郭冰玉，張 宇，回 薔，全亮亮，陶 凱

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·論著·

防己諾林堿抑制黑色素瘤的增殖轉(zhuǎn)移

郭冰玉，張 宇，回 薔，全亮亮，陶 凱*

目的 觀察防己諾林堿對(duì)黑色素瘤細(xì)胞生長轉(zhuǎn)移的影響。方法 通過MTT實(shí)驗(yàn)，觀察不同濃度防己諾林堿對(duì)A375細(xì)胞增殖的影響；利用Transwell實(shí)驗(yàn)，檢測(cè)不同濃度防己諾林堿對(duì)A375細(xì)胞轉(zhuǎn)移功能的影響。通過Western blot和RT-PCR，檢測(cè)防己諾林堿對(duì)A375生長轉(zhuǎn)移相關(guān)蛋白的影響。結(jié)果 防己諾林堿可以抑制黑色素瘤細(xì)胞A375的增殖轉(zhuǎn)移。10、20、40 μM/L防己諾林堿處理A375細(xì)胞后，對(duì)其增殖的抑制率分別為43.81%±1.53%、48.64%±4.65%、50.69%±4.99%。20、40 μM/L防己諾林堿處理A375細(xì)胞作用24 h后，對(duì)其轉(zhuǎn)移的抑制率分別為14.95%±4.31%、33.03%±5.46%。防己諾林堿顯著抑制細(xì)胞周期蛋白D1(Cyclin D1)、CDK4、CDK6、基質(zhì)金屬蛋白酶-2(MMP2)的表達(dá)(P<0.05)。結(jié)論 防己諾林堿能顯著抑制A375細(xì)胞的增殖轉(zhuǎn)移，可作為治療黑色素瘤的潛在化療藥物進(jìn)行深入研究。

防己諾林堿；增殖；轉(zhuǎn)移；A375；黑色素瘤

0 引言

黑色素瘤是一種惡性程度高、容易發(fā)生早期轉(zhuǎn)移、死亡率高的惡性皮膚腫瘤[1]。目前其發(fā)病機(jī)制尚未完全闡明，臨床上除了早期發(fā)現(xiàn)的手術(shù)治療外，尚無有效的治療藥物[2]。近年來，國內(nèi)外黑色素瘤的發(fā)病率急劇上升，死亡率居高不下，黑色素瘤的死亡率為皮膚腫瘤的80%[3]。雖然早期黑色素瘤可以通過手術(shù)獲得痊愈，但是由于其發(fā)病隱蔽，易發(fā)生轉(zhuǎn)移，且轉(zhuǎn)移后對(duì)大多數(shù)放化療藥物不敏感，所以黑色素瘤患者的5年生存率很難提高[4-6]。

防己諾林堿(Fangchinoline)又名漢防己、白木香，是防己科植物粉防己干燥的根，具有抗炎鎮(zhèn)痛、抗氧化、非特異性阻滯鈣離子通道、抑制組胺釋放、擴(kuò)張冠狀動(dòng)脈、減少心肌耗氧量、抑制血小板聚集、降血壓、降血糖等多種生物學(xué)功能[7-8]。另有研究證實(shí)，在某些細(xì)胞中，防己諾林堿可以通過阻滯細(xì)胞周期、誘導(dǎo)細(xì)胞自噬凋亡來實(shí)現(xiàn)對(duì)抗腫瘤的效果[8]。但是目前沒有明確的報(bào)道指出防己諾林堿對(duì)黑色素瘤是否有治療效果[9]。

本研究初步探討了防己諾林堿單體對(duì)人黑色素瘤細(xì)胞A375增殖與轉(zhuǎn)移的影響，發(fā)現(xiàn)防己諾林堿可以顯著抑制A375細(xì)胞的增殖與轉(zhuǎn)移功能，降低細(xì)胞周期蛋白D1(Cyclin D1)等細(xì)胞周期相關(guān)蛋白及基質(zhì)金屬蛋白酶-2(MMP2，這些物質(zhì)在黑色素瘤中的蛋白表達(dá)升高[10-11])，旨在為防己諾林堿在黑色素瘤中的治療提供新的理論依據(jù)。

1 材料與方法

1.1 材料 人黑色素瘤細(xì)胞A375(實(shí)驗(yàn)室凍存)，胎牛血清(天津?yàn)?，天?，DMEM培養(yǎng)基(Invitrogen，上海)，0.4%臺(tái)盼藍(lán)(經(jīng)科，上海)，防己諾林堿(Selleck，美國)，抗體：Cyclin D1，CDK4、CDK6、MMP2和GAPDH(Santa，美國)，SYBR Green (Promega，美國)。

1.2 方法

1.2.1 細(xì)胞培養(yǎng) 使用含10%胎牛血清的DMEM培養(yǎng)基培養(yǎng)人黑色素瘤細(xì)胞A375。

1.2.2 藥物處理濃度 選取10、20、40 μM/L防己諾林堿；對(duì)照組用生理鹽水處理。

1.2.3 MTT實(shí)驗(yàn) 以1×104/孔密度將A375細(xì)胞接種于96孔板中，12 h后添加不同濃度的防己諾林堿。藥物處理后分別在0、12、24、36、48 h加入MTT，孵育4 h后加入100 μL DMSO溶解MTT-甲臜結(jié)晶，15 min后，測(cè)量490 nm吸光值。藥物對(duì)細(xì)胞增殖的抑制率計(jì)算公式：抑制率=1-(劑量組平均OD值/對(duì)照組平均OD值)×100%。

1.2.4 Transwell實(shí)驗(yàn) 以1×105/孔密度將細(xì)胞接種于上室中，下室添加600 μL雙無培養(yǎng)基，12 h后添加不同處理因素。藥物作用24 h后，95%乙醇固定，0.4%臺(tái)盼藍(lán)染色。200倍顯微鏡下觀察。藥物對(duì)細(xì)胞轉(zhuǎn)移的抑制率計(jì)算公式：抑制率=1-(劑量組平均細(xì)胞數(shù)/對(duì)照組平均細(xì)胞數(shù))×100%。

1.2.5 Western blot實(shí)驗(yàn) 裂解藥物處理24 h后的細(xì)胞，取40 μg蛋白進(jìn)行SDS-PAGE電泳，轉(zhuǎn)膜封閉后，一抗4 ℃過夜，二抗室溫1 h。

1.2.6 RT-PCR實(shí)驗(yàn) 通過TRIZOL法提取細(xì)胞總RNA，反轉(zhuǎn)錄成cDNA，進(jìn)行RT-PCR反應(yīng)。所用引物為Cyclin D1，F(xiàn)(5′-3′)：CGAGGAGCTGCTGCAAATGG，R(5′-3′)：GAAATCGTGCGGGGTCATTGCG；CDK4，F(xiàn)(5′-3′):CTGGTGACAAGTGGTGGAAC，R(5′-3′):GGTCGGCTTCAGAGTTTCC；CDK6，F(xiàn)(5′-3′)：GTCTGATTACCTGCTCCGC，R(5′-3′)：CCTCGAAGCGAAGTCCTC；MMP2，F(xiàn)(5′-3′)：CGCATCTGGGGCTTTAAAC，R(5′-3′)：CAGCACAAACAGGTTGCAG；GAPDH，F(xiàn)(5′-3′)：CATCCCTTCTCCCCACACAC，R(5′-3′)：AGTCCCAGGGCTTTGATTTG.

2 結(jié)果

2.1 防己諾林堿對(duì)A375細(xì)胞增殖的影響 如圖1所示，MTT實(shí)驗(yàn)顯示，防己諾林堿處理后，A375細(xì)胞的增殖受到抑制。表1、圖2顯示，防己諾林堿對(duì)A375細(xì)胞增殖的抑制作用隨藥物濃度的增加而增加。

圖1 不同濃度防己諾林堿處理后A375細(xì)胞的增殖情況(n=3)

時(shí)間(h)10μM/L20μM/L40μM/L01.36±2.360.97±0.68-0.99±4.661233.37±0.83**34.55±2.48**36.84±2.99**2443.81±1.53**48.64±4.65**50.69±4.99**3648.72±0.86**53.91±1.15**58.28±1.81**4849.83±2.57**58.05±0.96**63.69±0.69**

注：與0 h比較，**P<0.01

圖2 防己諾林堿對(duì)A375細(xì)胞增殖的抑制率(n=3)

2.2 防己諾林堿對(duì)A375細(xì)胞增殖相關(guān)蛋白的影響 如圖3、圖4所示，防己諾林堿對(duì)Cyclin D1、CDK4與CDK6均有一定程度的抑制作用，抑制作用隨著藥物濃度的增加而增強(qiáng)。

2.3 防己諾林堿對(duì)A375細(xì)胞轉(zhuǎn)移的影響 見圖5、圖6。結(jié)果顯示，防己諾林堿可顯著抑制A375細(xì)胞的轉(zhuǎn)移功能，其抑制作用隨著藥物濃度增加而增加。作用24 h后，NaCl及20、40 μM/L防己諾林堿對(duì)A375細(xì)胞轉(zhuǎn)移的抑制率(n=3)分別為0、14.95%±4.31%、33.03%±5.46%。

2.4 防己諾林堿對(duì)MMP2的影響 如圖7、圖8所示，防己諾林堿可以在蛋白及RNA水平上下調(diào)MMP2，其下調(diào)作用隨藥物濃度的升高而增強(qiáng)。

圖3 不同濃度防己諾林堿作用下A375細(xì)胞蛋白的變化

圖4 不同濃度防己諾林堿處理后A375細(xì)胞蛋白R(shí)NA水平的影響

圖5 防己諾林堿作用下A375細(xì)胞的轉(zhuǎn)移情況(n=3)

圖6 防己諾林堿作用下A375細(xì)胞轉(zhuǎn)移數(shù)目(n=3)

圖7 防己諾林堿對(duì)MMP2的影響

圖8 防己諾林堿對(duì)MMP2的影響

3 討論

作為皮膚腫瘤的一種，黑色素瘤的發(fā)病率增長迅速[12-13]。黑色素瘤具有過度增殖、易轉(zhuǎn)移等特點(diǎn)，早期可以出現(xiàn)淋巴結(jié)、肝、腦等多處轉(zhuǎn)移。其治療棘手，預(yù)后差，已引起人們的廣泛關(guān)注[14]。黑色素瘤組織中經(jīng)常發(fā)現(xiàn)血管生成因子、MMP等蛋白的高表達(dá)[15]。黑色素瘤細(xì)胞的增殖與侵襲的信號(hào)轉(zhuǎn)導(dǎo)通路經(jīng)常會(huì)發(fā)生異常激活，因此，篩選出低毒、高效的靶向抑制劑成為研究黑色素瘤治療的發(fā)展趨勢(shì)。

研究表明，很多中藥不僅可以直接殺傷腫瘤細(xì)胞，也可以降低放化療產(chǎn)生的毒副作用[16]，同時(shí)，已經(jīng)有很多輔助化療中藥對(duì)人體的不良反應(yīng)較小且不易發(fā)生耐藥反應(yīng)[17]。最近研究表明，防己諾林堿是一種有效的抗腫瘤中藥成分，可以與其他化療藥物聯(lián)合使用治療腫瘤，具有降低化療藥物毒副作用的效果[17-19]。有報(bào)道，防己諾林堿可以通過抑制多種信號(hào)通路，實(shí)現(xiàn)抑制腫瘤細(xì)胞生長與侵襲轉(zhuǎn)移的功能，但是未見防己諾林堿在黑色素瘤治療中的研究報(bào)道[17]。

腫瘤的特點(diǎn)是細(xì)胞大量快速增殖[20-21]。這是由于腫瘤細(xì)胞中存在大量誘導(dǎo)細(xì)胞增殖的因子，即致癌基因。其中，Cyclin D1的前致癌基因在多種腫瘤中過量表達(dá)，直接導(dǎo)致基因擴(kuò)增或者移位的現(xiàn)象明顯。在肺癌、胃癌、乳腺癌、黑色素瘤等約80%的人類腫瘤中，也存在CDK4、CDK6的高表達(dá)現(xiàn)象，直接促使細(xì)胞增殖加快[22-23]。CDK4、CDK6可以與Cyclin D1結(jié)合，調(diào)節(jié)細(xì)胞G1向S期的轉(zhuǎn)換。Cyclin D1、CDK4及CDK6的異常表達(dá)，可以加速G1期向S期轉(zhuǎn)換的進(jìn)程，導(dǎo)致腫瘤細(xì)胞的過度增殖[24]。因此，多種針對(duì)Cyclin D1、CDK4及CDK6的靶向腫瘤藥物已開始應(yīng)用，其具有毒副作用小、藥物敏感性高、不易發(fā)生耐受等優(yōu)勢(shì)[25]。腫瘤的轉(zhuǎn)移包括以下步驟：早期原發(fā)灶形成，相關(guān)血管生成，細(xì)胞脫落，細(xì)胞進(jìn)入脈管形成癌栓，轉(zhuǎn)移灶生成[26-27]。MMP2在多種腫瘤中高表達(dá)，又稱明膠酶A，屬于Zn2+離子依賴性內(nèi)肽酶，結(jié)構(gòu)一般包括信號(hào)肽、前肽結(jié)構(gòu)域、催化結(jié)構(gòu)域和C末端血紅素結(jié)合蛋白樣結(jié)構(gòu)域(PEX)。其過度表達(dá)與侵襲轉(zhuǎn)移密切相關(guān)，針對(duì)后者的抗腫瘤治療是目前的研究熱點(diǎn)[28]。MMP2能夠降解基底膜的主要成分-Ⅳ型膠原，可以水解細(xì)胞外基質(zhì)來促進(jìn)腫瘤細(xì)胞的侵襲轉(zhuǎn)移。也有研究表明，MMP2可以釋放生長因子，促進(jìn)腫瘤細(xì)胞增殖。同時(shí)，MMP2通過降解血管基底膜蛋白，誘導(dǎo)血管生成。研究表明，抑制MMP2的活性可以有效抑制血管內(nèi)皮細(xì)胞和腫瘤細(xì)胞的侵襲轉(zhuǎn)移，進(jìn)而抑制腫瘤轉(zhuǎn)移和腫瘤的新生血管生成[27]。

本研究結(jié)果表明，防己諾林堿可以顯著抑制A375細(xì)胞的增殖與侵襲轉(zhuǎn)移。MTT實(shí)驗(yàn)表明，40 μM/L防己諾林堿作用A375細(xì)胞24 h時(shí)對(duì)細(xì)胞增殖的抑制率達(dá)50%。有報(bào)道，同等濃度的防己諾林堿對(duì)正常細(xì)胞的增殖抑制作用不明顯[29]。進(jìn)一步研究表明，防己諾林堿可以在蛋白、RNA水平上顯著抑制A375細(xì)胞中Cyclin D1、CDK4和CDK6的表達(dá)。同時(shí)，防己諾林堿可以通過抑制MMP2的蛋白、RNA水平來實(shí)現(xiàn)對(duì)A375細(xì)胞侵襲轉(zhuǎn)移的抑制功能。

綜上所述，防己諾林堿可以在蛋白、RNA水平上調(diào)控人黑色素瘤A375細(xì)胞的增殖與轉(zhuǎn)移，具有安全性高、毒副作用小的特點(diǎn)，可以作為臨床治療黑色素瘤的潛在藥物，值得進(jìn)一步研究。

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Fangchinoline inhibits the growth and migration of melanoma

GUO Bing-yu,ZHANG Yu,HUI Qiang,QUAN Liang-liang,TAO Kai*(Plastic Surgery,General Hospital of Shenyang Military Command,Shenyang 110840,China)

Objective To observe the effect of fangchinoline on growth and metastasis of melanoma cells.Methods MTT assay was used to observe the effect of different concentrations of fangchinoline on the proliferation of A375 cells.Transwell assay was used to observe the transfer function of A375 cells.Western blot and RT-PCR were used to detect the related proteins.Results Fangchinoline can inhibit the proliferation and migration of A375 cells.After being treated by 10,20 and 40 μM/L of fangchinoline,the inhibition rates of proliferation of A375 cells were 43.81%±1.53%,48.64%±4.65% and 50.69%±4.99%.After being treated by 20 and 40 μM/L of fangchinoline for 24 h,the inhibition rates of migration of A375 cells were 14.95%±4.31% and 33.03%±5.46%.Fangchinoline could significantly inhibit the expression of Cyclin D1,CDK4,CDK6 and MMP2.Conclusion As a potential chemotherapeutic agent for treatment of melanoma,fangchinoline can significantly inhibit the proliferation and migration of A375 cells.

Fangchinoline;Proliferation;Migration;A375;Melanoma

2016-02-05

沈陽軍區(qū)總醫(yī)院整形外科，沈陽 110840

*通信作者

10.14053/j.cnki.ppcr.201610002