壁虎活性單體衍生物腺嘌呤對(duì)人肝癌細(xì)胞作用的實(shí)驗(yàn)研究

韓 明，程 鑫，趙榮榮，張仕狀

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·論著·

壁虎活性單體衍生物腺嘌呤對(duì)人肝癌細(xì)胞作用的實(shí)驗(yàn)研究

韓 明，程 鑫，趙榮榮，張仕狀

目的 探討壁虎活性單體衍生物腺嘌呤對(duì)人肝癌細(xì)胞的抑制作用及機(jī)制。方法 2014年9月—2015年4月，收集對(duì)數(shù)生長(zhǎng)期的人肝癌Bel-7402細(xì)胞進(jìn)行實(shí)驗(yàn)，將96孔板的第2～9列作為腺嘌呤組〔加入腺嘌呤的濃度分別為0.500 00、0.250 00、0.125 00、0.062 50、0.031 25、0.015 63、0.007 81、0.003 91 mg/ml(分別記為A組～H組)〕，第10列作為對(duì)照組(加入含10%胎牛血清的RPMI-1640培養(yǎng)液)，第11列作為空白組。采用噻唑藍(lán)(MTT)法計(jì)算不同濃度腺嘌呤組不同時(shí)間(培養(yǎng)24、48、60 h)抑瘤率及腺嘌呤組半抑制濃度(IC50)。將對(duì)數(shù)生長(zhǎng)期的人肝癌Bel-7402細(xì)胞分為腺嘌呤組(加入0.500 00 mg/ml的腺嘌呤)和對(duì)照組(加入含10%胎牛血清的RPMI-1640培養(yǎng)液)，采用透射電鏡觀察細(xì)胞超微結(jié)構(gòu)。將對(duì)數(shù)生長(zhǎng)期的人肝癌Bel-7402細(xì)胞分為腺嘌呤組(加入0.500 00 mg/ml的腺嘌呤)和對(duì)照組(加入含10%胎牛血清的RPMI-1640培養(yǎng)液)，采用流式細(xì)胞儀檢測(cè)細(xì)胞周期。結(jié)果 24 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，E組～H組抑瘤率均低于D組，E組～G組抑瘤率均高于H組(P<0.05)；48 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，D組、E組抑瘤率均高于H組(P<0.05)；60 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，D組～F組抑瘤率均高于G組和H組(P<0.05)。C組、E組～H組24 h時(shí)抑瘤率均低于48 h時(shí)抑瘤率，A組～F組24 h時(shí)抑瘤率均低于60 h時(shí)抑瘤率，G組24 h時(shí)抑瘤率高于60 h時(shí)抑瘤率，A組～D組48 h時(shí)抑瘤率均低于60 h時(shí)抑瘤率，G組、H組48 h時(shí)抑瘤率高于60 h時(shí)抑瘤率(P<0.05)。不同時(shí)間IC50比較，差異有統(tǒng)計(jì)學(xué)意義(F=16.816，P<0.001)；48 h、60 h時(shí)IC50低于24 h時(shí)，60 h時(shí)IC50低于48 h時(shí)(P<0.05)。腺嘌呤作用48h后，肝癌細(xì)胞出現(xiàn)明顯的線粒體改變。對(duì)照組G0/G1期、G2/M期人肝癌Bel-7402細(xì)胞數(shù)高于腺嘌呤組，S期人肝癌Bel-7402細(xì)胞數(shù)低于腺嘌呤組(P<0.05)；腺嘌呤組S期人肝癌Bel-7402細(xì)胞數(shù)高于G0/G1期，G2/M期人肝癌Bel-7402細(xì)胞數(shù)低于S期(P<0.05)。結(jié)論 腺嘌呤抑制人肝癌細(xì)胞的增殖且呈一定的濃度、時(shí)間依賴性。腺嘌呤可引起人肝細(xì)胞線粒體改變，可將腫瘤細(xì)胞阻滯于S期并進(jìn)一步誘導(dǎo)其凋亡進(jìn)而發(fā)揮抗腫瘤作用。

肝腫瘤；壁虎活性單體；腺嘌呤；細(xì)胞凋亡

韓明，程鑫，趙榮榮，等.壁虎活性單體衍生物腺嘌呤對(duì)人肝癌細(xì)胞作用的實(shí)驗(yàn)研究[J].中國(guó)全科醫(yī)學(xué)，2015，18(27)：3308-3313.[www.chinagp.net]

Han M,Cheng X,Zhao RR,et al.Empirical research of gecko active monomer derivative adenine on human hepatic carcinoma cells[J].Chinese General Practice，2015，18(27)：3308-3313.

惡性腫瘤嚴(yán)重危害人類健康，高效低毒的抗腫瘤藥物的研發(fā)是優(yōu)化腫瘤治療的重要課題。目前核苷類抗腫瘤藥物種類眾多，大部分是將嘌呤、嘧啶等代謝物結(jié)構(gòu)進(jìn)行改造，通過干擾核酸的代謝，阻止腫瘤細(xì)胞的分裂和繁殖，以達(dá)到抗腫瘤的作用，如5-氟尿嘧啶、吉西他濱等[1-2]。目前，人體正常核苷成分的抗腫瘤作用國(guó)內(nèi)外鮮見報(bào)道，本研究觀察壁虎活性單體衍生物腺嘌呤的體外抑瘤作用、腫瘤細(xì)胞超微結(jié)構(gòu)及細(xì)胞周期改變等，初步探討腺嘌呤的抗腫瘤機(jī)制，為腺嘌呤的開發(fā)研究提供科學(xué)依據(jù)。

1 材料與方法

1.1 材料與儀器 人肝癌Bel-7402細(xì)胞購(gòu)自中國(guó)科學(xué)院典型培養(yǎng)物保藏委員會(huì)細(xì)胞庫(kù)。腺嘌呤(Sigma，美國(guó))，RPMI-1640 培養(yǎng)液、胰蛋白酶(Gibco，美國(guó))，胎牛血清(Solarbio，中國(guó))，噻唑藍(lán)(MTT，Amresco，美國(guó))。CO2培養(yǎng)箱(Thermo，美國(guó))，H-7500透射電鏡(HITACHI，日本)，CKX41倒置顯微鏡(OLYMPUS，日本)，F(xiàn)ACSCanto Ⅱ流式細(xì)胞儀(美國(guó)BD公司)。

1.2 研究方法

1.2.1 細(xì)胞復(fù)蘇與培養(yǎng) 2014年9月—2015年4月，從液氮罐中取出人肝癌Bel-7402細(xì)胞，37 ℃水浴中快

本研究背景：

由中藥壁虎展開研究，從壁虎中得到抗腫瘤活性單體，經(jīng)結(jié)構(gòu)鑒定為腺苷。由于腺苷的體內(nèi)代謝極其迅速，很難制成抗腫瘤藥物。根據(jù)核苷的結(jié)構(gòu)特點(diǎn)，本課題組對(duì)腺嘌呤進(jìn)行了進(jìn)一步的研究。腺嘌呤(又稱維生素B4)是一種臨床應(yīng)用的升白細(xì)胞藥物，有望成為一種新的抗腫瘤藥物。核苷是DNA和RNA的基本組成單位。目前，臨床常用的核苷類抗腫瘤藥物如5-氟尿嘧啶、氟達(dá)拉濱等，是通過將正常核苷的化學(xué)結(jié)構(gòu)進(jìn)行修飾，摻入DNA或RNA分子中干擾細(xì)胞復(fù)制，阻止細(xì)胞的分裂和繁殖，最終導(dǎo)致腫瘤細(xì)胞死亡。正常核苷類抗腫瘤藥物具有潛在的研究?jī)r(jià)值及廣闊的市場(chǎng)前景。

速搖動(dòng)凍存管，直至凍存液完全融化。將細(xì)胞懸液移入離心管，加入4 ml含10%胎牛血清的RPMI-1640培養(yǎng)液，以1 000 r/min離心5 min(離心半徑10 cm)，棄上清液，加入含10%胎牛血清的RPMI-1640培養(yǎng)液重懸細(xì)胞，轉(zhuǎn)移至培養(yǎng)瓶中，加適量含10%胎牛血清的RPMI-1640培養(yǎng)液，置于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)。每2～3 d傳代1次，收集對(duì)數(shù)生長(zhǎng)期的細(xì)胞進(jìn)行實(shí)驗(yàn)。

1.2.2 計(jì)算抑瘤率及半抑制濃度(IC50) 取處于對(duì)數(shù)生長(zhǎng)期的細(xì)胞，用胰蛋白酶消化后收集細(xì)胞，調(diào)整細(xì)胞濃度至2.5×104個(gè)/ml，接種于96孔培養(yǎng)板，每孔100 μl，37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)24 h。細(xì)胞貼壁后，棄去培養(yǎng)液。將96孔板的第2～9列作為腺嘌呤組，第10列作為對(duì)照組，第11列作為空白組，第1列和第12列僅加磷酸鹽緩沖液(PBS)以維持第2列和第11列的濕度，減少“邊緣效應(yīng)”[3]。腺嘌呤組用含10%胎牛血清的RPMI-1640培養(yǎng)液配制腺嘌呤溶液，并依次倍比稀釋，腺嘌呤的濃度分別為0.500 00、0.250 00、0.125 00、0.062 50、0.031 25、0.015 63、0.007 81、0.003 91 mg/ml(分別記為A組～H組)，對(duì)照組加入含10%胎牛血清的RPMI-1640培養(yǎng)液。分別于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)24、48、60 h后，采用CKX41倒置顯微鏡觀察細(xì)胞形態(tài)、數(shù)量，加入5 mg/ml的MTT 20 μl，繼續(xù)孵育4 h，小心吸棄全部上清液，每孔加入150 μl二甲基亞砜(DMSO)，輕微震蕩至結(jié)晶顆粒完全溶解。酶標(biāo)儀在570 nm處測(cè)定各組吸光度值(OD570)，設(shè)定空白組OD值為零。計(jì)算腺嘌呤對(duì)腫瘤細(xì)胞的抑制率，即抑瘤率〔抑瘤率(%)=(1-腺嘌呤組OD570平均值/對(duì)照組OD570平均值)×100%〕和IC50{IC50=lg-1〔Xm-i(ΣP-0.5)〕，其中Xm為設(shè)計(jì)的最大濃度的對(duì)數(shù)值，i為各組倍比濃度的對(duì)數(shù)值，ΣP為各組抑瘤率之和，0.5為經(jīng)驗(yàn)常數(shù)[4]}。實(shí)驗(yàn)共重復(fù)4次，取均值。

1.2.3 觀察人肝癌Bel-7402細(xì)胞超微結(jié)構(gòu) 用胰蛋白酶消化1.0×106個(gè)對(duì)數(shù)生長(zhǎng)期的人肝癌Bel-7402細(xì)胞后，收集細(xì)胞，鋪于25 cm2培養(yǎng)瓶中，2瓶細(xì)胞分別為腺嘌呤組及對(duì)照組，腺嘌呤組于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)24 h后加入濃度為0.500 00 mg/ml的腺嘌呤，對(duì)照組加入含10%胎牛血清的RPMI-1640培養(yǎng)液。48 h后用胰蛋白酶消化，收集細(xì)胞，2.5%戊二醛固定4 h，1%鋨酸4 ℃固定1 h、乙醇梯度脫水、丙酮脫水，包埋處理，超薄切片，經(jīng)醋酸鈾和枸櫞酸鉛雙重染色，將超薄切片置于H-7500透射電鏡下觀察細(xì)胞超微結(jié)構(gòu)并照相。

1.2.4 檢測(cè)人肝癌Bel-7402細(xì)胞周期 采用碘化丙啶(propidiumiodide，PI)單染法。胰蛋白酶消化并收集處于對(duì)數(shù)生長(zhǎng)期的人肝癌Bel-7402細(xì)胞，調(diào)整每瓶1.3×106個(gè)細(xì)胞，2瓶細(xì)胞分別為腺嘌呤組和對(duì)照組，腺嘌呤組培養(yǎng)24 h后加入濃度為0.500 00 mg/ml的腺嘌呤，對(duì)照組加入含10%胎牛血清的RPMI-1640培養(yǎng)液，每瓶6 ml。培養(yǎng)24 h后，胰蛋白酶消化，收集細(xì)胞于離心管內(nèi)，2 000 r/min離心5 min(離心半徑10 cm)，棄上清液；1 ml PBS洗2次，棄上清液，將殘余的PBS完全

在美國(guó)國(guó)防部和商務(wù)部列出的關(guān)鍵技術(shù)中，有80%是軍民重疊的技術(shù)。信息網(wǎng)絡(luò)技術(shù)、航空航天技術(shù)、新材料技術(shù)、新能源技術(shù)、核技術(shù)等典型的軍民兩用技術(shù)，不僅具有良好的經(jīng)濟(jì)技術(shù)效益，且能夠?qū)ο嚓P(guān)領(lǐng)域的科技發(fā)展起到巨大的帶動(dòng)作用，這就為推進(jìn)軍民融合發(fā)展提供十分難得的機(jī)遇。兩種資源的充分利用，會(huì)加快創(chuàng)新與發(fā)展，促進(jìn)核心競(jìng)爭(zhēng)力的提高。

本研究創(chuàng)新點(diǎn)：

腺嘌呤是一種臨床應(yīng)用的升白細(xì)胞藥物，關(guān)于其抗腫瘤作用鮮見報(bào)道。本研究探討壁虎活性單體衍生物腺嘌呤的體外抗腫瘤作用，發(fā)現(xiàn)腺嘌呤可抑制人肝癌細(xì)胞的增殖，并在一定程度上呈濃度與時(shí)間依賴性；腫瘤細(xì)胞出現(xiàn)明顯的線粒體改變；G0/G1、G2/M期細(xì)胞數(shù)減少，S期細(xì)胞數(shù)增加，表現(xiàn)出S期阻滯。由此可推測(cè)腺嘌呤通過誘導(dǎo)其凋亡進(jìn)而發(fā)揮抗腫瘤作用。

吸凈；將細(xì)胞置于4 ℃ 75%的乙醇(-20 ℃預(yù)冷)中固定1 h；2 500 r/min離心5 min(離心半徑3 cm)，棄固定液，PBS洗3次，加10 μl核糖核酸酶A(RNase A)至終濃度100 μg/ml，37 ℃孵育30 min；加入40 μl終濃度為100 μg/ml的PI，室溫避光孵育15 min；過濾后采用FACSCanto Ⅱ流式細(xì)胞儀檢測(cè)細(xì)胞周期。實(shí)驗(yàn)共重復(fù)3次，取均值。

2 結(jié)果

2.1 不同濃度腺嘌呤組抑瘤率及IC50比較 加入不同濃度的腺嘌呤后細(xì)胞出現(xiàn)不同程度的皺縮，貼壁細(xì)胞減少，體積變小，散在生長(zhǎng)，細(xì)胞呈多形性，細(xì)胞質(zhì)內(nèi)出現(xiàn)圓形透明顆粒，核分裂象減少，漂浮細(xì)胞增多，且隨著腺嘌呤濃度的增加、作用時(shí)間的延長(zhǎng)，細(xì)胞形態(tài)改變的程度越大。而對(duì)照組細(xì)胞形態(tài)呈上皮細(xì)胞樣，密集生長(zhǎng)，細(xì)胞質(zhì)內(nèi)未見透明顆粒。不同濃度腺嘌呤組不同時(shí)間抑瘤率比較，差異均有統(tǒng)計(jì)學(xué)意義(P<0.05)。24 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，E組～H組抑瘤率均低于D組，E組～G組抑瘤率均高于H組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；48 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，D組、E組抑瘤率均高于H組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；60 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，D組～F組抑瘤率均高于G組和H組，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。C組、E組～H組24 h時(shí)抑瘤率均低于48 h時(shí)抑瘤率，A組～F組24 h時(shí)抑瘤率均低于60 h時(shí)抑瘤率，G組24 h時(shí)抑瘤率高于60 h時(shí)抑瘤率，A組～D組48 h時(shí)抑瘤率均低于60 h時(shí)抑瘤率，G組、H組48 h時(shí)抑瘤率高于60 h時(shí)抑瘤率，差異有統(tǒng)計(jì)學(xué)意義(P<0.05，見表1)。24 h時(shí)IC50為(0.40±0.07)mg/ml，48 h時(shí)IC50為(0.34±0.04)mg/ml，60 h時(shí)IC50為(0.26±0.03)mg/ml，不同時(shí)間IC50比較，差異有統(tǒng)計(jì)學(xué)意義(F=16.816，P<0.001)；48 h、60 h時(shí)IC50低于24 h時(shí)，差異有統(tǒng)計(jì)學(xué)意義(q=3.764、8.193，P<0.05)；60 h時(shí)IC50低于48 h時(shí)，差異有統(tǒng)計(jì)學(xué)意義(q=4.428，P=0.013)。

Table 1 Comparison of tumor inhibition rate at different time points in different levels of adenine group

組別24h48h60hA組4368±4505142±8207818±725hiB組1719±232a2215±325a3294±307ahiC組1116±101ab1491±125abh1822±231abhiD組542±043abc596±046abc776±073abchiE組157±012abcd483±032abch455±033abchF組112±006abcd415±022abch412±028abchG組036±002abcd111±007abch-851±062abcdefhiH組-951±068abcdefg-450±038abcdeh-1089±103abcdefiχ2值305453046030324P值<005<005<005

注：與A組比較，aP<0.05；與B組比較，bP<0.05；與C組比較，cP<0.05；與D組比較，dP<0.05；與E組比較，eP<0.05；與F組比較，fP<0.05；與G組比較，gP<0.05；與24 h比較，hP<0.05；與48 h比較，iP<0.05

2.2 對(duì)照組和腺嘌呤組人肝癌Bel-7402細(xì)胞超微結(jié)構(gòu)比較 對(duì)照組人肝癌Bel-7402細(xì)胞結(jié)構(gòu)清晰，細(xì)胞器結(jié)構(gòu)完整，核內(nèi)染色質(zhì)分布均勻，線粒體較多，細(xì)胞表面有微絨毛及偽足突起。腺嘌呤作用48 h后，人肝癌Bel-7402細(xì)胞出現(xiàn)細(xì)胞核異染色質(zhì)增多、固縮、凝集于核膜邊緣，呈境界分明塊狀或新月狀，細(xì)胞核異形變甚至核碎裂，細(xì)胞微絨毛減少，細(xì)胞質(zhì)空泡化；線粒體改變最明顯，線粒體腫脹，發(fā)生空泡變、髓樣變；粗面內(nèi)質(zhì)網(wǎng)增粗、增寬，核糖體脫顆粒(見圖1)。

3 討論

中藥壁虎的臨床應(yīng)用歷史久遠(yuǎn)，壁虎對(duì)多種體外培養(yǎng)的腫瘤細(xì)胞有明顯的抑制作用[5-6]。本課題組選用山東濰坊地區(qū)的無蹼壁虎，在體外抑瘤實(shí)驗(yàn)示蹤下進(jìn)行抗腫瘤藥物篩選，提取得到了鮮無蹼壁虎抗腫瘤活性成分，制備出了鮮無蹼壁虎抗腫瘤活性成分脂質(zhì)體[7]，并獲得國(guó)家發(fā)明專利[8]。進(jìn)一步研究發(fā)現(xiàn)，壁虎活性成分可抑制人肝癌細(xì)胞Bel-7402、人肺癌細(xì)胞95-C的生長(zhǎng)[9]，并可以誘導(dǎo)人臍靜脈內(nèi)皮細(xì)胞ECV304、人肝癌細(xì)胞HepG-2的凋亡[10-11]，對(duì)小鼠結(jié)腸癌細(xì)胞CT-26體內(nèi)、外實(shí)驗(yàn)均顯示明顯的抑制作用[12-13]。在進(jìn)一步的體外藥篩示蹤下，對(duì)鮮無蹼壁虎抗腫瘤活性成分進(jìn)行梯級(jí)分離純化，得到抗腫瘤活性較強(qiáng)的單體化合物，經(jīng)鑒定為腺苷，與李雯等[14]、劉玉軍等[15]的研究結(jié)果一致。目前腺苷在臨床上被用來治療心律失常[16]。有關(guān)腺苷治療腫瘤的文獻(xiàn)報(bào)道較多，其抗腫瘤機(jī)制與激活A(yù)3受體有關(guān)[17-18]。腺苷的體內(nèi)代謝極其迅速[19-20]，很難制成抗腫瘤藥物。根據(jù)真核細(xì)胞核苷代謝和化學(xué)結(jié)構(gòu)組成的規(guī)律，本課題組推斷其他核苷及其正常衍生物可能有抗腫瘤作用。

Table 2 Comparison of human liver cancer Bel-7402 cell cycle between control group and adenine group

組別G0/G1期S期G2/M期對(duì)照組6460±1612415±1341125±112腺嘌呤組3226±1746774±193a001±000bt值236332131738P值<0001<0001<005

注：與G0/G1期比較，aP<0.05；與S期比較，bP<0.05

注：A為對(duì)照組(×8 000)，B為對(duì)照組(×20 000)，C為腺嘌呤組(×8 000)，D為腺嘌呤組(×20 000)

圖1 對(duì)照組與腺嘌呤組人肝癌Bel-7402細(xì)胞超微結(jié)構(gòu)(醋酸鈾和枸櫞酸鉛雙重染色)

Figure 1 The ultrastructure of human liver cancer Bel-7402 cells between control group and adenine group

腺嘌呤(又稱維生素B4)是一種臨床應(yīng)用的升白細(xì)胞藥物，本課題組希望能夠?qū)⑵渥罱K開發(fā)成全新機(jī)制的一類抗腫瘤藥物。本研究結(jié)果顯示，24 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，E組～H組抑瘤率均低于D組，E組～G組抑瘤率均高于H組；48 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，D組、E組抑瘤率均高于H組；60 h時(shí)，B組～H組抑瘤率均低于A組，C組～H組抑瘤率均低于B組，D組～H組抑瘤率均低于C組，D組～F組抑瘤率均高于G組和H組；C組、E組～H組24 h時(shí)抑瘤率均低于48 h時(shí)抑瘤率，A組～F組24 h時(shí)抑瘤率均低于60 h時(shí)抑瘤率，A組～D組48 h時(shí)抑瘤率均低于60 h時(shí)抑瘤率。提示腺嘌呤可顯著抑制人肝癌Bel-7402細(xì)胞的生長(zhǎng)，且具有一定的時(shí)效和量效關(guān)系。腺嘌呤對(duì)Bel-7402細(xì)胞作用后48 h、60 h時(shí)IC50低于24 h時(shí)，60 h時(shí)IC50低于48 h時(shí)，說明腺嘌呤對(duì)人肝癌Bel-7402細(xì)胞作用后不同時(shí)間點(diǎn)的IC50隨時(shí)間延長(zhǎng)而逐漸降低。

關(guān)于腫瘤發(fā)生機(jī)制的學(xué)說很多，經(jīng)典學(xué)說認(rèn)為，腫瘤是一類細(xì)胞周期紊亂和凋亡異常性疾病[21-22]。以調(diào)控細(xì)胞周期和誘導(dǎo)細(xì)胞凋亡為靶點(diǎn)，為抗腫瘤藥物的研究提供了新思路。

腫瘤不僅是增殖異常的疾病，同時(shí)也是凋亡異常的疾病。形態(tài)學(xué)觀察是檢測(cè)細(xì)胞凋亡最可靠的方法之一。凋亡發(fā)生的第一階段為凋亡的開始，形態(tài)學(xué)變化表現(xiàn)為細(xì)胞表面的特化結(jié)構(gòu)如微絨毛的減少，細(xì)胞膜仍完整，未失去選擇透過性；線粒體大致完整，核糖體逐漸從內(nèi)質(zhì)網(wǎng)上脫離，內(nèi)質(zhì)網(wǎng)囊腔膨脹，并逐漸與質(zhì)膜融合；染色質(zhì)固縮，形成新月形帽狀結(jié)構(gòu)等形態(tài)。第二階段為凋亡小體形成，核染色質(zhì)斷裂，細(xì)胞表面產(chǎn)生了許多泡狀或芽泡狀突起；隨后逐漸分離，形成單個(gè)的凋亡小體，并逐漸被鄰近的細(xì)胞所吞噬并消化[23]。本研究結(jié)果顯示，腺嘌呤作用48 h后，人肝癌Bel-7402細(xì)胞出現(xiàn)細(xì)胞核異染色質(zhì)增多、固縮、凝集于核膜邊緣，細(xì)胞核異形變甚至核碎裂，細(xì)胞微絨毛減少，細(xì)胞質(zhì)空泡化；線粒體改變最明顯，線粒體腫脹，發(fā)生空泡變、髓樣變。提示人肝癌Bel-7402細(xì)胞發(fā)生了凋亡的起始改變。腺嘌呤對(duì)腫瘤細(xì)胞作用的形態(tài)學(xué)表現(xiàn)符合細(xì)胞凋亡的形態(tài)學(xué)變化特征。

許多抗腫瘤藥物可使細(xì)胞周期停滯在G1或G2/M期，大部分為G2/M期阻滯[24-26]。G2/M期轉(zhuǎn)換在細(xì)胞周期進(jìn)展及凋亡啟動(dòng)中起重要作用，G2/M期阻滯的細(xì)胞容易發(fā)生凋亡，其增殖能力明顯降低。本實(shí)驗(yàn)采用PI單染法檢測(cè)腺嘌呤對(duì)人肝癌Bel-7402細(xì)胞周期分布的影響，結(jié)果顯示，對(duì)照組G0/G1期、G2/M期人肝癌Bel-7402細(xì)胞數(shù)高于腺嘌呤組，S期人肝癌Bel-7402細(xì)胞數(shù)低于腺嘌呤組，且腺嘌呤組S期人肝癌Bel-7402細(xì)胞數(shù)高于G0/G1期，G2/M期人肝癌Bel-7402細(xì)胞數(shù)低于S期，提示腺嘌呤對(duì)腫瘤細(xì)胞有S期阻滯作用，導(dǎo)致腫瘤細(xì)胞增殖周期停止，隨后發(fā)生細(xì)胞凋亡。

綜上所述，壁虎活性單體衍生物腺嘌呤可顯著抑制腫瘤細(xì)胞的增殖，且呈一定的濃度、時(shí)間依賴性。腺嘌呤作用腫瘤細(xì)胞48 h后，透射電鏡下呈現(xiàn)凋亡細(xì)胞的形態(tài)改變；可通過將腫瘤細(xì)胞阻滯于S期并進(jìn)一步誘導(dǎo)腫瘤細(xì)胞凋亡進(jìn)而發(fā)揮抗腫瘤作用，但其分子機(jī)制有待于進(jìn)一步研究。

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(本文編輯：崔麗紅)

Empirical Research of Gecko Active Monomer Derivative Adenine on Human Hepatic Carcinoma Cells

HANMing,CHENGXin,ZHAORong-rong,etal.

BasicMedicalCollege,WeifangMedicalUniversity,Weifang261053,China

Objective To study the inhibitory effect of gecko active monomer derivative adenine on human hepatic carcinoma cells.Methods Human liver cancer Bel-7402 cells in logarithmic phase and conducted experiments were collected in the study from September 2014 to April 2015.The 2nd to 9th columns of the 96 hole plate was assigned into the adenine group (adenine concentrations were 0.500 00,0.250 00,0.125 00,0.062 50,0.031 25,0.015 63,0.007 81 and 0.003 91 mg/ml,which were respectively recorded as group A to group H).The 10th column was taken as the control group (RPMI-1640 culture medium containing 10% fetal bovine serum).The 11th column was taken as the blank group.Thiazolyl blue method (MTT) was used to observe the tumor inhibition rate of each group (at 24 h,48 h and 60 h of the culture) and the 50% inhibiting concentration (IC50) of the adenine group.The human liver cancer Bel-7402 cells in logarithmic phase were divided into adenine group (0.500 00 mg/ml adenine) and control group (RPMI-1640 culture medium containing 10% fetal bovine serum),and the cell ultrastructure was observed by transmission electron microscope.The collected human liver cancer Bel-7402 cells in logarithmic phase were divided into adenine group (0.500 00 mg/ml adenine) and control group (RPMI-1640 culture medium containing 10% fetal bovine serum),and tumor cell cycle was detected by flow cytometry.Results At 24 h,tumor inhibition rates of group B-group H were lower than those of group A;tumor inhibition rates of group C-group H were lower than group B;tumor inhibition rates of group D-group H were lower than group C;tumor inhibition rates of group E-group H were lower than group D;tumor inhibition rates of group E-group G were higher than H group (P<0.05).At 48 h,tumor inhibition rates of group B -group H were lower than those of group A;tumor inhibition rates of group C-group H were lower than group B;tumor inhibition rates group D-group H were lower than group C;tumor inhibition rates of group D and group E were higher than group H (P<0.05).At 60 h,tumor inhibition rates of group B-group H were lower than that of group A;tumor inhibition rates of group C-group H were lower than group B;tumor inhibition rate of group D-group H were lower than group C;tumor inhibition rates of group D-group F were higher than group G and group H (P<0.05).Tumor inhibition rates of group C and group E-group H at 24 h were lower than those at 48 h.Tumor inhibition rates of group A-group F at 24 h were lower than those at 60 h.Tumor inhibition rate of group G at 24 h was higher than that at 60 h.Tumor inhibition rates of group A-group D at 48 h were lower than those at 60 h.Tumor inhibition rates of group G and goupr H at 48 h were higher than those at 60 h(P<0.05).IC50varied significantly at different time points (F=16.816,P=0.000).The IC50at 48 h and 60 h was lower than that at 24 h;the IC50at 60 h was lower than that at 48 h (P<0.05).After 48h,the changes of mitochondria in liver cancer cells were obvious.The numbers of cells in G0/G1and G2/M phase in control group were higher than those in adenine group;the number of cells in S phase in control group was lower than that in adenine group (P<0.05).In adenine group,the number of cells in S phrase was higher than that in G0/G1phrase,and the number of cells in G2/M phrase was lower than that in S phrase (P<0.05).Conclusion Adenine could inhibit the proliferation of human hepatic carcinoma cells in a time and dose dependent manner.Adenine lead to changes of mitochondria.Adenine could inhibit tumor cells in S phase and further induce tumor cell apoptosis,thus playing an anti-tumor effect.

Liver neoplasms;Gecko active monomer;Adenine;Apoptosis

國(guó)家自然科學(xué)基金資助項(xiàng)目(81202992，81102735)；山東省自然科學(xué)基金資助項(xiàng)目(ZR2011HQ023，ZR2014HP008)；山東省醫(yī)藥衛(wèi)生科技發(fā)展計(jì)劃項(xiàng)目(2011QZ026)

261053 山東省濰坊市，濰坊醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院(韓明)，醫(yī)學(xué)影像學(xué)系(程鑫，趙榮榮，張仕狀)

張仕狀，261053 山東省濰坊市，濰坊醫(yī)學(xué)院醫(yī)學(xué)影像學(xué)系；E-mail：zhangsz5712@163.com

R 735.7

A

10.3969/j.issn.1007-9572.2015.27.011

2015-05-14；

2015-07-02)