丹參經(jīng)肝動(dòng)脈灌注對(duì)大鼠移植性肝癌的治療及其肝功能保護(hù)作用研究

魏明琴,蔣建霞

(1.湖北醫(yī)藥學(xué)院附屬襄陽(yáng)醫(yī)院 腫瘤科，湖北 襄陽(yáng) 441000；2.南京醫(yī)科大學(xué)，江蘇 南京 210000)

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丹參經(jīng)肝動(dòng)脈灌注對(duì)大鼠移植性肝癌的治療及其肝功能保護(hù)作用研究

魏明琴1,蔣建霞2

(1.湖北醫(yī)藥學(xué)院附屬襄陽(yáng)醫(yī)院 腫瘤科，湖北 襄陽(yáng) 441000；2.南京醫(yī)科大學(xué)，江蘇 南京 210000)

目的 觀察丹參經(jīng)肝動(dòng)脈灌注對(duì)大鼠移植性肝癌的治療作用，并研究丹參在抗腫瘤的同時(shí)對(duì)移植性肝癌大鼠肝功能的保護(hù)作用。方法 選用Sprague-Dawley雄性大鼠150只，并成功建立Walker-256移植性肝癌模型120只。所有造模大鼠隨機(jī)分為對(duì)照組、絲裂霉素組和丹參組，每組40只。正常飼養(yǎng)10 d后，取各組大鼠血液檢測(cè)血清ALT、AST和ALP水平，剖腹測(cè)量計(jì)算移植性瘤體體積。同時(shí)行經(jīng)胃十二指腸動(dòng)脈至肝臟固有動(dòng)脈插管灌注治療：對(duì)照組大鼠灌注2 mL的注射用生理鹽水，絲裂霉素組大鼠灌注絲裂霉素(2.5 mg/kg)，丹參組灌注丹參(1.6 g/kg)。灌注1周后，取血液檢測(cè)ALT，AST和ALP水平，測(cè)量計(jì)算瘤體體積，并計(jì)算腫瘤增長(zhǎng)率和抑制率。同時(shí)處死所有大鼠，取出瘤體，顯微鏡下觀察腫瘤壞死范圍并判斷壞死程度。結(jié)果 灌注治療后，對(duì)照組、絲裂霉素組和丹參組大鼠分別剩余37只、37只和38只。絲裂霉素組和丹參組腫瘤體積及增長(zhǎng)率均明顯小于對(duì)照組(P<0.05)，絲裂霉素組和丹參組腫瘤體積及增長(zhǎng)率無(wú)明顯差異。絲裂霉素組和丹參組對(duì)移植性肝癌腫瘤的抑制率分別為22.32%和26.38%，差異無(wú)統(tǒng)計(jì)學(xué)意義。灌注后，丹參組和絲裂霉素組大鼠中度壞死明顯高于對(duì)照組，絲裂霉素組和丹參組輕、中、重腫瘤壞死分布無(wú)顯著性差異。灌注后，丹參組和絲裂霉素組大鼠血清ALT，AST和ALP的水平均明顯低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01，P<0.05)；丹參組上述指標(biāo)顯著低于絲裂霉素組，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論 丹參經(jīng)肝動(dòng)脈灌注對(duì)大鼠移植性肝癌有良好的抑制作用，其抑瘤活性與常用抗腫瘤藥物絲裂霉素的活性相當(dāng)。在抑制腫瘤的同時(shí)對(duì)移植性肝癌大鼠的肝功能也具有較好的保護(hù)作用。

丹參；肝動(dòng)脈灌注；移植性肝癌；肝功能保護(hù)

肝癌是一種十分常見(jiàn)的惡性消化道腫瘤，其病死率高、危害性大，已經(jīng)位居世界腫瘤性疾病的第3位，且有逐年升高的趨勢(shì)[1-4]。中國(guó)是肝癌的高發(fā)區(qū)，其發(fā)患者數(shù)約占全世界總發(fā)病數(shù)的一半，而其死亡率也位居世界之首。目前，治療肝癌的首選方法是進(jìn)行手術(shù)切除，但是大多數(shù)的患者在確診為肝癌時(shí)已經(jīng)錯(cuò)過(guò)了最佳的手術(shù)時(shí)機(jī)[5]。由于肝臟供血主要來(lái)自肝動(dòng)脈，且肝癌血供中有90%來(lái)自肝動(dòng)脈[6]。所以，對(duì)于不能進(jìn)行手術(shù)切除治療的患者，目前主要采用經(jīng)肝動(dòng)脈灌注化療或經(jīng)肝動(dòng)脈栓塞化療[7]。丹參作為一味傳統(tǒng)中藥，歸心、肝經(jīng)，有祛瘀止痛，活血通經(jīng)之功效[8]。近年來(lái)的研究表明丹參具有十分廣泛的藥理活性，如抑制血小板凝聚、擴(kuò)張冠狀動(dòng)脈等功效[9-10]。此外，丹參能夠顯著抑制多種腫瘤細(xì)胞的增殖。為了進(jìn)一步研究丹參在治療肝癌和肝功能保護(hù)方面的特殊作用，本研究建立了大鼠移植性肝癌模型，并對(duì)肝癌造模小鼠進(jìn)行丹參肝動(dòng)脈灌注治療，以觀察丹參對(duì)肝癌的治療效果及肝功能保護(hù)作用。

1 材料與方法

1.1 實(shí)驗(yàn)試劑 注射用丹參粉針劑(哈藥集團(tuán)中藥二廠，國(guó)藥準(zhǔn)字Z10970093)；注射用絲裂霉素粉針劑(浙江海正藥業(yè)股份有限公司，國(guó)藥準(zhǔn)字H33020786)；注射用生理鹽水(四川科倫藥業(yè)股份有限公司，國(guó)藥準(zhǔn)字H51021157)；75%醫(yī)用乙醇(衡陽(yáng)原野實(shí)業(yè)有限公司，湘衛(wèi)消證字第0009號(hào))；2%戊巴比妥鈉注射液用注射用生理鹽水自行配制。

1.2 實(shí)驗(yàn)動(dòng)物 Sprague-Dawley雄性大鼠150只，體質(zhì)量210～250 g，皮下荷瘤SD小鼠(Walker-256瘤株)，均購(gòu)自中國(guó)科學(xué)院斯萊克實(shí)驗(yàn)動(dòng)物中心(SCXK京2011-0012)，購(gòu)回后適應(yīng)性飼養(yǎng)1周再用于試驗(yàn)。本實(shí)驗(yàn)遵循《實(shí)驗(yàn)動(dòng)物保護(hù)條例》，對(duì)實(shí)驗(yàn)動(dòng)物進(jìn)行嚴(yán)格的檢驗(yàn)檢疫和飼養(yǎng)管理。

1.3 方法

1.3.1 大鼠移植性肝癌造模：將荷瘤SD小鼠處死，經(jīng)75%乙醇消毒后，取出瘤體，挑選無(wú)出血損壞的腫瘤組織，切割成0.5 mm2見(jiàn)方的瘤塊備用。用2%戊巴比妥鈉注射液(45 mg/kg)對(duì)150只大鼠進(jìn)行腹腔麻醉，然后以仰臥位固定于超凈臺(tái)，剪掉腹部毛發(fā)，用75%乙醇消毒手術(shù)區(qū)后，于腹部中央剪開(kāi)皮膚，暴露出肝臟，采用肝內(nèi)隧道植入法植入準(zhǔn)備好的Walker-256瘤塊。肝臟復(fù)位，腹部縫合。共有120只大鼠造模成功。

1.3.2 實(shí)驗(yàn)分組及給藥：將造模成功的120只大鼠隨機(jī)分為對(duì)照組、絲裂霉素組和丹參組，共3組，每組各40只。正常飼養(yǎng)10 d后，所有大鼠用2%戊巴比妥鈉注射液(45 mg/kg)行腹腔麻醉，取血備用，并再次切開(kāi)腹部，暴露出肝臟，測(cè)量腫瘤表面最大和最小徑。同時(shí)行經(jīng)胃十二指腸動(dòng)脈至肝臟固有動(dòng)脈插管灌注治療。對(duì)照組大鼠灌注2 mL的注射用生理鹽水，絲裂霉素組大鼠灌注絲裂霉素(2.5 mg/kg)，丹參組灌注丹參(1.6 g/kg)。灌注完成后，縫合切口，并涂抹抗生素藥膏。正常飼養(yǎng)1周后，2%戊巴比妥鈉行腹腔注射麻醉，再次取血，并測(cè)量腫瘤表面最大和最小徑。最后處死所有實(shí)驗(yàn)大鼠，取出瘤體，在顯微鏡下觀察3組大鼠腫瘤壞死程度。

1.3.3 測(cè)定移植腫瘤的體積及其生長(zhǎng)率：體積(V)=腫瘤最大徑(cm)×腫瘤最小徑(cm)2/2；增長(zhǎng)率(GR)=治療后體積/治療前體積；生長(zhǎng)抑制率=(GR1-GR2)/GR1×100%。

1.3.4 腫瘤壞死程度觀察：實(shí)驗(yàn)結(jié)束時(shí)，處死所有實(shí)驗(yàn)大鼠，取出瘤體，顯微鏡下觀察3組大鼠腫瘤壞死程度，按照輕、中、重程度劃分。因術(shù)后死亡，各組大鼠剩余數(shù)量分別為對(duì)照組37只，絲裂霉素組37只，丹參組38只。

1.3.5 大鼠肝功能檢測(cè)：2次所取大鼠血液經(jīng)離心后，取出上清液，用自動(dòng)生化儀檢測(cè)丙氨酸氨基轉(zhuǎn)移酶(ALT)、天門冬酸氨基轉(zhuǎn)移酶(AST)水平、堿性磷酸酶(ALP)的水平。

2 結(jié)果

2.1 大鼠移植性肝癌腫瘤體積、增長(zhǎng)率及抑制率比較 由表1可知，絲裂霉素組和丹參組腫瘤體積及增長(zhǎng)率均明顯小于對(duì)照組(P<0.05)，絲裂霉素和丹參組腫瘤體積及增長(zhǎng)率無(wú)明顯差異。絲裂霉素組和丹參組對(duì)移植性肝癌腫瘤抑制率的差異無(wú)統(tǒng)計(jì)學(xué)意義(見(jiàn)表1)。

表1 各組大鼠肝癌腫瘤體積、增長(zhǎng)率及抑制率比較±s)Tab.1 Comparison of the volume，growth rate and inhibition rate of ±s)

*P<0.05，與對(duì)照組相比較，compared with control group

2.2 大鼠移植性肝癌腫瘤壞死程度比較 由表2可知，灌注治療后，丹參組和絲裂霉素組大鼠中度壞死高于對(duì)照組。絲裂霉素組和丹參組輕，中，重腫瘤壞死分布無(wú)顯著性差異。

表2 各組大鼠腫瘤壞死程度比較Tab.2 Comparison of the degree of tumor necrosis among groups

2.3 大鼠血清中ALT，AST和ALP的測(cè)定比較 由表3可知，灌注治療后，丹參組和絲裂霉素組大鼠血清ALT，AST和ALP的水平均明顯低于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01，P<0.05)；丹參組上述指標(biāo)顯著低于絲裂霉素組，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05，見(jiàn)表3)。

表3 各組大鼠血清中ALT，AST和ALP的水平比較Tab.3 Comparison of the serum ALT，AST and ALP levels among ±s，μ/L)

*P<0.05，**P<0.01，與對(duì)照組比較，compared with control group；△P<0.05，與絲裂霉素組比較，compared with mitomycin group

3 討論

有研究表明[11]，不能接受手術(shù)治療的肝癌患者，采用化療藥物經(jīng)肝動(dòng)脈灌注，可直達(dá)肝癌組織，導(dǎo)致肝癌組織壞死，而對(duì)正常的肝組織損傷較小。近年來(lái)，已有絲裂霉素、順鉑等化療藥物經(jīng)肝動(dòng)脈灌注治療并取得理想療效的報(bào)道[12-13]。此外，傳統(tǒng)中藥丹參具逆轉(zhuǎn)肝病纖維化、誘導(dǎo)肝癌細(xì)胞分化、促進(jìn)肝癌細(xì)胞凋亡等作用。

本研究采用丹參經(jīng)肝動(dòng)脈灌注療法來(lái)治療大鼠移植性肝癌。研究發(fā)現(xiàn)，丹參經(jīng)肝動(dòng)脈灌注對(duì)于大鼠移植性肝癌有明顯的治療作用，能夠顯著抑制移植性瘤體的增長(zhǎng)，其抑瘤活性與常用抗腫瘤藥物絲裂霉素的活性相當(dāng)。ALT主要存在于肝細(xì)胞漿內(nèi)，AST主要分布于肝細(xì)胞線粒體內(nèi)，ALP也主要分布在肝細(xì)胞內(nèi)[14]。正常情況下，血清中ALT、AST和ALP的水平很低，只有當(dāng)肝細(xì)胞受到損傷壞死時(shí)，ALT、AST和ALP才會(huì)釋放進(jìn)入血液中，導(dǎo)致其血清水平升高。因此檢測(cè)血清ALT、AST和ALP水平可以判斷肝功能[15]。本研究發(fā)現(xiàn)經(jīng)丹參灌注治療后，大鼠血清ALT，AST和ALP的水平均明顯降低，表明丹參在抗腫瘤的同時(shí)，對(duì)移植性肝癌大鼠具有良好的肝保護(hù)作用。

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(編校：譚玲,王儼儼)

Efficacy of salvia miltiorrhiza on transplanted hepatoma in rats via hepatic artery perfusion and its protection of liver function

WEI Ming-qin1,JIANG Jian-xia2

(1.Department of Oncology, Xiangyang Hospital, Affiliated Hospital of Hubei University of Medicine, Xiangyang 441000, China; 2.Nanjing Medical University, Nanjing 210000, China)

ObjectiveTo observe the therapeutic effect of salvia miltiorrhiza on transplanted hepatoma in rats via hepatic artery perfusion therapy and research the liver protection of salvia miltiorrhiza.Methods150 Sprague-Dawley rats were selected to build Walker-256 transplanted hepatoma model with successful modeling of 120 rats.All rats were randomly divided into control group,mitomycin group,salvia miltiorrhiza group,and 40 rats in each group.After 10 days normal feeding,bloods of all rats were collected to detect levels of ALT,AST and ALP.Tumor volumes were measured at the same time.Hepatic arterial infusion therapy: 2 mL saline was injected into control group,mitomycin (2.5 mg/kg) was injected into mitomycin group and salvia miltiorrhiza (1.6 g/kg) was injected into salvia miltiorrhiza group.After a week,bloods were collected to detect levels of ALT,AST,ALP and tumor volumes were measured.The tumor growth rate and inhibition rate were calculated.At the same time,all rats were killed and tumor were removed.The scope of tumor necrosis and degree of tumor necrosis was observed and compared.ResultsThere were 37 rats left in control group,37 rats left in mitomycin group and 38 rats left in salvia miltiorrhiza group after perfusion.The tumor volume and growth rate of mitomycin group and salvia miltiorrhiza group were significantly smaller than those of control group(P<0.05),that had no significant difference between mitomycin group and salvia miltiorrhiza group.The tumor inhibition rate of mitomycin group and salvia miltiorrhiza group were 22.32% and 26.38%,and there was no statistical difference.After perfusion,the moderate degrees of tumor necrosis in mitomycin group and salvia miltiorrhiza group were higher than that in control group,and there were no significant difference in mitomycin group and salvia miltiorrhiza group in mild,moderate,and severe degrees of tumor necrosis.The levels of ALT,AST and ALP of mitomycin group and salvia miltiorrhiza group were lower than those of control group (P<0.01,P<0.05); the above indexes were significantly in salvia miltiorrhiza group lower than those in mitomycin group(P<0.05). ConclusionSalvia miltiorrhiza could inhibit transplanted hepatoma in rats via hepatic artery perfusion therapy and protect the liver function.

salvia miltiorrhiza; hepatic artery perfusion; transplanted hepatoma; protection of liver function

國(guó)家自然科學(xué)基金(81101800)

魏明琴，女，本科，主治醫(yī)師，研究方向：肺癌，E-mail：qch1821460051@163.com。

R969

A

1005-1678(2015)02-0042-03