脂肪細(xì)胞Rankl表達(dá)對破骨細(xì)胞發(fā)育和骨穩(wěn)態(tài)的調(diào)節(jié)作用
發(fā)布時間：2018-08-26 15:29
【摘要】：背景:很多研究證實(shí)肥胖會導(dǎo)致骨密度下降,造成骨量丟失,一般認(rèn)為脂肪細(xì)胞通過分泌多種脂肪因子間接的對骨代謝產(chǎn)生影響。但近年有體外研究提示脂肪細(xì)胞可表達(dá)RANKL,并直接調(diào)節(jié)破骨細(xì)胞的發(fā)育,對骨代謝產(chǎn)生影響,但目前缺少在體研究證據(jù)。為了進(jìn)一步揭示脂肪細(xì)胞是否通過RANKL直接調(diào)控破骨細(xì)胞發(fā)育,我們構(gòu)建了脂肪細(xì)胞特異性Rankl轉(zhuǎn)基因鼠和基因沉默鼠,分析其骨量變化和破骨細(xì)胞數(shù)目及功能變化等,闡明脂肪細(xì)胞中RANKL表達(dá)水平變化對破骨細(xì)胞發(fā)育的影響,為肥胖合并骨質(zhì)疏松的治療提供新思路。方法:1.實(shí)驗(yàn)室前期以脂肪細(xì)胞特異性Marker基因a P2的啟動子(5.4 kb)分別引導(dǎo)Rankl c DNA和基因沉默序列的表達(dá),構(gòu)建Rankl轉(zhuǎn)基因載體及Rankl基因沉默載體。由南京大學(xué)模式生物研究所通過顯微注射、受精卵移植構(gòu)建脂肪細(xì)胞Rankl轉(zhuǎn)基因小鼠(a P2-Rankl Tg)和脂肪細(xì)胞Rankl基因沉默小鼠(a P2-Rankl KD),并鑒定基因型。提取轉(zhuǎn)基因小鼠及陰性對照小鼠不同組織器官的總RNA,逆轉(zhuǎn)為c DNA后用特異性引物分析Rankl轉(zhuǎn)基因的組織學(xué)分布;提取轉(zhuǎn)基因鼠、基因沉默鼠及各自陰性對照鼠腹股溝脂肪組織總蛋白,檢測脂肪細(xì)胞中RANKL蛋白表達(dá)變化。2.X光及Micro-CT分別掃描檢測轉(zhuǎn)基因鼠、基因沉默鼠及各自陰性對照鼠骨密度;取小鼠骨組織制作石蠟切片,行阿爾新藍(lán)-蘇木素-橙黃G染色,比較骨量的變化;對轉(zhuǎn)基因鼠、基因沉默鼠及各自陰性對照鼠骨組織石蠟切片行抗酒石酸酸性磷酸酶(anti-tartaric acid phosphatase,TRAP)染色,比較骨髓腔中破骨細(xì)胞數(shù)量及吸收表面變化;分離小鼠骨髓細(xì)胞進(jìn)行體外培養(yǎng)并誘導(dǎo)破骨細(xì)胞分化,檢測破骨細(xì)胞標(biāo)志基因的表達(dá)變化,對體外誘導(dǎo)生成的破骨細(xì)胞行TRAP染色,比較轉(zhuǎn)基因鼠、基因沉默鼠及各自陰性對照鼠破骨細(xì)胞生成能力的不同;使用鈣黃綠素對小鼠骨組織進(jìn)行熒光標(biāo)記并行冰凍切片,比較骨動力學(xué)參數(shù)的改變;提取小鼠血清并用ELISA檢測血清中骨鈣素的變化。結(jié)果:1.基因型鑒定PCR證實(shí)a P2-Rankl Tg和a P2-Rankl KD小鼠基因型正確。采用Rankl轉(zhuǎn)基因特異性引物PCR檢測Rankl轉(zhuǎn)基因的組織學(xué)分布,證實(shí)Rankl轉(zhuǎn)基因在脂肪組織高表達(dá),在含骨髓脛骨組織有低水平表達(dá),在肝、脾、腦、去骨髓脛骨等組織不表達(dá);與陰性對照鼠相比,轉(zhuǎn)基因鼠腹股溝脂肪組織中RANKL蛋白表達(dá)上升,基因沉默鼠與陰性對照鼠相比,腹股溝脂肪組織中RANKL蛋白表達(dá)下降。這表明Rankl轉(zhuǎn)基因鼠與基因沉默鼠構(gòu)建成功。2.與陰性對照鼠相比,3月齡轉(zhuǎn)基因鼠脛骨干骺端骨小梁相對體積(BV/TV)減少了90%、小梁骨密度(BMD)減少了71%、骨小梁數(shù)量(Tb.N)減少了91%、骨小梁結(jié)構(gòu)模型指數(shù)(SMI)增加了180%,脛骨石蠟切片行TRAP染色,結(jié)果表明3月齡轉(zhuǎn)基因鼠破骨細(xì)胞數(shù)目增加77%、破骨細(xì)胞表面增加83%;體外誘導(dǎo)培養(yǎng)骨髓破骨細(xì)胞,轉(zhuǎn)基因鼠破骨細(xì)胞分化數(shù)目為對照組的1.73倍,破骨細(xì)胞特異性基因抗酒石酸酸性磷酸酶5b(anti-tartaric acid phosphatase 5b,TRAP-5b)、降鈣素受體(calcitonin receptor,CTR)和組織蛋白酶K(Cathepsin K)m RNA表達(dá)量分別比對照組上升了1.35倍、5.14倍和1.54倍;1月齡轉(zhuǎn)基因鼠骨礦化沉積率(MAR)增加了59%,1月齡轉(zhuǎn)基因鼠血清中骨鈣素水平增加了54%,這表明脂肪細(xì)胞Rankl轉(zhuǎn)基因鼠(a P2-Rankl Tg)骨髓破骨細(xì)胞生成增加,骨吸收加強(qiáng),骨轉(zhuǎn)換率加快,骨量減少。3.與陰性對照鼠相比,3月齡基因沉默鼠脛骨干骺端BV/TV增加67%、BMD增加28%、Tb.N增加45%、SMI下降17%,3月齡基因沉默鼠脛骨切片行TRAP染色提示破骨細(xì)胞數(shù)目下降54%、破骨細(xì)胞表面下降45%;體外誘導(dǎo)培養(yǎng)骨髓破骨細(xì)胞,基因沉默鼠破骨細(xì)胞分化數(shù)目為對照組的40%,破骨細(xì)胞特異性基因TRAP-5b、CTR和Ctsk m RNA表達(dá)量分別比對照組下降了70%、49%和63%;1月齡基因沉默鼠骨MAR下降了49%,1月齡基因沉默鼠血清中骨鈣素水平下降了31%;以上結(jié)果表明脂肪細(xì)胞Rankl基因沉默鼠(a P2-Rankl KD)骨髓破骨細(xì)胞生成減少,骨吸收減弱,骨轉(zhuǎn)換率降低,骨量增加。結(jié)論:1.首次構(gòu)建了脂肪細(xì)胞Rankl轉(zhuǎn)基因鼠和基因沉默鼠。2.脂肪細(xì)胞Rankl轉(zhuǎn)基因鼠(a P2-Rankl Tg)骨髓破骨細(xì)胞生成增加,骨吸收加強(qiáng),骨轉(zhuǎn)換率加快,骨量減少。3.脂肪細(xì)胞Rankl基因沉默鼠(a P2-Rankl KD)骨髓破骨細(xì)胞生成減少,骨吸收減弱,骨轉(zhuǎn)換率降低,骨量增加。4.脂肪細(xì)胞可通過表達(dá)RANKL調(diào)控破骨細(xì)胞生成,這可能是肥胖致骨丟失的一種機(jī)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R589.2;R580
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