Lefty1抗积水肾损伤及其机制研究

发布时间：2018-06-22 13:50

本文选题：leftyl蛋白 + 纤维化　；参考：《武汉大学》2014年博士论文

【摘要】：第一部分目的构建leftyl基因与pcDNA3.1(+)相融合的重组质粒,在leftyl基因的下游加上flag标签,并探讨重组质粒在体内外能否有效表达。方法设计并合成leftyl基因上下游引物,同时加上flag标签序列,PCR扩增基因,并将其连接到真核表达载体pcDNA3.1(+)上,双酶切图谱分析和扩增产物测序鉴定所构建的真核表达载体。脂质体转染法将重组质粒pcDNA3.1(+)和pcDNA3.1(+)-leftyl-flag转染至肾小管上皮细胞株HK-2,免疫印迹检测lefty1、 flag蛋白的表达。通过流体力学的方法将leftyl基因转至输尿管梗阻的小鼠体内,mRNA和蛋白水平检测leftyl,蛋白水平检测flag在小鼠肝脏和肾脏的表达,ELISA检测血循环中leftyl蛋白的水平。结果以leftyl基因质粒模板扩增得到的片段约1150bp,双酶切得到目的基因片段,将目的基因片段与pcDNA3.1(+)连接,双酶切分析及测序显示与leftyl基因序列相同。肾小管上皮细胞质粒转染后,pcDNA3.1(+)-lefty1-flag较pcDNA3.1(+)转染的肾小管上皮细胞leftyl表达水平上调,仅pcDNA3.1(+)-lefty1-flag转染组表达flag蛋白。小鼠体内基因转移后,pcDNA3.1(+)-lefty1-flag较pcDNA3.1(+)转染组肾脏和肝脏中leftyl表达水平升高明显,仅pcDN A3.1(+)-lefty1-flag转染组在肝脏和肾脏表达flag蛋白。ELISA法检测了循环中leftyl蛋白含量显示pcDNA3.1(+)-lefty1-flag较pcDNA3.1(+)转染组血液中leftyl蛋白含量明显增加。结论成功构建leftyl基因真核表达载体,在肾小管上皮细胞和小鼠体内pcDNA3.1(+)-lefty1-flag能成功转染真核细胞并表达leftyl蛋白。第二部分目的研究leftyl蛋白对肾小管损伤和间质纤维化的影响,并探讨可能机制。方法建立左侧输尿管完全梗阻肾损伤模型,通过流体力学的方法将pcDNA3.1(+)和pcDN A3.1(+)-lefty1-flag转至小鼠体内。HE染色和PAS染色检测leftyl蛋白对肾小管损伤的影响。肾脏PSR染色、qPCR和免疫组化检测胶原Ⅰ和纤维粘粘蛋白以检测leftyl蛋白对肾脏间质纤维沉积的影响。免疫组化和免疫印迹检测leftyl蛋白对肾小管上皮间质转化相关分子E-cadherin和a-SMA的影响。免疫印迹检测leftyl蛋白对肾脏TGF-pl和p-Smad3的影响。结果输尿管梗阻+pcDNA3.1(+)处理组肾小管损伤评分为2.244±0.72,PSR染色间质纤维面积百分比为12.58±1.98%,胶原Ⅰ和纤维粘粘蛋白相对表达量分别为6.44±1.57、8.59±2.10, E-cadherin和a-SMA的相对表达量分别为0.16±0.05、1.23±0.16, TGF-β1和p-Smad3的相对表达量分别为1.26±0.15、1.23±0.09,以上个各指标与假手术组相比P0.05。而输尿管梗阻+pcDNA3.1(+)-lefty1-flag处理组肾小管损伤评分为1.42±0.61,PSR染色间质纤维面积百分比为7.17±1.20%,胶原Ⅰ和纤维粘粘蛋白相对表达量分别为3.53±0.95、4.04±2.08,E-cadherin和α-SMA的相对表达量分别为0.52±0.11、0.72±0.27,TGF-β1和p-Smad3的相对表达量分别为0.68±0.10、0.74±0.16,以上各指标与输尿管梗阻+pcDNA3.1(+)处理组相比P0.05。结论Leftyl能拮抗输尿管梗阻引起的肾小管损伤和间质纤维化。其效果可能是由其抑制smad依赖性的TGF-β1信号通路所介导。第三部分目的研究leftyl蛋白的对肾脏小管间质炎症的影响,并探讨可能机制。方法建立左侧输尿管完全梗阻肾损伤模型,通过流体力学的方法将pcDNA3.1(+)和pcDNA3.1(+)-lefty1-flag转移至小鼠体内。免疫组化检测肾间质巨噬细胞和T淋巴细胞。免疫组化检测p65的核转位。免疫印迹检测p-p65、趋化因子CCL2和CCL5、细胞因子TNF-α和IL-1β的表达。结果输尿管梗阻+pcDNA3.1(+)处理组肾间质巨噬细胞和T淋巴细胞细胞数分别为23.90±6.31/视野、10.20±3.20/视野,肾脏p-p65阳性细胞数为52.40±11.27/视野,p-p65的相对表达量为0.13±0.04, CCL2和CCL5相对表达量分别为1.23±0.18、0.80±0.12, TNF-α和IL-1β相对表达量分别为1.17±0.10、1.21±0.11,以上个各指标与假手术组相比P0.05。而输尿管梗阻+pcDN A3.1(+)-lefty1-flag处理组肾脏间质巨噬细胞和T淋巴细胞细胞数分别为8.50±3.50/视野、5.50±1.90/视野,肾脏p-p65阳性细胞数为23.80±4.78/视野,p-p65的相对表达量为0.08±0.02,CCL2和CCL5相对表达量分别为0.64±0.17、0.23±0.06,TNF-α和IL-1β相对表达量分别为0.64±0.10、0.68±0.12,各指标与输尿管梗阻-pcDNA3.1(+)处理组相比P0.05。结论leftyl蛋白能拮抗输尿管梗阻引起的肾脏小管间质炎症,其机制可能与其抑制肾间质巨噬细胞和T淋巴细胞浸润有关。
[Abstract]:Part one
Objective to construct a recombinant plasmid containing leftyl gene and pcDNA3.1 (+), and to tag the downstream leftyl gene with flag, and to explore the effective expression of the recombinant plasmid in vivo and in vitro.
Methods the leftyl gene upstream and downstream primers were designed and synthesized. At the same time, the flag tagged sequence was added, and the gene was amplified by PCR, and was connected to the eukaryotic expression vector pcDNA3.1 (+). The eukaryotic expression vector was constructed by the analysis of the double enzyme cutting map and the sequencing of the amplified products. The recombinant plasmid pcDNA3.1 (+) and pcDNA3.1 (+) -leftyl-flag were transferred by the liposome transfection method. The renal tubular epithelial cell line HK-2 was dyed, and the expression of lefty1 and flag protein was detected by immunoblotting. The leftyl gene was transferred to the mice of ureteral obstruction by fluid mechanics. The mRNA and protein levels were detected by leftyl, the protein level was detected in the expression of flag in the liver and kidney of the mice, and the level of leftyl protein in the blood circulation was detected by ELISA.
Results the fragment of the leftyl gene plasmid template was 1150bp, the target gene fragment was cut by double enzyme, the target gene fragment was connected with the pcDNA3.1 (+). The double enzyme digestion analysis and sequencing showed that the leftyl gene sequence was the same. After the transfection of the renal tubular epithelial cell plasmid, the pcDNA3.1 (+) -lefty1-flag was thinner than the pcDNA3.1 (+) transfected renal tubule epithelium. The expression level of cell leftyl was up, only pcDNA3.1 (+) -lefty1-flag transfection group expressed flag protein. After gene transfer in mice, the expression level of leftyl (+) -lefty1-flag in the kidneys and liver of the transfected group was obviously higher than that of pcDNA3.1 (+) transfected group. Only pcDN A3.1 (+) -lefty1-flag transfer group expressed the flag protein in the liver and kidney to detect the circulation. The content of leftyl protein showed that pcDNA3.1 (+) -lefty1-flag increased significantly compared with pcDNA3.1 (+) transfection group.
Conclusion the eukaryotic expression vector of leftyl gene is successfully constructed. The eukaryotic cells and leftyl protein can be transfected successfully in the renal tubular epithelial cells and the pcDNA3.1 (+) -lefty1-flag in mice.
The second part
Objective to study the effect of leftyl protein on renal tubular injury and interstitial fibrosis, and to explore the possible mechanism.
Methods pcDNA3.1 (+) and pcDN A3.1 (+) -lefty1-flag were transferred to the mice with.HE staining and PAS staining to detect the effects of leftyl protein on renal tubule injury. Renal PSR staining, qPCR and immunohistochemical detection of collagen I and fibronectin were used to detect leftyl eggs. Effect of white on renal interstitial fibrous deposition. The effects of leftyl protein on renal tubuloepithelial mesenchymal transition related molecules E-cadherin and a-SMA were detected by immunohistochemistry and Western blot. The influence of leftyl protein on renal TGF-pl and p-Smad3 was detected by immunoblot.
Results the score of renal tubular injury in +pcDNA3.1 (+) treatment group was 2.244 + 0.72, the percentage of interstitial fiber area in PSR staining was 12.58 + 1.98%, the relative expression of collagen I and fibrin mucin was 6.44 + 1.57,8.59 + 2.10 respectively. The relative expressions of E-cadherin and a-SMA were 0.16 + 0.05,1.23 + 0.16, TGF- beta 1 and p-Smad3, respectively. The expression amount was 1.26 + 0.15,1.23 + 0.09 respectively. The above indexes were compared with the sham group P0.05., and the renal tubular injury score of the +pcDNA3.1 (+) -lefty1-flag treatment group was 1.42 + 0.61, the percentage of PSR staining interstitial fiber area was 7.17 + 1.20%, and the relative expression of collagen I and fibronectin was 3.53 + 0.95,4.04 + 2, respectively. 8, the relative expression of E-cadherin and alpha -SMA was 0.52 + 0.11,0.72 + 0.27, and the relative expression of TGF- beta 1 and p-Smad3 was 0.68 + 0.10,0.74 + 0.16 respectively. The above indexes were compared with the +pcDNA3.1 (+) treatment group of ureteral obstruction.
Conclusion Leftyl can antagonize renal tubule injury and interstitial fibrosis caused by ureteral obstruction. The effect may be mediated by its inhibition of Smad dependent TGF- beta 1 signaling pathway.
The third part
Objective to study the effect of leftyl protein on tubulointerstitial inflammation and explore the possible mechanism.
Methods pcDNA3.1 (+) and pcDNA3.1 (+) -lefty1-flag were transferred to the mice by fluid mechanics. Immunohistochemistry was used to detect renal interstitial macrophages and T lymphocytes. Immunohistochemistry was used to detect the nuclear transposition of p65. Immunological trace was used to detect p-p65, chemokine CCL2 and CCL5, and cytokine TNF-. The expression of alpha and IL-1 beta.
Results the number of renal interstitial macrophages and T lymphocyte cells in +pcDNA3.1 (+) treatment group of ureteral obstruction was 23.90 + 6.31/ visual field, 10.20 + 3.20/ field of vision, and the number of p-p65 positive cells in kidney was 52.40 + 11.27/, the relative expression of p-p65 was 0.13 + 0.04, CCL2 and CCL5 relative expressions were 1.23 + 0.18,0.80 + 0.12, TNF- alpha and IL-1 beta phase The expression amount was 1.17 + 0.10,1.21 + 0.11 respectively. The above indexes were compared with the sham group P0.05. and the number of renal interstitial macrophages and T lymphocyte cells in the +pcDN A3.1 (+) -lefty1-flag treatment group of ureter obstruction were 8.50 + 3.50/ visual field, 5.50 + 1.90/ visual field, and the number of p-p65 positive cells in the kidney was 23.80 + 4.78/ field of vision, and the relative table of p-p65 was The relative expression of CCL2 and CCL5 was 0.64 + 0.17,0.23 + 0.06 respectively, and the relative expression of TNF- - and IL-1 beta was 0.64 + 0.10,0.68 + 0.12, respectively, and the indexes were compared with the -pcDNA3.1 (+) treatment group of ureteral obstruction.
Conclusion leftyl protein can antagonize the renal tubulointerstitial inflammation caused by ureteral obstruction, and its mechanism may be related to its inhibition of interstitial macrophages and T lymphocyte infiltration.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.5

【共引文献】