ZIP1蛋白对酒精诱导的兔BMSCs成脂基因PPARγ、aP2的影响研究
发布时间：2018-09-08 18:19
【摘要】：目的:酒精性股骨头坏死是因为长期大量酗酒而引起的一种非创伤性的股骨头坏死,是以中青年男性患者居多的骨科常见疾病。脂质紊乱及脂肪分化异常是导致酒精性股骨头坏死的重要机制,锌铁调控蛋白1(ZIP1)作为锌离子的转运体,能将锌离子转运到细胞内,促进骨髓间充质干细胞的成骨分化,但ZIP1蛋白是否能通过影响骨髓间充质干细胞分化,从而抑制脂肪细胞的形成尚不明确,本实验将探讨ZIP1蛋白对兔骨髓间充质干细胞成脂分化的影响,从而为酒精性股骨头坏死的防治提供思路。方法:1、取兔骨髓间充质干细胞进行复苏,再用完全培养基培养,传代培养后获得状态良好的骨髓间充质干细胞,定向诱导骨髓间充质干细胞并进行油红“O”染色,分析骨髓间充质干细胞成脂分化能力;设计合成ZIP1siRNA1、2、3三条序列,并兔转染骨髓间充质干细胞,RT-PCR检测骨髓间充质干细胞中ZIP1m RNA表达量,筛选ZIP1siRNA最佳序列;同时设计合成ZIP1目的片段,构建ZIP1表达载体,予FD Kpn I和FD Eco R限制性内切酶进行双切验证和重组载体测序,将成功构建的重组载体转染兔骨髓间充质干细胞。2、先将骨髓间充质干细胞分为9组,即正常对照组、模型组(0.03mol/L酒精组、0.09mol/L酒精组、0.15 mol/L酒精组、0.21mol/L酒精组)、实验组(0.03mol/L酒精+ZIP1siRNA组、0.09mol/L酒精+ZIP1siRNA组、0.15mol/L酒精+ZIP1siRNA组、0.21mol/L酒精+ZIP1siRNA组)。正常对照组不做任何处理正常培养,模型组分别用含有0.03mol/L、0.09mol/L、0.15mol/L、0.21mol/L酒精浓度的培养基进行培养,实验组予转染ZIP1siRNA序列后分别用含有0.03mol/L、0.09mol/L、0.15mol/L、0.21mol/L酒精浓度的培养基进行培养,每组均培养6h。采用试剂盒检测各组细胞内甘油三酯含量,记录甘油三酯含量变化,Western blotting分别检测各组骨髓间充质干细胞中PPARγ,aP2蛋白表达的含量。3、将骨髓间充质干细胞分为3组,正常对照组、ZIP1表达组、ZIP1siRNA组。正常对照组未经任何处理正常培养,表达转染组予ZIP1表达载体转染后正常培养,ZIP1siRNA转染组予ZIP1siRNA序列转染后正常培养,每组均培养6h。采用Western blotting方法分别检测各组骨髓间充质干细胞中PPARγ,aP2蛋白表达的含量。结果:1、在浓度为siRNA 25n M情况下ZIP1 siRNA序列1、2、3组与ZIP1 siRNA空转染序列组比较,差异具有统计学意义(p㩳0.05),但ZIP1siRNA序列2组ZIP1m RNA表达含量明显升高;ZIP1siRNA序列1、3组ZIP1m RNA表达含量明显降低,相比较ZIP1siRNA序列3组ZIP1m RNA表达含量更低。ZIP1表达载体经FD Kpn I和FD Eco RI双酶切和测序鉴定后,目的片段和重组载体插入片段完全一致重组载体构建成功。2、0.03mol/L酒精组、0.09mol/L酒精组、0.15mol/L酒精组、0.21mol/L酒精组中aP2、PPARγ蛋白表达量明显升高,与正常对照组比较差异有统计学意义(P㩳0.05);甘油三酯含量在0.03mol/L酒精组与正常对照组比较时无明显升高,差异无统计学意义(p㧐0.05)外,其他组与正常组比较差异均有统计学意义(P㩳0.05),其中0.21mol/L酒精组aP2、PPARγ蛋白表达量及甘油三酯含量最高,与其他组比较差异均有统计学意义(P㩳0.05);而在同一浓度为0.21 mol/L酒精培养下,0.21mol/L酒精+ZIP1siRNA组aP2、PPARγ蛋白表达含量及甘油三酯含量较0.21mol/L酒精组明显升高,差异均有统计学意义(P㩳0.05)。3、ZIP1表达组aP2、PPARγ蛋白表达含量明显降低,与正常对照组比较差异有统计学意义(P㩳0.05),ZIP1siRNA组aP2、PPARγ蛋白表达含量明显升高,与正常对照组比较差异有统计学意义(P㩳0.05)。结论:1、成功合成并筛选ZIP1siRNA序列3为最佳抑制序列,能够明显抑制ZIP1m RNA的表达,成功构建了表达ZIP1基因的载体,经酶切和测序鉴定正确。2、酒精能够诱导骨髓间充质干细胞成脂分化,在一定范围内随着酒精浓度增加骨髓间充质干细胞成脂分化的能力增强,并且在酒精作用下ZIPsiRNA能促进骨髓间充质干细胞成脂分化。3、表达ZIP1基因能够抑制骨髓间充质干细胞成脂分化,沉默ZIP1基因能够促进骨髓间充质干细胞成脂分化。
[Abstract]:Objective: Alcoholic femoral head necrosis is a non-traumatic osteonecrosis of the femoral head caused by long-term heavy drinking. It is a common orthopaedic disease in young and middle-aged men. Lipid disorders and abnormal adipogenesis are important mechanisms leading to alcoholic head necrosis. Zinc-iron regulatory protein 1 (ZIP1) acts as a transporter of zinc ions. Zinc ions can be transported into cells to promote the osteogenic differentiation of bone marrow mesenchymal stem cells, but whether ZIP1 protein can inhibit the formation of adipocytes by affecting the differentiation of bone marrow mesenchymal stem cells is not clear. This experiment will explore the effect of ZIP1 protein on adipogenic differentiation of bone marrow mesenchymal stem cells in rabbits, so as to alcoholic femoral head. Methods: 1. Rabbit bone marrow mesenchymal stem cells (BMSCs) were resuscitated and cultured in complete medium. After subculture, BMSCs in good condition were obtained. BMSCs were induced to differentiate into adipogenic cells by oil red "O" staining, and ZIP1 was designed and synthesized. The expression of ZIP1m RNA in bone marrow mesenchymal stem cells was detected by RT-PCR, and the optimal sequence of ZIP1siRNA was screened. At the same time, the ZIP1 target fragment was designed and synthesized, and the ZIP1 expression vector was constructed. The recombinant vector was sequenced by restriction endonuclease of FD Kpn I and FD Eco R. Rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with recombinant vector. The BMSCs were divided into 9 groups: normal control group, model group (0.03 mol/L alcohol group, 0.09 mol/L alcohol group, 0.15 mol/L alcohol group, 0.21 mol/L alcohol group), experimental group (0.03 mol/L alcohol + ZIP1 siRNA group, 0.09 mol/L alcohol + ZIP1 siRNA group, 0.15 mol/L alcohol + IP1 RNA group, 0.2 mol/L alcohol + ZIP1 siRNA group). 1 mol/L alcohol + ZIP1 siRNA group). Normal control group was not treated with normal culture. Model group was cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. Experimental group was transfected with ZIP1 siRNA sequence and then cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. The content of intracellular triglyceride was detected by kit and the content of triglyceride was recorded. The expression of PPAR-gamma and aP2 protein in bone marrow mesenchymal stem cells was detected by Western blotting. The bone marrow mesenchymal stem cells were divided into three groups: normal control group, ZIP1 expression group and ZIP1 siRNA group. After normal culture, the expression transfection group was transfected with ZIP1 expression vector, and the ZIP1 siRNA transfection group was transfected with ZIP1 siRNA sequence. Each group was cultured for 6 hours. In the case of ZIP1 siRNA sequence 1,2,3 groups and ZIP1 siRNA empty transfection sequence group, the difference was statistically significant (p?0.05), but ZIP1 siRNA expression in two groups of ZIP1 siRNA sequence was significantly higher; ZIP1 siRNA expression in 1,3 groups of ZIP1 siRNA sequence was significantly lower than ZIP1 siRNA expression in three groups of ZIP1 siRNA sequence. After digestion and sequencing of I and FD Eco RI, the recombinant vectors were successfully constructed. 2,0.03 mol/L ethanol group, 0.09 mol/L ethanol group, 0.15 mol/L ethanol group, 0.21 mol/L ethanol group, the expression of aP2 and PPAR gamma protein was significantly higher than the normal control group (P?0.05). The content of triglyceride in 0.03 mol/L alcohol group was not significantly higher than that in the normal control group, and there was no significant difference (p?0.05). There was significant difference between the other groups and the normal group (P?0.05). Among them, the expression of aP2, PPAR-gamma protein and triglyceride content in 0.21 mol/L alcohol group were the highest, and there was significant difference between the other groups (P?0.05). In the same concentration of 0.21 mol/L alcohol culture, 0.21 mol/L alcohol + ZIP1 siRNA group aP2, PPAR gamma protein expression and triglyceride content were significantly higher than 0.21 mol/L alcohol group, the difference was statistically significant (P?0.05). 3, ZIP1 expression group aP2, PPAR gamma protein expression was significantly lower than the normal control group, the difference was statistically significant. Significance (P? 0.05), ZIP1 siRNA group aP2, PPAR gamma protein expression significantly increased, compared with the normal control group was statistically significant (P? 0.05). Conclusion: 1, ZIP1 siRNA sequence 3 was successfully synthesized and screened as the best inhibitory sequence, can significantly inhibit the expression of ZIP1m RNA, successfully constructed the expression vector of ZIP1 gene, identified by enzyme digestion and sequencing. Indeed, alcohol can induce adipogenic differentiation of bone marrow mesenchymal stem cells, and the ability of adipogenic differentiation of bone marrow mesenchymal stem cells increases with the increase of alcohol concentration in a certain range. ZIPsiRNA can promote adipogenic differentiation of bone marrow mesenchymal stem cells under the effect of alcohol. 3. Expression of ZIP1 gene can inhibit adipogenic differentiation of bone marrow mesenchymal stem cells. Silencing ZIP1 gene can promote the adipogenic differentiation of bone marrow mesenchymal stem cells.
【学位授予单位】：广西中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R274.9
本文编号：2231316
[Abstract]:Objective: Alcoholic femoral head necrosis is a non-traumatic osteonecrosis of the femoral head caused by long-term heavy drinking. It is a common orthopaedic disease in young and middle-aged men. Lipid disorders and abnormal adipogenesis are important mechanisms leading to alcoholic head necrosis. Zinc-iron regulatory protein 1 (ZIP1) acts as a transporter of zinc ions. Zinc ions can be transported into cells to promote the osteogenic differentiation of bone marrow mesenchymal stem cells, but whether ZIP1 protein can inhibit the formation of adipocytes by affecting the differentiation of bone marrow mesenchymal stem cells is not clear. This experiment will explore the effect of ZIP1 protein on adipogenic differentiation of bone marrow mesenchymal stem cells in rabbits, so as to alcoholic femoral head. Methods: 1. Rabbit bone marrow mesenchymal stem cells (BMSCs) were resuscitated and cultured in complete medium. After subculture, BMSCs in good condition were obtained. BMSCs were induced to differentiate into adipogenic cells by oil red "O" staining, and ZIP1 was designed and synthesized. The expression of ZIP1m RNA in bone marrow mesenchymal stem cells was detected by RT-PCR, and the optimal sequence of ZIP1siRNA was screened. At the same time, the ZIP1 target fragment was designed and synthesized, and the ZIP1 expression vector was constructed. The recombinant vector was sequenced by restriction endonuclease of FD Kpn I and FD Eco R. Rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with recombinant vector. The BMSCs were divided into 9 groups: normal control group, model group (0.03 mol/L alcohol group, 0.09 mol/L alcohol group, 0.15 mol/L alcohol group, 0.21 mol/L alcohol group), experimental group (0.03 mol/L alcohol + ZIP1 siRNA group, 0.09 mol/L alcohol + ZIP1 siRNA group, 0.15 mol/L alcohol + IP1 RNA group, 0.2 mol/L alcohol + ZIP1 siRNA group). 1 mol/L alcohol + ZIP1 siRNA group). Normal control group was not treated with normal culture. Model group was cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. Experimental group was transfected with ZIP1 siRNA sequence and then cultured with medium containing 0.03 mol/L, 0.09 mol/L, 0.15 mol/L, 0.21 mol/L alcohol concentration respectively. The content of intracellular triglyceride was detected by kit and the content of triglyceride was recorded. The expression of PPAR-gamma and aP2 protein in bone marrow mesenchymal stem cells was detected by Western blotting. The bone marrow mesenchymal stem cells were divided into three groups: normal control group, ZIP1 expression group and ZIP1 siRNA group. After normal culture, the expression transfection group was transfected with ZIP1 expression vector, and the ZIP1 siRNA transfection group was transfected with ZIP1 siRNA sequence. Each group was cultured for 6 hours. In the case of ZIP1 siRNA sequence 1,2,3 groups and ZIP1 siRNA empty transfection sequence group, the difference was statistically significant (p?0.05), but ZIP1 siRNA expression in two groups of ZIP1 siRNA sequence was significantly higher; ZIP1 siRNA expression in 1,3 groups of ZIP1 siRNA sequence was significantly lower than ZIP1 siRNA expression in three groups of ZIP1 siRNA sequence. After digestion and sequencing of I and FD Eco RI, the recombinant vectors were successfully constructed. 2,0.03 mol/L ethanol group, 0.09 mol/L ethanol group, 0.15 mol/L ethanol group, 0.21 mol/L ethanol group, the expression of aP2 and PPAR gamma protein was significantly higher than the normal control group (P?0.05). The content of triglyceride in 0.03 mol/L alcohol group was not significantly higher than that in the normal control group, and there was no significant difference (p?0.05). There was significant difference between the other groups and the normal group (P?0.05). Among them, the expression of aP2, PPAR-gamma protein and triglyceride content in 0.21 mol/L alcohol group were the highest, and there was significant difference between the other groups (P?0.05). In the same concentration of 0.21 mol/L alcohol culture, 0.21 mol/L alcohol + ZIP1 siRNA group aP2, PPAR gamma protein expression and triglyceride content were significantly higher than 0.21 mol/L alcohol group, the difference was statistically significant (P?0.05). 3, ZIP1 expression group aP2, PPAR gamma protein expression was significantly lower than the normal control group, the difference was statistically significant. Significance (P? 0.05), ZIP1 siRNA group aP2, PPAR gamma protein expression significantly increased, compared with the normal control group was statistically significant (P? 0.05). Conclusion: 1, ZIP1 siRNA sequence 3 was successfully synthesized and screened as the best inhibitory sequence, can significantly inhibit the expression of ZIP1m RNA, successfully constructed the expression vector of ZIP1 gene, identified by enzyme digestion and sequencing. Indeed, alcohol can induce adipogenic differentiation of bone marrow mesenchymal stem cells, and the ability of adipogenic differentiation of bone marrow mesenchymal stem cells increases with the increase of alcohol concentration in a certain range. ZIPsiRNA can promote adipogenic differentiation of bone marrow mesenchymal stem cells under the effect of alcohol. 3. Expression of ZIP1 gene can inhibit adipogenic differentiation of bone marrow mesenchymal stem cells. Silencing ZIP1 gene can promote the adipogenic differentiation of bone marrow mesenchymal stem cells.
【学位授予单位】：广西中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R274.9
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