脂肪細胞Rankl表達對破骨細胞發(fā)育和骨穩(wěn)態(tài)的調節(jié)作用
發(fā)布時間：2018-08-26 15:29
【摘要】：背景:很多研究證實肥胖會導致骨密度下降,造成骨量丟失,一般認為脂肪細胞通過分泌多種脂肪因子間接的對骨代謝產生影響。但近年有體外研究提示脂肪細胞可表達RANKL,并直接調節(jié)破骨細胞的發(fā)育,對骨代謝產生影響,但目前缺少在體研究證據(jù)。為了進一步揭示脂肪細胞是否通過RANKL直接調控破骨細胞發(fā)育,我們構建了脂肪細胞特異性Rankl轉基因鼠和基因沉默鼠,分析其骨量變化和破骨細胞數(shù)目及功能變化等,闡明脂肪細胞中RANKL表達水平變化對破骨細胞發(fā)育的影響,為肥胖合并骨質疏松的治療提供新思路。方法:1.實驗室前期以脂肪細胞特異性Marker基因a P2的啟動子(5.4 kb)分別引導Rankl c DNA和基因沉默序列的表達,構建Rankl轉基因載體及Rankl基因沉默載體。由南京大學模式生物研究所通過顯微注射、受精卵移植構建脂肪細胞Rankl轉基因小鼠(a P2-Rankl Tg)和脂肪細胞Rankl基因沉默小鼠(a P2-Rankl KD),并鑒定基因型。提取轉基因小鼠及陰性對照小鼠不同組織器官的總RNA,逆轉為c DNA后用特異性引物分析Rankl轉基因的組織學分布;提取轉基因鼠、基因沉默鼠及各自陰性對照鼠腹股溝脂肪組織總蛋白,檢測脂肪細胞中RANKL蛋白表達變化。2.X光及Micro-CT分別掃描檢測轉基因鼠、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-蘇木素-橙黃G染色,比較骨量的變化;對轉基因鼠、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胞數(shù)量及吸收表面變化;分離小鼠骨髓細胞進行體外培養(yǎng)并誘導破骨細胞分化,檢測破骨細胞標志基因的表達變化,對體外誘導生成的破骨細胞行TRAP染色,比較轉基因鼠、基因沉默鼠及各自陰性對照鼠破骨細胞生成能力的不同;使用鈣黃綠素對小鼠骨組織進行熒光標記并行冰凍切片,比較骨動力學參數(shù)的改變;提取小鼠血清并用ELISA檢測血清中骨鈣素的變化。結果:1.基因型鑒定PCR證實a P2-Rankl Tg和a P2-Rankl KD小鼠基因型正確。采用Rankl轉基因特異性引物PCR檢測Rankl轉基因的組織學分布,證實Rankl轉基因在脂肪組織高表達,在含骨髓脛骨組織有低水平表達,在肝、脾、腦、去骨髓脛骨等組織不表達;與陰性對照鼠相比,轉基因鼠腹股溝脂肪組織中RANKL蛋白表達上升,基因沉默鼠與陰性對照鼠相比,腹股溝脂肪組織中RANKL蛋白表達下降。這表明Rankl轉基因鼠與基因沉默鼠構建成功。2.與陰性對照鼠相比,3月齡轉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%、骨小梁結構模型指數(shù)(SMI)增加了180%,脛骨石蠟切片行TRAP染色,結果表明3月齡轉基因鼠破骨細胞數(shù)目增加77%、破骨細胞表面增加83%;體外誘導培養(yǎng)骨髓破骨細胞,轉基因鼠破骨細胞分化數(shù)目為對照組的1.73倍,破骨細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表達量分別比對照組上升了1.35倍、5.14倍和1.54倍;1月齡轉基因鼠骨礦化沉積率(MAR)增加了59%,1月齡轉基因鼠血清中骨鈣素水平增加了54%,這表明脂肪細胞Rankl轉基因鼠(a P2-Rankl Tg)骨髓破骨細胞生成增加,骨吸收加強,骨轉換率加快,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染色提示破骨細胞數(shù)目下降54%、破骨細胞表面下降45%;體外誘導培養(yǎng)骨髓破骨細胞,基因沉默鼠破骨細胞分化數(shù)目為對照組的40%,破骨細胞特異性基因TRAP-5b、CTR和Ctsk m RNA表達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%;以上結果表明脂肪細胞Rankl基因沉默鼠(a P2-Rankl KD)骨髓破骨細胞生成減少,骨吸收減弱,骨轉換率降低,骨量增加。結論:1.首次構建了脂肪細胞Rankl轉基因鼠和基因沉默鼠。2.脂肪細胞Rankl轉基因鼠(a P2-Rankl Tg)骨髓破骨細胞生成增加,骨吸收加強,骨轉換率加快,骨量減少。3.脂肪細胞Rankl基因沉默鼠(a P2-Rankl KD)骨髓破骨細胞生成減少,骨吸收減弱,骨轉換率降低,骨量增加。4.脂肪細胞可通過表達RANKL調控破骨細胞生成,這可能是肥胖致骨丟失的一種機制。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R589.2;R580
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