本文選題：基因敲除技術　切入點：腫瘤壞死因子　出處：《寧夏醫(yī)科大學》2017年碩士論文

【摘要】：目的構建靶向TNF-α基因的shRNA載體,觀察其對脊柱結核破骨細胞形成的影響,為后續(xù)脊柱結核骨質破壞的干預性研究奠定基礎。方法結核菌素純蛋白衍生物(PPD)誘導小鼠RAW264.7細胞,采用細胞增殖與毒性(CCK)試驗檢測PPD對細胞增殖的影響,抗酒石酸酸性磷酸酶(TRAP)染色觀察破骨細胞的形成,比較不同劑量(0、2、20、50、100μL)及誘導時間(1、4、7天)條件下,破骨細胞形成的差異。通過q PCR和Western Blotting檢測PPD誘導1、4、7天TNF-α的表達。雙酶切法構建靶向TNF-α基因的shRNA,經(jīng)脂質體轉染RAW264.7細胞,熒光顯微鏡下觀察轉染效率,RT-PCR觀察轉染后1、3、5、7天TNF-α基因的表達,收集轉染后第三天的細胞,q PCR檢測轉染后TNF-α和RANK基因的表達,Western Blotting檢測TNF-α蛋白的表達,TRAP染色計數(shù)轉染后第7天破骨細胞形成的數(shù)量。結果PPD抑制RAW264.7細胞的增殖;TRAP染色可見破骨細胞形成;不同劑量PPD誘導破骨細胞形成的數(shù)量:PPD 0μL組為(58.067.1±)個、2μL組(58.067.4±)個、20μL組(52.067.16±)個、50μL組(89.267.10±)個、100μL組(58.033.1±)個;20μL PPD誘導1、4、7天破骨細胞形成的數(shù)量:1天組為(1.50±0.55)個、4天組為(7.50±1.87)個、7天組為(17.33±2.07)個;PPD誘導1、4、7天TNF-α基因的表達量為:對照組:(1±0),1天組:(4.267±0.737),4天組:(13.60±0.648),7天組:(14.07±1.013),TNF-α蛋白的表達量,對照組為:(0.066±0.004),1天組:(0.081±0.005),4天組:(0.162±0.003),7天組:(0.179±0.005);轉染前后TNF-α基因的表達量:轉染前為(1.426±0.086),轉染后為(0.464±0.029);轉染前后RANK基因的表達量:轉染前為(1.393±0.007),轉染后為(1.154±0.006);轉染前后TNF-α蛋白的表達量:轉染前為(82.72±1.843),轉染后為(55.34±0.824);轉染前后破骨細胞形成的數(shù)量:轉染前為(56.67±3.786)個,轉染后為(19.33±1.528)個。結論PPD可以誘導破骨細胞形成,且破骨細胞形成與PPD之間只有時間依賴性,無劑量依賴性;TNF-α在脊柱結核破骨細胞的形成中起著重要的作用,TNF-α基因shRNA能使TNF-α的表達下調(diào),并抑制破骨細胞的形成。
[Abstract]:Objective to construct shRNA vector targeting TNF- 偽 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- 偽 was detected by Q PCR and Western Blotting. The expression of TNF- 偽 was induced by PPD for 7 days. ShRNAs targeting TNF- 偽 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- 偽 gene was detected by RT-PCR on the 7th day after transfection. The expression of TNF- 偽 and RANK genes was detected by PCR on the third day after transfection. The expression of TNF- 偽 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 鹵2 渭 L, 58.067.4 鹵) 20 渭 L, 52.067.16 鹵) 50 渭 L, 58.033.1 鹵) 20 渭 L PPD induced osteoclast formation on the 1st day, 1.50 鹵0.55), 7.50 鹵1.87, 7.50 鹵1.87, 17.33 鹵2.07, respectively. The expression of TNF- 偽 gene in the control group was 4.267 鹵0.737 and the expression of TNF- 偽 protein in the control group was 4.267 鹵0.737 and the expression of TNF- 偽 protein in the control group was 14.07 鹵1.013 and the expression of TNF- 偽 protein in the control group was 14.07 鹵1.013 and the expression of TNF- 偽 protein in the control group was 14.07 鹵1.013 and the expression of TNF- 偽 protein in the control group was 4.267 鹵0.737. The expression of TNF- 偽 gene before and after transfection was 1.426 鹵0.086 and 0.464 鹵0.029 before and after transfection. The expression of RANK gene before and after transfection was 1.393 鹵0.007 and 1.154 鹵66.The expression of TNF- 偽 protein before and after transfection was 1.393 鹵0.007 and 1.154 鹵6 respectively before and after transfection: the expression of TNF- 偽 gene before and after transfection was 1. 393 鹵0. 007, and before and after transfection, the expression of TNF- 偽 gene in the control group was 0. 066 鹵0. 004 鹵0. 029; before and after transfection, the expression of TNF- 偽 gene was 1. 426 鹵0. 086 and 0. 464 鹵0. 029; before and after transfection, the expression of TNF- 偽 protein:. The number of osteoclasts before and after transfection was 82.72 鹵1.843 and 55.34 鹵0.824, and the number of osteoclasts before and after transfection was 56.67 鹵3.786, respectively. Conclusion PPD can induce osteoclast formation, and there is only a time dependent relationship between osteoclast formation and PPD. TNF- 偽 plays an important role in the formation of osteoclasts in spinal tuberculosis. ShRNA of TNF- 偽 gene can down-regulate the expression of TNF- 偽 and inhibit the formation of osteoclasts.
【學位授予單位】：寧夏醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R529.2

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