hAG（K1～3）联合肿瘤坏死因子相关凋亡诱导配体基因真核表达质粒对裸鼠胃癌皮下移植瘤的作用

Anti-tumor effect of eukaryotic expressing plasmid containing soluble tumor necrotic factor-related apoptosis inducing ligand combined with human angiostatin Kringle （1 -3 ） genes on human gastric cancer xenografts in nude mice

目的探讨hAG（K1～3）联合肿瘤坏死因子相关凋亡诱导配体（TRAIL）基因在体内对裸鼠胃癌皮下移植瘤的生长抑制作用。方法通过亚克隆法构建重组质粒pBud—hAG及pBud—hAG-TRAIL，将人胃癌细胞SGC-7901注射裸鼠皮下形成移植瘤，并将移植瘤小鼠随机分为实验组和对照组。实验组包括pBud—hAG组和pBud—hAG—TRAIL组，各5只；对照组包括空质粒（pBud）组和生理盐水组，各5只。然后分别将2种重组质粒分次多点注入实验组移植瘤体内，并与对照组比较，通过检测移植瘤的体积和肿瘤微血管密度（MVD）研究其对肿瘤生长抑制情况及对肿瘤血管密度的影响。结果pBud—hAG组和pBud—hAG-TRAIL组移植瘤的MVD分别是（4．6±1．2）个／高倍视野（HP）和（4．8±0．9）个/HP（P〉0．05），而pBud组和生理盐水组分别是（17．4±2．4）个／HP和（18．2±2．7）个／HP，实验组和对照组之间差异有统计学意义（P〈0．05）。pBud—hAG组和pBud-hAG-TRAIL组的sTRAIL和hAGmRNA及蛋白表达均为阳性，其移植瘤体积分别是（1．862±0．017）cm^3和（1．325±0．012）cm^3（P〈0．05），而pBud组和生理盐水组分别是（3．637±0．032）cm^3和（3．521±0．028）cm^3，实验组与对照组差异有统计学意义（P〈0．05）。结论hAG（KI-3）通过抑制血管内皮细胞的生长而抑制肿瘤血管的生成，而TRAIL则通过诱导肿瘤细胞凋亡，从而抑制肿瘤细胞生长，因此hAG（K1—3）联合TRAIL具有更强的抗肿瘤作用。

Objective To investigate the anti-tumor effect of eukaryotic expressing plasmid containing human angiostatin Kringle （ 1 - 3 ） [ hAG （ K1 - 3 ） ] combined with soluble tumor necrotic factorrelated apoptosis inducing ligand （sTRAIL） genes on human gastric cancer xenografts in nude mice. Methods Recombinant plasmids of pBud-hAG and pBud-hAG-TRAIL were constructed by subcloning technique. Twenty nude BALB/c mice were inoculated with human gastric cancer ceils of the line BGC-823 subcutaneously into the back. One week later after the appearance of implanted tumors the mice were randomly divided into 4 groups with pBud-hAG, pBud-hAG-TRAIL, pBud blank plasmid, and normal saline （NS） injected into the tumors respectively once the other day for 7 times. The size of tumor was observed. 7 days later the mice were killed with their tumors taken out. RT-PCR was used to detect the expression of hAG and sTRAIL. The microvessel density （MVD） of tumor was observed and recorded by detecting the protein of CD34 with immunohistiochemitry on the microscopy. Results The MVD of tumor in the pBud- hAG-TRAIL and pBud-hAG groups were （4. 8 ±0.9）/HP and （4. 6 ± 1.2）/HP respectively, significantly lower than those of the pBud and NS groups [ （ 17.4 ± 2.4 ）/HP and （ 18.2 ± 2.7 ）/HP respectively, all P 〈 0. 05 ] , but there was no significant difference between the pBud-hAG-TRAIL and pBud-hAG groups. mRNA expression and protein expression of sTRAIL and hAG were positive in the pBud-hAG-TRAIL and pBud-hAG groups. The tumor volumes of tumors of the pBud-hAG-TRAIL and pBud-hAG groups were （ 1. 325 ± 0. 012 ） cm^3 and （ 1. 862 ± 0. 017 ） cm^3 respectively, both significantly lower than those of the pBud and NS groups [ （3.637 ±0. 032） cm^3 and （3. 521 ±0. 028） ems respectively, all P 〈0. 05]. Conclusion Angiostatin inhibits tumor angiogenesis through inhibiting the growth of vascular endothelial cells, and TRAIL induces tumor cell apoptosis, hAG （ K1 - 3 ） combined with TRAIL can inhibit tumor growth more efficiently.