文輝才，眭云鵬，簡(jiǎn)雪平，廖懷偉，馬麗，徐桂珍，劉燕平，萬珺

(南昌大學(xué)第一附屬醫(yī)院，南昌330006)

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體外培養(yǎng)的人惡性黑色素瘤球體細(xì)胞干細(xì)胞特性觀察

文輝才，眭云鵬，簡(jiǎn)雪平，廖懷偉，馬麗，徐桂珍，劉燕平，萬珺

(南昌大學(xué)第一附屬醫(yī)院，南昌330006)

目的觀察體外培養(yǎng)的人惡性黑色素瘤球體細(xì)胞的干細(xì)胞特性。方法利用無血清培養(yǎng)基培養(yǎng)人惡性黑色素瘤A375細(xì)胞系，獲得黑色素瘤球體細(xì)胞(球體組)和普通貼壁細(xì)胞(貼壁組)。采用Transwell小室檢測(cè)兩組細(xì)胞遷移能力。采用免疫熒光法檢測(cè)兩組細(xì)胞中的CD20、CD133。取BALB/c小鼠16只，分實(shí)驗(yàn)組和對(duì)照組(各8只)，于小鼠肩胛骨處皮下分別注射球體組、貼壁組細(xì)胞各1 mL(104個(gè)細(xì)胞)，觀察并比較兩組細(xì)胞致瘤能力。6周后處死小鼠，取瘤。結(jié)果A375細(xì)胞接種于無血清培養(yǎng)基后，可見小的類圓形懸浮細(xì)胞球形成并逐漸變大，球內(nèi)細(xì)胞連接緊密，折光性好。球體組細(xì)胞遷移能力及細(xì)胞中CD20、CD133表達(dá)強(qiáng)度均高于貼壁組。注射細(xì)胞6周后，實(shí)驗(yàn)組小鼠均長(zhǎng)出較大腫瘤，對(duì)照組小鼠均未見腫瘤生長(zhǎng)。取實(shí)驗(yàn)組小鼠的腫瘤組織行HE染色，病理診斷為惡性黑色素瘤。結(jié)論體外培養(yǎng)的人惡性黑色素瘤球體細(xì)胞具有干細(xì)胞特性，遷移能力和致瘤能力強(qiáng)，高表達(dá)免疫標(biāo)記CD20、CD133，惡性黑色素瘤存在腫瘤干細(xì)胞。

黑色素瘤；腫瘤干細(xì)胞；無血清培養(yǎng)基

惡性黑色素瘤是起源于皮膚黑色素細(xì)胞的高度惡性腫瘤[1]，其惡性程度高、易復(fù)發(fā)、進(jìn)展快，手術(shù)、放療、化療等傳統(tǒng)治療方法療效均較差[2,3]，需要尋找新的治療手段[4]。新近研究[5]發(fā)現(xiàn)，黑色素瘤細(xì)胞體外培養(yǎng)可形成球體細(xì)胞，球體細(xì)胞形成實(shí)驗(yàn)可富集干細(xì)胞。2014年9月～2015年4月，我們以無血清培養(yǎng)基(SFM，由DMEM/F12、10 ng/mL bFGF、B27、20 ng/mL EGF組成)培養(yǎng)惡性黑色素瘤A375細(xì)胞系，分離、擴(kuò)增出黑色素瘤球體細(xì)胞并觀察其干細(xì)胞特性，現(xiàn)報(bào)告如下。

1　材料與方法

1.1主要實(shí)驗(yàn)材料人惡性黑色素瘤A375細(xì)胞系購自上海中國科學(xué)院細(xì)胞庫。BALB/c小鼠均為雌性，體質(zhì)量18～20 g，4～6周齡，飼養(yǎng)于SPF級(jí)環(huán)境中。DMEM/F12(1∶1)細(xì)胞培養(yǎng)液，優(yōu)級(jí)胎牛血清，0.25%胰酶消化液，B27，堿性成纖維細(xì)胞因子(bFGF)，表皮生長(zhǎng)因子(EGF)，超低吸附培養(yǎng)板，超凈工作臺(tái)，CO2培養(yǎng)箱。

1.2惡性黑色素瘤球體細(xì)胞的獲取與培養(yǎng)A375細(xì)胞用含10%胎牛血清的DMEM/F12(1∶1)細(xì)胞培養(yǎng)液在37 ℃、5% CO2、飽和濕度條件下培養(yǎng)，當(dāng)細(xì)胞融合達(dá)80%時(shí)，胰蛋白酶消化細(xì)胞，1∶2傳代。取貼壁培養(yǎng)的對(duì)數(shù)生長(zhǎng)期A375細(xì)胞用PBS液清洗，胰酶消化，經(jīng)臺(tái)盼藍(lán)染色并計(jì)數(shù)后重懸于SFM中，以1×104/mL接種于6孔超低吸附培養(yǎng)板，放入37 ℃、5% CO2、95%濕度的培養(yǎng)箱中培養(yǎng)。每隔1 d在每孔加入0.5 mL新鮮SFM。培養(yǎng)2周可形成球體細(xì)胞，球體細(xì)胞形成3～4 d后，收集細(xì)胞并機(jī)械吹打成單細(xì)胞懸液，重懸于SFM，按1∶3比例傳代培養(yǎng)，倒置顯微鏡下觀察細(xì)胞形態(tài)變化。將球體細(xì)胞納入球體組，貼壁細(xì)胞納入貼壁組。

1.3惡性黑色素瘤細(xì)胞遷移能力檢測(cè)將含10%血清的DMEM/F12培養(yǎng)基按600 μL/孔加入到24孔板中，取出Transwell小室放入24孔板中，將貼壁組和球體組細(xì)胞在無血清DMEM/F12培養(yǎng)基重懸后加入未鋪膠的小室上層，置入37 ℃孵箱培養(yǎng)12 h，4%多聚甲醛固定20 min，結(jié)晶紫染色，用棉簽拭去小室上層細(xì)胞，清洗后置于顯微鏡下觀察。

1.4惡性黑色素瘤細(xì)胞免疫表型檢測(cè)分別將貼壁組和球體組細(xì)胞種植于蓋玻片上培養(yǎng)，等細(xì)胞爬滿蓋玻片后用4%多聚甲醛固定20 min，PBS洗3次，用0.3% Triton X-100室溫下穿孔，PBS洗3次，3% BSA室溫下封閉，加入熒光素標(biāo)記的鼠抗人CD133、CD20抗體，DAPI染色，抗淬滅封片劑封片，在激光共聚焦顯微鏡下觀察、照相。

1.5惡性黑色素瘤細(xì)胞成瘤實(shí)驗(yàn)取BALB/c小鼠16只，分實(shí)驗(yàn)組和對(duì)照組，每組8只。在實(shí)驗(yàn)組和對(duì)照組小鼠肩胛骨處皮下分別注射球體組、貼壁組細(xì)胞各1 mL(104個(gè)細(xì)胞)。觀察并比較兩組細(xì)胞致瘤能力。6周后處死小鼠，取瘤。

2　結(jié)果

A375細(xì)胞接種于無血清培養(yǎng)基后2～3 d，大部分細(xì)胞死亡崩解，少數(shù)細(xì)胞存活，懸浮生長(zhǎng)，細(xì)胞逐漸變大，呈卵圓形或圓形，折光性好，一周后可見小的類圓形懸浮細(xì)胞球形成并逐漸變大，球內(nèi)細(xì)胞連接緊密，無法區(qū)分細(xì)胞間界限，折光性好，新生的單個(gè)細(xì)胞常以“出芽”方式連接在球體表面。2周時(shí)細(xì)胞球體積達(dá)到最大。見圖1。

圖1　無血清懸浮培養(yǎng)黑色素瘤球體細(xì)胞(200×)

2.1球體細(xì)胞遷移能力球體組遷移細(xì)胞數(shù)目為(158.3±5.0)個(gè)/視野、貼壁組為(75.3±4.4)個(gè)/視野，兩組相比，P<0.05。

2.2球體細(xì)胞CD20、CD133表達(dá)情況球體組與貼壁組細(xì)胞中均檢測(cè)到CD20、CD133，且球體組細(xì)胞CD20、CD133表達(dá)強(qiáng)度高于貼壁組細(xì)胞。見圖2、3。

2.3球體細(xì)胞致瘤能力實(shí)驗(yàn)組小鼠注射球體組細(xì)胞后2周看到有腫瘤凸起皮面，對(duì)照組小鼠未見腫瘤形成。注射細(xì)胞6周后，實(shí)驗(yàn)組小鼠均長(zhǎng)出較大腫瘤，對(duì)照組小鼠均未見腫瘤生長(zhǎng)(見圖4)。取實(shí)驗(yàn)組腫瘤組織，病理診斷為惡性黑色素瘤。

注：A為球體組細(xì)胞，B為貼壁組細(xì)胞。

圖2球體組與貼壁組細(xì)胞中CD20表達(dá)情況

注：A為球體組細(xì)胞，B為貼壁組細(xì)胞。

圖3球體組與貼壁組細(xì)胞中CD133表達(dá)情況

注：A為實(shí)驗(yàn)組，B為對(duì)照組。

圖4實(shí)驗(yàn)組與對(duì)照組小鼠注入細(xì)胞6周后瘤體形成情況

3　討論

腫瘤干細(xì)胞具有很強(qiáng)的分化潛能和自我更新能力，具有耐藥性，是腫瘤增殖、轉(zhuǎn)移、復(fù)發(fā)的重要原因[6，7]。Fang等[8]用人胚胎干細(xì)胞的培養(yǎng)基培養(yǎng)原代惡性黑色素瘤細(xì)胞，發(fā)現(xiàn)約20%的惡性黑色素瘤細(xì)胞能形成懸浮腫瘤球，這些懸浮球體細(xì)胞具有可塑性[9]，能在適當(dāng)培養(yǎng)條件下分化成多種類型的細(xì)胞，如骨細(xì)胞[10]、軟骨細(xì)胞[11]、脂肪細(xì)胞[12]等。Mansur等[13]從惡性黑色素瘤中分離出表達(dá)CD133的細(xì)胞亞群，并發(fā)現(xiàn)所有的CD133陽性細(xì)胞均可在NOD/SCID小鼠體內(nèi)成瘤，而CD133陰性細(xì)胞則不能成瘤。上述研究成果提供了惡性黑色素瘤干細(xì)胞存在的直接證據(jù)。

腫瘤干細(xì)胞研究的首要工作是分離出腫瘤干細(xì)胞[14]。最新研究[15]證實(shí)，含EGF和bFGF的SFM可分離出各種實(shí)體瘤的腫瘤干細(xì)胞。我們利用含EGF、bFGF和B27添加劑的SFM分離人惡性黑色素瘤A375細(xì)胞系的腫瘤干細(xì)胞，結(jié)果顯示，A375細(xì)胞在SFM中能夠形成球體細(xì)胞，延長(zhǎng)培養(yǎng)時(shí)間，球體細(xì)胞數(shù)量可增多、體積增大，證實(shí)無血清環(huán)境能刺激腫瘤干細(xì)胞分裂增殖。

腫瘤細(xì)胞遷移性越強(qiáng)，轉(zhuǎn)移能力也就越強(qiáng)[16]。我們通過Transwell小室檢測(cè)球體組與貼壁組細(xì)胞的遷移能力，結(jié)果顯示球體組細(xì)胞遷移能力明顯強(qiáng)于貼壁組。CD20是B細(xì)胞表面常見的標(biāo)志物，CD20陽性的黑色素瘤細(xì)胞具備腫瘤干細(xì)胞的特征。CD133廣泛存在于實(shí)體瘤干細(xì)胞表面，為腦腫瘤、結(jié)腸癌、急性粒細(xì)胞性白血病的腫瘤起始細(xì)胞表面標(biāo)志物。球體組細(xì)胞與貼壁組細(xì)胞均可表達(dá)CD20、CD133，且球體組細(xì)胞表達(dá)強(qiáng)度更高，這進(jìn)一步表明黑色素瘤A375細(xì)胞中存在腫瘤干細(xì)胞，其遷移能力更強(qiáng)。我們將兩種細(xì)胞分別注入BALB/c小鼠皮下，發(fā)現(xiàn)注射細(xì)胞6周后實(shí)驗(yàn)組小鼠均成瘤，而對(duì)照組小鼠無一成瘤，取實(shí)驗(yàn)組小鼠腫瘤組織行HE染色，均證實(shí)為惡性黑色素瘤，這提示富集干細(xì)胞的球體細(xì)胞比普通貼壁細(xì)胞具有更強(qiáng)的致瘤能力。我們認(rèn)為，無血清培養(yǎng)的人惡性黑色素瘤球體細(xì)胞具有干細(xì)胞特性，遷移能力和致瘤能力強(qiáng)，惡性黑色素瘤存在腫瘤干細(xì)胞。這為惡性黑色素瘤的治療提供了新的研究方向。

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Stem cell characteristics of human malignant melanoma sphere cells cultured in vitro

WEN Huicai,SUI Yunpeng,JIAN Xueping,LIAO Huaiwei,MA Li,XU Guizhen,LIU Yanping,WAN Jun

(The First Affiliated Hospital of Nanchang University,Nanchang 330006,China)

ObjectiveTo observe the stem cell properties of human malignant melanoma cells cultured in serum-free medium.MethodsHuman malignant melanoma A375 cell line was cultured in serum-free medium,and the melanoma sphere cells (sphere group) and common adherent cells (adherent group) were obtained.Cell migration was detected using Transwell chamber.CD20 and CD133 were detected by immunofluorescence.Sixteen BALB/c mice were divided into the experimental group and control group (8 rats in each group),and the sphere cells and the adherent cells of 1 mL (104) were injected into the mouse shoulder blade by subcutaneous injection in the sphere and the adherent groups,respectively.The tumor cells were observed and compared between the two groups.Six weeks later,the mice were killed to obtain the tumor tissues.ResultsA375 cells were seeded in serum-free medium,visible small round ball suspension cells formed and gradually becomes larger,the ball inside the cells were closely connected with good refraction.The cell migration ability and the expression intensity of CD20 and CD133 in the sphere group were higher than those in the adherent group.After 6 weeks of injection,the mice in the experimental group had a large tumor,and no tumor growth was found in the control group.The tumor tissues of the experimental group were stained with HE,and the malignant melanoma was pathologically diagnosed.ConclusionsHuman malignant melanoma cells cultured in serum-free medium have the characteristics of stem cells with high migration ability and tumorigenesis.High expression of immune markers CD20,CD133 is found and malignant melanoma exists in the tumor stem cells.

melanoma; tumor stem cells; serum-free medium

江西省科技支撐計(jì)劃項(xiàng)目(2010BSA14600)。

文輝才(1973-)，男，醫(yī)學(xué)博士，副主任醫(yī)師，副教授，碩士生導(dǎo)師，主要研究方向?yàn)槠つw體表腫瘤、器官再造，改臉型。E-mail: whcjxmc@163.com

10.3969/j.issn.1002-266X.2016.19.007

R739.5

A

1002-266X(2016)19-0022-03

2015-12-14)