簡捷，劉利珍，黃緣，李雙，鄧峰

(1南昌大學(xué)第三附屬醫(yī)院，南昌 330008；2上海市嘉定區(qū)中心醫(yī)院；3南昌大學(xué)第二附屬醫(yī)院)

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人參皂苷Rg3對人結(jié)腸癌細(xì)胞株SW480增殖的影響及其作用機(jī)制

簡捷1，劉利珍2，黃緣3，李雙1，鄧峰1

(1南昌大學(xué)第三附屬醫(yī)院，南昌 330008；2上海市嘉定區(qū)中心醫(yī)院；3南昌大學(xué)第二附屬醫(yī)院)

摘要：目的觀察人參皂苷Rg3對人結(jié)腸癌細(xì)胞株SW480增殖的影響，并探討其可能作用機(jī)制。方法取對數(shù)生長期SW480細(xì)胞，分別加入含160、80、40、0 μmol/L人參皂苷Rg3的儲備液(分別計為A、B、C、D組)培養(yǎng)24 h，采用MTT法檢測人參皂苷Rg3對SW480細(xì)胞增殖活性(OD值)的影響，倒置顯微鏡觀察人參皂苷Rg3誘導(dǎo)SW480細(xì)胞凋亡形態(tài)學(xué)改變，流式細(xì)胞術(shù)檢測SW480細(xì)胞凋亡率，RT-PCR法檢測細(xì)胞間黏附分子1(ICAM-1)、閉鎖蛋白(Occludin)mRNA，Western blotting法檢測ICAM-1、Occludin蛋白。結(jié)果與D組比較，A、B、C組細(xì)胞OD值下降，凋亡率增加，ICAM-1 mRNA、蛋白表達(dá)降低，Occludin mRNA、蛋白表達(dá)升高(P均<0.05)。結(jié)論 人參皂苷Rg3可抑制SW480細(xì)胞增殖，促進(jìn)其凋亡，機(jī)制可能與ICAM-1表達(dá)下調(diào)、Occludin表達(dá)上調(diào)有關(guān)。

關(guān)鍵詞：人參皂苷Rg3；結(jié)腸癌細(xì)胞株SW480；細(xì)胞間黏附分子1；閉鎖蛋白

人參皂苷Rg3是從人參根的浸出液中分離出的一種有效成分，是一種微量四環(huán)三萜皂苷，可抑制腫瘤細(xì)胞新生血管形成，誘導(dǎo)腫瘤細(xì)胞凋亡，并能選擇性抑制腫瘤細(xì)胞轉(zhuǎn)移，提高機(jī)體免疫功能[1～3]。細(xì)胞間黏附分子(ICAM)在腫瘤生長、侵襲和轉(zhuǎn)移中起重要作用，能介導(dǎo)異型細(xì)胞間及細(xì)胞-細(xì)胞外基質(zhì)間的黏附作用，并參與免疫、炎癥、腫瘤轉(zhuǎn)移等一系列重要生理和病理過程。腫瘤的浸潤、轉(zhuǎn)移與上皮細(xì)胞的通透性密切相關(guān)，而閉鎖蛋白(Occludin)是上皮細(xì)胞緊密連接中最重要的結(jié)構(gòu)蛋白，可通過外環(huán)以拉鏈?zhǔn)浇Y(jié)合產(chǎn)生嚴(yán)密的細(xì)胞旁封閉，還能與不同的分子結(jié)合，參與緊密連接形成的信號調(diào)節(jié)。本研究觀察了人參皂苷Rg3對人結(jié)腸癌細(xì)胞株SW480增殖的影響，并探討其可能機(jī)制。

1材料與方法

1.1細(xì)胞、藥物及試劑SW480細(xì)胞購自上海滬尚生物科技有限公司。人參皂苷Rg3購自吉林亞泰制藥股份有限公司，MTT和胰酶購自美國Sigma公司，Annexin V-FITC試劑盒購自美國Beckman Coulter公司，引物由上海Sangon公司合成，TRIzol試劑、逆轉(zhuǎn)錄試劑盒均購自美國Invitrogen公司，抗ICAM-1及Occludin抗體均購自英國Abcam公司，辣根過氧化物酶(HRP)標(biāo)記的羊抗兔二抗購自北京全式金生物技術(shù)有限公司，RPMI1640培養(yǎng)基購自美國Gibco公司，胎牛血清購自杭州四季青公司。

1.2SW480細(xì)胞培養(yǎng)及人參皂苷Rg3配制細(xì)胞常規(guī)培養(yǎng)于含100 mL/L胎牛血清的RPMI1640培養(yǎng)液中。人參皂苷Rg3用二甲基亞砜(DMSO)溶解，配制成80 mmol/L的儲備液，用培養(yǎng)液稀釋至所需濃度，DMSO在各組培養(yǎng)液中的終濃度均為1 mL/L。

1.3人參皂苷Rg3對SW480細(xì)胞增殖影響觀察取對數(shù)生長期SW480細(xì)胞，常規(guī)胰酶消化接種于96孔板，每孔4×103個細(xì)胞，培養(yǎng)24 h后，加入人參皂苷Rg3儲備液，使其稀釋后終濃度分別為160、80、40、0 μmol/L(分別計為A、B、C、D組)，每組設(shè)6個平行孔，培養(yǎng)24 h后加入5 g/L的MTT共20 μL，再培養(yǎng)4 h后倒盡板中培養(yǎng)液，各孔加100 μL的DMSO，輕輕振蕩后用酶標(biāo)儀(波長490 nm)測定各孔的光密度值(OD值)，以每組6個孔的平均值作為各組的平均值。實驗重復(fù)3次。

1.4人參皂苷Rg3對SW480細(xì)胞凋亡影響觀察常規(guī)培養(yǎng)SW480細(xì)胞，按1×105個/孔接種于24孔細(xì)胞培養(yǎng)板。分別用160、80、40、0 μmol/L人參皂苷Rg3處理SW480細(xì)胞24 h(分別計為A、B、C、D組)，倒置顯微鏡下觀察其形態(tài)變化。收集SW480細(xì)胞，PBS洗滌1次，400 μL Binding Buffer重懸細(xì)胞。取200 μL細(xì)胞懸液加入Annexin V 5 μL，PI 10 μL，避光20 min，加入200 μL Binding Buffer上機(jī)檢測細(xì)胞凋亡率。用Cellquest軟件獲取數(shù)據(jù)，用Mod Fit軟件進(jìn)行數(shù)據(jù)分析。

1.5SW480細(xì)胞ICAM-1、Occludin mRNA檢測采用RT-PCR法。常規(guī)收集經(jīng)160、80、40、0 μmol/L人參皂苷Rg3處理24 h的SW480細(xì)胞(分別計為A、B、C、D組)，總RNA提取按照TRIzol試劑說明書的操作步驟進(jìn)行。逆轉(zhuǎn)錄為cDNA后使用LA Taq擴(kuò)增ICAM-1、Occludin編碼區(qū)域。PCR產(chǎn)物全部加入10 g/L低熔點瓊脂糖凝膠進(jìn)行電泳檢測。利用Bandleader3.0軟件分析PCR產(chǎn)物條帶密度。結(jié)果以(目的基因OD值/β-actin基因OD值)×100%表示。

1.6SW480細(xì)胞ICAM-1、Occludin蛋白檢測采用Western blotting法。常規(guī)收集經(jīng)160、80、40、0 μmol/L人參皂苷Rg3處理24 h的SW480細(xì)胞(分別計為A、B、C、D組)，提取細(xì)胞總蛋白，用Bradford法測定蛋白濃度。取5×106細(xì)胞，與2×SDS凝膠加樣緩沖液等體積混合，100 ℃加熱5 min后分裝于-70 ℃凍存。細(xì)胞總蛋白每泳道上樣30 μg，12.5%SDS凝膠電泳，1 mA/cm2半干轉(zhuǎn)100 min，以含5%脫脂奶粉的TBST室溫封閉2 h，分別加入一抗(ICAM-1單抗、Occludin單抗，1∶400稀釋)4 ℃孵育過夜，二抗(IgG-HRP，1∶2 000稀釋)37 ℃孵育1 h，ECL顯影，凝膠成像系統(tǒng)掃描各條帶灰度值，以此表示ICAM-1、Occludin蛋白的相對表達(dá)量。

2結(jié)果

2.1各組細(xì)胞OD值、細(xì)胞凋亡率比較結(jié)果見表1。

表1　各組細(xì)胞OD值、細(xì)胞凋亡率比較±s)

注：與D組比較，*P<0.05，△P<0.01。

2.2各組細(xì)胞ICAM-1 mRNA、Occludin mRNA、ICAM-1蛋白、Occludin蛋白比較結(jié)果見表2。

表2　各組細(xì)胞ICAM-1 mRNA、Occludin mRNA、ICAM-1蛋白、Occludin蛋白比較±s)

注：與D組比較，*P<0.05，△P<0.01。

3討論

結(jié)腸癌是一種高發(fā)的惡性腫瘤，浸潤和轉(zhuǎn)移是影響患者預(yù)后的主要原因，但其轉(zhuǎn)移擴(kuò)散的具體機(jī)制尚不清楚。人參皂苷Rg3是中藥中的一種抗腫瘤成分，目前其抗癌作用引起廣泛關(guān)注。人參皂苷Rg3可以抑制腫瘤細(xì)胞的黏附、浸潤、增殖，與化療藥聯(lián)合應(yīng)用可提高抗腫瘤作用。體內(nèi)外實驗及流行病學(xué)研究顯示，人參皂苷Rg3對多種腫瘤均有增殖抑制作用。而通過體外抗浸潤試驗證明，人參皂甙Rg3能明顯抑制小鼠腹水肝癌細(xì)胞、人小細(xì)胞肺癌和人胰腺癌細(xì)胞的單層浸潤。研究[4,5]還發(fā)現(xiàn)，人參皂苷Rg3對高轉(zhuǎn)移性的小鼠黑色素瘤細(xì)胞肺轉(zhuǎn)移及小鼠結(jié)腸癌細(xì)胞肺轉(zhuǎn)移具有抑制作用。本研究顯示，A、B、C組細(xì)胞OD值較D組下降，凋亡率較D組增加，提示人參皂苷Rg3對SW480細(xì)胞有明顯的抑制作用，并誘導(dǎo)其凋亡。

ICAM-1是由人第19對染色體編碼的單鏈糖蛋白，其表達(dá)水平與腫瘤淋巴結(jié)轉(zhuǎn)移、TNM分期密切相關(guān)[6,7]。惡性腫瘤的轉(zhuǎn)移擴(kuò)散，需借助于其細(xì)胞表面的黏附分子和細(xì)胞外基質(zhì)及其他細(xì)胞的一系列相互作用，近年研究[8,9]表明ICAM-1與肝癌、胰腺癌、胃癌、結(jié)腸癌等腫瘤轉(zhuǎn)移密切相關(guān)。上皮細(xì)胞是細(xì)胞黏膜屏障的主要組織結(jié)構(gòu)基礎(chǔ)，細(xì)胞間的連接復(fù)合體參與維持上皮細(xì)胞的完整性，其中緊密連接是細(xì)胞間最重要的連接方式。近來研究發(fā)現(xiàn)，腫瘤的浸潤、轉(zhuǎn)移與上皮細(xì)胞的通透性密切相關(guān)。上皮的通透性有兩個途徑，即跨上皮途徑和細(xì)胞旁通路(緊密連接)。細(xì)胞旁通路是一組復(fù)雜的結(jié)構(gòu)，主要由上皮細(xì)胞之間的緊密連接控制。細(xì)胞旁通路對內(nèi)毒素和細(xì)菌的其他產(chǎn)物等大分子物質(zhì)的通過起到關(guān)鍵性調(diào)控作用[10]。緊密連接和黏附連接與細(xì)胞的增殖和腫瘤的形成密切相關(guān)[11～13]。Occludin是緊密連接中最重要的組成蛋白，其人類基因定位于5q13．1，mRNA全長2 379 bp，編碼522個氨基酸[14]。多數(shù)學(xué)者認(rèn)為，Occludin表達(dá)的減少使緊密連接的滲透性增加，導(dǎo)致上皮間質(zhì)轉(zhuǎn)化，隨后通道開放促進(jìn)腫瘤的進(jìn)展[15]。本研究發(fā)現(xiàn)，A、B、C組ICAM-1 mRNA、蛋白表達(dá)較D組降低，Occludin mRNA、蛋白表達(dá)較D組升高。提示人參皂苷Rg3可抑制SW480細(xì)胞增殖，促進(jìn)其凋亡，機(jī)制可能與ICAM-1表達(dá)下調(diào)、Occludin表達(dá)上調(diào)有關(guān)。

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Effect of ginsenoside Rg3 on cell proliferation of human colon cancer cell line SW480 and its mechanism

JIANJie1,LIULizhen,HUANGYuan,LIShuang,DENGFeng

(1TheThirdAffiliatedHospitalofNanchangUniversity,Nanchang330008,China)

Abstract:ObjectiveTo observe the effect of ginsenoside Rg3 on the proliferation of human colon cancer cell line SW480, and to investigate its mechanism.Methods SW480 cells in the logarithmic phase were exposed to 160, 80, 40 and 0 μmol/L ginsenoside Rg3 for 24 hours (marked as groups A, B, C and D). MTT assay was used to measure the effect of ginsenoside Rg3 on the cell proliferative activity (OD value). Invert microscope was used to observe the morphological changes of SW480 cells, and flow cytometry was employed to measure the apoptosis of SW480 cells. The intercellular adhesion molecule 1 (ICAM-1) and occludin mRNA was measured by reverse transcription polymerase chain reaction (RT-PCR), and Western blotting was used to detect the expression of ICAM-1 and occludin protein.ResultsThe OD values were decreased, the apoptosis rates were increased, the expression of ICAM-1 mRNA and protein was decreased, and the expression of occludin mRNA and protein was increased in the groups A, B and C as compared with those of group D (all P<0.05).ConclusionGinsenoside Rg3 can inhibit the proliferation of SW480 cells and induce apoptosis, and its mechanism may be related with the down-regulated expression of ICAM-1 and up-regulated expression of occludin.

Key words:ginsenoside Rg3; colon cancer cell line SW480; intercellular adhesion molecule 1; occludin

(收稿日期:2015-09-15)

作者簡介：第一簡捷(1984-)，男，碩士，主治醫(yī)師，主要研究方向為消化道腫瘤相關(guān)基因。E-mail: 9957813@qq.com

中圖分類號：R735.3

文獻(xiàn)標(biāo)志碼：A

文章編號：1002-266X(2016)03-0008-03

doi：10.3969/j.issn.1002-266X.2016.03.003