本文選題：eIF6 + TGF-β1�。� 參考：《第三軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：纖維化是一個多因素共同參與的復(fù)雜過程，是各種器官組織不可逆損傷修復(fù)的共同標(biāo)志性病理變化。其具體表現(xiàn)為組織間質(zhì)成纖維細(xì)胞增生，成纖維細(xì)胞表達(dá)α-SMA（α-smooth muscle actin，α-平滑肌肌動蛋白），分化成肌成纖維細(xì)胞，分泌大量細(xì)胞外基質(zhì)，并導(dǎo)致組織結(jié)構(gòu)破壞，器官功能喪失。目前的研究發(fā)現(xiàn)，TGF-β1（Transforminggrowth factor-β1，轉(zhuǎn)化生長因子β1）是一個在組織纖維化進(jìn)程中十分重要的細(xì)胞因子，它參與了各個器官炎癥反應(yīng)及纖維化。迄今為止，臨床上尚無治療組織纖維化的有效藥物。因此，研究纖維化分子機(jī)制、研制控制纖維化的有效藥物是臨床迫切需要解決的重大科學(xué)問題。 有文獻(xiàn)報道eIF6(eukaryotic initiation factor6，真核啟動因子6)可能參與調(diào)控組織纖維化過程。eIF6作為一種核基質(zhì)蛋白，參與核糖體的裝配，并可轉(zhuǎn)位至細(xì)胞核中參與翻譯起始調(diào)控，并廣泛表達(dá)于成纖維細(xì)胞及內(nèi)皮細(xì)胞等。本研究擬通過eIF6+/-基因敲降小鼠的皮膚和腎臟纖維化模型，研究eIF6在組織纖維化中可能的作用。 研究方法： eIF6+/-基因敲降小鼠SPF（Specific pathogen Free，無特定病原體）級eIF6+/-小鼠（引進(jìn)于San Raffaele Scientific Institute, Milan, Italy）和對照野生型小鼠，由第三軍醫(yī)大學(xué)附屬大坪醫(yī)院動物所飼養(yǎng)。每次實(shí)驗(yàn)3-8對小鼠。選取體重20±2g，8-10周齡，雄性小鼠。eIF6+/-小鼠因基因表型呈褐色，野生型呈黑色，均為C57BL/6背景。 動物模型 2.1小鼠皮膚缺損模型 小鼠麻醉后（n=8），刮去小鼠背部毛發(fā)，70%乙醇消毒皮膚；以4.5mm為直徑，用打孔器造對稱2個圓形創(chuàng)面，全層切除小鼠皮膚；以數(shù)碼相機(jī)垂直創(chuàng)面，同時在創(chuàng)面旁放置參照物，數(shù)碼相機(jī)鏡頭距離創(chuàng)面15cm，微距模式下采取創(chuàng)面圖像；采用Image-Pro Plus6.0軟件(美國Media Cybernetics軟件公司)測量創(chuàng)面面積。每個時間點(diǎn)的創(chuàng)面實(shí)際大小利用參照物校正。愈合過程中，創(chuàng)面的變化用不同時相點(diǎn)的創(chuàng)面面積與原始創(chuàng)面面積之比的百分?jǐn)?shù)表示。 2.2.小鼠皮膚線性模型 小鼠麻醉后（n=6），刮去小鼠背部毛發(fā)，酒精消毒皮膚；在小鼠側(cè)腹壁，垂直于脊柱平面，標(biāo)記2個對稱的8mm長線性創(chuàng)口，無菌手術(shù)刀全層劃開小鼠皮膚；以數(shù)碼相機(jī)垂直創(chuàng)面，同時在創(chuàng)面旁放置參照物，鏡頭距離創(chuàng)面15cm下采取創(chuàng)面圖像；采用IPP6.0軟件測量創(chuàng)面面積，愈合過程中，創(chuàng)面的變化用不同時相點(diǎn)的創(chuàng)面面積與原始創(chuàng)面面積之比的百分?jǐn)?shù)表示。 2.3小鼠UUO模型 腹腔注射麻醉小鼠（n=6），腹部脫毛，酒精消毒。通過腹部正中切口，暴露左側(cè)輸尿管；縫線分別結(jié)扎上1/3和2/3分界處2次，完全阻斷左側(cè)輸尿管；復(fù)位腎臟及腸管，分層縫合肌肉、皮膚，結(jié)扎后10天處死小鼠，取材（具體見研究方法3）。假手術(shù)對照組分離出左側(cè)輸尿管后不結(jié)扎,復(fù)位腎臟及腸管，10天后處死小鼠，取材。 3.組織樣本制作與觀察 3.1HE和Masson染色：皮膚創(chuàng)面組織取中間1/3,腎臟組織取垂直長軸方向的中間1/3，標(biāo)本置于4%多聚甲醛固定，24小時后進(jìn)行石蠟包埋，5μm厚度切片，蘇木素伊紅和Masson染色,光鏡下觀察病變情況，每張切片在200倍視野下隨機(jī)選取5個視野獲取圖像，IPP6.0軟件對圖像進(jìn)行定量測量,結(jié)果進(jìn)行統(tǒng)計分析。 3.2TGFβ1、α-SMA免疫組織染色和定量分析 皮膚創(chuàng)面組織取中間1/3,腎臟組織取垂直長軸方向的中間1/3，4%多聚甲醛固定標(biāo)本，24小時后常規(guī)脫水、石蠟包埋，5μm切片。切片常規(guī)脫蠟至水。分別進(jìn)行免疫組織化學(xué)染色（TGF-β1稀釋度：1:200；α-SMA稀釋度：1:400）。小鼠腎臟組織的每張切片在400倍視野下隨機(jī)選取5個組織的視野獲取圖像，IPP分別定量分析TGF-β1和α-SMA的表達(dá)強(qiáng)度，以AOD（Average optical density，平均光密度）表示陽性物質(zhì)的含量，AOD的均值代表每只小鼠的表達(dá)量，結(jié)果進(jìn)行統(tǒng)計分析。 3.3Western Blot檢測TGFβ1、α-SMA、eIF6的表達(dá) TGFβ1、α-SMA的檢測以創(chuàng)面為中心取0.5×0.5cm組織，eIF6的檢測取正常組織。提取蛋白，BCA法行蛋白定量，蛋白/泳道上樣10μg，SDS-PAGE電泳分離，電轉(zhuǎn)移至醋酸纖維薄膜，5%脫脂奶粉封閉90min，分別加入兔抗鼠單克隆抗體（TGF-β1,1:2000; α-SMA，1:2000; eIF6，1:1000），恒溫冰箱4℃孵育過夜，洗膜，加入HRP標(biāo)記的羊抗兔二抗（稀釋度1:1500），室溫孵育90min，顯影，拍照。IPP6.0軟件進(jìn)行灰度掃描，IOD值代表?xiàng)l帶信號的強(qiáng)弱，進(jìn)行統(tǒng)計分析。 4.統(tǒng)計學(xué)處理 計量資料數(shù)據(jù)以x±s表示,用SPSS17.0統(tǒng)計軟件進(jìn)行t檢驗(yàn),以p 0.05為差異有統(tǒng)計學(xué)意義。 實(shí)驗(yàn)結(jié)果 1.基因敲降小鼠基因型鑒定和eIF6蛋白表達(dá)檢測 1.1小鼠基因型的鑒定 小鼠DNA凝膠電泳結(jié)果中，小鼠DNA凝膠電泳在320bp和650bp兩處同時顯示亮帶，，提示其基因型為eIF6+/-。DNA凝膠電泳僅在320bp顯示亮帶，提示其基因型為eIF6+/+。 1.2基因敲降小鼠正常皮膚和腎臟中eIF6的表達(dá) WB結(jié)果驗(yàn)證了基因敲降小鼠正常皮膚中eIF6表達(dá)下降63%，腎臟下降50%。 2.低表達(dá)eIF6對小鼠皮膚損傷愈合的影響 2.1低表達(dá)eIF6導(dǎo)致創(chuàng)面的收縮增強(qiáng) 為了評估小鼠皮膚缺損模型的創(chuàng)面愈合情況，實(shí)驗(yàn)人員每24小時對小鼠的創(chuàng)面進(jìn)行一次拍照，并用IPP6.0圖像分析軟件進(jìn)行定量分析；在創(chuàng)傷后24小時，eIF6+/-組創(chuàng)面收縮顯著高于對照組，p=0.045，n=8，有統(tǒng)計學(xué)差異；說明eIF6低表達(dá)導(dǎo)致創(chuàng)面的收縮增強(qiáng)，但兩組均在10天完全愈合，在愈合時間上并沒有差異。 2.2低表達(dá)eIF6導(dǎo)致肉芽組織的過度形成 在建立線性切口模型后第6天處死小鼠，取創(chuàng)面中間1/3組織，4%多聚甲醛，石蠟包埋，進(jìn)行HE染色檢測切面愈合、肉芽形成情況和炎性反應(yīng)等。HE染色結(jié)果提示：實(shí)驗(yàn)組肉芽組織在創(chuàng)面大量形成，對照組在創(chuàng)面形成肉芽組織較實(shí)驗(yàn)組較少。IPP6.0軟件定量測量肉芽組織面積，發(fā)現(xiàn)eIF6+/-組較eIF6+/+組肉芽組織面積顯著升高（p=0.02，n=6）。 2.3低表達(dá)eIF6導(dǎo)致創(chuàng)面TGF-β1表達(dá)增強(qiáng) 為了觀察小鼠皮膚缺損模型TGF-β1的表達(dá)情況，實(shí)驗(yàn)人員采用了免疫組織化學(xué)和WB，對創(chuàng)傷后第1天和第6天創(chuàng)傷皮膚組織進(jìn)行TGF-β1的免疫組織化學(xué)染色和定量分析，測量TGF-β1表達(dá)的分布和TGF-β1的表達(dá)量。創(chuàng)傷組織中TGF-β1表達(dá)于創(chuàng)緣表皮細(xì)胞，皮脂腺，新生肉芽組織。WB結(jié)果提示，在新生肉芽組織和創(chuàng)緣中，eIF6+/-組表達(dá)TGF-β1量較eIF6+/+組顯著增多（p=0.017，n=3）。 2.4低表達(dá)eIF6導(dǎo)致創(chuàng)面肉芽組織α-SMA的表達(dá)增強(qiáng) 實(shí)驗(yàn)人員取創(chuàng)傷后第6天創(chuàng)面組織，進(jìn)行免疫組織化學(xué)和WB檢測。免疫組織化學(xué)提示，eIF6低表達(dá)后α-SMA在肉芽組織中高表達(dá)。WB檢測結(jié)果提示, eIF6+/-組小鼠肉芽組織中α-SMA的量較eIF6+/+組顯著增多（p=0.040，n=3）。 3. eIF6對UUO模型中腎間質(zhì)纖維化的影響 3.1成功建立UUO模型，低表達(dá)eIF6后，小鼠腎纖維化程度增加。 對照組小鼠腎臟組織結(jié)構(gòu)正常，無明顯病理改變。UUO模型組，eIF6+/+小鼠和eIF6+/-小鼠的腎間質(zhì)可見炎細(xì)胞浸潤，腎臟組織周邊可見嗜伊紅蛋白沉積，遠(yuǎn)端腎小管管腔擴(kuò)大，腎小管上皮細(xì)胞脫落，出現(xiàn)空泡樣變性，腎實(shí)質(zhì)萎縮。Masson染色見，對照組eIF6+/-小鼠和eIF6+/+小鼠均無明顯膠原沉積，UUO模型小鼠腎小管間質(zhì)，膠原沉積，纖維增生明顯，eIF6+/-小鼠較eIF6+/+小鼠膠原顯著增多（p＜0.001，n=6）。 3.2在UUO模型中，低表達(dá)eIF6后，小鼠α-SMA和TGF-β1的表達(dá)上調(diào) 免疫組織化學(xué)結(jié)果顯示，行輸尿管結(jié)扎10天后，α-SMA在腎血管內(nèi)皮細(xì)胞和腎小管間質(zhì)成纖維細(xì)胞明顯表達(dá)。免疫組織化學(xué)IPP定量分析α-SMA表達(dá)的結(jié)果顯示，eIF6+/-實(shí)驗(yàn)組α-SMA表達(dá)量較eIF6+/+實(shí)驗(yàn)組顯著增多（p＜0.001，n=6）。同時，免疫組織化學(xué)提示，在UUO模型組中可見TGF-β1表達(dá)于炎癥細(xì)胞、腎間質(zhì)纖維化組織和腎小管上皮細(xì)胞。對免疫組織化學(xué)結(jié)果，運(yùn)用IPP6.0軟件，進(jìn)行定量分析顯示TGF-β1在eIF6+/-實(shí)驗(yàn)組較eIF6+/+實(shí)驗(yàn)組表達(dá)增高（p＜0.001，n=6）。 實(shí)驗(yàn)結(jié)論 1.低表達(dá)eIF6導(dǎo)致了小鼠創(chuàng)面收縮增強(qiáng)，肉芽組織形成增加，α-SMA和TGF-β1的上調(diào)。證明了低表達(dá)eIF6增加了小鼠皮膚愈合過程中纖維化程度，eIF6參與了小鼠皮膚纖維化。提示eIF6可能能夠下調(diào)TGF-β1和抑制小鼠成纖維細(xì)胞向肌成纖維細(xì)胞分化的發(fā)生發(fā)展。 2.低表達(dá)eIF6促進(jìn)了小鼠腎膠原形成，上調(diào)了腎臟組織TGF-β1和α-SMA的表達(dá)。提示eIF6參與了小鼠腎纖維化過程。 eIF6的低表達(dá)導(dǎo)致了小鼠皮膚和腎臟纖維化程度增加。eIF6可能通過降低TGF-β1表達(dá)和抑制成纖維細(xì)胞等向肌成纖維細(xì)胞分化，參與抑制皮膚纖維化和腎纖維化過程。eIF6可能是未來防治組織纖維化藥物新靶點(diǎn)。
[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 , 偽 - 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 偽 - SMA : 1 : 400 ) . The expression intensity of TGF - 尾1 and 偽 - 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 偽 - 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 , 偽 - SMA and eIF6 by Western Blot

The detection of TGF - 尾1 , 偽 - 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 ; 偽 - 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 鹵 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

灝忛紶DNA鍑濊兌鐢墊吵緇撴灉涓

本文編號：1805829