基于脂肪源性干细胞的alphastatin肽胶质瘤靶向载体的构建及其对血管生成的抑制作用

Construction of an adipose derived stem cell-based alphastatin peptide glioma targeting vector and its inhibitory effect on angiogenesis

目的 构建基于脂肪源性干细胞的alphastatin肽胶质瘤靶向载体并检测其体外抗血管生成作用。方法 构建alphastatin肽慢病毒表达载体并转染脂肪源性干细胞(adipose derived stem cell, ADSCs),构建基于ADSCs的alphastatin肽胶质瘤靶向载体,流式细胞仪检测alphastatin肽胶质瘤靶向载体干细胞表面标志物表达情况,Western blotting及ELISA法分别检测alphastatin肽胶质瘤靶向载体中及细胞培养上清液中alphastatin肽表达情况;细胞迁移实验检测alphastatin肽胶质瘤靶向载体对CD133+胶质瘤干细胞趋向性;体外管腔形成实验检测alphastatin肽胶质瘤靶向载体在体外对内皮细胞血管生成的影响。结果 经转染后alphastatin肽胶质瘤靶向载体表达干细胞表面标志物与普通脂肪源性干细胞相比无明显变化;Western blotting结果显示,alphastatin肽胶质瘤靶向载体能够成功表达alphastatin肽;ELISA结果显示,在alphastatin肽胶质瘤靶向载体培养上清液中可检出alphastatin肽分泌[FLAG质量浓度(54.65±5.63)mg/L];细胞迁移实验显示,alphastatin肽胶质瘤靶向载体与普通脂肪源性干细胞对CD133+胶质瘤干细胞趋向性差异无统计学意义[迁移细胞数(106.67±2.41)个/HPF vs.(109.47±2.55)个/HPF,P>0.05];管腔形成实验显示,alphastatin肽胶质瘤靶向载体在体外能抑制内皮细胞介导的血管生成[管腔计数(7.27±0.31)个/HPF vs.(19.40±1.71)个/HPF,P<0.01]。结论 在构建基于ADSCs的alphastatin肽胶质瘤靶向载体过程中,靶向载体干细胞生物学特性及肿瘤趋向性无明显改变,且能够稳定表达并分泌alphastatin肽并在体外对内皮细胞介导的血管生成具有抑制作用。

Objective To construct an adipose derived stem cell-based alphastatin peptide glioma targeting vector and detect its anti-angiogenesis effect in vitro. Methods The adipose derived stem cell-based alphastatin peptide glioma targeting vector(Al-ADSCs) was constructed by transfecting the alphastatin peptide lentivirus vector into adipose derived stem cells(ADSCs). The expressions of stem cell markers on the surface of targeted vector were detected by flow cytometry. The expression of alphastatin peptide in the targeted vector and in the cell culture supernatant of the targeted vector were detected by Western blotting and ELISA, respectively. Cell migration assay was used to detect the tendency of the targeted vector toward CD133+ glioma stem cells, and lumen formation assay was used to detect the effect of the targeted vector on endothelial cell angiogenesis in vitro. Results After transfection, the surface markers of stem cells expressed by targeted vector did not significantly change compared with ordinary adipose derived stem cells. Western blotting showed that the targeted vector could successfully express alphastatin peptide. ELISA showed that the alphastatin peptide was detected in the cell culture supernatant of targeted vector [FLAG concentration(54.65±5.63]mg/L]. Cell migration test showed no significant difference in the tendency of CD133+ glioma stem cells between the targeted vector and ordinary adipose derived stem cells [ADSCs group(109.47±2.55) cells/HPF, Al-ADSCs(106.67±2.41) cells/HPF, P>0.05]. Lumen formation experiment showed that the targeted vector could inhibit endothelial cell-mediated angiogenesis in vitro [Lumen count: Control group(13.33±0.76)/HPF, ADSCs group(19.40±1.71)/HPF, Al-ADSCs group(7.27±0.31)/HPF, P<0.01]. Conclusion In the process of constructing the adipose derived stem cell-based alphastatin peptide glioma targeting vector, the stem cell biological characteristics and tumor tendency of targeted vector have no significant changes. This targeted vector can stably express and secrete alphastatin peptide and inhibit endothelial cell-mediated angiogenesis in vitro.