陸錄　朱文偉　陳進(jìn)宏　賈戶亮

(復(fù)旦大學(xué)附屬華山醫(yī)院普外科，上海　200040)

?

·論著·

AMD3100對(duì)索拉非尼治療肝細(xì)胞肝癌的療效影響

陸錄朱文偉陳進(jìn)宏賈戶亮

(復(fù)旦大學(xué)附屬華山醫(yī)院普外科，上海200040)

摘要目的：觀察基質(zhì)細(xì)胞衍生因子1-α(stromal cell derived factor 1-α, SDF1-α)受體抑制劑AMD3100聯(lián)用索拉非尼對(duì)肝細(xì)胞肝癌(HCC)裸鼠的療效。方法： 建立肝細(xì)胞肝癌原位裸鼠模型，分為對(duì)照組、索拉非尼組、索拉非尼+AMD3100 組，每組6只。索拉非尼組給予索拉非尼30 mg/(kg·d)灌胃；索拉非尼+AMD3100組在給予索拉非尼灌胃的基礎(chǔ)上，給予AMD3100 2.5 mg/(kg·d)腹腔注射；對(duì)照組給予0.9%氯化鈉液灌胃。比較三組的腫瘤肝內(nèi)播散情況、腫瘤大小、肺轉(zhuǎn)移情況和裸鼠的生存時(shí)間。結(jié)果：在索拉非尼治療的基礎(chǔ)上，聯(lián)用AMD3100能夠明顯抑制索拉非尼引起的HCC肝內(nèi)侵襲和轉(zhuǎn)移，進(jìn)一步減小腫瘤體積，減少肺轉(zhuǎn)移，延長(zhǎng)荷瘤裸鼠的生存期。結(jié)論：SDF1-α受體抑制劑AMD3100能夠增強(qiáng)索拉非尼對(duì)HCC的療效。

關(guān)鍵詞AMD3100；索拉菲尼；肝細(xì)胞肝癌

索拉非尼(sorafenib)是目前晚期肝細(xì)胞肝癌(HCC)的一線治療藥物[1]。但是，研究[2-4]發(fā)現(xiàn)，索拉非尼在抑制原發(fā)瘤的同時(shí)促進(jìn)了轉(zhuǎn)移瘤的生長(zhǎng)。我們的前期研究發(fā)現(xiàn)，應(yīng)用索拉非尼后，腫瘤組織和癌旁微環(huán)境中基質(zhì)細(xì)胞衍生因子1-α(stromal cell derived factor 1-α, SDF1-α)的表達(dá)明顯增加。因此，本研究將SDF1-α受體抑制劑AMD3100與索拉非尼聯(lián)合應(yīng)用于治療HCC裸鼠模型，探討AMD3100協(xié)同索拉非尼治療HCC的療效。

1資料與方法

1.1實(shí)驗(yàn)動(dòng)物及藥物18只雄性BALB/c裸鼠，6周齡，體質(zhì)量15~20 g，購(gòu)自中國(guó)科學(xué)院上海藥物研究所，在25℃恒溫的SPF級(jí)層流室中飼養(yǎng)，12 h光照和12 h黑暗交替，自由進(jìn)食標(biāo)準(zhǔn)顆粒飼料及飲水。索拉非尼購(gòu)自拜耳(中國(guó))有限公司， AMD3100購(gòu)自德國(guó)Sigma-Aldrich公司。

1.2裸鼠原位HCC模型的建立無(wú)菌條件下將培養(yǎng)的1×107的人HCC LM3細(xì)胞注射到裸鼠皮下。待3~4周皮下瘤成瘤后，取瘤原，浸入冷0.9%氯化鈉液中，剪成2 mm×1 mm×1 mm大小備用。以2.5 mg/kg戊巴比妥腹腔注射麻醉裸鼠，消毒手術(shù)野，在左肋緣下作長(zhǎng)約8 mm的斜切口，將肝葉取出，斜形切開(kāi)肝表面，植入備好的瘤原組織塊，將肝葉放回原位，用 6-0 線縫合肝切緣，用5-0線雙層縫合腹壁和皮膚。

1.3給藥方法及分組將18只HCC模型裸鼠分為3組，每組6只。模型建立后2周開(kāi)始給藥。將索拉非尼400 mg溶于100 mL的溶劑(蓖麻油∶乙醇∶dH2O=12.5∶12.5∶75)中，AMD3100溶于0.9%氯化鈉液中。索拉非尼組給予裸鼠索拉非尼30 mg/(kg·d)灌胃；索拉非尼+AMD3100組在給予索拉非尼的基礎(chǔ)上給予裸鼠AMD3100 2.5 mg/(kg·d)腹腔注射。對(duì)照組給予0.9%氯化鈉液 0.1mL/d 灌胃。

1.4觀察指標(biāo)給藥4周后處死裸鼠，取出腫瘤，測(cè)量最長(zhǎng)徑a和最短徑b，腫瘤體積=ab2/2；取雙肺，石蠟包埋并連續(xù)切片，HE染色后讀取每隔5張切片中的肺轉(zhuǎn)移灶數(shù)目。

1.5統(tǒng)計(jì)學(xué)處理如連續(xù)變量呈正態(tài)分布，采用t檢驗(yàn)(Students’t-test)；如連續(xù)變量呈非正態(tài)分布，采用秩和檢驗(yàn)(Mann-Whitney U test)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2結(jié)果

2.1AMD3100對(duì)腫瘤體積及侵襲轉(zhuǎn)移能力的影響對(duì)照組腫瘤平均體積為4.4 cm3。索拉非尼組腫瘤平均體積為2.7 cm3，明顯小于對(duì)照組(P=0.02)，但腫瘤向肝內(nèi)播散。與索拉非尼組比較，AMD3100+索拉非尼組腫瘤體積更小，平均為1.3 cm3，差異有統(tǒng)計(jì)學(xué)意義(P=0.04)；腫瘤邊界清晰，肝內(nèi)播散被明顯抑制。見(jiàn)圖1、2A。

圖1　HCCLM3裸鼠原位瘤模型大體標(biāo)本

2.2AMD3100對(duì)腫瘤肺轉(zhuǎn)移的影響索拉非尼組裸鼠的肺轉(zhuǎn)移灶數(shù)目較對(duì)照組明顯減少(10個(gè)比18個(gè)，P=0.01)，索拉非尼+AMD3100組肺轉(zhuǎn)移灶的數(shù)目進(jìn)一步減少(4個(gè)，P=0.02)，見(jiàn)圖2B。

圖2A：裸鼠原位肝細(xì)胞肝癌模型腫瘤體積比較；B：裸鼠原位肝細(xì)胞肝癌模型肺轉(zhuǎn)移數(shù)目的比較；C：原位肝細(xì)胞肝癌模型裸鼠生存時(shí)間的比較

2.3AMD3100對(duì)裸鼠生存期的影響索拉非尼組的裸鼠平均生存時(shí)間為76 d，長(zhǎng)于對(duì)照組(69 d)；索拉非尼+AMD3100組裸鼠的平均生存時(shí)間達(dá) 91 d。見(jiàn)圖2C。

3討論

抗血管生成在HCC的治療中非常重要[5-6]。索拉非尼是目前臨床上治療晚期HCC的一線用藥，它主要通過(guò)抑制血管內(nèi)皮生長(zhǎng)因子(VEGF)發(fā)揮抗血管生成的作用[7]。但是兩個(gè)大型的臨床研究[8-9]顯示，其對(duì)于HCC的治療效果不佳，對(duì)患者的中位生存期延長(zhǎng)不足3個(gè)月。

SDF1又稱為CXCL12，是一種趨化因子，主要由成纖維細(xì)胞、炎性細(xì)胞、內(nèi)皮細(xì)胞等分泌[10]。SDF1-α通過(guò)與其受體CXCR4結(jié)合，促進(jìn)腫瘤的生長(zhǎng)[10]。有文獻(xiàn)[11]報(bào)道，AMD3100作為CXCR4的特異性抑制劑，能夠增強(qiáng)前列腺癌對(duì)于放療的敏感性。AMD3100能夠抑制囊腺癌的嗜神經(jīng)侵襲[12]。然而，AMD3100應(yīng)用于肝癌的報(bào)道比較少見(jiàn)。本研究將CXCR4的特異性抑制劑AMD3100與索拉非尼聯(lián)用，發(fā)現(xiàn)AMD3100能夠明顯抑制索拉非尼引起的腫瘤肝內(nèi)播散，并可進(jìn)一步減小腫瘤體積、抑制腫瘤肺轉(zhuǎn)移、延長(zhǎng)荷瘤裸鼠的生存時(shí)間，說(shuō)明AMD3100能夠增強(qiáng)索拉非尼的療效。

參考文獻(xiàn)

[1]Llovet JM, Bisceglie AM, Bruix J, et al. Design and endpoints of clinical trials in hepatocellular carcinoma[J]. J Natl Cancer Inst, 2008, 100(10): 698-711.

[2]Ebos JM, Lee CR, Cruz MW, et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis[J]. Cancer Cell, 2009, 15(3): 232-239.

[3]Paez-Ribes RM, Allen E, Hudock J, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis[J]. Cancer Cell, 2009, 15(3): 220-231.

[4]Zhang W, Sun HC, Wang WQ, et al. Sorafenib down-regulates expression of HTATIP2 to promote invasiveness and metastasis of orthotopic hepatocellular carcinoma tumors in mice[J]. Gastroenterology, 2012, 143(6): 1641-1649.

[5]Farazi PA, DePinho RA, Hepatocellular carcinoma pathogenesis: from genes to environment[J]. Nat Rev Cancer, 2006, 6(9): 674-687.

[6]Pang R, Poon RT, Angiogenesis and antiangiogenic therapy in hepatocellular carcinoma[J]. Cancer Lett, 2006, 242(2): 151-167.

[7]de Lope CR, Tremosini S, Forner A, et al. Management of HCC[J]. J Hepatol, 2012, 56 Suppl: S75-S87.

[8]Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepatocellular carcinoma[J]. N Engl J Med, 2008, 359(4): 378-390.

[9]Cheng AL, Kang YK, Chen Z, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial[J]. Lancet Oncol, 2009, 10(1): 25-34.

[10]Sun X, Cheng G, Hao M, et al. CXCL12/CXCR4/CXCR7 chemokine axis and cancer progression[J]. Cancer Metastasis Rev, 2010, 29(4): 709-722.

[11]Domanska UM, Boer JC, Timmer-Bosscha H, et al. CXCR4 inhibition enhances radiosensitivity, while inducing cancer cell mobilization in a prostate cancer mouse model[J]. Clin Exp Metastasis, 2014, 31(7): 829-839.

[12]Jeong WJ, Choi IJ, Park MW, et al. CXCR4 antagonist inhibits perineural invasion of adenoid cystic carcinoma[J]. J Clin Pathol, 2014, 67(11): 992-998.

The Effect of AMD3100 on the Efficacy of Sorafenib in the Treatment of Hepatocellular Carcinoma

LULuZHUWenweiCHENJinhongJIAHuliang

DepartmentofSurgery，HuaShanHospital,FudanUniversity,Shanghai200040,China

AbstractObjective：To investigate the effect of AMD3100, an inhibitor of receptor of stromal cell derived factor 1-α (SDF1-α), combined with sorafenib on nude mice with hepatocellular carcinoma (HCC). Methods:Nude mice models of HCC in situ was established and the mice were divided into control group, sorafenib group and sorafenib combined with AMD3100 group, with six mice in each group. In sorafenib group, sorafenib 30 mg/(kg·d) was given by garage. In sorafenib combined with AMD3100 group, AMD3100 2.5 mg/(kg·d) was administrated by intraperitoneal injection based on the method of sorafenib group. In the control group, 0.9% sodium chloride solution was given by garage. The intrahepatic invasion, the tumor volume, the situation of pulmonary metastasis and the survival time of nude mice, were compared among the three groups. Results: When sorafenib was combined with AMD3100, the intrahepatic invasion and metastasis of HCC caused by sorafenib could be significantly inhibited. Thus the tumor volume was decreased and the pulmonary metastasis could be reduced. The survival time was prolonged. Conclusions: AMD3100, a SDF1-α receptor inhibitor, can enhance the efficacy of sorafenib.

Key WordsAMD3100；Sorafenib；Hepatocellular carcinoma

中圖分類號(hào)R735.7

文獻(xiàn)標(biāo)識(shí)碼A

通訊作者賈戶亮，E-mail：jbl-1@163.com

基本項(xiàng)目：國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：81101564)