薛成俊 周忠海 陳復(fù)興 呂小婷 李瑩 費(fèi)素娟

·論著·

氫化可的松對(duì)人NK細(xì)胞增殖及其對(duì)胰腺癌SW1990細(xì)胞殺傷效率的影響

薛成俊 周忠海 陳復(fù)興 呂小婷 李瑩 費(fèi)素娟

目的探討氫化可的松對(duì)人外周血NK細(xì)胞增殖及其對(duì)胰腺癌SW1990細(xì)胞殺傷力的影響。方法分離健康人外周血單個(gè)核細(xì)胞加入到含IL-15細(xì)胞因子的NK細(xì)胞培養(yǎng)基中誘導(dǎo)培養(yǎng)NK細(xì)胞。當(dāng)NK細(xì)胞純度達(dá)到70%以上時(shí)，加入10-6、10-5、10-4、10-3μmol/L氫化可的松繼續(xù)培養(yǎng)7 d。以未加氫化可的松的NK細(xì)胞作為對(duì)照組。采用錐蟲藍(lán)染色計(jì)數(shù)細(xì)胞；采用流式細(xì)胞術(shù)檢測(cè)CD3-CD56+NK細(xì)胞含量及其穿孔素、顆粒酶B和IFN-γ的表達(dá)；以20∶1的效靶比將NK細(xì)胞與SW1990細(xì)胞共培養(yǎng)，用細(xì)胞增殖-毒性檢驗(yàn)法(CCK-8)檢測(cè)NK細(xì)胞對(duì)SW1990細(xì)胞的殺傷效應(yīng)。結(jié)果用氫化可的松處理7 d后NK細(xì)胞量平均達(dá)到70.72%～76.39%，與對(duì)照組的(72.61±3.76)%差異無統(tǒng)計(jì)學(xué)意義；10-6、10-5、10-4、10-3μmol/L氫化可的松處理組NK細(xì)胞的增殖倍數(shù)分別為(9.13±0.94)、(9.67±1.51)、(10.33±1.07)、(8.40±1.47)倍，均顯著高于對(duì)照組的(4.23±0.82)倍(P值均<0.01)；NK細(xì)胞對(duì)SW1990細(xì)胞的殺傷效率分別為(58.58±4.89)%、(62.27±5.63)%、(64.02±5.79)%、(63.88±3.61)%，均較對(duì)照組的(57.46±5.11)%增強(qiáng)，其中10-4μmol/L組的差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)；10-4、10-3μmol/L氫化可的松處理組NK細(xì)胞穿孔素表達(dá)分別為(96.71±3.04)%、(97.56±2.18)%，顯著高于對(duì)照組的(92.40±3.53)%(P<0.05或0.01)；10-5μmol/L氫化可的松處理組NK細(xì)胞的顆粒酶B表達(dá)為(78.23±2.94)%，顯著高于對(duì)照組(73.68±3.52)%(P<0.05)；10-5、10-4、10-3μmol/L 氫化可的松處理組NK細(xì)胞IFN-γ表達(dá)分別為(96.61±2.04)%、(97.58±2.17)%、(98.00±1.77)%，均顯著高于對(duì)照組的(92.44±2.74)%(P值均<0.01)。結(jié)論氫化可的松可促進(jìn)IL-15活化的NK細(xì)胞增殖，適當(dāng)濃度條件下增強(qiáng)對(duì)SW1990細(xì)胞殺傷活性，其機(jī)制可能與其穿孔素、顆粒酶B和IFN-γ表達(dá)上調(diào)有關(guān)。

氫化可的松; 殺傷細(xì)胞，天然; 胰腺腫瘤; 細(xì)胞增殖; 殺傷活性

NK細(xì)胞是機(jī)體固有免疫重要的淋巴細(xì)胞，它通過分泌IFN-γ、IL-2和TNF-α等細(xì)胞因子和趨化因子啟始和調(diào)節(jié)機(jī)體的免疫反應(yīng)[1]，無需通過抗原預(yù)先刺激即可直接殺傷腫瘤和病毒感染的靶細(xì)胞，是腫瘤免疫治療重要的效應(yīng)細(xì)胞[1-2]。氫化可的松(hydrocortisone, HC)屬糖皮質(zhì)激素，不僅具有免疫抑制作用，而且對(duì)免疫系統(tǒng)可能有正性調(diào)節(jié)作用，這取決于免疫系統(tǒng)淋巴細(xì)胞的活化狀態(tài)[3]。因此，本研究觀察HC對(duì)NK細(xì)胞體外增殖及殺傷胰腺癌SW1990細(xì)胞效率的影響，為培養(yǎng)更高數(shù)量和功能活性的NK細(xì)胞用于腫瘤過繼性免疫治療提供實(shí)驗(yàn)依據(jù)。

材料與方法

一、外周血NK細(xì)胞培養(yǎng)

8例外周血標(biāo)本采自健康獻(xiàn)血員，其中男性5人，女性3人，年齡23～48歲，中位年齡35歲。經(jīng)人淋巴細(xì)胞分離液梯度密度離心獲得外周血單個(gè)核細(xì)胞(PBMCs)，用磷酸鹽緩沖液(PBS)洗滌2次，調(diào)整細(xì)胞密度為5×105/ml，加入含IL-15 (廈門特寶生物工程公司)500 U、5%自身血清的NK細(xì)胞培養(yǎng)基中培養(yǎng)10 d，收集細(xì)胞進(jìn)行CD3-CD56+NK細(xì)胞表型鑒定，獲取有效擴(kuò)增的NK細(xì)胞。調(diào)整有效擴(kuò)增的NK細(xì)胞密度為5×105/ml，分別加入10-6、10-5、10-4、10-3μmol/L的HC，置37℃、5% CO2培養(yǎng)箱中培養(yǎng)7 d。以未加HC的作為對(duì)照組。

二、NK細(xì)胞表型鑒定

收集各組細(xì)胞，用PBS洗滌2次，計(jì)數(shù)細(xì)胞，調(diào)整細(xì)胞密度為1×107/ml。取100 μl 細(xì)胞懸液，分別加入20 μl 異硫氰酸熒光素(FITC)標(biāo)記的anti-CD56和藻紅蛋白(PE)標(biāo)記的anti-CD3室溫避光孵育15 min，PBS洗滌2遍，加入400 μl PBS重懸細(xì)胞，流式細(xì)胞術(shù)檢測(cè)各組NK細(xì)胞的百分含量。

三、NK細(xì)胞增殖倍數(shù)

收集各組NK細(xì)胞，用PBS洗滌后錐蟲藍(lán)染色計(jì)數(shù)細(xì)胞總數(shù)，結(jié)合流式細(xì)胞術(shù)細(xì)胞純度檢測(cè)結(jié)果，NK細(xì)胞增殖倍數(shù)=HC干預(yù)后細(xì)胞數(shù)×NK細(xì)胞含量(%)/HC干預(yù)前細(xì)胞數(shù)×NK細(xì)胞含量(%)。

四、NK細(xì)胞穿孔素、顆粒酶B和IFN-γ表達(dá)的檢測(cè)

收集各組NK細(xì)胞，用PBS洗滌2次，調(diào)整細(xì)胞密度為1×107/ml。每組取3管100 μl 細(xì)胞懸液，加入20 μl FITC標(biāo)記的anti-CD56和PerCP-Cy5.5標(biāo)記的anti-CD3(BD公司)，室溫避光孵育15 min，加入100 μl破膜試劑A(Invitrogen公司)室溫避光孵育15 min，PBS洗滌后再加入100 μl破膜試劑B(Invitrogen公司)，同時(shí)在穿孔素測(cè)定管中加入5 μl PE標(biāo)記的anti-Perforin(eBioscience公司)，顆粒酶B測(cè)定管中加入20 μl PE標(biāo)記的anti-Granzyme B(eBioscience公司)，IFN-γ測(cè)定管中加入5 μl 別藻青蛋白(APC)標(biāo)記的anti-IFN-γ(BD公司)，室溫避光孵育15 min，PBS洗滌，以400 μl PBS重懸細(xì)胞，上流式細(xì)胞儀檢測(cè)穿孔素、顆粒酶B和IFN-γ的表達(dá)，結(jié)果以百分值表示。

五、NK細(xì)胞對(duì)SW1990細(xì)胞殺傷效率的檢測(cè)

人胰腺癌SW1990細(xì)胞購(gòu)自中國(guó)科學(xué)院上海生命科學(xué)研究所細(xì)胞資源中心，常規(guī)培養(yǎng)、傳代。取對(duì)數(shù)生長(zhǎng)期SW1990細(xì)胞作為靶細(xì)胞，以5×103/孔的密度接種于96孔板，以20∶1的效靶比分別加入不同濃度HC干預(yù)培養(yǎng)7 d的NK細(xì)胞，總體積為200 μl/孔，同時(shí)設(shè)各濃度組的空白對(duì)照組、效應(yīng)細(xì)胞對(duì)照組和靶細(xì)胞對(duì)照組，每組設(shè)3個(gè)復(fù)孔。效應(yīng)細(xì)胞與靶細(xì)胞共孵育24 h后加入CCK-8溶液(碧云天生物技術(shù)公司)20 μl/孔，繼續(xù)孵育4 h后上酶聯(lián)免疫分析儀測(cè)每孔450 nm處吸光度值(A450值)。殺傷活性(%)=[1-(實(shí)驗(yàn)組A450值-效應(yīng)細(xì)胞對(duì)照組A450值)/ 靶細(xì)胞對(duì)照組A450值]×100%。

六、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、NK細(xì)胞培養(yǎng)表型鑒定

10-6、10-5、10-4、10-3μmol/L HC處理7 d后，CD3-CD56+的NK細(xì)胞量分別為(72.14±3.51)%、(71.75±2.36)%、(70.83±2.90)%、(69.88±4.14)%，與對(duì)照組的(72.61±3.76)%差異無統(tǒng)計(jì)學(xué)意義(t值分別為0.258，0.548，1.060，1.381，P值均>0.05，圖1)。

圖1對(duì)照組(左)、10-5μmol/L氫化可的松處理組(右)CD3-CD56+的NK細(xì)胞量

二、NK細(xì)胞增殖力的變化

10-6、10-5、10-4、10-3μmol/L HC處理7 d后，NK細(xì)胞增殖倍數(shù)分別為(9.13±0.94)、(9.67±1.51)、(10.33±1.07)、(8.40±1.47)倍，較對(duì)照組的(4.23±0.82)倍顯著增高(t值分別為11.111，8.955，12.799，7.007，P值均<0.01)。

三、NK細(xì)胞對(duì)SW1990細(xì)胞的殺傷效率

10-6、10-5、10-4、10-3μmol/L HC處理7 d后，NK細(xì)胞對(duì)SW1990細(xì)胞的殺傷效率在效靶比20∶1時(shí)分別為(58.58±4.89)%、(62.27±5.63)%、(64.02±5.79)%、(63.88±3.61)%，較對(duì)照組的(57.46±5.11)%增強(qiáng)，其中10-4μmol/L HC處理組的差異具有統(tǒng)計(jì)學(xué)意義(t值分別為0.448，1.789，2.403，2.902，P值均<0.05)。

四、NK細(xì)胞穿孔素、顆粒酶B和IFN-γ的表達(dá)

10-5μmol/L HC處理組NK細(xì)胞的顆粒酶B和IFN-γ表達(dá)顯著高于對(duì)照組，10-4、10-3μmol/L HC處理組NK細(xì)胞穿孔素和IFN-γ的表達(dá)均顯著高于對(duì)照組(表1、圖2)。

組別穿孔素顆粒酶BIFN-γ對(duì)照組92.40±3.5373.68±3.5292.44±2.7410-6μmol/LHC組90.59±5.8275.03±3.1995.03±2.5210-5μmol/LHC組92.67±3.4978.23±2.94a96.61±2.04b10-4μmol/LHC組96.71±3.04a77.75±3.8497.58±2.17b10-3μmol/LHC組97.56±2.18b74.28±4.1598.00±1.77b

注：與對(duì)照組比較，aP<0.05，bP<0.01

圖2同型對(duì)照組(左)、對(duì)照組(中)、10-4μmol/L氫化可的松處理組(右)NK細(xì)胞的穿孔素(上)、顆粒酶B(中)和INF-γ(下)表達(dá)

討 論

NK細(xì)胞是不同于T、B細(xì)胞的一類大顆粒淋巴細(xì)胞亞群，在外周血中含量相對(duì)較少，約占外周血淋巴細(xì)胞10%～15%，具有抗腫瘤、抗感染和免疫調(diào)節(jié)等功能，且不受組織相容性復(fù)合物(MHC)的限制，是機(jī)體重要的固有免疫細(xì)胞，同時(shí)還具有適應(yīng)性免疫的特性[4]。動(dòng)物實(shí)驗(yàn)和臨床腫瘤過繼免疫治療研究結(jié)果表明，基于NK細(xì)胞的過繼性免疫治療是安全有效的[5-7]，有望成為臨床腫瘤細(xì)胞免疫治療的重要手段。

鑒于體外擴(kuò)增的NK細(xì)胞具有較高的抗腫瘤活性[8-10]和HC對(duì)免疫系統(tǒng)的雙相調(diào)節(jié)作用，本研究先選擇IL-15活化擴(kuò)增NK細(xì)胞，然后用不同濃度的HC處理NK細(xì)胞7 d，結(jié)果經(jīng)HC處理各組NK細(xì)胞百分含量并沒有顯著性的變化，但增殖倍數(shù)卻顯著高于未加HC處理的對(duì)照組，這表明HC促進(jìn)了IL-15活化的NK細(xì)胞的體外擴(kuò)增，其作用機(jī)制可能是HC增強(qiáng)了NK細(xì)胞表面IL-15R的表達(dá)，從而放大了IL-15及其受體介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)途徑[11]。

穿孔素是NK細(xì)胞胞質(zhì)中的細(xì)胞毒顆粒，可在靶細(xì)胞膜上形成管狀跨膜通道，致靶細(xì)胞裂解。顆粒酶B是NK細(xì)胞顆粒中重要的絲氨酸蛋白酶，通過穿孔素在靶細(xì)胞膜上形成的通道進(jìn)入靶細(xì)胞，激活凋亡相關(guān)酶系統(tǒng)致細(xì)胞凋亡。穿孔素/顆粒酶途徑和IFN-γ等細(xì)胞因子產(chǎn)生和分泌是NK細(xì)胞殺傷靶細(xì)胞的重要機(jī)制[12-13]。本研究結(jié)果表明，HC與IL-15共同誘導(dǎo)培養(yǎng)后，NK細(xì)胞穿孔素、顆粒酶B及IFN-γ的表達(dá)增強(qiáng)，提示HC處理后的IL-15活化的NK細(xì)胞殺傷腫瘤細(xì)胞的功能沒有受到負(fù)面的影響，而是不同程度地增強(qiáng)。

胰腺癌是消化系統(tǒng)常見的惡性腫瘤，發(fā)病率呈逐年上升趨勢(shì)，高居惡性腫瘤死亡病因的第5位，手術(shù)切除后的生存率偏低[14]，因此本研究選擇胰腺癌SW1990細(xì)胞進(jìn)行體外殺傷研究。結(jié)果表明不同濃度HC處理后NK細(xì)胞對(duì)SW1990細(xì)胞的殺傷能力增強(qiáng)，其中10-4μmol/L HC處理組與無HC處理的對(duì)照組的差異具有統(tǒng)計(jì)學(xué)意義，與Moustaki等[15]結(jié)果基本一致。HC處理的NK細(xì)胞保持了原有的腫瘤細(xì)胞殺傷活性并略有增加，這可能與HC和IL-15共培養(yǎng)后NK細(xì)胞穿孔素、顆粒酶B及IFN-γ的表達(dá)上調(diào)有關(guān)，是否影響其他與NK細(xì)胞殺傷腫瘤細(xì)胞相關(guān)的分子機(jī)制以及HC干預(yù)培養(yǎng)的NK細(xì)胞用于臨床治療的安全性和有效性還有待于進(jìn)一步研究。

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EffectofhydrocortisoneonproliferationandkillingactivityofNKcellsagainstSW1990cells

XUECheng-jun,ZHOUZhong-hai,CHENFu-xing,LüXiao-ting,LIYing,FEISu-juan.

DepartmentofGastroenterology,AffiliatedHospitalofXuzhouMedicalCollege,Xuzhou221002,China

Correspondingauthor:FEISu-juan,Email:feisj1031@yahoo.com.cn

ObjectiveTo investigate the effects of hydrocortisone (HC) on proliferation and killing activity of NK cells against pancreatic cancer SW1990 cells in vitro.MethodsPeripheral blood mononuclear cells of healthy people were isolated and cultured with NK cells medium containing IL-15. When the purity of NK cells reached above 70%, different concentrations of HC (10-6, 10-5, 10-4, 10-3μmol/L) were added and co-cultured with NK cells for 7 days. And NK cells without HC were used as control. CD3-CD56+NK cell numbers of each group were countered by trypan blue staining. Perforin, granzyme B and IFN-γ expression of CD3-CD56+NK cells were verified by flow cytometry. NK cells and SW1990 cells were co-cultured with a 20∶1 effector to target ratio, then the cytotoxic activity of NK cells against SW1990 cells were analyzed by CCK-8 kit.ResultsAfter treatment with different concentration of HC for 7 days, NK cells purity of each group reached 70.72%～76.39%, and it was not significantly different with that in control group [(72.61±3.76)%]. The proliferation folds of NK cells treated with 10-6, 10-5, 10-4, 10-3μmol/L HC were (9.13±0.94), (9.67±1.51), (10.33±1.07), (8.40±1.47) times, respectively, while it was (4.23±0.82) times in control group (allP<0.01). The killing effects of NK cells on SW1990 cells were (58.58±4.89)%, (62.27±5.63)%, (64.02±5.79)%, (63.88±3.61)%, which were higher than that in control group [(57.46±5.11)%], moreover, the difference between NK cells of 10-4μmol/L HC treatment group and control group was statistically significant(P<0.05). The expressions of perforins of 10-4, 10-3μmol/L HC treatment group were (96.71±3.04)%, (97.56±2.18)%, which were significantly higher than that in control group [(92.40±3.53)%,P<0.05 or 0.01]. The expression of granzyme B in 10-5μmol/L HC treatment group was (78.23±2.94)%, which were significantly higher than that in control group [(73.68±3.52)%,P<0.05]. The expressions of IFN-γ in 10-5, 10-4, 10-3μmol/L HC treatment group were (96.61±2.04)%, (97.58±2.17)%, (98.00±1.77)%, which were significantly higher than that in control group [(92.44±2.74)%,P<0.01].ConclusionsHC can promote IL-15 activated NK cells proliferation and enhance NK cells mediated killing activity against SW1990 cells with proper concentration, and up-regulation of perforin, granzyme B and IFN-γ expression may be the main mechanisms.

Hydrocortisone; Killer cell, natural; Pancreatic neoplasms; Cell proliferation; Killing activity

2013-03-20)

(本文編輯：呂芳萍)

10.3760/cma.j.issn.1674-1935.2013.03.009

221002 江蘇徐州，徐州醫(yī)學(xué)院附屬醫(yī)院消化內(nèi)科(薛成俊、費(fèi)素娟)；南通大學(xué)附屬建湖醫(yī)院消化內(nèi)科(薛成俊)；解放軍第97醫(yī)院中心實(shí)驗(yàn)室(周忠海、陳復(fù)興、呂小婷、李瑩)

費(fèi)素娟,Email: feisj1031@yahoo.com.cn