基质金属蛋白酶-26在人脑胶质瘤血管新生中的作用

Effect of matrix metalloproteinase-26 on human glioma angiogenesis

目的探讨MMP-26在人脑胶质瘤血管新生中的作用及可能的作用机制。方法用MMP-26质粒和pc-DNA3.1空质粒稳定转染人胶质瘤细胞株U251,构建裸鼠异种移植瘤模型,进而构建体外基于人肿瘤组织块的三维血管生成模型,观察MMP-26转染组(U251-MMP-26)、空载组(U251-pc DNA3.1)和未转染组(U251)的新生血管,统计有血管生成孔所占的百分比(I%),以及根据新生血管的长度和密度用半定量法对新生血管进行评分(血管生成分数AI,0~16分);采用RT-PCR法和免疫组化法测定U251-MMP-26、U251-pc DNA3.1和U251中MMP-26和VEGF mRNA和蛋白的表达;免疫组化法测定CD31在纤维蛋白-凝血酶胶基质血管内皮细胞中的定位。结果免疫组化检测内皮细胞标志物CD31为阳性,证明侵入纤维蛋白原-凝血酶胶基质中的成分为内皮细胞来源;U251-MMP-26组血管长、密度大,所占面积大,而U251-pc DNA3.1组和U251组血管短,密度小,所占面积小;U251-MMP-26组在第14天的I%和AI高于U251-pc DNA3.1组和U251组(P<0.05);U251-MMP-26组在14天中I%和AI的发展趋势较U251-pc DNA3.1组和U251组明显;MMP-26的mRNA和蛋白在U251-MMP-26中高表达,VEGF的mRNA和蛋白在U251-MMP-26中的表达水平明显强于U251-pc DNA3.1组和U251组(P<0.01)。结论 MMP-26可能通过增强VEGF的表达促进人脑胶质瘤的血管新生,可作为抗肿瘤治疗的靶点。

Purpose To investigate the effect of MMP-26 on human glioma angiogenesis and the possible mechanism. Methods The MMP-26 plasmid and empty plasmid pcDNA3.1 were stably transfected into U251 cells to establish a nude mice xenograft model, and then an in vitro human tumor tissue-based three-dimensional angiogenic model. Tissue disks were visually assessed over time to determine the percentage of wells that developed an angiogenic response（ I% ） and the density and length of neovessel growth were graded at intervals using a semiquanti- tative visual growth-rating scheme （ angiogenic index, AI, 0-16 scale） in groups of MMP-26 transfected U251 cells （U251- MMP-26）, pcDNA3.1 vector-transfected U251 cells （U251- pcDNA3.1 ） and non-transfected U251 cells （ U251 ）. RT-PCR and immunohistochemistry were used to detect the expression of mRNA and protein of MMP-26 and VEGF in groups of U251- MMP-26, U251-pcDNA3. 1 and U251. Immunohistochemical localization of CD31 was determined in the endothelial tubes invading the fibrin-thrombin clot matrix. Results Immunohistochemical endothelial cell markers CD31 was positive in the vascular tubes invading the fibrin-thrombin clot matrix, confirming their endothelial origin. The angiogenesis results showed that difference of length of micro capillaries, density of branches, and the area occupied between U251-MMP-26 groups and con- trol groups were significant. The percentage of tumor implants that developed invasion （ I% ） and the angiogenic index AI in U251-MMP-26 group on day 14 were higher than those of U251- pcDNA3.1 group and U251 group （P 〈 0. 05 ）. The trends of I% and AI in 14 days were significant compared with those in control groups. The expression of mRNA and protein of MMP-26 and VEGF in U251-MMP-26 group was significantly higher in U251-MMP-26 group than those in U251-pcDNA3. 1 group and U251 group （ P 〈 0. 01 ）. Conclusion The effect of MMP-26 on promoting glioma angiogenesis may be related to the increased expression of VEGF, which can be used as targets for anti-tumor therapy.