TNF-α基因shRNA影响脊柱结核破骨细胞形成的实验研究

发布时间：2018-03-30 14:04

  本文选题：基因敲除技术　切入点：肿瘤坏死因子　出处：《宁夏医科大学》2017年硕士论文

【摘要】：目的构建靶向TNF-α基因的shRNA载体,观察其对脊柱结核破骨细胞形成的影响,为后续脊柱结核骨质破坏的干预性研究奠定基础。方法结核菌素纯蛋白衍生物(PPD)诱导小鼠RAW264.7细胞,采用细胞增殖与毒性(CCK)试验检测PPD对细胞增殖的影响,抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞的形成,比较不同剂量(0、2、20、50、100μL)及诱导时间(1、4、7天)条件下,破骨细胞形成的差异。通过q PCR和Western Blotting检测PPD诱导1、4、7天TNF-α的表达。双酶切法构建靶向TNF-α基因的shRNA,经脂质体转染RAW264.7细胞,荧光显微镜下观察转染效率,RT-PCR观察转染后1、3、5、7天TNF-α基因的表达,收集转染后第三天的细胞,q PCR检测转染后TNF-α和RANK基因的表达,Western Blotting检测TNF-α蛋白的表达,TRAP染色计数转染后第7天破骨细胞形成的数量。结果PPD抑制RAW264.7细胞的增殖;TRAP染色可见破骨细胞形成;不同剂量PPD诱导破骨细胞形成的数量:PPD 0μL组为(58.067.1±)个、2μL组(58.067.4±)个、20μL组(52.067.16±)个、50μL组(89.267.10±)个、100μL组(58.033.1±)个;20μL PPD诱导1、4、7天破骨细胞形成的数量:1天组为(1.50±0.55)个、4天组为(7.50±1.87)个、7天组为(17.33±2.07)个;PPD诱导1、4、7天TNF-α基因的表达量为:对照组:(1±0),1天组:(4.267±0.737),4天组:(13.60±0.648),7天组:(14.07±1.013),TNF-α蛋白的表达量,对照组为:(0.066±0.004),1天组:(0.081±0.005),4天组:(0.162±0.003),7天组:(0.179±0.005);转染前后TNF-α基因的表达量:转染前为(1.426±0.086),转染后为(0.464±0.029);转染前后RANK基因的表达量:转染前为(1.393±0.007),转染后为(1.154±0.006);转染前后TNF-α蛋白的表达量:转染前为(82.72±1.843),转染后为(55.34±0.824);转染前后破骨细胞形成的数量:转染前为(56.67±3.786)个,转染后为(19.33±1.528)个。结论PPD可以诱导破骨细胞形成,且破骨细胞形成与PPD之间只有时间依赖性,无剂量依赖性;TNF-α在脊柱结核破骨细胞的形成中起着重要的作用,TNF-α基因shRNA能使TNF-α的表达下调,并抑制破骨细胞的形成。
[Abstract]:Objective to construct shRNA vector targeting TNF- 伪 gene and observe its effect on osteoclast formation of spinal tuberculosis, and to lay a foundation for further study on bone destruction of spinal tuberculosis. Methods the purified protein derivative of tuberculin induced RAW264.7 cells in mice. The effect of PPD on cell proliferation was detected by cell proliferation and toxicity test, and osteoclast formation was observed by tartrate-resistant acid phosphatase (TRAP) staining. The difference of osteoclast formation. The expression of TNF- 伪 was detected by Q PCR and Western Blotting. The expression of TNF- 伪 was induced by PPD for 7 days. ShRNAs targeting TNF- 伪 gene were constructed by double enzyme digestion and transfected into RAW264.7 cells by liposome. The transfection efficiency was observed under fluorescence microscope and the expression of TNF- 伪 gene was detected by RT-PCR on the 7th day after transfection. The expression of TNF- 伪 and RANK genes was detected by PCR on the third day after transfection. The expression of TNF- 伪 protein was detected by Western Blotting. The number of osteoclasts formed on the 7th day after transfection was counted. Results PPD inhibited the proliferation of RAW264.7 cells by trap staining. Osteoclast formation could be seen in color. The number of osteoclast formation induced by different doses of PPD was 58.067.1 卤2 渭 L, 58.067.4 卤) 20 渭 L, 52.067.16 卤) 50 渭 L, 58.033.1 卤) 20 渭 L PPD induced osteoclast formation on the 1st day, 1.50 卤0.55), 7.50 卤1.87, 7.50 卤1.87, 17.33 卤2.07, respectively. The expression of TNF- 伪 gene in the control group was 4.267 卤0.737 and the expression of TNF- 伪 protein in the control group was 4.267 卤0.737 and the expression of TNF- 伪 protein in the control group was 14.07 卤1.013 and the expression of TNF- 伪 protein in the control group was 14.07 卤1.013 and the expression of TNF- 伪 protein in the control group was 14.07 卤1.013 and the expression of TNF- 伪 protein in the control group was 4.267 卤0.737. The expression of TNF- 伪 gene before and after transfection was 1.426 卤0.086 and 0.464 卤0.029 before and after transfection. The expression of RANK gene before and after transfection was 1.393 卤0.007 and 1.154 卤66.The expression of TNF- 伪 protein before and after transfection was 1.393 卤0.007 and 1.154 卤6 respectively before and after transfection: the expression of TNF- 伪 gene before and after transfection was 1. 393 卤0. 007, and before and after transfection, the expression of TNF- 伪 gene in the control group was 0. 066 卤0. 004 卤0. 029; before and after transfection, the expression of TNF- 伪 gene was 1. 426 卤0. 086 and 0. 464 卤0. 029; before and after transfection, the expression of TNF- 伪 protein:. The number of osteoclasts before and after transfection was 82.72 卤1.843 and 55.34 卤0.824, and the number of osteoclasts before and after transfection was 56.67 卤3.786, respectively. Conclusion PPD can induce osteoclast formation, and there is only a time dependent relationship between osteoclast formation and PPD. TNF- 伪 plays an important role in the formation of osteoclasts in spinal tuberculosis. ShRNA of TNF- 伪 gene can down-regulate the expression of TNF- 伪 and inhibit the formation of osteoclasts.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R529.2

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