早期糖尿病大鼠視網(wǎng)膜Notch-1表達(dá)變化的初步研究

高娜

·實(shí)驗(yàn)研究·

早期糖尿病大鼠視網(wǎng)膜Notch-1表達(dá)變化的初步研究

高娜

目的 研究發(fā)現(xiàn)Notch受體與其Delta樣配體4的信號(hào)通路參與糖尿病視網(wǎng)膜病變病程進(jìn)展,但其信號(hào)通路在糖尿病視網(wǎng)膜具體作用并未得到具體研究。故本次研究SD大鼠糖尿病模型早期視網(wǎng)膜內(nèi)Notch-1表達(dá)變化。方法 2月齡雄性SD大鼠40只,隨機(jī)分為4組(對(duì)照1月組,對(duì)照2月組,糖尿病1月組,糖尿病2月組),糖尿病(diabetes mellitus,DM)模型組均予以60mg/Kg體重鏈脲佐菌素(STZ,streptozocin)腹腔注射,造模成功后對(duì)比模型組和對(duì)照1月以及2月組視網(wǎng)膜內(nèi)Notch-1 受體表達(dá)變化。結(jié)果 免疫組化結(jié)果顯示Notch-1在大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層分布最為集中,且比正常對(duì)照組表達(dá)明顯增強(qiáng)。免疫印跡結(jié)果顯示DM 1月和2月組Notch-1蛋白表達(dá)均明顯高于對(duì)應(yīng)正常對(duì)照組,但DM 1月和2 月組間未見(jiàn)顯著差異。結(jié)論 早期糖尿病大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層Notch-1表達(dá)明顯增強(qiáng),其在糖尿病視網(wǎng)膜病變可能有著重要的干預(yù)作用。

糖尿??； 糖尿病視網(wǎng)膜病變； Notch-1

目前,Delta-Notch信號(hào)通路被認(rèn)為是調(diào)節(jié)新生血管發(fā)生的另一個(gè)重要機(jī)制,VEGF和Delta-Notch信號(hào)通路在多方面相互作用[1],在腫瘤血管的研究中發(fā)現(xiàn)聯(lián)合抑制VEGF和Delta-Notch號(hào)通路可協(xié)同抑制腫瘤生長(zhǎng)[2]。DII4/Notch信號(hào)通路有望成為針對(duì)新生血管的又一新靶點(diǎn),而新生血管作為糖尿病視網(wǎng)膜病變最嚴(yán)重的并發(fā)癥,此信號(hào)通路的研究將為糖尿病視網(wǎng)膜病變的治療提供新方向。所以本次實(shí)驗(yàn)初步研究早期糖尿病SD大鼠模型Notch-1信號(hào)通路改變,為臨床治療提供理論依據(jù)。

1 實(shí)驗(yàn)材料及方法

1.1 實(shí)驗(yàn)材料

清潔級(jí)2月齡雄性SD大鼠40只(成都中醫(yī)藥大學(xué)實(shí)驗(yàn)動(dòng)物中心提供),體重200～250克。實(shí)驗(yàn)動(dòng)物均適應(yīng)性喂養(yǎng)一周,予以自由飲食和飲水,室內(nèi)溫度為24±3°C,相對(duì)濕度為50±15%,其余按照實(shí)驗(yàn)室飼養(yǎng)標(biāo)準(zhǔn)進(jìn)行。實(shí)驗(yàn)材料及設(shè)備主要包括：STZ,十二烷基磺酸鈉、過(guò)硫酸銨、四甲基二乙胺、甘氨酸、Tris-base(美國(guó)Sigma)；檸檬酸緩沖液,免疫組化試劑盒 (武漢博士德),血糖儀及試紙(美國(guó)強(qiáng)生)；HRP標(biāo)記二抗羊抗兔,Notch 兔抗大鼠抗體(美國(guó)Santa Cruz ),ECL發(fā)光液 (Thermo scientific)；RIPA裂解液、BCA蛋白濃度測(cè)定試劑盒、SDS上樣緩沖液液體(5X)(北京碧云天)；電泳儀電源,垂直電泳儀,垂直轉(zhuǎn)膜儀(美國(guó)bio-rad)；高速低溫離心機(jī) (Thermo scientific)。

1.2 實(shí)驗(yàn)方法

SD大鼠(200～250g)禁食12h,腹腔注射60 mg STZ后(對(duì)照組注射生理鹽水),自由飲食,72小時(shí)后再次測(cè)空腹血糖,血糖濃度超過(guò)13.8mmol/L認(rèn)為造模成功,自由飲食1月,每周剪尾測(cè)空腹血糖一次,剔除血糖低于13.8mol/L大鼠。1月組和2月組均在相應(yīng)時(shí)間予以3%戊巴比妥鈉 30mg/Kg麻醉,摘除一側(cè)眼球后顯微鏡下取出視網(wǎng)膜至于液氮中,隨后右心灌注PBS后繼續(xù)灌注4%多聚甲醛,取眼球后4%多聚甲醛進(jìn)行固定,后期行視網(wǎng)膜切片。視網(wǎng)膜切片石蠟包埋后視神經(jīng)標(biāo)識(shí)切片,厚度為5μm,貼片脫蠟復(fù)水后檸檬酸緩沖液抗原修復(fù),Notch-1 兔抗大鼠抗體(1:50) 4℃孵育過(guò)夜,PBS沖洗后羊抗兔1:500室溫孵育1h,DAB顯色,鏡下觀察拍照。視網(wǎng)膜免疫印跡主要步驟為視網(wǎng)膜組織裂解勻漿,BCA法檢測(cè)蛋白含量,按比例加入SDS-PAGE上樣緩沖液,95℃充分變性后-20℃保存。7.5%分離膠,5%濃縮膠,每孔上20微升細(xì)胞裂解上樣液,邊孔加入5微升預(yù)染Marker,80V電泳,100mA低溫轉(zhuǎn)膜。轉(zhuǎn)膜后5%脫脂奶粉室溫封閉1h,Notch-1 兔抗大鼠抗體(1:500)室溫下孵育2h,或者予以β-actin內(nèi)參室溫孵育2h,其后予以HRP標(biāo)記二抗羊抗兔(1:3000)室溫孵育1h,TBST再次洗膜,ECL發(fā)光,X光片中曝光。Gel-pro analyze 4.5凝膠分析系統(tǒng)檢測(cè)目的條帶及內(nèi)參IOD值,每次分別測(cè)量3次后記錄兩者比值。

1.3 統(tǒng)計(jì)學(xué)方法

采用SPSS 23．0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,免疫印跡IOD平均值±S表示。組間比較采用獨(dú)立樣本t檢驗(yàn)。

2 結(jié)果

2.1 免疫組化切片結(jié)果

本次實(shí)驗(yàn)各組免疫組化切片DAB染色結(jié)果圖1至圖4,圖中可見(jiàn)Notch-1免疫組織化學(xué)染色陽(yáng)性表達(dá)主要集中在神經(jīng)節(jié)細(xì)胞層。與正常對(duì)照組 Control 1M 和Control 2M比較,糖尿模型組DM 1M 和DM 2M 組的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層Notch-1陽(yáng)性表達(dá)明顯增強(qiáng),DM 1M組和DM 2M組之間比較Notch-1表達(dá)略顯增強(qiáng)。

圖1 Control 1M Notch-l免疫組織化學(xué)染色結(jié)果(400×)

圖2 Control 2M Notch-l免疫組織化學(xué)染色結(jié)果(400×)

圖3 DM 1M Notch-l免疫組織化學(xué)染色結(jié)果(400×)

圖4 DM 2M Notch-l免疫組織化學(xué)染色結(jié)果(400×)

2.2 免疫印跡結(jié)果

由圖5可見(jiàn)Western blot條帶經(jīng)Gel-pro analyze 4.5分析顯示糖尿病1月組和2月組Notch-1表達(dá)均明顯高于正常組(P=0.003),但糖尿病1,2月組Notch-1表達(dá)無(wú)明顯差異(P=0.56)。

圖5 Western blot 結(jié)果圖注Control 1M 為正常對(duì)照1月組,Control 2M 為正常對(duì)照2組,DM 1M 為糖尿病1月組,DM 2M 為糖尿病2月組,Beta-actin為內(nèi)參,*為與Control 1M 和2M 比較差異有統(tǒng)計(jì)學(xué)意義(P=0.003),#為與DM 1M組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.56)

3 討論

因血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor,VEGF)在臨床所帶來(lái)的良好的治療效果,所以大量研究幾乎全部集中與與血管生成相關(guān)細(xì)胞因子的信號(hào)通路。但是臨床發(fā)現(xiàn),仍有部分病例使用抗VEGF后不能完全阻斷視網(wǎng)膜新生血管的形成,另外還存在藥物有效作用時(shí)間短、局部及全身副作用等問(wèn)題,導(dǎo)致患者使用抗VEGF藥物后療效不佳[3-5]。近期研究發(fā)現(xiàn)Notch信號(hào)通路與各類型腫瘤發(fā)生發(fā)展有密切的聯(lián)系,腫瘤細(xì)胞內(nèi)有大量表達(dá)的Notch受體和配體,Notch信號(hào)通路的激活對(duì)于腫瘤的血管形成也有重要意義[1,6],所以Notch受體與配體的研究也成為抑制視網(wǎng)膜新生血管化的新的熱點(diǎn)。糖尿環(huán)境下VEGF表達(dá)升高機(jī)理類似,Notch-1表達(dá)增加也與缺氧、氧化應(yīng)激等有關(guān)[7]。另外研究也發(fā)現(xiàn)抑制Notch-1表達(dá)也能抑制VEGF表達(dá)升高所導(dǎo)致的糖尿病腎臟損害[8]。

本次研究雖然只是對(duì)早期糖尿病大鼠視網(wǎng)膜中Notch-1表達(dá)變化進(jìn)行了簡(jiǎn)單研究,但發(fā)現(xiàn)正常對(duì)照組與糖尿病模型組的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層見(jiàn)到陽(yáng)性表達(dá),且隨糖尿病造模時(shí)間延長(zhǎng)Notch-1表達(dá)有不斷增加的趨勢(shì)。本次研究同時(shí)發(fā)現(xiàn)Notch-1受體表達(dá)最為集中的在視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層,推斷糖尿病早期對(duì)于眼部的損害最先損傷的也應(yīng)該是視網(wǎng)膜神經(jīng)節(jié)細(xì)胞層,所以下一步研究的主要是針對(duì)其對(duì)應(yīng)配體Delta-like ligand 4在視網(wǎng)膜內(nèi)層變化的研究。

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[3] Boyer DS,Hopkins JJ,Sorof J,Ehrlich JS.Anti-vascular endothelial growth factor therapy for diabetic macular edema.[J].Ther Adv Endocrinol Metab,2013,4:151-169.

[4] Falavarjani KG,Nguyen QD.Adverse events and complications associated with intravitreous injection of anti-VEGF agents:a review of literature.[J].Eye (Lond),2013,27:787-794.

[5] Cheung N,Wong IY,Wong TY.Ocular anti-VEGF therapy for diabetic retinopathy:overview of clinical efficacy and evolving applications[J].Diabetes Care,2014,37:900-905.

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Expression of Notch-1 in retina of diabetic rats at early stage

GAONa

(ChengduUniversityofTraditionalChineseMedicine,Chengdu,Sichuan,610075)

Objective Notch receptors and the Delta-like ligand 4 signaling pathway was shown to participate in the process of diabetic retinopathy.However,the function of this signaling pathway in diabetic retinopathy was still unknown.So this study was designed to investigate the expression change of Notch-1 in early stage of diebetes mellitus (DM) of SD rats’ retina.Methods DM was induced in 2-month-old male 40 Sprague-Dawley rats,which were randomly divided into 4 groups (control 1 and 2 month group,DM model 1 and 2 month group).DM animal models were all set with 60mg/Kg streptozocin intraperitoneal injection.After 1 and 2 month,Notch-1 expression in retinal was detected with immunohistochemical stainning and western blot,and compared with that of 1 and 2 month control group.Results The immunohistochemical results showed that Notch-1 expressed intensly in diabetic rats retinal ganglion cell layer,which is expressed significantly higher than in normal rat．Western blot showed that the expression of Notch-1 protein were also higher in DM 1 month group and DM 2 month group than that in normal group,while there were no significant difference between the two DM groups.Conclusion The expression of Notch-1 increased in retinal ganglion cell layer of the early stage in diabetic rats,which may play an essential role in the process of diabetic retinopathy．

Diabetic mellitus; Diabetic retinopathy; Notch-1

610075,四川成都,成都中醫(yī)藥大學(xué)

高娜,E-mail:346508571@qq.com

10.3969/j.issn.1674-9006.2016.04.003

R774