南虎松, 尹明姬, 金春姬
(延邊大學(xué)附屬醫(yī)院兒科, 吉林 延吉 133000)
CFTR對TF1細(xì)胞活力和凋亡的影響及相關(guān)機(jī)制
南虎松△, 尹明姬, 金春姬
(延邊大學(xué)附屬醫(yī)院兒科, 吉林 延吉 133000)
目的探討囊性纖維化跨膜傳導(dǎo)調(diào)節(jié)因子(CFTR)在急性髓系白血病中的表達(dá)情況并探討其對人紅白血病細(xì)胞株TF1生物學(xué)功能的影響及潛在作用機(jī)制。方法Real-time PCR技術(shù)檢測急性髓系白血病患兒骨髓單個核細(xì)胞中CFTR的表達(dá)水平。常規(guī)培養(yǎng)的TF1細(xì)胞中加入CFTR特異性抑制劑CFTRinh-172,然后分別采用CCK-8法和流式細(xì)胞術(shù)檢測細(xì)胞活力、細(xì)胞周期及細(xì)胞凋亡情況;Western blot檢測Wnt信號通路相關(guān)蛋白的表達(dá)水平。結(jié)果CFTR在急性髓系白血病患者及白血病細(xì)胞中均呈高表達(dá)。TF1細(xì)胞中加入CFTR特異性抑制劑后,細(xì)胞的活力下降,G0/G1期細(xì)胞比例顯著升高而S期細(xì)胞比例降低,細(xì)胞凋亡率顯著增加,細(xì)胞中β-catenin、c-Myc及cyclin D1的蛋白表達(dá)水平顯著降低(P<0.05)。結(jié)論CFTR在急性髓系白血病中表達(dá)顯著升高;抑制CFTR可通過經(jīng)典的Wnt信號通路抑制白血病細(xì)胞株TF1的生長,并促進(jìn)其凋亡。
囊性纖維化跨膜傳導(dǎo)調(diào)節(jié)因子; 急性髓系白血?。?細(xì)胞活力; 細(xì)胞周期; 細(xì)胞凋亡; Wnt信號通路
急性髓系白血病(acute myeloid leukemia,AML)是一組血液系統(tǒng)的惡性腫瘤,起源于多能造血干細(xì)胞的惡性克隆,通過抑制正常造血和免疫功能,造成發(fā)熱、貧血和感染等臨床事件,嚴(yán)重威脅了人類的生命健康[1]。而其治療方式仍以聯(lián)合化療為主,總的完全緩解率及生存率依舊較低[1-3],尋找更有效的治療手段已成為目前亟待解決的問題之一。
人紅白血病細(xì)胞系TF1、人急性單核細(xì)胞白血病細(xì)胞系THP-1及人支氣管上皮樣細(xì)胞HBE由本實驗室凍存;淋巴細(xì)胞分離液Ficoll購自GE Healthcare;胎牛血清(fetal bovine serum,F(xiàn)BS)、RPMI-1640培養(yǎng)基及DMEM培養(yǎng)基均購于Gibco;CFTR特異性抑制劑CFTRinh-172購于Sigma; 抗CFTR抗體購于Abcam;抗β-catenin、c-Myc、cyclin D1及β-actin抗體購于CST;CCK-8細(xì)胞活力分析試劑盒購于廣州賴德生物技術(shù)有限公司;細(xì)胞周期檢測試劑盒及細(xì)胞凋亡檢測試劑盒購于南京凱基公司;SYBR Green I Real-time PCR Kit由上海吉瑪制藥技術(shù)有限公司提供。
2.1臨床標(biāo)本制備 收集由本院收診的32例經(jīng)MICM分型診斷的初診AML患兒(實驗組,AML組)和10例健康志愿者(對照組,control組)的骨髓樣本。所選取的AML患兒依據(jù)FAB分型包括M1(7例)、M2(14例)、M3(10例)和M5(1例)。所有實驗均經(jīng)家屬知情同意,并報醫(yī)院倫理委員會批準(zhǔn)。骨髓穿刺抽取初診或者臨床化療前患兒新鮮骨髓2~4 mL,置于EDTA抗凝管中。依據(jù)說明書要求采用Ficoll法分離患者骨髓中的單個核細(xì)胞,步驟簡述如下:取離心管按照1∶2或者2∶3的比例加入淋巴細(xì)胞分離液Ficoll和骨髓液,100×g離心20 min,吸取界面層的單個核細(xì)胞,而后PBS洗2次。收集細(xì)胞凍存于-80 ℃冰箱中備用。
2.2細(xì)胞培養(yǎng)、分組及處理 實驗用TF1細(xì)胞和THP-1細(xì)胞常規(guī)培養(yǎng)于預(yù)先配制好并濾過除菌的含10% FBS RPMI-1640 完全培養(yǎng)基中,實驗用HBE細(xì)胞常規(guī)培養(yǎng)于預(yù)先配制好并濾過除菌的含10% FBS DMEM完全培養(yǎng)基中;培養(yǎng)箱條件設(shè)置為飽和濕度、37 ℃、5% CO2,間隔1~2 d換液。
2.3CCK-8法測定細(xì)胞活力 將細(xì)胞以2×108/L的密度接種于96孔板,按0、50、100、150和200 μmol/L的濃度梯度依次加入CFTRinh-172,于培養(yǎng)箱中孵育22 h。之后按照說明書要求,加入10 μL CCK-8試劑,置于5% CO2培養(yǎng)箱中繼續(xù)反應(yīng)2 h,經(jīng)酶標(biāo)儀測定450 nm波長處的吸光度(A)值。每組設(shè)置3個復(fù)孔取均值,另設(shè)單孔只加入培養(yǎng)基作空白對照。
2.4流式細(xì)胞術(shù)檢測細(xì)胞周期 各組細(xì)胞經(jīng)CFTRinh-172(0、50和100 μmol/L)孵育24 h后,離心收集細(xì)胞,后用預(yù)冷的PBS漂洗并再次離心棄上清;加70%的冰乙醇于4 ℃固定24 h,1 000×g離心洗去乙醇。依據(jù)試劑盒說明書,先后加入RNase(37 ℃反應(yīng)30 min)及碘化丙啶(propidium iodide,PI)染色液(4 ℃避光孵育30 min),用流式細(xì)胞儀檢測激發(fā)波長488 nm處紅色熒光強度,分析DNA周期及各期細(xì)胞比例。
2.5Annexin V/PI 雙染色法檢測細(xì)胞凋亡 各組細(xì)胞經(jīng)CFTRinh-172(0、50和100 μmol/L)處理后,依據(jù)說明書要求,加不含EDTA的胰酶消化并離心收集細(xì)胞,而后加Binding Buffer重懸細(xì)胞。先后加入Annexin V-FITC(室溫避光孵育10 min)和PI染液(室溫避光孵育5 min)。用流式細(xì)胞儀檢測激發(fā)波長488 nm、發(fā)射波長530 nm處的熒光強度,分析細(xì)胞的凋亡率。
2.6Western blot測定蛋白水平 加RIPA裂解液提取細(xì)胞蛋白,BCA試劑盒進(jìn)行蛋白定量后,調(diào)整各組上樣蛋白總量至80 μg,加入4倍體積的Loading Buffer,95 ℃ 5 min變性,然后進(jìn)行SDS-PAGE。結(jié)束后將蛋白在200 mA恒流的條件下電轉(zhuǎn)至PVDF膜上。用5%脫脂牛奶于搖床上室溫封閉2 h,依據(jù)說明書要求加入相應(yīng)的 I 抗4 ℃孵育過夜,次日復(fù)溫后,吸去 I 抗孵育液并加TBST洗滌3次,每次5 min;而后加入對應(yīng)的 II 抗室溫孵育2 h,TBST洗滌3次,每次10 min,暗室中加ECL發(fā)光液顯影、定影,沖洗膠片后經(jīng)ImageJ行蛋白半定量灰度分析。
2.7Real-time PCR實驗 依據(jù)Trizol一步法說明書提取各組樣品的總RNA,并測定所提樣品的純度。定量后取2 μg總RNA逆轉(zhuǎn)錄至終體積20 μL的cDNA,然后依照SYBR Green實時熒光PCR試劑盒說明檢測CFTR mRNA的表達(dá)。CFTR的上游引物為5’-TGAACACAGGATAGAAGCGATGTTG-3’,下游引物為 5’-GGAGCTAATGGCCTGCTGGA-3’; β-actin為內(nèi)參照,上游引物為5’-ACAGAGCCTCGCCTTTGCCGATC-3’,下游引物為5’-ATCCTTCTGACCCAT GCCCACCA-3’。PCR反應(yīng)條件為: 95 ℃ 預(yù)變性2 min; 95 ℃ 15 s, 60 ℃ 20 s, 72 ℃ 20 s, 40個循環(huán)。采用2-ΔΔCt法分析基因相對表達(dá)量。
所有數(shù)據(jù)錄入SPSS 17.0軟件進(jìn)行統(tǒng)計學(xué)分析。實驗結(jié)果以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。兩組計量資料的組間差異采用t檢驗,多組計量資料行單因素方差分析,同時采用Bonferroni校正的t檢驗進(jìn)行均數(shù)組間的兩兩比較,計數(shù)資料的比較采用2檢驗。 以P<0.05為差異有統(tǒng)計學(xué)意義。
Real-time PCR的檢測結(jié)果顯示,急性髓系白血病患兒體內(nèi)CFTR的平均表達(dá)水平是對照組的(1.87±0.11)倍(P<0.05),見圖1A。同時相對于陰性對照組HBE細(xì)胞,白血病細(xì)胞系TF1及THP-1中CFTR的mRNA及蛋白表達(dá)均明顯升高(P<0.05),見圖1B、C。上述結(jié)果表明CFTR在急性髓系白血病中高表達(dá)。
Figure 1. CFTR expression in acute myeloid leukemia (AML) patients and leukemia cell lines. A: the mRNA expression of CFTR in AML patients; B: the mRNA expression of CFTR in leukemia cell lines; C: the protein expression of CFTR in leukemia cell lines. Mean±SD.n=6.#P<0.05vsnormal group;*P<0.05vsHBE cells.
圖1CFTR在急性髓系白血病患者和白血病細(xì)胞系中的表達(dá)
在常規(guī)培養(yǎng)的TF1細(xì)胞中加入0~200 μmol/L的CFTR特異性抑制劑CFTRinh-172,采用CCK-8法檢測細(xì)胞活力的變化。結(jié)果顯示,加入100 μmol/L 的CFTRinh-172后,TF1細(xì)胞活力降低至(58.47±4.65)%(P<0.05),而HBE細(xì)胞活力沒有顯著變化,見圖2。進(jìn)一步采用流式細(xì)胞術(shù)檢測了TF1細(xì)胞的周期分布情況,結(jié)果顯示, 100 μmol/L的CFTRinh-172孵育24 h后,G0/G1期的細(xì)胞所占百分比增加(P<0.05),S期細(xì)胞所占百分比減少(P<0.05),說明CFTRinh-172可抑制TF1細(xì)胞周期由G0/G1期向S期轉(zhuǎn)化,見圖3。
細(xì)胞凋亡檢測顯示,100 μmol/L的CFTRinh-172孵育24 h后,TF1細(xì)胞的早期凋亡率由(7.21±0.62)%增加至(29.47±1.95)%(P<0.05),說明CFTRinh-172可促進(jìn)TF1細(xì)胞凋亡,見圖4。
Figure 2. The effects of CFTR specific inhibitor CFTRinh-172 on the viability of TF1 cells. Mean±SD.n=6.*P<0.05vs0 μmol/L group.
圖2CFTR特異性抑制劑CFTRinh-172對TF1細(xì)胞活力的影響
相較于對照組,100 μmol/L的CFTRinh-172孵育24 h后,TF1細(xì)胞中β-catenin、c-Myc及cyclin D1 的蛋白水平均顯著降低(P<0.05),提示CFTRinh-172可抑制經(jīng)典的Wnt信號通路的激活,見圖5。
隨著對AML發(fā)病機(jī)制的深入研究,針對特定基因的靶向治療給AML的診療帶來了新的方向。目前在AML中發(fā)現(xiàn)了包括酪氨酸激酶(tyrosine kinases, TKs)相關(guān)基因及轉(zhuǎn)錄抑制因子AML1-ETO在內(nèi)的超過100種基因異常表達(dá),但是仍有大量病例缺乏特征性的標(biāo)記分子[6-7];而相當(dāng)多的研究顯示[8-11],CFTR在腫瘤中起著類似原癌基因或者抑癌基因的作用。本研究檢測了急性髓系白血病中CFTR的表達(dá)情況,結(jié)果顯示急性髓系白血病患兒體內(nèi)及白血病細(xì)胞系TF1和THP-1中CFTR的表達(dá)均明顯升高,提示CFTR可能參與調(diào)控急性髓系白血病的發(fā)生發(fā)展進(jìn)程。眾所周知,腫瘤的發(fā)生發(fā)展通常與腫瘤細(xì)胞生物學(xué)特性的改變密切相關(guān),大量研究顯示,腫瘤細(xì)胞的惡性增殖是腫瘤形成及發(fā)展的關(guān)鍵[12]。本實驗進(jìn)一步借助CFTR特異性抑制劑CFTRinh-172探討了CFTR對白血病TF1細(xì)胞活力、細(xì)胞周期及凋亡等生物學(xué)特性的影響。CCK-8細(xì)胞活力檢測和流式細(xì)胞周期檢測的結(jié)果顯示,CFTRinh-172可降低TF1細(xì)胞活力,并抑制細(xì)胞周期由G0/G1期向S期轉(zhuǎn)化;同時細(xì)胞凋亡檢測結(jié)果顯示,CFTRinh-172可促進(jìn)TF1細(xì)胞凋亡。結(jié)果提示,在急性髓系白血病中,CFTR可通過影響腫瘤細(xì)胞的生長及凋亡,調(diào)控白血病的發(fā)展進(jìn)程。
Figure 3. The effects of CFTR specific inhibitor CFTRinh-172 on cell cycle distribution of TF1 cells. Mean±SD.n=6.*P<0.05vs0 μmol/L group.
圖3CFTR特異性抑制劑CFTRinh-172對TF1細(xì)胞周期的影響
Figure 4. The effects of CFTR specific inhibitor CFTRinh-172 on the apoptosis of TF1 cells. Mean±SD.n=6.*P<0.05vs0 μmol/L group.
圖4CFTR特異性抑制劑CFTRinh-172對TF1細(xì)胞凋亡的影響
Figure 5. The effects of CFTR specific inhibitor CFTRinh-172 on the expression of Wnt signaling pathway-related proteins. Mean±SD.n=6.*P<0.05vs0 μmol/L group.
圖5CFTR特異性抑制劑CFTRinh-172對Wnt信號通路相關(guān)蛋白的影響
細(xì)胞生物學(xué)功能通常是受到細(xì)胞內(nèi)復(fù)雜信號通路的調(diào)控,Wnt信號通路就是其中之一。根據(jù)是否依據(jù)依賴β-catenin,Wnt信號通路可分為經(jīng)典和非經(jīng)典通路[13]。經(jīng)典的Wnt通路被激活后可抑制效應(yīng)物β-catenin的降解,進(jìn)而激活下游通路;研究顯示,該通路與造血干細(xì)胞的自我更新有著密切的關(guān)系,且在各類白血病中均出現(xiàn)異常[14-16]。此外,Le Henaff等[17]發(fā)現(xiàn) ΔF508-CFTR突變小鼠體內(nèi)成骨細(xì)胞分化及功能異常的出現(xiàn)與Wnt/β-catenin信號通路活性降低密切相關(guān)。故而本研究進(jìn)一步分析了在白血病細(xì)胞系TF1中,CFTR與Wnt/β-catenin信號通路之間的關(guān)系。結(jié)果顯示, CFTRinh-172可下調(diào)TF1細(xì)胞中β-catenin、c-Myc及cyclin D1 的蛋白水平,提示CFTRinh-172可抑制經(jīng)典的Wnt信號通路的激活。由此我們推測,CFTR對白血病細(xì)胞TF1生長及凋亡的影響可能與Wnt信號通路相關(guān),但是兩者之間是否存在直接的相互作用以及其中具體的作用機(jī)制還需進(jìn)一步深入的研究。
綜上所述,CFTR在急性髓系白血病患者及白血病細(xì)胞系TF1和THP-1中表達(dá)顯著升高。抑制CFTR可抑制白血病細(xì)胞株TF1的生長,并促進(jìn)其凋亡;其作用機(jī)制可能與經(jīng)典的Wnt信號通路相關(guān)。
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Effect of CFTR on viability and apoptosis of acute leukemia cells and its related mechanisms
NAN Hu-song, YIN Ming-ji, JIN Chun-ji
(DepartmentofPediatrics,YanbianUniversityHospital,Yanji133000,China.E-mail: 1569657202@qq.com)
AIM: To investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in acute myeloid leukemia (AML) and its effect on the biological function of human erythroleukemia cell line TF1, and to explore the underlying mechanism.METHODSThe abundance of CFTR in the bone marrow mononuclear cells of patients with AML was measured by real-time PCR. After TF1 cells were incubated with CFTR specific inhibitor CFTRinh-172, cell viability, cell cycle distribution and cell apoptosis were analyzed by CCK-8 assay and flow cytometry. The Wnt signaling pathway-related proteins were determined by Western blot.RESULTSCFTR was highly expressed in both patients with AML and leukemia cell lines. After incubated with CFTRinh172, the viability of TF1 cells was decreased, the proportion of the cells in G0/G1phase was increased, while that in S phase declined (P<0.05). Furthermore, the cells treated with CFTRinh-172 exhibited higher apoptotic rate, accompanied with lower protein expression of β-catenin, c-Myc and cyclin D1 (P<0.05).CONCLUSIONCFTR expression is dramatically increased in AML. Inhibition of CFTR restrains the growth and promotes the apoptosis of TF1 cells via classical Wnt signaling pathway.
Cystic fibrosis transmembrane conductance regulator; Acute myeloid leukemia; Cell viability; Cell cycle; Apoptosis; Wnt signaling pathway
1000- 4718(2017)12- 2202- 06
2017- 08- 23
2017- 11- 20
△通訊作者 Tel: 0433-2660083; E-mail: 1569657202@qq.com
R733.71; R363
A
10.3969/j.issn.1000- 4718.2017.12.014
(責(zé)任編輯: 陳妙玲, 羅 森)