脂肪细胞Rankl表达对破骨细胞发育和骨稳态的调节作用
发布时间：2018-08-26 15:29
【摘要】：背景:很多研究证实肥胖会导致骨密度下降,造成骨量丢失,一般认为脂肪细胞通过分泌多种脂肪因子间接的对骨代谢产生影响。但近年有体外研究提示脂肪细胞可表达RANKL,并直接调节破骨细胞的发育,对骨代谢产生影响,但目前缺少在体研究证据。为了进一步揭示脂肪细胞是否通过RANKL直接调控破骨细胞发育,我们构建了脂肪细胞特异性Rankl转基因鼠和基因沉默鼠,分析其骨量变化和破骨细胞数目及功能变化等,阐明脂肪细胞中RANKL表达水平变化对破骨细胞发育的影响,为肥胖合并骨质疏松的治疗提供新思路。方法:1.实验室前期以脂肪细胞特异性Marker基因a P2的启动子(5.4 kb)分别引导Rankl c DNA和基因沉默序列的表达,构建Rankl转基因载体及Rankl基因沉默载体。由南京大学模式生物研究所通过显微注射、受精卵移植构建脂肪细胞Rankl转基因小鼠(a P2-Rankl Tg)和脂肪细胞Rankl基因沉默小鼠(a P2-Rankl KD),并鉴定基因型。提取转基因小鼠及阴性对照小鼠不同组织器官的总RNA,逆转为c DNA后用特异性引物分析Rankl转基因的组织学分布;提取转基因鼠、基因沉默鼠及各自阴性对照鼠腹股沟脂肪组织总蛋白,检测脂肪细胞中RANKL蛋白表达变化。2.X光及Micro-CT分别扫描检测转基因鼠、基因沉默鼠及各自阴性对照鼠骨密度;取小鼠骨组织制作石蜡切片,行阿尔新蓝-苏木素-橙黄G染色,比较骨量的变化;对转基因鼠、基因沉默鼠及各自阴性对照鼠骨组织石蜡切片行抗酒石酸酸性磷酸酶(anti-tartaric acid phosphatase,TRAP)染色,比较骨髓腔中破骨细胞数量及吸收表面变化;分离小鼠骨髓细胞进行体外培养并诱导破骨细胞分化,检测破骨细胞标志基因的表达变化,对体外诱导生成的破骨细胞行TRAP染色,比较转基因鼠、基因沉默鼠及各自阴性对照鼠破骨细胞生成能力的不同;使用钙黄绿素对小鼠骨组织进行荧光标记并行冰冻切片,比较骨动力学参数的改变;提取小鼠血清并用ELISA检测血清中骨钙素的变化。结果:1.基因型鉴定PCR证实a P2-Rankl Tg和a P2-Rankl KD小鼠基因型正确。采用Rankl转基因特异性引物PCR检测Rankl转基因的组织学分布,证实Rankl转基因在脂肪组织高表达,在含骨髓胫骨组织有低水平表达,在肝、脾、脑、去骨髓胫骨等组织不表达;与阴性对照鼠相比,转基因鼠腹股沟脂肪组织中RANKL蛋白表达上升,基因沉默鼠与阴性对照鼠相比,腹股沟脂肪组织中RANKL蛋白表达下降。这表明Rankl转基因鼠与基因沉默鼠构建成功。2.与阴性对照鼠相比,3月龄转基因鼠胫骨干骺端骨小梁相对体积(BV/TV)减少了90%、小梁骨密度(BMD)减少了71%、骨小梁数量(Tb.N)减少了91%、骨小梁结构模型指数(SMI)增加了180%,胫骨石蜡切片行TRAP染色,结果表明3月龄转基因鼠破骨细胞数目增加77%、破骨细胞表面增加83%;体外诱导培养骨髓破骨细胞,转基因鼠破骨细胞分化数目为对照组的1.73倍,破骨细胞特异性基因抗酒石酸酸性磷酸酶5b(anti-tartaric acid phosphatase 5b,TRAP-5b)、降钙素受体(calcitonin receptor,CTR)和组织蛋白酶K(Cathepsin K)m RNA表达量分别比对照组上升了1.35倍、5.14倍和1.54倍;1月龄转基因鼠骨矿化沉积率(MAR)增加了59%,1月龄转基因鼠血清中骨钙素水平增加了54%,这表明脂肪细胞Rankl转基因鼠(a P2-Rankl Tg)骨髓破骨细胞生成增加,骨吸收加强,骨转换率加快,骨量减少。3.与阴性对照鼠相比,3月龄基因沉默鼠胫骨干骺端BV/TV增加67%、BMD增加28%、Tb.N增加45%、SMI下降17%,3月龄基因沉默鼠胫骨切片行TRAP染色提示破骨细胞数目下降54%、破骨细胞表面下降45%;体外诱导培养骨髓破骨细胞,基因沉默鼠破骨细胞分化数目为对照组的40%,破骨细胞特异性基因TRAP-5b、CTR和Ctsk m RNA表达量分别比对照组下降了70%、49%和63%;1月龄基因沉默鼠骨MAR下降了49%,1月龄基因沉默鼠血清中骨钙素水平下降了31%;以上结果表明脂肪细胞Rankl基因沉默鼠(a P2-Rankl KD)骨髓破骨细胞生成减少,骨吸收减弱,骨转换率降低,骨量增加。结论:1.首次构建了脂肪细胞Rankl转基因鼠和基因沉默鼠。2.脂肪细胞Rankl转基因鼠(a P2-Rankl Tg)骨髓破骨细胞生成增加,骨吸收加强,骨转换率加快,骨量减少。3.脂肪细胞Rankl基因沉默鼠(a P2-Rankl KD)骨髓破骨细胞生成减少,骨吸收减弱,骨转换率降低,骨量增加。4.脂肪细胞可通过表达RANKL调控破骨细胞生成,这可能是肥胖致骨丢失的一种机制。
[Abstract]:BACKGROUND: Many studies have confirmed that obesity leads to bone density decline and bone mass loss. It is generally believed that adipocytes indirectly affect bone metabolism by secreting a variety of adipokines. However, recent studies in vitro have suggested that adipocytes can express RANKL and directly regulate the development of osteoclasts, which has an impact on bone metabolism. In order to further reveal whether adipocytes directly regulate osteoclast development through RANKL, adipocyte-specific Rankl transgenic mice and gene-silenced mice were constructed. The changes of bone mass, number and function of osteoclasts were analyzed to clarify the changes of RANKL expression in adipocytes. Methods: 1. The promoter of adipocyte-specific Marker gene a P2 (5.4 kb) was used to induce the expression of Rankl C DNA and gene silencing sequence respectively in the early stage of the laboratory to construct Rankl transgenic vector and Rankl gene silencing vector. The genotypes of Rankl transgenic mice (a P2-Rankl Tg) and Rankl silenced mice (a P2-Rankl KD) were identified by microinjection. Total RNA from different tissues and organs of transgenic mice and negative control mice was extracted and reversed to C DNA. The histological distribution of Rankl gene was analyzed by specific primers. The expression of RANKL protein in adipocytes of transgenic mice, gene-silenced mice and their negative control mice was detected. 2. Bone mineral density of transgenic mice, gene-silenced mice and their negative control mice were detected by X-ray and micro-CT, respectively. Anti-tartaric acid phosphatase (TRAP) staining was performed on the paraffin sections of bone tissue from transgenic mice, gene silenced mice and their respective negative control mice to compare the number of osteoclasts in bone marrow cavity and the changes of absorption surface. The osteoclasts induced in vitro were stained with TRAP to compare the osteoclast formation ability of transgenic mice, gene silenced mice and their respective negative control mice. Results: 1. Genotype identification PCR confirmed that the genotypes of a P2-Rankl Tg and a P2-Rankl KD mice were correct. Rankl specific primer PCR was used to detect the histological distribution of Rankl transgenic gene, which confirmed that Rankl transgenic gene was highly expressed in adipose tissue and in bone marrow-bearing tibia group. The expression of RANKL protein in inguinal adipose tissue of transgenic mice was higher than that of negative control mice, and the expression of RANKL protein in inguinal adipose tissue of transgenic mice was lower than that of negative control mice. Compared with negative control mice, the relative trabecular volume (BV / TV) of tibial metaphysis in 3-month-old transgenic mice decreased by 90%, the trabecular bone density (BMD) decreased by 71%, the number of trabecular bone (Tb. N) decreased by 91%, the trabecular structural model index (SMI) increased by 180%, and the number of osteoclasts in 3-month-old transgenic mice were stained by TRAP. The number of osteoclasts differentiated by transgenic mice was 1.73 times as much as that of the control group, and the tartaric acid phosphatase 5b (TRAP-5b), calcitonin receptor (CTR) and cathepsin K (Ca) were the specific genes of osteoclasts. The expression of thepsin K m RNA increased by 1.35 times, 5.14 times and 1.54 times, respectively, compared with the control group; the bone mineralization rate (MAR) increased by 59% in the 1-month-old transgenic mice, and the serum osteocalcin level increased by 54% in the 1-month-old transgenic mice, which indicated that osteoclasts in bone marrow of the adipocyte Rankl transgenic mice (a P2-Rankl Tg) increased, bone resorption strengthened and bone turnover. Compared with negative control mice, 3-month-old gene-silenced mice showed 67% increase in BV/TV, 28% increase in BMD, 45% increase in Tb.N, and 17% decrease in SMI, and 54% decrease in the number of osteoclasts and 45% decrease in the surface of osteoclasts. The number of osteoclasts differentiated into 40% of the control group, and the expression of TRAP-5b, CTR and CTKM RNA decreased by 70%, 49% and 63% respectively. The bone MAR of 1-month-old gene silenced mice decreased by 49% and the serum osteocalcin of 1-month-old gene silenced mice decreased by 31%. Bone marrow osteoclasts in a P2-Rankl KD mice were reduced, bone resorption was weakened, bone turnover rate was decreased, and bone mass was increased. Conclusion: 1. Fat cell Rankl transgenic mice and gene silenced mice were constructed for the first time. 2. Bone marrow osteoclasts in a P2-Rankl transgenic mice (a P2-Rankl Tg) were increased, bone resorption was strengthened, bone turnover was accelerated, and bone mass was decreased. Fat cell Rankl gene silenced mice (a P2-Rankl KD) bone marrow osteoclast production decreased, bone resorption decreased, bone turnover rate decreased, bone mass increased. 4. Adipocytes can regulate osteoclast production through the expression of RANKL, which may be a mechanism of obesity-induced bone loss.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2;R580
[Abstract]:BACKGROUND: Many studies have confirmed that obesity leads to bone density decline and bone mass loss. It is generally believed that adipocytes indirectly affect bone metabolism by secreting a variety of adipokines. However, recent studies in vitro have suggested that adipocytes can express RANKL and directly regulate the development of osteoclasts, which has an impact on bone metabolism. In order to further reveal whether adipocytes directly regulate osteoclast development through RANKL, adipocyte-specific Rankl transgenic mice and gene-silenced mice were constructed. The changes of bone mass, number and function of osteoclasts were analyzed to clarify the changes of RANKL expression in adipocytes. Methods: 1. The promoter of adipocyte-specific Marker gene a P2 (5.4 kb) was used to induce the expression of Rankl C DNA and gene silencing sequence respectively in the early stage of the laboratory to construct Rankl transgenic vector and Rankl gene silencing vector. The genotypes of Rankl transgenic mice (a P2-Rankl Tg) and Rankl silenced mice (a P2-Rankl KD) were identified by microinjection. Total RNA from different tissues and organs of transgenic mice and negative control mice was extracted and reversed to C DNA. The histological distribution of Rankl gene was analyzed by specific primers. The expression of RANKL protein in adipocytes of transgenic mice, gene-silenced mice and their negative control mice was detected. 2. Bone mineral density of transgenic mice, gene-silenced mice and their negative control mice were detected by X-ray and micro-CT, respectively. Anti-tartaric acid phosphatase (TRAP) staining was performed on the paraffin sections of bone tissue from transgenic mice, gene silenced mice and their respective negative control mice to compare the number of osteoclasts in bone marrow cavity and the changes of absorption surface. The osteoclasts induced in vitro were stained with TRAP to compare the osteoclast formation ability of transgenic mice, gene silenced mice and their respective negative control mice. Results: 1. Genotype identification PCR confirmed that the genotypes of a P2-Rankl Tg and a P2-Rankl KD mice were correct. Rankl specific primer PCR was used to detect the histological distribution of Rankl transgenic gene, which confirmed that Rankl transgenic gene was highly expressed in adipose tissue and in bone marrow-bearing tibia group. The expression of RANKL protein in inguinal adipose tissue of transgenic mice was higher than that of negative control mice, and the expression of RANKL protein in inguinal adipose tissue of transgenic mice was lower than that of negative control mice. Compared with negative control mice, the relative trabecular volume (BV / TV) of tibial metaphysis in 3-month-old transgenic mice decreased by 90%, the trabecular bone density (BMD) decreased by 71%, the number of trabecular bone (Tb. N) decreased by 91%, the trabecular structural model index (SMI) increased by 180%, and the number of osteoclasts in 3-month-old transgenic mice were stained by TRAP. The number of osteoclasts differentiated by transgenic mice was 1.73 times as much as that of the control group, and the tartaric acid phosphatase 5b (TRAP-5b), calcitonin receptor (CTR) and cathepsin K (Ca) were the specific genes of osteoclasts. The expression of thepsin K m RNA increased by 1.35 times, 5.14 times and 1.54 times, respectively, compared with the control group; the bone mineralization rate (MAR) increased by 59% in the 1-month-old transgenic mice, and the serum osteocalcin level increased by 54% in the 1-month-old transgenic mice, which indicated that osteoclasts in bone marrow of the adipocyte Rankl transgenic mice (a P2-Rankl Tg) increased, bone resorption strengthened and bone turnover. Compared with negative control mice, 3-month-old gene-silenced mice showed 67% increase in BV/TV, 28% increase in BMD, 45% increase in Tb.N, and 17% decrease in SMI, and 54% decrease in the number of osteoclasts and 45% decrease in the surface of osteoclasts. The number of osteoclasts differentiated into 40% of the control group, and the expression of TRAP-5b, CTR and CTKM RNA decreased by 70%, 49% and 63% respectively. The bone MAR of 1-month-old gene silenced mice decreased by 49% and the serum osteocalcin of 1-month-old gene silenced mice decreased by 31%. Bone marrow osteoclasts in a P2-Rankl KD mice were reduced, bone resorption was weakened, bone turnover rate was decreased, and bone mass was increased. Conclusion: 1. Fat cell Rankl transgenic mice and gene silenced mice were constructed for the first time. 2. Bone marrow osteoclasts in a P2-Rankl transgenic mice (a P2-Rankl Tg) were increased, bone resorption was strengthened, bone turnover was accelerated, and bone mass was decreased. Fat cell Rankl gene silenced mice (a P2-Rankl KD) bone marrow osteoclast production decreased, bone resorption decreased, bone turnover rate decreased, bone mass increased. 4. Adipocytes can regulate osteoclast production through the expression of RANKL, which may be a mechanism of obesity-induced bone loss.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2;R580
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