夏佳佳，鄭曉雅，劉嬋，胡鈺梅，任偉

沉默TCF7L2對胰島素抵抗HepG2細(xì)胞胰島素降解酶表達(dá)的調(diào)控作用觀察

夏佳佳，鄭曉雅，劉嬋，胡鈺梅，任偉

目的探討沉默轉(zhuǎn)錄因子7類似物2(TCF7L2)對胰島素抵抗(IR)HepG2細(xì)胞胰島素降解酶(IDE)表達(dá)的調(diào)控作用及可能機(jī)制。方法將HepG2細(xì)胞分為空白組、TCF7L2干擾組、空載體組、IR組、IR+TCF7L2干擾組、IR+空載體組。采用高濃度胰島素(5×10-6mol/L)持續(xù)作用24h誘導(dǎo)IR模型(IR-HepG2細(xì)胞)生成。以人TCF7L2 mRNA編碼序列為干擾靶點(diǎn)構(gòu)建TCF7L2特異性小干擾RNA慢病毒載體(LV-TCF7L2-siRNA)轉(zhuǎn)染空白組及IR組細(xì)胞，空載體病毒轉(zhuǎn)染空載體組及IR+空載體組細(xì)胞。qRT-PCR法檢測各組細(xì)胞TCF7L2及IDE mRNA的表達(dá)，Western blotting檢測各組細(xì)胞TCF7L2、IDE、胰島素刺激后蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)蛋白表達(dá)的變化，流式細(xì)胞術(shù)檢測各組2-脫氧-D-葡萄糖(2-NBDG)熒光葡萄糖攝取率。結(jié)果與空白組比較，IR組細(xì)胞葡萄糖消耗量及2-NBDG攝取率均明顯降低(P<0.01)，證明IR細(xì)胞模型建立成功。qRT-PCR及Western blotting結(jié)果顯示，IR組TCF7L2及IDE mRNA種蛋白表達(dá)水平均明顯低于空白組(P<0.05)，TCF7L2干擾組TCF7L2、IDE mRNA和蛋白表達(dá)水平較空白組、空載體組明顯下降，IR+TCF7L2干擾組TCF7L2、IDE mRNA和蛋白表達(dá)水平較IR組、IR+空載體組均明顯下降(P<0.05)。生理劑量胰島素刺激后，IR組、IR+TCF7L2干擾組p-AKT蛋白水平較空白組明顯下降(P<0.01)，各組總AKT水平差異無統(tǒng)計(jì)學(xué)意義。TCF7L2干擾組2-NBDG熒光葡萄糖攝取率較空白組和空載體組明顯下降，IR+TCF7L2干擾組2-NBDG熒光葡萄糖攝取率較IR組、IR+空載體組明顯下降(P<0.01)。結(jié)論TCF7L2聯(lián)合IDE致肝細(xì)胞IR，其機(jī)制可能與減少胰島素信號通路關(guān)鍵酶p-AKT蛋白的表達(dá)有關(guān)。

糖尿病，2型；胰島素抗藥性；轉(zhuǎn)錄因子7樣蛋白2；胰島素溶酶

2型糖尿病(type 2 diabetes mellitus，T2DM)是遺傳、環(huán)境及個(gè)體易感性相互作用所致的慢性疾病。全基因組關(guān)聯(lián)研究(genome-wide association studies，GWAS)發(fā)現(xiàn)轉(zhuǎn)錄因子7類似物2(transcription factor 7-like 2，TCF7L2)及胰島素降解酶(insulin degrading enzyme，IDE)基因突變可顯著增加T2DM的發(fā)病風(fēng)險(xiǎn)。因此，減少風(fēng)險(xiǎn)基因的致病作用具有重要的臨床意義。

TCF7L2是Wnt信號通路的重要組成部分，是迄今發(fā)現(xiàn)的與T2DM相關(guān)性最強(qiáng)的易感基因[1-2]，攜帶TCF7L2風(fēng)險(xiǎn)基因可使葡萄糖刺激胰島素分泌減少、肝糖異生及肝糖輸出增加，最終使血糖水平升高[3-4]。IDE能特異性地降解胰島素，維持體內(nèi)胰島素濃度的穩(wěn)定。IDE基因突變或蛋白功能改變會影響胰島素的代謝和降解，繼而影響胰島β細(xì)胞分泌及肝細(xì)胞降解胰島素，導(dǎo)致胰島素抵抗(insulin resistance，IR)及胰島β細(xì)胞功能失常，從而導(dǎo)致T2DM[5]。Morrison等[6]檢測了ob/ob糖尿病小鼠模型體內(nèi)多個(gè)T2DM風(fēng)險(xiǎn)基因的表達(dá)，肝臟及脂肪組織中TCF7L2的表達(dá)及脂肪組織中IDE的表達(dá)與較瘦小鼠相比明顯降低。本課題小組前期通過免疫共沉淀(chromatin immunoprecipitation，ChIP)技術(shù)發(fā)現(xiàn)TCF7L2與IDE基因啟動子存在結(jié)合位點(diǎn)[7]，但兩者在致細(xì)胞IR過程中的相互影響及TCF7L2風(fēng)險(xiǎn)基因聯(lián)合IDE易感基因的確切機(jī)制尚不清楚，且無相關(guān)研究報(bào)道。本研究通過轉(zhuǎn)染外源性人TCF7L2小干擾RNA下調(diào)TCF7L2表達(dá)，探討下調(diào)TCF7L2對肝細(xì)胞IDE表達(dá)的影響，并初步驗(yàn)證TCF7L2與IDE之間的相互關(guān)系和作用機(jī)制，為主要風(fēng)險(xiǎn)基因等致T2DM的發(fā)病機(jī)制研究及防治提供新的思路。

1 材料與方法

1.1 實(shí)驗(yàn)材料與試劑 豬胰島素(Sigma公司)；2-脫氧-D-葡萄糖(2-NBDG，Invitrogen公司)；葡萄糖測定試劑盒(南京建成公司)；細(xì)胞培養(yǎng)基RPMI 1640、胰酶(Gibco公司)；胎牛血清(Hyclone公司)；PBS緩沖液(博士德公司)；TCF7L2、磷酸化蛋白激酶B(p-AKT)及蛋白激酶B(AKT)抗體(Cell Signaling Technology公司)；IDE抗體(Abcam公司)；β-actin一抗(Protein-tech公司)；鼠、兔二抗(中杉金橋公司)；總RNA提取、反轉(zhuǎn)錄、PCR擴(kuò)增試劑盒及引物(TaKaRa公司)；總蛋白提取試劑盒(碧云天公司)。

1.2 細(xì)胞培養(yǎng)及HepG2-IR模型建立 人肝癌HepG2細(xì)胞株由重慶醫(yī)科大學(xué)生命科學(xué)院提供，在含10%FBS的RPMI 1640培養(yǎng)基37℃、5%CO2、95%空氣條件下培養(yǎng)，取對數(shù)生長期細(xì)胞置96孔板用于實(shí)驗(yàn)，待細(xì)胞生長至80%～90%融合度，用無血清RPMI 1640培養(yǎng)液饑餓12h。空白組為無細(xì)胞及血清的RPMI 1640培養(yǎng)液；對照組為無血清培養(yǎng)的細(xì)胞；實(shí)驗(yàn)組為胰島素終濃度分別為5×10-9、5×10-8、5×10-7、5×10-6、5×10-5mol/L的培養(yǎng)液。各組細(xì)胞孵育24h，收集細(xì)胞培養(yǎng)上清液，用葡萄糖測定試劑盒(God-Pod法)檢測上清液的葡萄糖濃度。以空白組為參照，計(jì)算24h各組細(xì)胞的葡萄糖消耗量。根據(jù)所得結(jié)果，選取形成IR且對細(xì)胞傷害最小的胰島素濃度，觀察該濃度胰島素作用不同時(shí)間對HepG2細(xì)胞IR的影響。

1.3 HepG2細(xì)胞IR模型的鑒定 用GOD-POD法檢測培養(yǎng)液上清的葡萄糖含量[8]。以空白組為參照，計(jì)算24h葡萄糖消耗量。每組6孔，實(shí)驗(yàn)重復(fù)3次。用熒光標(biāo)記的2-NBDG檢測兩組細(xì)胞的葡萄糖攝取率。每組3孔，實(shí)驗(yàn)重復(fù)3次。用熒光標(biāo)記的2-NBDG進(jìn)行葡萄糖攝取實(shí)驗(yàn)，方法參考文獻(xiàn)[9]并稍作改進(jìn)。按1×104/孔密度將HepG2細(xì)胞接種到96孔板，對照組給予RPMI 1640培養(yǎng)液，IR組給予含5×10-6mol/L胰島素RPMI 1640培養(yǎng)液，分別孵育24h后可進(jìn)行2-NBDG檢測。孵育24h后，移除原有細(xì)胞培養(yǎng)液，用KRH溶液洗滌3次，用熒光酶標(biāo)儀(激發(fā)波長488nm，發(fā)射波長520nm)測定熒光值，記為Fa。用2-NBDG(50μmol/L)于37℃孵育細(xì)胞15min，用KRH洗滌5次至2-NBDG液變澄清，用熒光酶標(biāo)儀測定熒光值，記為Fb。用含100nmol/L胰島素的2-NBDG(50μmol/L)于37℃孵育15min，用KRH洗滌5次至2-NBDG液變澄清，用熒光酶標(biāo)儀測定熒光值，記為Fc。最后用MTT比色法測定細(xì)胞密度，用普通酶標(biāo)儀測定其吸光度(A)值。葡萄糖攝取率計(jì)算公式為：(Fc-Fb)/(Fb-Fa)/A[10]。

1.4 TCF7L2基因干擾載體的構(gòu)建、鑒定及慢病毒包裝 根據(jù)人TCF7L2基因序列(GenBank NM-001198525)、siRNA原理設(shè)計(jì)并合成siRNA。正義鏈：5'-CCGGTAGCTGAGTGCACGTTGAAAGCTC GAGCTTTCAACGTGCACTCAGCTATTTTTG-3'；反義鏈：5'-AATTCAAAAATAGCTGAGTGCACG TTGAAAGCTCGAGCTTTCAACGTGCACTCAGC TA-3'。由上海生博生物工程有限公司合成干擾質(zhì)粒并用慢病毒包裝，同時(shí)合成對照慢病毒，并驗(yàn)證序列的正確性。

1.5 TCF7L2慢病毒轉(zhuǎn)染細(xì)胞 將細(xì)胞以1×105/ml密度接種于6孔板中(1ml/孔)進(jìn)行IR誘導(dǎo)，培養(yǎng)24h，根據(jù)病毒滴度及最佳MOI值，分別加入TCF7L2基因RNAi慢病毒及空載體病毒上清液，與HepG2及IR-HepG2細(xì)胞共培養(yǎng)12h后換液，48h后加入嘌呤霉素(1.0μg/ml)建立穩(wěn)定細(xì)胞株，用熒光顯微鏡觀察轉(zhuǎn)染效率。轉(zhuǎn)染后提取各組細(xì)胞總RNA及蛋白用于實(shí)驗(yàn)。實(shí)驗(yàn)分組：空白組(A組)，TCF7L2干擾組(LV-TCF7L2-siRNA轉(zhuǎn)染組，B組)，空載體組(亂序慢病毒轉(zhuǎn)染組，C組)，IR組(用含5×10-6mol/L胰島素處理組，D組)，IR+TCF7L2干擾組(IR+ LVTCF7L2-siRNA轉(zhuǎn)染組，E組)，IR+空載體組(IR+亂序慢病毒轉(zhuǎn)染組，F(xiàn)組)。生理劑量胰島素刺激后重新分組為：空白組(a組)，TCF7L2干擾組+胰島素刺激組(b組)，TCF7L2干擾組-胰島素刺激組(c組)，IR+胰島素刺激組(d組)，IR+TCF7L2干擾+胰島素刺激組(e組)，IR+TCF7L2干擾組-胰島素刺激組(f組)。

1.6 實(shí)時(shí)定量PCR檢測各組細(xì)胞TCF7L2及IDE mRNA的表達(dá)量 根據(jù)GenBank中TCF7L2、IDE及GAPDH基因序列設(shè)計(jì)引物。人TCF7L2：上游5'-TGCGTTCGCTACATACAAGGTG-3'，下游5'-TCTGTGACTTGGCGTCTCGG-3'，產(chǎn)物長度127bp；人IDE：上游5'-GCTCCTCAAAGACTCACT CAACG-5'，下游5'-TTGGCTGCTTGTCATTGTAAC CT-3'，產(chǎn)物長度125bp；人G A PDH：上游5'-CTTTGGTATCGTGGAAGGACTC-3'，下游5'-GTAGAGGCAGGGATGATGTTCT-3'，產(chǎn)物長度323bp。Trizol法提取細(xì)胞總RNA，測RNA濃度、純度，反轉(zhuǎn)錄合成cDNA，以cDNA為模板進(jìn)行擴(kuò)增，以GAPDH為內(nèi)參。用SYBR Green熒光定量PCR進(jìn)行分析。擴(kuò)增條件：95℃ 30s，95℃ 5s，58℃ 30s；72℃ 45s，共40個(gè)循環(huán)；72℃ 10min。分析熔解及擴(kuò)增曲線。采用Opticon2-Real-time-PCR儀(美國Bio-Rad公司)檢測TCF7L2及IDE表達(dá)量(2-ΔΔCt值)。實(shí)驗(yàn)重復(fù)3次。

1.7 Western blotting檢測各組細(xì)胞目的蛋白的表達(dá)量 提取各組細(xì)胞總蛋白，采用BCA試劑盒測定蛋白濃度。蛋白定量后行SDS-PAGE電泳，將蛋白電轉(zhuǎn)至硝酸纖維素膜，5%BSA封閉后加入一抗(TCF7L2、IDE、p-AKT Rabbit mAb及AKT Rabbit mAb)，4℃過夜后，TBST洗膜，與相應(yīng)二抗孵育2h，ECL化學(xué)發(fā)光法顯影。采用Quantity-One軟件分析圖像灰度值。ATCF7L2/Aβ-actin值表示TCF7L2蛋白的相對表達(dá)量，AIDE/Aβ-actin值表示IDE蛋白的相對表達(dá)量。

1.8 流式細(xì)胞術(shù)檢測各組細(xì)胞2-NBDG攝取率按5×105/孔密度將HepG2細(xì)胞接種到6孔板，孵育24h，給予相應(yīng)處理因素后，移除細(xì)胞原有培養(yǎng)液，分別加入配好的2-NBDG溶液，孵育2h后移除2-NBDG溶液，用KRH溶液洗滌3次直至2-NBDG澄清，胰酶消化各組細(xì)胞，終止，離心，用1ml PBS重懸于EP管中，用流式細(xì)胞儀檢測各組細(xì)胞2h的2-NBDG攝取率。

1.9 統(tǒng)計(jì)學(xué)處理 采用SPSS 17.0軟件進(jìn)行統(tǒng)計(jì)分析。計(jì)量資料數(shù)據(jù)以表示，多組間比較采用單因素方差分析(One-way ANOVA)，兩組間比較采用獨(dú)立樣本Tukey檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 不同濃度胰島素對各組HepG2細(xì)胞葡萄糖消耗量的影響 采用GOD-POD法檢測各組葡萄糖消耗量，孵育24h后，各組間差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。胰島素濃度由5×10-9mol/L增加至5×10-5mol/L時(shí)，細(xì)胞培養(yǎng)液中葡萄糖含量依次增加，即細(xì)胞葡萄糖消耗量逐漸減少，5×10-6mol/L時(shí)達(dá)到最小(P<0.01，圖1)。

圖1 不同濃度胰島素對HepG2細(xì)胞糖消耗的影響(n=6，±s)Fig.1 Effects of different concentrations insulin on the glucose consumption of HepG2 cells (n=6,±s)

2.2 胰島素作用不同時(shí)間對HepG2細(xì)胞葡萄糖消耗的影響 以5×10-6mol/L的胰島素培養(yǎng)HepG2細(xì)胞，隨作用時(shí)間延長，胰島素組葡萄糖消耗與對照組比較明顯降低，IR狀態(tài)從24h開始持續(xù)到60h，差異均有統(tǒng)計(jì)學(xué)意義(P<0.01，圖2)。

圖2 胰島素作用不同時(shí)間對HepG2細(xì)胞葡萄糖消耗的影響(n=6，±s)Fig.2 Effect of the acting time of insulin on glucose consumption of HepG2 cells (n=6,±s)

2.3 兩組HepG2細(xì)胞葡萄糖攝取率比較 2-NBDG法檢測顯示，與對照組(82.5%±8.2%)相比，IR組細(xì)胞葡萄糖攝取率(37.4%±6.5%)明顯減少，熒光顯微鏡顯示空白細(xì)胞胞內(nèi)攝取2-NBDG明顯增加，IR-HepG2細(xì)胞內(nèi)2-NBDG顯著減少，都集中在胞膜上，與正常對照組細(xì)胞比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05，n=3，圖3)。

圖3 兩組細(xì)胞2-NBDG葡萄糖攝取率(熒光顯微鏡)Fig.3 2-NBDG glucose uptake rate of cells in two groups (Fluorescence microscope)

2.4 TCF7L2干擾慢病毒轉(zhuǎn)染細(xì)胞效率觀察 采用熒光顯微鏡觀察TCF7L2干擾慢病毒對兩組細(xì)胞的轉(zhuǎn)染效果，結(jié)果顯示，轉(zhuǎn)染慢病毒并用嘌呤霉素篩選后，幾乎所有細(xì)胞均帶有GFP綠色熒光蛋白，表明轉(zhuǎn)染效果良好(圖4)。

圖4 重組慢病毒轉(zhuǎn)染HepG2的熒光圖像(×200)Fig.4 Fluoroscopic image of HepG2 infected with recombinant lentivirus (×200)

2.5 HepG2細(xì)胞內(nèi)TCF7L2、IDE mRNA的表達(dá)與空白組相比，IR組TCF7L2、IDE mRNA表達(dá)明顯減少(P<0.01)；與空白組、空載體及IR組、IR+空載體組相比，TCF7L2干擾組及IR+TCF7L2干擾組TCF7L2、IDE mRNA表達(dá)明顯減少(P<0.01，圖5)。

2.6 沉默TCF7L2對HepG2細(xì)胞蛋白表達(dá)影響 與空白組相比，IR組TCF7L2、IDE蛋白表達(dá)明顯增加(P<0.01)；與空白組、空載體組相比，TCF7L2干擾組TCF7L2、IDE蛋白表達(dá)明顯減少(P<0.05，P<0.01)；與IR組、IR+空載體組相比，IR+TCF7L2干擾組TCF7L2、IDE蛋白表達(dá)明顯減少(P<0.01，圖6)。生理劑量胰島素(100nmol/L)刺激后，與空白組相比，IR組、TCF7L2干擾組及IR+TCF7L2干擾組p-AKT表達(dá)均明顯減少(P<0.01)；與IR組相比，IR+TCF7L2干擾組p-AKT表達(dá)明顯減少(P<0.01)；各組之間總AKT表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P>0.05，圖7)。

2.7 各組細(xì)胞2-NBDG攝取率比較 流式細(xì)胞儀檢測結(jié)果顯示，與空白組、空載體組相比，TCF7L2干擾組2-NBDG熒光葡萄糖攝取率明顯降低(P<0.01)；與IR組、IR+空載體組相比，IR+TCF7L2干擾組2-N B D G熒光葡萄糖攝取率明顯降低(P<0.01)；與空白組相比，IR組、IR+空載體組、IR+TCF7L2干擾組2-NBDG熒光葡萄糖攝取率明顯降低(P<0.01，圖8)。

圖5 HepG2細(xì)胞TCFL2、IDE mRNA表達(dá)Fig.5 Expressions of TCF7L2 and IDE mRNA in HepG2 cells

圖6 各組HepG2細(xì)胞TCF7L2、IDE蛋白的表達(dá)Fig.6 Expressions of TCF7L2 and IDE protein in HepG2 cells

圖7 各組HepG2細(xì)胞p-AKT、AKT蛋白的表達(dá)Fig.7 Expressions of p-AKT and AKT protein in HepG2 cells

圖8 流式細(xì)胞儀檢測各組細(xì)胞2-NBDG攝取率Fig.8 2-NBDG uptake rate in each group of HepG2 cells (Flow cytometry)

3 討 論

隨著生活水平的不斷提高，糖尿病發(fā)病率逐年升高，并呈年輕化趨勢，嚴(yán)重威脅人類的生命健康及生活質(zhì)量[11]。IR即胰島素敏感性下降，是T2DM主要的發(fā)病機(jī)制之一[12]，表現(xiàn)為體內(nèi)胰島素介導(dǎo)的葡萄糖利用率下降.肝細(xì)胞是形成IR的主要場所，并已在多種人群中得到驗(yàn)證[13-15]。GWAS發(fā)現(xiàn)多種基因參與了IR形成，如TCF7L2及IDE基因突變會增加T2DM的發(fā)病風(fēng)險(xiǎn)。

TCF7L2基因又稱T細(xì)胞轉(zhuǎn)錄因子4(TCF-4)，位于人類染色體10q25.3[16]。TCF7L2基因突變可致胰島β細(xì)胞分化、成熟障礙，并阻斷前胰島素轉(zhuǎn)化為生物活性胰島素，使血胰島素分泌減少，血糖升高。Takamoto等[17]研究發(fā)現(xiàn)，TCF7L2基因突變小鼠體內(nèi)胰島β細(xì)胞體積及胰島素分泌顯著減少，證實(shí)TCF7L2通過調(diào)節(jié)β細(xì)胞數(shù)量而在糖代謝中發(fā)揮重要作用。Oh等[18]研究發(fā)現(xiàn)，IR小鼠TCF7L2表達(dá)量明顯減少，下調(diào)TCF7L2可增加肝糖異生基因的表達(dá)，使血糖水平升高，過表達(dá)TCF7L2可增加肝胰島素信號通路中PI3K/AKT磷酸化水平，使肝葡萄糖攝取增加，從而提高肝臟合成糖原的能力。本研究結(jié)果顯示，IR-HepG2細(xì)胞TCF7L2 mRNA及蛋白表達(dá)量均明顯減少，胰島素刺激后的p-AKT水平也顯著降低，與上述研究結(jié)果一致，進(jìn)一步證實(shí)了TCF7L2在肝糖調(diào)節(jié)中發(fā)揮著重要作用。

IDE基因位于人類染色體10q23-q25，IDE蛋白可高度特異性地降解胰島素，促進(jìn)胰島素受體再循環(huán)和新的胰島素分泌，維持體內(nèi)胰島素濃度穩(wěn)定[19-20]。肝臟是胰島素降解的主要器官，IDE表達(dá)減少可引起胰島素降解障礙，增加高胰島素血癥、IR等T2DM表型的發(fā)生[21]。Galagovsky等[22]對果蠅的研究證實(shí)，IDE是胰島素信號通路中的重要調(diào)節(jié)分子，IDE表達(dá)下降使AKT磷酸化水平降低，導(dǎo)致胰島素敏感性下降。Abdul-Hay等[23]認(rèn)為IDE表達(dá)減少使肝胰島素受體受損，其機(jī)制可能與胰島素信號通路中活性成分抑制致高胰島素血癥有關(guān)。Maianti等[24]發(fā)現(xiàn)了可特異性阻斷IDE降解的小分子化合物并闡明了其分子結(jié)構(gòu)，IDE抑制劑可提高糖耐量、降低血糖水平，故IDE抑制劑可成為糖尿病治療的一個(gè)靶點(diǎn)。本研究結(jié)果顯示，IR-HepG2細(xì)胞中IDE mRNA及蛋白表達(dá)量均明顯減少，再次驗(yàn)證了IDE減少與IR密切相關(guān)。

單個(gè)T2DM易感基因的致病作用是微弱的，近年來的研究熱點(diǎn)開始轉(zhuǎn)為易感基因的聯(lián)合作用。TCF7L2和IDE均位于人類10號染色體上，全基因組關(guān)聯(lián)研究顯示兩者有強(qiáng)大的連鎖效應(yīng)，在致T2DM上有聯(lián)合作用。Nordman等[25]研究發(fā)現(xiàn)TCF7L2基因rs7903146與IDE基因rs2251101聯(lián)合作用可顯著增加T2DM的易感性(P=0.0257，OR=1.398)。Kirkpatrick等[26]研究表明，在人的胰島β細(xì)胞中TCF7L2和IDE基因的mRNA和蛋白表達(dá)水平呈正相關(guān)(r分別為0.509、0.720，P<0.05)。本課題小組在前期研究中采用染色質(zhì)免疫共沉淀技術(shù)(ChIP)，在HepG2細(xì)胞中發(fā)現(xiàn)并證實(shí)特異性TCF7L2抗體可以與IDE基因轉(zhuǎn)錄啟動子的特異區(qū)域結(jié)合，并采用RNA干擾技術(shù)特異性沉默TCF7L2基因的表達(dá)，證實(shí)TCF7L2表達(dá)減少可引起IDE mRNA及蛋白表達(dá)水平明顯下降，從而證實(shí)TCF7L2是IDE基因表達(dá)調(diào)控中的重要轉(zhuǎn)錄因子[27]。以上研究均提示TCF7L2與IDE關(guān)系密切，但目前TCF7L2和IDE基因之間的聯(lián)合作用及具體機(jī)制尚不完全清楚。

本研究采用特異性TCF7L2-siRNA慢病毒載體，分別在對照及IR細(xì)胞下調(diào)TCF7L2的表達(dá)后觀察IDE的變化，結(jié)果顯示：IR細(xì)胞模型TCF7L2及IDE均明顯低于對照組；下調(diào)TCF7L2基因的表達(dá)后，對照組及IR組IDE的表達(dá)明顯減少；采用生理劑量胰島素刺激后，下調(diào)TCF7L2表達(dá)可使p-AKT水平降低，使肝臟主要胰島素信號通路之一受阻；IR組及下調(diào)TCF7L2表達(dá)可明顯降低細(xì)胞葡萄糖攝取率。正常生理?xiàng)l件下，血糖調(diào)節(jié)依賴胰島素水平，糖負(fù)荷增加促使胰島β細(xì)胞分泌胰島素，胰島素與靶腺上的胰島素受體結(jié)合，從而激活PI3K/AKT信號通路磷酸化，增加糖攝取、糖儲存、糖利用[28]。故PI3K-AKT信號通路是肝臟調(diào)節(jié)葡萄糖轉(zhuǎn)運(yùn)的重要調(diào)控點(diǎn)。本研究下調(diào)TCF7L2表達(dá)后，IDE表達(dá)量明顯減少，使胰島素降解減少，導(dǎo)致外周IR，胰島素介導(dǎo)的AKT激活被抑制，p-AKT蛋白表達(dá)水平下降，致下游信號分子與靶細(xì)胞胰島素受體結(jié)合，從而抑制酪氨酸磷酸化激活，使細(xì)胞葡萄糖攝取減少。該現(xiàn)象提示TCF7L2聯(lián)合IDE是調(diào)控胰島素信號通路的一個(gè)關(guān)鍵因子。

綜上所述，TCF7L2可通過IDE表達(dá)水平影響IR的形成，TCF7L2表達(dá)下降可能參與IR發(fā)生的始動環(huán)節(jié)，而采用生物學(xué)方法干預(yù)TCF7L2表達(dá)，改變IDE水平則可能有助于改善IR。

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Regulation effect of TCF7L2 gene silence on the expression of insulin degrading enzyme in insulin resistant HepG2 cells

XIA Jia-jia, ZHENG Xiao-ya, LIU Chan, HU Yu-mei, REN Wei*
Department of Endocrinology, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

*Corresponding author, E-mail: weiren67@aliyun.com

This work was supported by the Medical Science and Technology Research Project of Chongqing Health Bureau (2008-2-96), the Ministry of Education Fund Project (20135503120001), and the National Key Clinical Specialties Construction Program of China (2011)

ObjectiveTo evaluate the effects of transcription factor 7-like 2 (TCF7L2) silence on the expression of insulin degrading enzyme (IDE) in insulin resistance (IR) model HepG2 cells and its possible mechanism.MethodsThe HepG2 cells were divided into blank group, TCF7L2 interference group, empty vector group, IR group, IR+TCF7L2 interference group and IR+empty vector group. IR-HepG2 cell model was induced byin vitrocultivation of the cells in high concentration of insulin (5×10-6mol/L) for 24 hours; GOD-POD and 2-NBDG method was used to verify successful reproduction of IR-cell model. TCF7L2 specific siRNA lentivirus vector (LV-TCF7L2-siRNA) was constructed with TCF7L2 mRNA coding sequence as the interference target, and it was used to transfect the cells in blank group and IR group. Empty vector virus was used to transfect the cells in empty vector group and IR+empty vector group. The expressions of TCF7L2 and IDE mRNA were detected by qRT-PCR, and the changes in the expression of TCF7L2, IDE, insulin stimulated protein kinase B(AKT) and phosphorylated protein kinase B(p-AKT) were detected by Western blotting. The uptake rate of 2-deoxy-D-glucose (2-NBDG) was analyzed by flow cytometry.ResultsCompared with that in control group, the glucose consumption and the uptake rate of 2-NBDG significantly decreased in IR group (P<0.01), provingthat the IR cell model had been reproduced successfully. Western blo tt ing and qRT-PCR revealed that the expression levels of TCF7L2 and IDE mRNA and protein were obviously decreased in IR group compared with that in blank group (P<0.05), in TCF7L2 interference group than in blank group and empty vector group, and in IR+TCF7L2 interference group than in blank group and IR+empty vector group (P<0.05). A ft er physiological insulin stimulation, the expression levels of p-AKT protein decreased more signi fi cantly in IR group and IR+TCF7L2 interference group than in blank group (P<0.01), while no statistically signi fi cant di ff erence in the total AKT protein level was found among all the groups. 2-NBDG uptake rate was significantly decreased in TCF7L2 interference group as compared with that in blank group and empty vector group, and also in IR+TCF7L2 interference group than in IR group and IR+empty vector group, respectivelyP<0.01.ConclusionThe mechanism of IR induced by the interaction of TCF7L2 and IDE might be related to the decreased expression of the insulin signaling pathway key enzyme p-AKT protein.

diabetes mellitus, type 2; insulin resistance; transcription factor 7-like 2 protein; insulinase

R587.1

A

0577-7420(2015)02-0110-07

10.11855/j.issn.0577-7402.2015.02.05

?2014-09-16；

2014-12-08)

(責(zé)任編輯：張小利)

重慶市衛(wèi)生局醫(yī)學(xué)科學(xué)技術(shù)項(xiàng)目(2008-2-96)；教育部基金項(xiàng)目(20135503120001)；國家臨床重點(diǎn)?？平ㄔO(shè)項(xiàng)目(2011)

夏佳佳，碩士研究生。主要從事成人生長激素缺乏癥及2型糖尿病胰島素抵抗方面的研究

400016 重慶 重慶醫(yī)科大學(xué)附屬第一醫(yī)院內(nèi)分泌科(夏佳佳、鄭曉雅、劉嬋、胡鈺梅、任偉)

任偉，E-mail：weiren67@aliyun.com