溫和高溫下胰腺癌細(xì)胞株P(guān)ANC1對(duì)吉西他濱耐藥性的影響

劉明浩 李淑德 李兆申 高軍 吳洪玉 龔艷芳

·論著·

溫和高溫下胰腺癌細(xì)胞株P(guān)ANC1對(duì)吉西他濱耐藥性的影響

劉明浩 李淑德 李兆申 高軍 吳洪玉 龔艷芳

目的觀察溫和高溫下胰腺癌細(xì)胞株P(guān)ANC1對(duì)吉西他濱敏感性的影響。方法應(yīng)用1 μmol/L吉西他濱處理胰腺癌PANC1細(xì)胞1 h后，分別置37、42、45℃水浴鍋孵育1 h，再繼續(xù)培養(yǎng)0、24、48 h。采用CCK-8法檢測(cè)細(xì)胞增殖，AV/PI染色后上流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率。結(jié)果42℃及45℃孵育細(xì)胞24、48 h后的細(xì)胞增殖均較37℃孵育的細(xì)胞顯著降低(0.96±0.05、0.88±0.03比1.05±0.02；1.28±0.04、0.94±0.04比1.49±0.09；t值分別為4.367、25.120、3.510、12.101，P值均<0.05)，且45℃孵育的細(xì)胞增殖顯著低于42℃孵育的細(xì)胞(t值分別為3.348、11.732，P值均<0.05)。37、42、45℃孵育的吉西他濱處理的PANC1細(xì)胞48 h后的細(xì)胞凋亡率分別為(7.125±0.064)%、(9.985±0.615)%、(14.845±1.987)%，3組間的差異均具有統(tǒng)計(jì)學(xué)意義(t值分別為10.320、9.832、4.575，P值均<0.05)。結(jié)論溫和高溫可以增加PANC1細(xì)胞對(duì)吉西他濱的敏感性。

胰腺腫瘤； 溫和高溫； 吉西他濱； 藥物療法

胰腺癌患者手術(shù)切除率低，對(duì)放、化療不敏感，生存期短[1]。文獻(xiàn)報(bào)道，溫和高溫(mild temperature hyperthermia)(40～45℃,1～2 h)處理腫瘤細(xì)胞可提高其對(duì)化療藥物的敏感性[2-3]。近期文獻(xiàn)報(bào)道，局部高溫處理(42℃，30 min)腫瘤聯(lián)合化療治療的胰腺癌患者平均生存期長(zhǎng)于僅化療的患者[4]。為此，本研究觀察溫和高溫聯(lián)合吉西他濱對(duì)胰腺癌細(xì)胞耐藥性的影響，為溫和高溫的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。

材料和方法

一、溫和高溫聯(lián)合吉西他濱處理PANC1細(xì)胞

PANC1胰腺癌細(xì)胞株購(gòu)于中國(guó)科學(xué)院細(xì)胞庫(kù)，常規(guī)培養(yǎng)傳代。取對(duì)數(shù)生長(zhǎng)期細(xì)胞接種于96孔板，每孔加入5×104個(gè)細(xì)胞(100 μl)，應(yīng)用1 μmol/L吉西他濱(法國(guó)禮來(lái)公司生產(chǎn)，每支200 mg,批號(hào)：A674393A)處理細(xì)胞。將處理后細(xì)胞分別置于37、42、45℃水浴鍋孵育1 h，再繼續(xù)常規(guī)培養(yǎng)0、24、48 h。每組每時(shí)間點(diǎn)設(shè)6個(gè)復(fù)孔。

二、細(xì)胞增殖檢測(cè)

將上述各組各時(shí)間點(diǎn)細(xì)胞去培養(yǎng)液，每孔加入10 μl CCK-8(Dojindo公司)溶液，再加入90 μl DMEM培養(yǎng)液繼續(xù)孵育4 h，應(yīng)用自動(dòng)酶標(biāo)儀測(cè)各孔450 nm處吸光度值(A450)。

三、細(xì)胞凋亡檢測(cè)

將溫和高溫聯(lián)合吉西他濱處理的細(xì)胞繼續(xù)培養(yǎng)48 h，收集細(xì)胞，調(diào)整細(xì)胞數(shù)為5×106/ml。應(yīng)用70%乙醇固定后置-20℃過(guò)夜，用PBS洗滌2次后應(yīng)用Annexin V-fluorescein isothiocyanate/propidium iodide(eBioscienc)試劑處理細(xì)胞，上流式細(xì)胞儀分析細(xì)胞凋亡率。

四、統(tǒng)計(jì)學(xué)分析

結(jié) 果

一、胰腺癌PANC1細(xì)胞增殖的改變

37℃孵育組PANC1細(xì)胞在0、24、48 h時(shí)的A450值分別為0.68±0.01、1.05±0.02、1.49±0.09；42℃孵育組分別為0.68±0.02、0.96±0.05、1.28±0.04；45℃孵育組分別為0.68±0.02、0.88±0.03、0.94±0.04。42℃及45℃孵育24 h后的細(xì)胞增殖較37℃顯著降低(t=4.367,P<0.05;t=25.120,P<0.01)；孵育48 h的細(xì)胞增殖亦較37℃顯著降低(t=3.510,P<0.05;t=12.101,P<0.01)。同時(shí)，45℃孵育的細(xì)胞增殖顯著低于42℃孵育的細(xì)胞(t=3.348,P<0.05;t=11.732,P<0.01)。

二、胰腺癌PANC1細(xì)胞凋亡的變化

顯微鏡下見(jiàn)42、45℃孵育組的懸浮細(xì)胞明顯多于37℃組(圖1)。37、42、45℃孵育48 h后的PANC1細(xì)胞的凋亡率分別為(7.125±0.064)%、(9.985±0.615)%、(14.845±1.987)%(圖2)。37℃孵育組的細(xì)胞凋亡率顯著低于42℃組(t=10.320,P<0.05)及45℃組(t=9.832,P<0.05);42℃組又顯著低于45℃組(t=4.575,P<0.05)。

圖1不同溫度孵育吉西他濱處理的PANC1細(xì)胞48 h的細(xì)胞形態(tài)(×100)

圖237(a)、42(b)、45℃(c)孵育吉西他濱處理的PANC1細(xì)胞48 h的細(xì)胞凋亡 (流式細(xì)胞儀)

討 論

溫度對(duì)腫瘤影響的研究可以追溯到1970年。當(dāng)時(shí)發(fā)現(xiàn)當(dāng)達(dá)到一定溫度時(shí)細(xì)胞會(huì)被阻滯在不同細(xì)胞周期，進(jìn)而死亡。導(dǎo)致細(xì)胞死亡的溫度是有特異性的，不同細(xì)胞可以耐受不同的溫度。之后發(fā)現(xiàn)細(xì)胞的死亡主要原因是高溫促使蛋白變性。如果使腫瘤組織蛋白變性壞死，溫度至少要達(dá)到或超過(guò)60℃，但臨床上使腫瘤組織達(dá)到或超過(guò)該溫度并不容易，且該溫度作用于人體的安全性較差。為此，有學(xué)者對(duì)溫和高溫處理腫瘤組織是否可以提高化療的敏感性進(jìn)行了研究,結(jié)果顯示溫和高溫聯(lián)合化療處理軟組織肉瘤可以提高患者平均生存期。將發(fā)生轉(zhuǎn)移的軟組織肉瘤患者整體溫度提高到41.8℃維持1 h可以使患者對(duì)化療藥物的敏感性提高24%[5-6]，維持機(jī)體39～40℃ 4～6 h可以達(dá)到抗腫瘤的效果[7]。Adachi等[8]發(fā)現(xiàn)，高溫(43℃ 1 h)可以誘導(dǎo)胰腺癌細(xì)胞熱休克蛋白70表達(dá)，從而提高腫瘤細(xì)胞對(duì)化療藥物的敏感性。有學(xué)者報(bào)道，提高機(jī)體溫度可以增加腫瘤組織血流及血管通透性，使化療藥物更容易作用于腫瘤細(xì)胞，從而達(dá)到增加其對(duì)化療藥物的敏感性。但有學(xué)者認(rèn)為，高溫對(duì)微血管的損傷及增加細(xì)胞間質(zhì)的壓力可能會(huì)抵消這種血流增加的作用[9]。因而溫度對(duì)提高化療藥物敏感性的機(jī)制尚未十分明確。

本研究結(jié)果顯示，溫和高溫聯(lián)合吉西他濱處理能明顯抑制胰腺癌細(xì)胞的增殖，增加細(xì)胞的凋亡率，表明溫和高溫聯(lián)合吉西他濱能提高胰腺癌PANC1細(xì)胞對(duì)吉西他濱的敏感性。雖然，45℃較42℃的效果更好，但臨床將患者局部溫度加熱到45℃時(shí)的安全性較差。42℃高溫相比傳統(tǒng)高溫(>45℃)應(yīng)用于臨床有其明顯的優(yōu)勢(shì)。首先，42℃高溫可以作用患者全身，不會(huì)出現(xiàn)傳統(tǒng)高溫治療腫瘤所發(fā)生的遺漏腫瘤細(xì)胞，導(dǎo)致腫瘤復(fù)發(fā)；其次，42℃高溫操作簡(jiǎn)便，可以使用水浴及紅外設(shè)備直接加熱患者，不必進(jìn)行有創(chuàng)操作；最后，42℃高溫可以提高腫瘤患者對(duì)化療藥物的敏感性，延長(zhǎng)患者的預(yù)后，有效地平衡了治療的有效性及安全性。

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[3] Westermann AM, Wiedemann GJ, Jager E, et al. A Systemic Hyperthermia Oncologic Working Group trial. Ifosfamide, carboplatin, and etoposide combined with 41.8 degrees C whole-body hyperthermia for metastatic soft tissue sarcoma. Oncology,2003,64:312-321.

[4] Maluta S, Schaffer M, Pioli F, et al. Regional hyperthermia combined with chemoradiotherapy in primary or recurrent locally advanced pancreatic cancer: an open-label comparative cohort trial. Strahlenther Onkol, 2011,187:619-625.

[5] Fiegl M, Schlemmer M, Wendtner CM, et al. Ifosfamide, carboplatin and etoposide (ICE) as second-line regimen alone and in combination with regional hyperthermia is active in chemo-pre-treated advanced soft tissue sarcoma of adults. Int J Hyperthermia,2004,20:661-670.

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[7] Kraybill WG, Olenki T, Evans SS, et al. A phase I study of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumours: correlation with mouse models. Int J Hyperthermia,2002,18:253-266.

[8] Adachi S, Kokura S, Okayama T, et al. Effect of hyperthermia combined with gemcitabine on apoptotic cell death in cultured human pancreatic cancer cell lines. Int J Hyperthermia,2009,25:210-219.

[9] Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science,2005,307:58-62.

MildhyperthermiaaffectschemoresistanceofgemcitabineinpancreaticcancerPANC1cellline

LIUMing-hao,LIShu-de,LIZhao-shen,GAOJun,WUHong-yu,GONGYan-fang.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

LIZhao-shen,Email:zhaoshenli@hotmail.com；LIShu-de,Email:lishude57@126.com

ObjectiveTo investigate the effect of mild hyperthermia on chemoresistance of gemcitabine in the pancreatic cancer cell line PANC1.MethodsThe PANC1 cell was treated with gemcitabine (1 μmol/L) for 1 h, then was heated at 37℃, 42℃ and 45℃ for 1 hour, and was cultured for 0 h, 24 h, 48 h. Cell growth was analyzed by CCK-8 assay. Cell apoptosis was analyzed by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining.ResultsThe proliferation of cells at 42℃ and 45℃ for 24 h and 48 h were significantly lower than that at 37℃(0.96±0.05,0.88±0.03vs1.05±0.02；1.28±0.04,0.94±0.04vs1.49±0.09；t=4.367, 25.120,P<0.05). The proliferation of cells at 45℃ was significantly lower than that at 42℃(t=3.348, 11.732,P<0.05).The cell apoptosis rates at 37℃, 42℃, 45℃ after 48 h were (7.125±0.064)%, (9.985±0.615)%, (14.845±1.987)%, the difference among the 3 groups was statistically significant (t=10.320, 9.832, 4.575,P<0.05).ConclusionsMild hyperthermia can reduce chemoresistance of gencitabine in pancreatic cancer cell PANC1.

Pancreatic neoplasms; Mild hyperthermia; Gemcitabine; Drug therapy

10.3760/cma.j.issn.1674-1935.2012.03.014

上海衛(wèi)生局科研基金

200433 上海，第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院消化內(nèi)科

李兆申，Email:zhaoshenli@hotmail.com；李淑德，Email:lishude57@126.com

2011-12-19)

(本文編輯：屠振興)