人参皂苷免疫纳米对人胃癌裸鼠移植瘤生长抑制机制探讨

Effect of Ginsenoside Rg3 immune-nanoemulsion on growth and lymphangiogenesis of human gastric cancer cell transplantation tumor in nude mice

目的研究人参皂苷免疫纳米(VEGFR3-mediated immune-nanoemulsion of ginsenoside Rg3,VRIN)对胃癌生长及其淋巴管生成的机制。方法通过外科原位移植能表达红色荧光蛋白人胃癌NUGC-4细胞的方法建立裸鼠胃癌模型,将32只Balb/c无胸腺裸鼠随机分为生理盐水组、5-氟尿嘧啶(5-FU)组(20mg/kg)、VRIN高剂量组(1mg/kg)和VRIN低剂量组(0.1mg/kg)4组,每组8只。实验终点处死所有受试裸鼠,在开放荧光系统下观察肿瘤生长及转移情况,切除移植肿瘤测瘤质量和瘤体积。免疫组织化学法检测肿瘤组织中微淋巴管密度(lymphatic microvessel density,LMVD),Real time-PCR和免疫组织化学法检测VEGF-C蛋白和mRNA表达。结果实验终点开放体内荧光成像显示,胃癌NUGC-4细胞肿瘤生长抑制率VRIN高剂量为72.9%,低剂量组为14.5%,5-FU组为69.2%,VRIN高剂量组和5-FU组较生理盐水组瘤质量明显减轻,P<0.001。生理盐水组淋巴转移率为87.5%(7/8),5-FU组为50.0%(4/8),VRIN高剂量组为12.5%(1/8),低剂量组为37.5%(3/8),VRIN高剂量组与生理盐水组差异有统计学意义,P=0.01。LMVD计数结果显示,生理盐水组为9.29±2.06,5-FU组为6.42±1.38,VRIN高剂量组为4.25±1.08,差异有统计学意义,F=20.895,P<0.001;VRIN高剂量组VEGF-C蛋白表达量为0.190±0.059,与生理盐水组(0.269±0.051)差异有统计学意义,P=0.014;VEGF-C mRNA表达结果显示,生理盐水组为1.05±0.19,5-FU组为0.62±0.25,VRIN高剂量组为0.49±0.19,差异有统计学意义,F=8.190,P=0.002。结论 VRIN可抑制人胃癌细胞祼鼠移植瘤生长及淋巴结转移,并通过下调VEGF-C表达抑制肿瘤淋巴管生成。

OBJECTIVE . To investigate whether Ginsenoside Rg3 immune-nanoemulsion （VRIN） can inhibit growth and lymphangiogenesis of gastric cancer in vivo. METHODS A gastric cancer model was developed by surgical orthotopic implantation of red fluorescent protein （RFP）-expressing NUGC-4 tumor in nude mice. Thirty-two Balb/c nude mice were randomly divided into 4 groups：saline treated group, 5-FU treated group （20 mg/kg）, high dose（1 mg/kg） VRIN treated group and low dose（0.1 mg/kg） VRIN treated group. At the end of the experiment, all mice were sacrificed and open fluorescent images of tumors and metastasis expressing RFP were acquired, and the tumor was removed from each mouse. The size of transplantation tumor and tumor weight were measured; gene and protein expressions of VEGF-C in tumor tissues were detected by Real time-PCR and immunohistochemistry; lymphatic microvessel density （LMVD） in tumors was measured with immunohistochemistry. RESULTS At the end of the experiment, open RFP imaging demonstrated that tumor weight decreased by 72.9%, 14. 5% and 69.2% in high dosage VRIN treated group （1 mg/kg）, low dosage VRIN treated group （0.1 mg/kg） and 5-FU treated group （20 mg/kg）. Compared to saline treated group, tumor weight decreased significantly in high dosage VRIN treated group and 5-FU treated group （both P values〈0. 001）. The metastasis ratio of lymph nodes in saline treated group, 5-FU treated group, high dosage VRIN treated group （1 mg/kg） and low dosage VRIN treated group （0.1 mg/kg） were 87.5%（7/8）, 50.0%（4/8）, 12.5%（1/8） and 37.5%（3/8） respectively. Compared with saline treated group, lymph node metastasis was significantly inhibited （P=0.01） in high dosage （1 mg/kg） VRIN treated group. LMVD in tumor tissues treated with saline treated group, 5-FU treated group, high dose VRIN treated group （1 mg/kg） were 9.29±2.06,6.42±1.38,4.25±1.08,respectively. LMVD in tumor tissues treated with high dose VRIN （1 mg/kg） and 5-FU （20 mg/kg） were both statistically significantly lower than that in the saline treated group（F= 20. 895,P〈0. 001）. In regard to tumor VEGF-C expressions in saline groups（0. 269±0. 051） and VRIN at the dose of 1 mg/kg （0. 190±0. 059）, the difference between VRIN treated group and saline treated group was statistically significant （P= 0. 014）. VEGF-C mRNA expressions in saline treated group, 5-FU （at 20 mg/kg） treated group and VRIN （at the dose of 1 mg/kg） treated group were 1.05±0.19, 0. 62±0. 25 and 0.49±0.19 respectively. Compared with the group treated with saline, the mRNA expression of VEGF-C were significantly lower in the group treated with VRIN at the close of 1 mg/kg and in 5-FU treated group （F=8. 190, P=0. 002）. CONCLUSIONS VRIN can inhibit the growth and lymphatic metastasis of human gastric cancer cell transplantation tumor in nude mice. It inhibits tumor lymphangiogenesis through down-regulating VEGF-C expressions.