抗血管內(nèi)皮生長因子抗體對肝癌的放射增敏作用

1、抗血管內(nèi)皮生長因子抗體對肝癌的放射增敏作用         07-08-22 10:29:00     編輯：studa20                   作者：鄭青平,陳龍華,石玉生 【關(guān)鍵詞】  肝腫瘤;放射療法;抗VEGF單抗;放射增敏Radiosensibility of h

2、uman hepatocellular carcinoma xenografts enhanced by antivascular endothelial growth factor monoclonal antibody【Abstract】 AIM: To investigate the inhibiting effects of combining antivascular endothelial growth factor (VEGF) monoclonal antibody (mAb) therapy with radiotherapy on the growth of hepatoc

3、ellular carcinoma xenografts in vivo. METHODS: Human hepatocellular carcinoma cell line, SMMC7721, was implanted sc in mice. The mice with xenografts were divided into 4 groups, radiation (RT) 20 Gy group, RT 20 Gy plus mAb group, RT 30 Gy plus mAb group and control group. AntiVEGF mAb was injected

4、ip on alternate days for a total of 6 injections at a dosage of 50 g/mouse. Single radiation doses were given 24 h after the 4th injection of mAb. Tumor diameters were measured and tumor volume was calculated. Mice were sacrificed 2 wk after the 6th injection and the intratumoral microvessel density

5、 was determined by counting the stained vessels with immunohistochemistry. RESULTS: Radiation significantly reduced the growth and the microvessel density of tumors. The inhibitory effects were more significant when radiation was combined with antiVEGF mAb, and RT 30 Gy plus mAb was more effective t

6、han RT 20 Gy plus mAb. For RT 20 Gy group, RT 20 Gy plus mAb group and RT 30 Gy plus mAb group, the inhibitory rates of tumor weight were 75.3%, 83.9% and 94.7%, respectively. CONCLUSION: AntiVEGF mAb enhances the radiosensibility of human hepatocellular carcinoma xenografts.The strategy is one of t

7、he approaches for hepatocellular carcinoma treatment.【Keywords】 hepatocellular carcinoma; radiotherapy; antiVEGF mAb; radiosensibility【摘要】 目的: 觀察抗血管內(nèi)皮生長因子抗體(VEGF mAb)與不同劑量放射聯(lián)合對肝癌裸鼠移植瘤生長的抑制作用. 方法: 將SMMC7721肝癌細(xì)胞種植于裸鼠皮下,成瘤動(dòng)物分為單純放射20 Gy組、放射20 Gy+抗體組、放射30 Gy+抗體組和對照組,腹腔給予抗VEGF mAb 50 g/只，隔日1次,共6次,放射劑量一次性給

8、予.測量腫瘤直徑并計(jì)算瘤體積,抗體給藥結(jié)束后2 wk處死動(dòng)物,免疫組化法測腫瘤微血管密度(MVD). 結(jié)果: 放射能抑制腫瘤生長,減少腫瘤MVD,放射與抗體結(jié)合對腫瘤生長的抑制作用更顯著，且放射30 Gy+抗體比放射20 Gy+抗體效果更好.單純放射20 Gy組、放射20 Gy+抗體組和放射30 Gy+抗體組的瘤質(zhì)量抑制率分別為75.3%,83.9%和94.7%,差異有統(tǒng)計(jì)學(xué)意義. 結(jié)論: 抗VEGF mAb對放射治療肝癌移植瘤有增敏作用,是肝癌綜合治療的有效途徑之一.【關(guān)鍵詞】 肝腫瘤;放射療法;抗VEGF單抗;放射增敏0引言放射治療腫瘤的目的是盡可能提高腫瘤的放射劑量以殺滅瘤細(xì)胞,但同時(shí)正

9、常組織和器官所受輻射量也相應(yīng)提高,增加了放射治療的副反應(yīng),因而限制了通過提高放射劑量來控制腫瘤的作用.肝癌是放射欠敏感腫瘤,而正常肝組織對放射線較敏感.單純放射治療肝癌的療效并不理想1.有報(bào)道2抗血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)抗體可以抑制肝癌裸鼠移植瘤生長,我們觀察抗VEGF mAb與不同放射劑量聯(lián)合對肝癌移植瘤生長的抑制作用,以了解兩者聯(lián)合應(yīng)用的機(jī)制與方法.1材料和方法1.1材料Balb/cnu/nu小鼠20只(南方醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心),56 wk齡,雄性,體質(zhì)量19.022.0 g, SPF層流柜分籠飼養(yǎng); SMMC7

10、721人肝癌細(xì)胞株（武漢大學(xué)中國典型培養(yǎng)物保藏中心）; Monoclonal antiVEGF antibody, produced in mouse, clone 26503.11（Sigma公司）;培養(yǎng)箱（37,50 mL/L CO2）,倒置顯微鏡(XDP1,上海光學(xué)儀器廠),Varian 23EX醫(yī)用直線加速器（美國）,RPMI1640細(xì)胞培養(yǎng)液（含100 mL/L胎牛血清,青霉素1×105 U/g,鏈霉素100 mg/L）,胰蛋白酶,免疫組化SABC法相關(guān)試劑（武漢博士德公司）.1.2方法1.2.1癌細(xì)胞培養(yǎng)SMMC7721人肝癌細(xì)胞株于25 mL培養(yǎng)瓶中繁殖傳代,細(xì)胞貼壁長

11、滿瓶壁后移入250 mL培養(yǎng)瓶繼續(xù)傳代繁殖,取指數(shù)生長期細(xì)胞胰酶消化,2000 r/min離心2 min,收集細(xì)胞用生理鹽水懸浮,顯微鏡下以細(xì)胞計(jì)數(shù)板計(jì)算細(xì)胞密度,生理鹽水定容細(xì)胞密度為2×1010/L.1.2.2癌細(xì)胞種植參考文獻(xiàn)3方法,裸鼠于飼養(yǎng)環(huán)境適應(yīng)1 d后種植癌細(xì)胞,取裸鼠右后肢大腿外側(cè)皮下為種植點(diǎn),1 mL注射器抽取0.2 mL細(xì)胞懸液（含4×106個(gè)細(xì)胞）注入種植點(diǎn)皮下.1.2.3動(dòng)物分組及腫瘤生長觀察腫瘤種植后1 wk, 可觀察到全部動(dòng)物有明顯皮下腫塊形成,第10日腫塊直徑約5 mm.將動(dòng)物隨機(jī)分為4組,即單純放射20 Gy組、放射20 Gy+抗體組、放射3

12、0 Gy+抗體組和對照組,每組5只.用游標(biāo)卡尺測量腫塊長徑（a）及橫徑（b）,隔日測量1次,腫瘤體積(V)=a×b2/2.1.2.4抗體給予及放射治療以0.2 m針式濾器過濾除菌的PBS液溶解抗VEGF mAb至100 mg/L,于腫瘤種植后10 d分組后腹腔注射抗VEGF mAb, 50 g/只,隔日1次,共6次.單純放射組及對照組同樣途徑給予等體積量PBS.第4次給予抗VEGF mAb后的第2日行放射治療,放療機(jī)房紫外線消毒,受照動(dòng)物四肢以細(xì)繩固定在小木板上,照射野大小3 cm×3 cm,源皮距100 cm,6 MeV電子線,劑量率4 Gy/min,單純放射組劑量20 Gy,放射+抗體組劑量分別為20 Gy和30 Gy,全部放射劑量一次性給予.1.2.5瘤體稱質(zhì)量抗體給藥結(jié)束后繼續(xù)觀察腫瘤生長2 wk,于腫瘤種植后34 d處死動(dòng)物,仔細(xì)分離腫塊周邊皮膚及非腫瘤組織,瘤體稱質(zhì)量,瘤質(zhì)量抑制率(%)=（1實(shí)驗(yàn)組平均瘤質(zhì)量/對照組平均瘤質(zhì)量）×100.1.2.6免疫組化微血管密度(MVD)測定將腫瘤組織切成3 mm厚片狀,40 g/L甲醛固定24 h,脫水石蠟包埋,切片厚5 m.免疫組化SABC法:一抗為多克隆兔抗鼠CD34抗