本文選題：leftyl蛋白 + 纖維化�。� 參考：《武漢大學》2014年博士論文

【摘要】：第一部分 目的構(gòu)建leftyl基因與pcDNA3.1(+)相融合的重組質(zhì)粒,在leftyl基因的下游加上flag標簽,并探討重組質(zhì)粒在體內(nèi)外能否有效表達。 方法設(shè)計并合成leftyl基因上下游引物,同時加上flag標簽序列,PCR擴增基因,并將其連接到真核表達載體pcDNA3.1(+)上,雙酶切圖譜分析和擴增產(chǎn)物測序鑒定所構(gòu)建的真核表達載體。脂質(zhì)體轉(zhuǎn)染法將重組質(zhì)粒pcDNA3.1(+)和pcDNA3.1(+)-leftyl-flag轉(zhuǎn)染至腎小管上皮細胞株HK-2,免疫印跡檢測lefty1、 flag蛋白的表達。通過流體力學的方法將leftyl基因轉(zhuǎn)至輸尿管梗阻的小鼠體內(nèi),mRNA和蛋白水平檢測leftyl,蛋白水平檢測flag在小鼠肝臟和腎臟的表達,ELISA檢測血循環(huán)中l(wèi)eftyl蛋白的水平。 結(jié)果以leftyl基因質(zhì)粒模板擴增得到的片段約1150bp,雙酶切得到目的基因片段,將目的基因片段與pcDNA3.1(+)連接,雙酶切分析及測序顯示與leftyl基因序列相同。腎小管上皮細胞質(zhì)粒轉(zhuǎn)染后,pcDNA3.1(+)-lefty1-flag較pcDNA3.1(+)轉(zhuǎn)染的腎小管上皮細胞leftyl表達水平上調(diào),僅pcDNA3.1(+)-lefty1-flag轉(zhuǎn)染組表達flag蛋白。小鼠體內(nèi)基因轉(zhuǎn)移后,pcDNA3.1(+)-lefty1-flag較pcDNA3.1(+)轉(zhuǎn)染組腎臟和肝臟中l(wèi)eftyl表達水平升高明顯,僅pcDN A3.1(+)-lefty1-flag轉(zhuǎn)染組在肝臟和腎臟表達flag蛋白。ELISA法檢測了循環(huán)中l(wèi)eftyl蛋白含量顯示pcDNA3.1(+)-lefty1-flag較pcDNA3.1(+)轉(zhuǎn)染組血液中l(wèi)eftyl蛋白含量明顯增加。 結(jié)論成功構(gòu)建leftyl基因真核表達載體,在腎小管上皮細胞和小鼠體內(nèi)pcDNA3.1(+)-lefty1-flag能成功轉(zhuǎn)染真核細胞并表達leftyl蛋白。 第二部分 目的研究leftyl蛋白對腎小管損傷和間質(zhì)纖維化的影響,并探討可能機制。 方法建立左側(cè)輸尿管完全梗阻腎損傷模型,通過流體力學的方法將pcDNA3.1(+)和pcDN A3.1(+)-lefty1-flag轉(zhuǎn)至小鼠體內(nèi)。HE染色和PAS染色檢測leftyl蛋白對腎小管損傷的影響。腎臟PSR染色、qPCR和免疫組化檢測膠原Ⅰ和纖維粘粘蛋白以檢測leftyl蛋白對腎臟間質(zhì)纖維沉積的影響。免疫組化和免疫印跡檢測leftyl蛋白對腎小管上皮間質(zhì)轉(zhuǎn)化相關(guān)分子E-cadherin和a-SMA的影響。免疫印跡檢測leftyl蛋白對腎臟TGF-pl和p-Smad3的影響。 結(jié)果輸尿管梗阻+pcDNA3.1(+)處理組腎小管損傷評分為2.244±0.72,PSR染色間質(zhì)纖維面積百分比為12.58±1.98%,膠原Ⅰ和纖維粘粘蛋白相對表達量分別為6.44±1.57、8.59±2.10, E-cadherin和a-SMA的相對表達量分別為0.16±0.05、1.23±0.16, TGF-β1和p-Smad3的相對表達量分別為1.26±0.15、1.23±0.09,以上個各指標與假手術(shù)組相比P0.05。而輸尿管梗阻+pcDNA3.1(+)-lefty1-flag處理組腎小管損傷評分為1.42±0.61,PSR染色間質(zhì)纖維面積百分比為7.17±1.20%,膠原Ⅰ和纖維粘粘蛋白相對表達量分別為3.53±0.95、4.04±2.08,E-cadherin和α-SMA的相對表達量分別為0.52±0.11、0.72±0.27,TGF-β1和p-Smad3的相對表達量分別為0.68±0.10、0.74±0.16,以上各指標與輸尿管梗阻+pcDNA3.1(+)處理組相比P0.05。 結(jié)論Leftyl能拮抗輸尿管梗阻引起的腎小管損傷和間質(zhì)纖維化。其效果可能是由其抑制smad依賴性的TGF-β1信號通路所介導。 第三部分 目的研究leftyl蛋白的對腎臟小管間質(zhì)炎癥的影響,并探討可能機制。 方法建立左側(cè)輸尿管完全梗阻腎損傷模型,通過流體力學的方法將pcDNA3.1(+)和pcDNA3.1(+)-lefty1-flag轉(zhuǎn)移至小鼠體內(nèi)。免疫組化檢測腎間質(zhì)巨噬細胞和T淋巴細胞。免疫組化檢測p65的核轉(zhuǎn)位。免疫印跡檢測p-p65、趨化因子CCL2和CCL5、細胞因子TNF-α和IL-1β的表達。 結(jié)果輸尿管梗阻+pcDNA3.1(+)處理組腎間質(zhì)巨噬細胞和T淋巴細胞細胞數(shù)分別為23.90±6.31/視野、10.20±3.20/視野,腎臟p-p65陽性細胞數(shù)為52.40±11.27/視野,p-p65的相對表達量為0.13±0.04, CCL2和CCL5相對表達量分別為1.23±0.18、0.80±0.12, TNF-α和IL-1β相對表達量分別為1.17±0.10、1.21±0.11,以上個各指標與假手術(shù)組相比P0.05。而輸尿管梗阻+pcDN A3.1(+)-lefty1-flag處理組腎臟間質(zhì)巨噬細胞和T淋巴細胞細胞數(shù)分別為8.50±3.50/視野、5.50±1.90/視野,腎臟p-p65陽性細胞數(shù)為23.80±4.78/視野,p-p65的相對表達量為0.08±0.02,CCL2和CCL5相對表達量分別為0.64±0.17、0.23±0.06,TNF-α和IL-1β相對表達量分別為0.64±0.10、0.68±0.12,各指標與輸尿管梗阻-pcDNA3.1(+)處理組相比P0.05。 結(jié)論leftyl蛋白能拮抗輸尿管梗阻引起的腎臟小管間質(zhì)炎癥,其機制可能與其抑制腎間質(zhì)巨噬細胞和T淋巴細胞浸潤有關(guān)。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R692.5

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