裸鼠胰腺癌種植瘤乙醇瘤內(nèi)注射的療效觀察

張文穎 吳洪玉 郭妍 龔艷芳 高軍 金震東 李兆申

·論著·

裸鼠胰腺癌種植瘤乙醇瘤內(nèi)注射的療效觀察

張文穎 吳洪玉 郭妍 龔艷芳 高軍 金震東 李兆申

目的評(píng)價(jià)不同濃度乙醇瘤內(nèi)注射對(duì)裸鼠胰腺癌種植瘤的療效，為臨床選擇合適的乙醇濃度治療胰腺癌提供實(shí)驗(yàn)依據(jù)。方法將人胰腺癌SW1990細(xì)胞接種于裸鼠皮下建立胰腺癌種植瘤模型。選取腫瘤大小相近的裸鼠48只，按隨機(jī)表法隨機(jī)分為20%、40%、60%、80%、95%乙醇注射組及注射生理鹽水的對(duì)照組。治療前后用游標(biāo)卡尺測(cè)量腫瘤最長(zhǎng)徑(a)和最短徑(b)，計(jì)算腫瘤體積(TV)、相對(duì)腫瘤體積(RTV)和相對(duì)腫瘤體積增殖率(T/C)。治療后第8天處死裸鼠，取腫瘤組織行病理組織學(xué)檢查。結(jié)果20%乙醇注射組與對(duì)照組的RTV差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.212)，40%、60%、80%、95%乙醇注射組的RTV均較對(duì)照組顯著縮小(P值均<0.01)，其中60%、80%、95%乙醇注射組的RTV<1，T/C<30%，且隨著乙醇濃度的增加RTV及T/C值越小。80%和95%乙醇注射組的RTV值顯著低于60%乙醇注射組(P值分別為0.003、0.009)，而80%和95%乙醇注射組的RTV差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.819)。病理組織學(xué)檢查顯示對(duì)照組無(wú)腫瘤壞死，20%、40%乙醇注射組種植瘤內(nèi)可見(jiàn)少量壞死， 60%、80%和95%乙醇注射組的種植瘤體內(nèi)出現(xiàn)大面積凝固性壞死。結(jié)論選擇80%乙醇瘤內(nèi)注射治療裸鼠皮下胰腺癌種植瘤是安全、有效的。

胰腺腫瘤； 乙醇； 超聲檢查，內(nèi)鏡； 注射，病灶內(nèi)

胰腺癌是一種病情兇險(xiǎn)、治愈率低、預(yù)后極差的消化道腫瘤。盡管吉西他濱已成為治療晚期胰腺癌患者的一線化療藥物，一定程度上延長(zhǎng)了患者的生存期，但其1年生存率僅為20%左右[1]。內(nèi)鏡超聲(endoscopic ultrasound，EUS)作為一種成熟的內(nèi)鏡技術(shù)已廣泛應(yīng)用于胰腺疾病的診斷。隨著凸面線陣型超聲內(nèi)鏡的出現(xiàn)，EUS引導(dǎo)下的介入治療也已逐步開(kāi)展，包括放射性粒子植入、腹腔神經(jīng)叢(節(jié))阻滯及基因重組人溶瘤腺病毒植入等。超聲引導(dǎo)下無(wú)水乙醇注射在肝、腎囊腫及原發(fā)性肝癌等治療方面已取得較好的療效。Matthes等[2]在EUS引導(dǎo)下對(duì)豬正常胰腺進(jìn)行不同濃度的乙醇注射(0、20%、40%、60%、80%、100%)，發(fā)現(xiàn)20%乙醇注射不能導(dǎo)致胰腺組織壞死，而40%及以上濃度可使胰腺組織發(fā)生壞死，并且壞死面積與乙醇濃度呈正相關(guān)。為此，本研究應(yīng)用不同濃度乙醇注射于裸鼠胰腺癌種植瘤內(nèi)，觀察其療效，為臨床應(yīng)用EUS引導(dǎo)下乙醇注射治療胰腺癌提供實(shí)驗(yàn)依據(jù)。

材料與方法

一、裸鼠皮下種植性腫瘤模型建立及分組

BalB/c-nu裸小鼠4～6周齡，體重約18～20 g，購(gòu)自中科院斯萊克動(dòng)物中心，均獲國(guó)家動(dòng)物品系合格證。人胰腺癌細(xì)胞株SW1990 購(gòu)自中科院上海細(xì)胞庫(kù)，常規(guī)培養(yǎng)、傳代。待細(xì)胞生長(zhǎng)至70%～80%融合時(shí)，胰蛋白酶消化，制成細(xì)胞懸液。調(diào)整細(xì)胞密度至1×107個(gè)/ ml，取0.2 ml接種于裸鼠胸肋部皮下。以腫瘤體積長(zhǎng)至100～300 mm3為成瘤標(biāo)準(zhǔn)，接種成功率為100%。給藥前各組裸鼠飲食、行為均表現(xiàn)正常。

選取腫瘤大小相近的荷瘤裸鼠48只，按隨機(jī)表法將其隨機(jī)分為20%、40%、60%、80%、95%乙醇注射組及注射生理鹽水的對(duì)照組，每組8只，采用單點(diǎn)注射。乙醇注射劑量參考肝臟腫瘤乙醇消融的回歸方程[3]，根據(jù)預(yù)實(shí)驗(yàn)結(jié)果進(jìn)行修正，Y=2.885X/8=0.36X[Y為每次注射劑量(ml)，X為腫瘤最大直徑(cm)]。

二、方法

1．療效判斷：治療前和治療后第2、4、6、8天分別用游標(biāo)卡尺測(cè)量腫瘤最長(zhǎng)徑(a)和最短徑(b)，同時(shí)稱裸鼠的體重，計(jì)算腫瘤體積(tumor volume，TV)、相對(duì)腫瘤體積(relative tumor volume，RTV)及相對(duì)腫瘤增殖率T/C。TV =ab2/2，RTV=Vt/V0(V0為治療前測(cè)量的腫瘤體積，Vt為t時(shí)間測(cè)量的腫瘤體積)，T/C=RTV治療組/RTV對(duì)照組×100%[4]。

2．種植瘤病理組織學(xué)檢查：治療后第8天將裸鼠全部處死，取腫瘤組織，常規(guī)染色，光鏡下觀察病理改變。

三、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、裸鼠存活情況

注射治療后，60%、80%乙醇注射組裸鼠各死亡1只，95%乙醇注射組死亡2只，其余各組裸鼠均可耐受治療。

二、療效

60%、80%、95%乙醇注射組裸鼠皮下種植瘤組織在治療后24 h即出現(xiàn)部分壞死。其中60%乙醇注射組在治療后第4天有1只裸鼠的種植瘤組織完全壞死，80%乙醇注射組在治療后第4天有4只裸鼠的種植瘤組織完全壞死，95%乙醇注射組在治療后第4天有2只裸鼠的種植瘤組織完全壞死，第6天有1只裸鼠的種植瘤組織完全壞死(圖1)。

20%乙醇注射組與對(duì)照組的RTV差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.212)， 40%、60%、80%、95%乙醇注射組的RTV均較對(duì)照組明顯縮小(P值均<0.01)，其中60%、80%、95%乙醇注射組的RTV<1，T/C<30%，且隨著乙醇濃度的增加RTV及T/C值越小。80%和95%乙醇注射組的RTV值顯著低于60%乙醇注射組(P值分別為0.003、0.009)，而80%和95%乙醇注射組的RTV差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.819，表1，圖2)。

表1 不同濃度乙醇對(duì)裸鼠種植瘤的治療效果

注：與對(duì)照組比較，aP<0.01；與60%乙醇注射組比較，bP<0.01

圖1對(duì)照組(a)和20%(b)、40%(c)、60%(d)、80%(e)、95%(f)乙醇注射組治療前(左)及治療后1周(右)的大體標(biāo)本

圖2 對(duì)照組及各濃度乙醇注射組的RTV

三、種植瘤病理組織學(xué)改變

對(duì)照組未見(jiàn)腫瘤壞死，20%和40%乙醇組腫瘤瘤體內(nèi)可見(jiàn)少量壞死，而60%、80%和95%乙醇組的瘤體內(nèi)出現(xiàn)大面積凝固性壞死(圖3)。

圖3對(duì)照組(a)和20%(b)、40%(c)、60%(d)、80%(e)、95%(f)乙醇注射組治療后1周種植瘤的病理改變(HE ×100)

討 論

經(jīng)皮乙醇消融在肝、腎囊腫及某些實(shí)體腫瘤如原發(fā)性肝癌、腎上腺腫瘤等方面治療已取得較好的療效[5-8]。但EUS引導(dǎo)下乙醇消融對(duì)胰腺癌治療的報(bào)道較少。為了評(píng)價(jià)EUS引導(dǎo)下乙醇消融治療胰腺癌的安全性，Aslanian等[9]在EUS引導(dǎo)下對(duì)豬的正常胰腺進(jìn)行乙醇注射，結(jié)果發(fā)現(xiàn)，豬均可耐受乙醇，并無(wú)胰腺炎的發(fā)生，僅有輕度的淀粉酶升高，在胰腺注射部位可見(jiàn)炎細(xì)胞浸潤(rùn)、壞死和纖維化。2005年Gan等[10]報(bào)道，用EUS引導(dǎo)下細(xì)針瘤內(nèi)注射乙醇治療胰腺囊性腫瘤是安全可行的。Oh等[11-12]報(bào)道一項(xiàng)前瞻性研究，在EUS引導(dǎo)下行乙醇加紫杉醇注射治療胰腺囊性腫瘤，結(jié)果完全緩解率達(dá)62%，僅1例并發(fā)輕度胰腺炎，1例并發(fā)脾靜脈栓塞，是安全有效的治療方法。Jürgensen等[13]報(bào)道1例EUS引導(dǎo)下給予95%乙醇8 ml注射治療胰島素瘤，治療期間僅出現(xiàn)輕微的胰腺炎，3 d后恢復(fù)正常，治療后患者低血糖癥狀明顯改善，隨訪34個(gè)月，再次行EUS檢查發(fā)現(xiàn)原發(fā)部位的腫塊已消失。另外治療1例胰腺內(nèi)2個(gè)神經(jīng)內(nèi)分泌瘤，腫塊大小分別是11 mm和7 mm，在EUS引導(dǎo)下給予40%乙醇2 ml注射治療，隨訪18個(gè)月，患者血管活性腸肽及鉻粒素A水平均恢復(fù)正常，MRI檢查胰腺呈陰性[14]。

本研究結(jié)果顯示，隨著注射的乙醇濃度增加，裸鼠皮下胰腺癌種植瘤的壞死面積增大，瘤體積明顯縮小。80%和95%乙醇注射組的療效優(yōu)于60%乙醇注射組，這2種濃度的療效接近，但80%乙醇注射組死亡1只，95%乙醇注射組死亡2只，表明瘤內(nèi)注射80%乙醇治療裸鼠皮下胰腺癌種植瘤是較安全、有效的。

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Theeffectsofintra-tumoralinjectionofethanolonnudemicewithimplantedpancreaticcancer

ZHANGWen-ying,WUHong-yu,GUOYan,GONGYan-fang,GAOJun,JINZhen-dong,LIZhao-shen.

DepartmentofGastroenterology,ChanghaiHospital,SecondMilitaryMedicalUniversity,Shanghai200433,China

Correspondingauthor:JINZhen-dong,Email:zhendjin@126.com

ObjectiveTo study the efficacy of intra-tumoral injection of different concentrations of ethanol for nude mice with implanted pancreatic cancer and provide evidence for choosing appropriate concentration of ethanol for clinical treatment of pancreatic cancer.MethodsA subcutaneous xenograft mouse model of human pancreatic cancer SW1990 was established. Forty-eight nude mice with similar tumor size were randomly divided into 20%, 40%, 60%, 80%, 95% ethanol injection groups and saline injection group. The longest (a) and the shortest diameters (b) of tumor of nude mice were measured. Tumor volume (TV), relative tumor volume (RTV) and the relative rate of tumor proliferation (T/C%) were calculated. Eight days later the nude mice were sacrificed. The tumor tissue was harvested for pathologic examinations.ResultsRTV in 20% ethanol injection group was similar that of saline injection group (P=0.212). RTV in 40%, 60%, 80% and 95% ethanol injection groups were significantly lower than that in saline injection group (P<0.01). RTV was less than 1 and T/C% was less than 30% in 60%, 80% and 95% ethanol injection groups. The values of RTV and T/C% decreased with the increase of ethanol concentration. RTV in 80% and 95% ethanol injection groups were significantly lower than that of 60% ethanol injection group (P=0.003 andP=0.009). RTV was similar in 80% and 95% ethanol injection groups (P=0.819). The pathologic examinations showed no tumor necrosis in saline injection group, while small amounts of necrosis in implanted pancreatic cancer was observed in 20% and 40% ethanol injection groups, while a large area of coagulation necrosis could be found in 60%, 80% and 95% ethanol injection groups.ConclusionsIntra-tumoral injection of 80% ethanol is feasible therapy method for nude mice with human pancreatic cancer xenografts.

Pancreatic neoplasms; Ethanol; Ultrasonography, endoscopic; Injections, intralesional

2013-01-11)

(本文編輯：屠振興)

10.3760/cma.j.issn.1674-1935.2013.03.012

上海市科學(xué)技術(shù)委員會(huì)(11D21921605)

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

金震東，Email：zhendjin@126. com