADK f/f, ADK f/flysmcre/cre, ADK f/flysmcre/+ three gene knockout mice. Osteoclasts were induced by M-CSF and RANKL stimulation. Primary bone marrow mononuclear cells of wild type BL/6J mice were collected and interfered with ADK inhibitor (ABT-702), tartrate-resistant acid phosphatase specific Trap staining, bone resorption test, TRAP, NFATc1 related to bone metabolism were performed. The effects of myelomonocyte knockout ADK on osteoclast differentiation and bone resorption in vitro were evaluated by RT-PCR. 3. Bone morphometry of 4-month-old ADK f/f, ADK f/flysmcre/cre, ADK f/flysmcre/+ male mice was compared with that of 4-month-old ADK f/f, ADK f/flysmcre/+ male mice. HE staining was used to observe the pathomorphological changes of bone tissue in mice. The changes of bone mass in mice were analyzed by bone histostatic analysis system. 4. The effect of FTZ on osteoclast differentiation and bone resorption in vitro. The primary bone marrow mononuclear cells of wild type BL/6J mice were collected and added into the culture system of osteoclasts in vitro. Drug serum intervention, tartrate-resistant acid phosphatase-specific TRAP staining, bone resorption test, Bone Metabolism-Related TRAP, NFATc1, c-Fos, MMP-9, Cts K, c-Src, CTR gene RT-PCR assay were used to evaluate the effect of FTZ on osteoclast differentiation and absorption function. [Results] 1. The knockout efficiency of ADKf/smcre/cre/ADflyK was higher than that of ADKf/fLysms/+mice. ADK f / flysmcre / CRE mice had a clear band at 350 bp. ADK f / flysmcre / CRE mice had a clear band at 700 bp. ADK f / flysmcre / + mice had two bands at 350 BP and 700 bp. (2) Western blot was used to detect ADK protein level in myeloid monocytes. Compared with ADK f/f group, ADK protein expression in ADK f/fLysmcre/cre group was decreased by 69.77% (P 0.01), and ADK protein expression in ADK f/fLysmcre/+ group was decreased by 36.83% (P 0.05). 2. Osteoclast differentiation and bone resorption were increased in ADK-knockout mice. (1) Osteoclast differentiation was increased in ADK-knockout mice: no The results of osteoclasts culture in vitro showed that the number of osteoclasts in ADKf/fLysmcre/cre group was significantly higher than that in ADKf/fLysmcre/cre group (P 0.01), but there was no significant change in the number of osteoclasts in ADKf/fLysmcre/+ group (P 0.05). The results showed that the number of osteoclasts in 100nM group was higher than that in control group and 5% Et OH group (P 0.01). The expression of NFATc1 (P 0.05) gene in ADKf / fLysmcre / CRE group was higher than that in ADKf / F group by RT-PCR. There was no significant difference in the expression of OS gene (P 0.05), but there was no significant change in the above genes in ADKf / fLysmcre /+ group (P 0.05). Compared with ADKf / fLysmcre / cre group, the expression of TRAP and NFATc1 gene in ADKf / fLysmcre /+ group was significantly different (P 0.05), and there was no difference in the expression of c-Fos gene (P 0.05). The results of RT-PCR showed that ABT-702 intervened osteoclast differentiation-related genes. Compared with 5% Et OH group, the expression of TRAP and NFATc1 m RNA in 100N M group increased, with statistical difference (P 0.05); the expression of c-Fos gene had no significant difference (P 0.05). (2) Myelogenic monocyte knockout ADK enhanced osteoclast resorption function: The results of osteoclast bone resorption in vitro in different genotypes of mice showed that ADK f was significantly higher than that in ADK f/f group. Osteoclast lacunae area and number of osteoclasts cultured in / fLysmcre / CRE group increased significantly (P 0.01) and increased significantly (P 0.05). There was no significant difference in lacuna area and number of osteoclasts cultured in ADKf / fLysmcre /+ group compared with ADKf / fLysmcre / CRE group (P 0.05). ABT-702 intervention osteoclasts bone resorption experiment results showed that compared with 5% Et OH group, 100nM bone lacuna area increased and the number increased, there was a significant difference (P 0.01). Osteoclasts absorption function related gene RT-PCR detection results, compared with ADKf/fLysmcre/cre group, ADKf/fLysmcre/cre group MMP-9, Cts K, CTR gene expression increased, with a significant difference. There was no significant difference in the expression of c-Src gene (P 0.05) between ADKf/f Lysmcre/+ group and ADKf/f Lysmcre/cre group (P 0.05). Compared with ADKf/f Lysmcre/cre group, the expression of MMP-9, Cts-K and CTR gene in ADKf/f Lysmcre/+ group was significantly different (P 0.05). The results of ABT-702 intervention osteoclast bone resorption related gene showed that the expression of ADKf/f Lysmcre/f Lysmcre/cre group was significantly different from that of ADKf Compared with 5% Et OH group, the expression of MMP-9 (P 0.01) and Cts K (P 0.05) increased in 100N M group, but the expression of c-Src and CTR genes did not change significantly (P 0.05). 3. Bone mass of ADK mice with myelomonocyte knockout was the lightest in body weight and femoral wet weight in ADK f/smcre/cre mice, with significant difference (P 0.01). Compared with ADKf/fLysmcre/cre mice, the body weight and femoral weight of ADKf/fLysmcre/+ mice were significantly different (P In ADKf / fLysmcre /+ group, the trabecular bone structure was between the two groups, and the trabecular bone was slender and partially broken. Static parameters of bone tissue showed that ADKf / flysmcre / CRE mice were significantly different from ADKf / F mice, ADKf / flysmcre /+ mice,% Tb. Ar, Tb. Th, Tb. N,% Ob. S / BS, Ob. N / BS, Tb. Sp,% Oc. N / BS (P 0.05), respectively. Segment analysis showed that compared with the other two groups,% Ct. Ar,% Ma. Ar parameters changed significantly (P 0.01). 4. FTZ inhibited the osteoclast differentiation and absorption function at different doses. Compared with the control group, the number of osteoclasts (P 0.01), the area of bone lacunae (P 0.01) and the number of osteoclasts (P 0.01) in the Alendronate group, FTZ-H group and FTZ-M group were inhibited. The number of osteoclasts and lacunae in FTZ-L group was significantly lower than that in FTZ-L group (P 0.05), and the number of osteoclasts and lacunae in FTZ-H group (P 0.01) and FTZ-M group (P 0.05). The expression of FATc1 gene was significantly decreased (P 0.05), but the expression of c-Fos gene was not statistically significant (P 0.05). The expression of MMP-9 gene in FTZ-H group was significantly lower than that in control group (P 0.01). The expression of Cts K, c-Src and CTR gene was not significantly changed in FTZ-H group (P 0.05). [Conclusion] Cell knockout ADK mice enhanced osteoclast differentiation. 2, myelomonocyte knockout ADK mice enhanced osteoclast absorption. 3, myelomonocyte knockout ADK mice showed decreased bone mass and abnormal bone structure. 4, FTZ improved osteoporosis by inhibiting osteoclast differentiation and absorption.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580
【参考文献】
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1 林泽苗;钟佳贤;贾欢欢;吴玉娥;陈s，
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