乙酰肝素酶抑制剂对裸鼠人食管鳞癌移植瘤的抑制作用

背景与目的:研究乙酰肝素酶(heparanase,Hpa)2种抑制剂硫酸化磷酸甘露戊糖(phosphomannopentaosesulfate,PI-88)和硫代磷酸寡脱氧核苷酸(antisense oligodeoxynucleotide,ASODN)对食管癌裸鼠移植瘤的体内抑瘤作用。材料与方法:采用低分化食管鳞癌TE-13细胞接种裸鼠皮下构建食管癌皮下侵袭模型,随机分为4组,30mg/kg PI-88组、2mg/kg ASODN组、20mg/kg PI-88+1mg/kgASODN联合用药组和无菌盐水对照组。模型建立后,各组于肿瘤接种区按上述剂量注射受试物,每天注射1次,注射15d后处死裸鼠,观察肿瘤体积和肿瘤微血管密度(micro vessel density,MVD),采用RT-PCR和免疫组化染色检测移植瘤中Hpa的表达。结果:接种后第6d,所有裸鼠在接种部位均长出肿瘤。接种后第21d,ASODN组、PI-88组和联合用药组肿瘤体积较对照组明显缩小,差异有统计学意义(P<0.01),联合用药组体积小于ASODN组和PI-88组(P均<0.01),ASODN组与PI-88两组体积差异无统计意义(P>0.05)。抑瘤率:ASODN组为49.67%,PI-88组为51.44%,联合用药组为83.00%。ASODN组、PI-88组和联合用药组MVD较对照组明显降低(P<0.01)。PI-88组和联合用药组MVD小于ASODN组(P<0.01),但前两组间差异无统计意义(P>0.05)。Hpa基因表达:ASODN组0.45±0.12,PI-88组1.22±0.01,联合用药组0.52±0.05,对照组1.33±0.05;Hpa蛋白表达:ASODN组43.40±7.80,PI-88组114.40±14.47,联合用药组37.40±7.20,对照组121.20±11.90。ASODN和联合用药组Hpa表达较对照组明显降低(Hpa基因:P<0.01;Hpa蛋白:P<0.01),PI-88组和对照组Hpa基因和蛋白表达差异无统计意义(P>0.05),ASODN和联合用药组Hpa基因和蛋白表达差异无统计意义(P>0.05)。结论:单独应用ASODN或联合PI-88用药,均能抑制肿瘤生长,PI-88的作用可能主要在于抑制肿瘤血管新生。ASODN的作用可能主要在于抑制肿瘤Hpa基因和蛋白合成。两者联合用药可以产生协同作用。

Inhibitory Effect of Heparanase Inhibitor on Growth and Angiogenesis of Xenograft Esophageal Squamous Gell Carcinoma in Nude Mice

BACKGROUND AND AIM: Heparanase(Hpa) is a kind of endo-D-glucuronidase that degrades heparin sulfate proteoglycans.It played important roles in malignant tumor’s invasion and metastasis.Both phosphomannopentaosesulfate(PI-88) and antisense oligodeoxynucleotide(ASODN) are Hpa inhibitors.This study was designed to observe inhibitory effects of PI-88 and Hpa ASODN on growth and angiogenesis of esophageal squamous cell carcinoma(ESCC) xenograft tumor in nude mice.MATERIALS AND METHODS: ESCC cell line TE-13 was injected subcutaneously in nude mice to establish the ESCC subcutaneous invasive model.The nude mice were randomized into 30 mg/kg PI-88 group,2 mg/kg Hpa ASODN group,20 mg/kg PI-88 and 1 mg/kg ASODN combined group and sterile saline control group.Each group contained 5 mice.Tumor volume and micro vessel density(MVD) were evaluated.Hpa expression was detected by RT-PCR and immunohistochemistry.RESULTS: On the 6th day after injection,all mice had palpable tumors.On the 21st day after injection,the tumor volume was significantly smaller in ASODN group,PI-88 group and combined group than in control group(P<0.01).The tumor volume in the combined group were less than in PI-88 alone and ASODN alone groups(P<0.01),and there was no difference between the latter two groups(P>0.05).The raitio of suppression in each group was 49.67%(ASODN),51.44%(PI-88) and 83.00%(combined group).The MVD was significantly less in ASODN,PI-88 and combined group than in control group(P<0.01).The MVD in PI-88 and combined group were less than in ASODN group(P<0.01),and there was no difference between PI-88 and combined group(P>0.05).Hpa gene expression in ASODN group was 0.45±0.12,PI-88 group 1.22±0.01,combined group 0.52±0.05,control group 1.33±0.05.Hpa protein expression in ASODN group was 43.40±7.80,PI-88 group 114.40±14.47,combined group 37.40±7.20,control group 121.20±11.90.Hpa gene and protein in ASODN and combined groups was less than in control group(Hpa gene: P<0.01,Hpa protein: P<0.01).But the expression of PI-88 group showed no difference compared with control group(P>0.05),and similarly between the ASODN and combined groups(P>0.05).CONCLUSION: Administratoring ASODN and PI-88 alone or in combination could all reduce the tumor growth.PI-88 could reduce the growth of tumor and angiogenesis,but had no effects on Hpa expression in vivo.Hpa ASODN could effectively reduce the expression of heparanase to slow down the growth of xenograft and angiogenesis.Combining the two showed synergistic effect,resulting in a much better result.