eIF6对小鼠纤维化影响的实验研究

发布时间：2018-04-26 11:28

  本文选题：eIF6 + TGF-β1　； 参考：《第三军医大学》2014年硕士论文

【摘要】：纤维化是一个多因素共同参与的复杂过程，是各种器官组织不可逆损伤修复的共同标志性病理变化。其具体表现为组织间质成纤维细胞增生，成纤维细胞表达α-SMA（α-smooth muscle actin，α-平滑肌肌动蛋白），分化成肌成纤维细胞，分泌大量细胞外基质，并导致组织结构破坏，器官功能丧失。目前的研究发现，TGF-β1（Transforminggrowth factor-β1，转化生长因子β1）是一个在组织纤维化进程中十分重要的细胞因子，它参与了各个器官炎症反应及纤维化。迄今为止，临床上尚无治疗组织纤维化的有效药物。因此，研究纤维化分子机制、研制控制纤维化的有效药物是临床迫切需要解决的重大科学问题。 有文献报道eIF6(eukaryotic initiation factor6，真核启动因子6)可能参与调控组织纤维化过程。eIF6作为一种核基质蛋白，参与核糖体的装配，并可转位至细胞核中参与翻译起始调控，并广泛表达于成纤维细胞及内皮细胞等。本研究拟通过eIF6+/-基因敲降小鼠的皮肤和肾脏纤维化模型，研究eIF6在组织纤维化中可能的作用。 研究方法： eIF6+/-基因敲降小鼠SPF（Specific pathogen Free，无特定病原体）级eIF6+/-小鼠（引进于San Raffaele Scientific Institute, Milan, Italy）和对照野生型小鼠，由第三军医大学附属大坪医院动物所饲养。每次实验3-8对小鼠。选取体重20±2g，8-10周龄，雄性小鼠。eIF6+/-小鼠因基因表型呈褐色，野生型呈黑色，均为C57BL/6背景。 动物模型 2.1小鼠皮肤缺损模型 小鼠麻醉后（n=8），刮去小鼠背部毛发，70%乙醇消毒皮肤；以4.5mm为直径，用打孔器造对称2个圆形创面，全层切除小鼠皮肤；以数码相机垂直创面，同时在创面旁放置参照物，数码相机镜头距离创面15cm，微距模式下采取创面图像；采用Image-Pro Plus6.0软件(美国Media Cybernetics软件公司)测量创面面积。每个时间点的创面实际大小利用参照物校正。愈合过程中，创面的变化用不同时相点的创面面积与原始创面面积之比的百分数表示。 2.2.小鼠皮肤线性模型 小鼠麻醉后（n=6），刮去小鼠背部毛发，酒精消毒皮肤；在小鼠侧腹壁，垂直于脊柱平面，标记2个对称的8mm长线性创口，无菌手术刀全层划开小鼠皮肤；以数码相机垂直创面，同时在创面旁放置参照物，镜头距离创面15cm下采取创面图像；采用IPP6.0软件测量创面面积，愈合过程中，创面的变化用不同时相点的创面面积与原始创面面积之比的百分数表示。 2.3小鼠UUO模型 腹腔注射麻醉小鼠（n=6），腹部脱毛，酒精消毒。通过腹部正中切口，暴露左侧输尿管；缝线分别结扎上1/3和2/3分界处2次，完全阻断左侧输尿管；复位肾脏及肠管，分层缝合肌肉、皮肤，结扎后10天处死小鼠，取材（具体见研究方法3）。假手术对照组分离出左侧输尿管后不结扎,复位肾脏及肠管，10天后处死小鼠，取材。 3.组织样本制作与观察 3.1HE和Masson染色：皮肤创面组织取中间1/3,肾脏组织取垂直长轴方向的中间1/3，标本置于4%多聚甲醛固定，24小时后进行石蜡包埋，5μm厚度切片，苏木素伊红和Masson染色,光镜下观察病变情况，每张切片在200倍视野下随机选取5个视野获取图像，IPP6.0软件对图像进行定量测量,结果进行统计分析。 3.2TGFβ1、α-SMA免疫组织染色和定量分析 皮肤创面组织取中间1/3,肾脏组织取垂直长轴方向的中间1/3，4%多聚甲醛固定标本，24小时后常规脱水、石蜡包埋，5μm切片。切片常规脱蜡至水。分别进行免疫组织化学染色（TGF-β1稀释度：1:200；α-SMA稀释度：1:400）。小鼠肾脏组织的每张切片在400倍视野下随机选取5个组织的视野获取图像，IPP分别定量分析TGF-β1和α-SMA的表达强度，以AOD（Average optical density，平均光密度）表示阳性物质的含量，AOD的均值代表每只小鼠的表达量，结果进行统计分析。 3.3Western Blot检测TGFβ1、α-SMA、eIF6的表达 TGFβ1、α-SMA的检测以创面为中心取0.5×0.5cm组织，eIF6的检测取正常组织。提取蛋白，BCA法行蛋白定量，蛋白/泳道上样10μg，SDS-PAGE电泳分离，电转移至醋酸纤维薄膜，5%脱脂奶粉封闭90min，分别加入兔抗鼠单克隆抗体（TGF-β1,1:2000; α-SMA，1:2000; eIF6，1:1000），恒温冰箱4℃孵育过夜，洗膜，加入HRP标记的羊抗兔二抗（稀释度1:1500），室温孵育90min，显影，拍照。IPP6.0软件进行灰度扫描，IOD值代表条带信号的强弱，进行统计分析。 4.统计学处理 计量资料数据以x±s表示,用SPSS17.0统计软件进行t检验,以p 0.05为差异有统计学意义。 实验结果 1.基因敲降小鼠基因型鉴定和eIF6蛋白表达检测 1.1小鼠基因型的鉴定 小鼠DNA凝胶电泳结果中，小鼠DNA凝胶电泳在320bp和650bp两处同时显示亮带，，提示其基因型为eIF6+/-。DNA凝胶电泳仅在320bp显示亮带，提示其基因型为eIF6+/+。 1.2基因敲降小鼠正常皮肤和肾脏中eIF6的表达 WB结果验证了基因敲降小鼠正常皮肤中eIF6表达下降63%，肾脏下降50%。 2.低表达eIF6对小鼠皮肤损伤愈合的影响 2.1低表达eIF6导致创面的收缩增强 为了评估小鼠皮肤缺损模型的创面愈合情况，实验人员每24小时对小鼠的创面进行一次拍照，并用IPP6.0图像分析软件进行定量分析；在创伤后24小时，eIF6+/-组创面收缩显著高于对照组，p=0.045，n=8，有统计学差异；说明eIF6低表达导致创面的收缩增强，但两组均在10天完全愈合，在愈合时间上并没有差异。 2.2低表达eIF6导致肉芽组织的过度形成 在建立线性切口模型后第6天处死小鼠，取创面中间1/3组织，4%多聚甲醛，石蜡包埋，进行HE染色检测切面愈合、肉芽形成情况和炎性反应等。HE染色结果提示：实验组肉芽组织在创面大量形成，对照组在创面形成肉芽组织较实验组较少。IPP6.0软件定量测量肉芽组织面积，发现eIF6+/-组较eIF6+/+组肉芽组织面积显著升高（p=0.02，n=6）。 2.3低表达eIF6导致创面TGF-β1表达增强 为了观察小鼠皮肤缺损模型TGF-β1的表达情况，实验人员采用了免疫组织化学和WB，对创伤后第1天和第6天创伤皮肤组织进行TGF-β1的免疫组织化学染色和定量分析，测量TGF-β1表达的分布和TGF-β1的表达量。创伤组织中TGF-β1表达于创缘表皮细胞，皮脂腺，新生肉芽组织。WB结果提示，在新生肉芽组织和创缘中，eIF6+/-组表达TGF-β1量较eIF6+/+组显著增多（p=0.017，n=3）。 2.4低表达eIF6导致创面肉芽组织α-SMA的表达增强 实验人员取创伤后第6天创面组织，进行免疫组织化学和WB检测。免疫组织化学提示，eIF6低表达后α-SMA在肉芽组织中高表达。WB检测结果提示, eIF6+/-组小鼠肉芽组织中α-SMA的量较eIF6+/+组显著增多（p=0.040，n=3）。 3. eIF6对UUO模型中肾间质纤维化的影响 3.1成功建立UUO模型，低表达eIF6后，小鼠肾纤维化程度增加。 对照组小鼠肾脏组织结构正常，无明显病理改变。UUO模型组，eIF6+/+小鼠和eIF6+/-小鼠的肾间质可见炎细胞浸润，肾脏组织周边可见嗜伊红蛋白沉积，远端肾小管管腔扩大，肾小管上皮细胞脱落，出现空泡样变性，肾实质萎缩。Masson染色见，对照组eIF6+/-小鼠和eIF6+/+小鼠均无明显胶原沉积，UUO模型小鼠肾小管间质，胶原沉积，纤维增生明显，eIF6+/-小鼠较eIF6+/+小鼠胶原显著增多（p＜0.001，n=6）。 3.2在UUO模型中，低表达eIF6后，小鼠α-SMA和TGF-β1的表达上调 免疫组织化学结果显示，行输尿管结扎10天后，α-SMA在肾血管内皮细胞和肾小管间质成纤维细胞明显表达。免疫组织化学IPP定量分析α-SMA表达的结果显示，eIF6+/-实验组α-SMA表达量较eIF6+/+实验组显著增多（p＜0.001，n=6）。同时，免疫组织化学提示，在UUO模型组中可见TGF-β1表达于炎症细胞、肾间质纤维化组织和肾小管上皮细胞。对免疫组织化学结果，运用IPP6.0软件，进行定量分析显示TGF-β1在eIF6+/-实验组较eIF6+/+实验组表达增高（p＜0.001，n=6）。 实验结论 1.低表达eIF6导致了小鼠创面收缩增强，肉芽组织形成增加，α-SMA和TGF-β1的上调。证明了低表达eIF6增加了小鼠皮肤愈合过程中纤维化程度，eIF6参与了小鼠皮肤纤维化。提示eIF6可能能够下调TGF-β1和抑制小鼠成纤维细胞向肌成纤维细胞分化的发生发展。 2.低表达eIF6促进了小鼠肾胶原形成，上调了肾脏组织TGF-β1和α-SMA的表达。提示eIF6参与了小鼠肾纤维化过程。 eIF6的低表达导致了小鼠皮肤和肾脏纤维化程度增加。eIF6可能通过降低TGF-β1表达和抑制成纤维细胞等向肌成纤维细胞分化，参与抑制皮肤纤维化和肾纤维化过程。eIF6可能是未来防治组织纤维化药物新靶点。
[Abstract]:TGF - 尾1 ( TGF - 尾1 , transforming growth factor - 尾1 ) is an important cytokine in the process of tissue fibrosis .

It is reported that eIF6 ( eukaryotic initiation factor 6 , eukaryotic initiation factor 6 ) may participate in the regulation and regulation of tissue fibrosis . eIF6 participates in the assembly of ribosome and can be transferred to the nucleus to participate in translation initiation and regulation , and can be widely expressed in fibroblasts and endothelial cells . The study is to study the possible role of eIF6 in tissue fibrosis by knocking down the skin and kidney fibrosis model of mice by eIF6 + / - gene .

Study method :

eIF6 + / - knockout mice SPF ( Specific pathogen Free , no specific pathogen ) grade eIF6 + / - mice ( introduced in San Jose aele Scientific Institute , Milan , Italy ) and control wild - type mice were bred by the animals of the Third Military Medical University Affiliated to Daping Hospital .

animal model

2.1 Mouse Skin Defect Model

After the mice were anesthetized ( n = 8 ) , the hair of the back of the mice was scraped , and the skin was disinfected with 70 % ethanol ;
With a diameter of 4.5 mm , 2 round wounds were made by using a hole puncher , and the skin of the mouse was removed by the whole layer .
taking the digital camera to be perpendicular to the wound surface and placing a reference object beside the wound surface , wherein the lens of the digital camera is 15 cm away from the wound surface , and the wound image is taken in the micro - distance mode ;
The area of wound was measured with Image - Pro Plus6.0 software ( American Media CyberKnife software company ) . The actual size of the wound at each time point was corrected by reference . During the healing process , the change of wound surface was expressed as a percentage of the ratio of wound area to the area of the original wound .

2.2 . Mouse Skin Linear Model

After the mice were anesthetized ( n = 6 ) , the hair of the back of the mice was scraped and the skin was disinfected by alcohol ;
Mouse side abdominal wall , perpendicular to the spine plane , marked 2 symmetric 8 mm long linear wounds , sterile surgical knife all - layer scratched the mouse skin ;
taking the digital camera to be perpendicular to the wound surface , placing a reference object beside the wound surface , and taking a wound image at the distance of 15 cm from the wound surface ;
The area of wound surface was measured by IPP6.0 software . During the healing process , the change of wound surface was expressed as a percentage of the ratio of wound area to the area of the original wound .

2.3 Mouse UUO Model

Intraperitoneal injection of anesthesia mice ( n = 6 ) , abdominal hair removal and alcohol disinfection . The left ureter was exposed through median incision .
The suture was ligated at 1 / 3 and 2 / 3 respectively at 1 / 3 and 2 / 3 , completely blocking the left ureter ;
Mice were sacrificed 10 days after reduction of renal and intestinal canals , layered suture of muscles , skin , ligation , and the mice were sacrificed 10 days after ligation . The left ureter was separated from the sham operation control group without ligation , the kidneys and the intestinal tube were reset , and the mice were sacrificed after 10 days .

3 . Tissue Sample Preparation and Observation

3 . 1 / 3 of the middle 1 / 3 of the skin wound tissue , 1 / 3 of the vertical long axis of the renal tissue was taken , the specimens were placed in 4 % polyformaldehyde , paraffin - embedded , 5 - 渭m thick sections , suxylin eosin and eosin staining were carried out after 24 hours , and the lesions were observed under light microscope . The images were randomly selected in five fields of view with the microscope under 200 - fold field of view . The IPP6.0 software quantitatively measured the images and the results were analyzed statistically .

3.2TGF - 尾1 , 伪 - SMA immunohistochemical staining and quantitative analysis

The middle 1 / 3 of the tissue was taken from the skin , 1 / 3 of the vertical long axis was taken from the tissue of the kidney and 4 % of the polyformaldehyde was fixed . After 24 hours , the specimens were dehydrated , paraffin - embedded and 5.mu . m slices . The paraffin sections were paraffin - embedded and 5 渭m . The paraffin sections were routinely removed from wax to water . The immunohistochemical staining was performed ( TGF - 尾1 dilution : 1 : 200 ) .
Dilution of 伪 - SMA : 1 : 400 ) . The expression intensity of TGF - 尾1 and 伪 - SMA was quantitatively analyzed by IPP . The mean optical density and the mean optical density were used to represent the expression of TGF - 尾1 and 伪 - SMA . The mean value of aod represents the expression level of each mouse , and the results are statistically analyzed .

3 . Expression of TGF - 尾1 , 伪 - SMA and eIF6 by Western Blot

The detection of TGF - 尾1 , 伪 - SMA was 0.5 脳 0.5 cm in the center of the wound and normal tissue was detected by eIF6 . The samples were separated by SDS - PAGE electrophoresis . The rabbit anti - mouse monoclonal antibody ( TGF - 尾1 , 1 : 2000 ; 伪 - SMA , 1 : 2000 ; eIF6 , 1 : 1000 ) was added to the rabbit anti - mouse monoclonal antibody ( diluted 1 : 1500 ) , incubated at room temperature for 90 min , developed and photographed .

4 . Statistical treatment

The data of measurement data was expressed by x 卤 s , t - test was performed by SPSS 17.0 statistical software , and the difference of p 0.05 was statistically significant .

experimental results

1 . Genotypic Identification of Knockdown Mice and Detection of eIF6 Protein Expression

1.1 Identification of mouse genotypes

灏忛紶DNA鍑濊兌鐢垫吵缁撴灉涓

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