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目的:探讨活血益气方及其拆方含药血清对含有血管内皮生长因子(VEGF)165的重组质粒pcDNA3.1-VEGF165转染人脐静脉内皮细胞(HUVEC)分泌基质金属蛋白酶(MMP)2,9的影响。方法:构建pcDNA3.1-VEGF165重组质粒,采用Fugene HD将质粒瞬时转染至HUVEC中。制备活血益气方及其拆方含药大鼠血清、生理盐水正常血清,以5%药物血清作用于转染后的HUVEC,Western blot法检测VEGF的表达,ELISA法测定MMP-2,MMP-9的表达。结果:(1)活血益气方组VEGF蛋白表达高于生理盐水组,与之比较有统计学差异(P<0.01)。拆方各组则低于活血益气方组,与之比较有统计学差异(P<0.01)。(2)活血益气方组、活血方组MMP-2含量高于生理盐水组,与之比较有统计学差异(P<0.05,P<0.01)。拆方各组中仅益气方组MMP-2含量低于活血益气方组,与之比较有统计学差异(P<0.01)。(3)活血益气方及其拆方各组MMP-9含量均高于生理盐水组,仅活血益气方组、活血方组与之比较有统计学差异(P<0.01,P<0.05)。拆方各组MMP-9含量均低于活血益气方组,与之比较具有统计学差异(P<0.01)。结论:活血益气方可以促进转染后HUVEC表达VEGF及分泌MMP-2,MMP-9,活血方药在这方面起主要作用。推测其治疗性血管生成作用机制可能与促进内皮细胞分泌MMP,从而促进内皮细胞迁移,提高血管通透性有关。
OBJECTIVE: To investigate the effect of Huoxueyiqi and its disassembled drug-containing serum on the secretion of matrix metalloproteinase (MMP-2) from human umbilical vein endothelial cells (HUVECs) transfected with the recombinant plasmid pcDNA3.1-VEGF165 containing vascular endothelial growth factor (VEGF) , 9 of the impact. Methods: The pcDNA3.1-VEGF165 recombinant plasmid was constructed and transiently transfected into HUVEC by Fugene HD. The serum and normal saline normal serum of Huoxue Yiqi prescription and its disassembled prescription were prepared and the expression of VEGF was detected by Western blot with 5% serum of the transfected HUVEC. The expressions of MMP-2 and MMP- 9 expression. Results: (1) The expression of VEGF protein in Huoxue Yiqi prescription group was higher than that in normal saline group (P <0.01). Demolition of each group was lower than Huoxue Yiqi group, compared with a statistically significant difference (P <0.01). (2) The content of MMP-2 in Huoxue Yiqi Prescription and Huoxue Prescription was higher than that of saline group (P <0.05, P <0.01). In each group, the MMP-2 level of Yiqi Recipe group was lower than that of Huoxue Yiqi Recipe group (P <0.01). (3) The content of MMP-9 in Huoxue Yiqi Prescription and its disassembled groups were all higher than those in normal saline group, and there was significant difference between Huoxue Yiqi Prescription and Huoxue Prescription only (P <0.01, P <0.05) . The content of MMP-9 in each group was significantly lower than that in Huoxue Yiqi group (P <0.01). Conclusion: Huoxue Yiqi Prescription can promote the expression of VEGF and the secretion of MMP-2, MMP-9 and Huoxue Recipe in HUVEC after transfection play a major role in this aspect. It is speculated that the mechanism of therapeutic angiogenesis may be related to the promotion of endothelial cell secretion of MMP, thereby promoting endothelial cell migration and increasing vascular permeability.