细胞联合培养杯膜的孔径对血小板源性生长因子通透的影响

Effect from different pore sizes of co-culture inserts on the permeability of platelet-derived growth factor

目的探讨细胞联合培养杯膜的孔径对生物大分子通透的影响,解决血管细胞分子力学生物学实验的关键技术问题。方法以杯底PET膜孔径为0.4μm和1.0μm的两种型号细胞联合培养杯作为研究对象,将大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)和内皮细胞(endothelial cells,ECs)分别种植于联合培养杯底PET膜的内外侧面。实验分为EC/VSMC联合培养施加低切应力组、PET膜未接种细胞静止组、PET膜单侧接种细胞静止组和PET膜双侧接种细胞静止组。ELISA检测低切应力组ECs侧和VSMCs侧培养液中血小板源性生长因子(platelet-derived growth factor BB,PDGF-BB)含量,Western blotting检测重组PDGF-BB(rPDGF-BB)刺激后,各组细胞内信号转导分子p-ERK1/2和p-Akt以及核骨架蛋白Lamin A的表达变化。结果 EC/VSMC联合培养施加0.5 Pa层流低切应力12 h后,0.4μm联合培养杯VSMCs侧PDGF-BB浓度显著高于ECs侧。0.4μm和1.0μm两种孔径的PET膜未接种细胞时,rPDGF-BB上调了隔开培养细胞的p-ERK1/2和p-Akt表达,并下调Lamin A表达。PET膜外侧面接种单层细胞时,rPDGF-BB上调了对侧细胞的p-ERK1/2和p-Akt表达,并下调Lamin A表达。PET膜内外侧面均接种细胞时,rPDGF-BB仅能影响0.4μm PET膜同侧细胞的p-ERK1/2、p-Akt和Lamin A表达,对对侧细胞无明显作用;而1.0μm PET膜两侧细胞的p-ERK1/2、p-Akt和Lamin A表达无显著性差异。结论两种有孔PET材料本身均允许生物大分子的通透,而细胞接种会影响有孔PET膜对生物大分子的通透。0.4μm孔径PET膜两侧均接种细胞时,对生物大分子的通透明显减弱,更接近于在体情况。

Objective To investigate the effect from different pore sizes of co-culture inserts on the permeability of biomacromolecules through polyethylene terephthalate（PET） membrane so as to solve the key technology problem in mechanobiology experiment on vascular cells.Methods Inserts with 0.4 μm and 1.0 μm pores on the PET membrane were studied using flow chamber system.Low shear stress was subjected to the co-cultured system of endothelial cell（EC）/vascular smooth muscle cell（VSMC） and the concentration of platelet-derived growth factor BB（PDGF-BB） was detected by ELISA.Under the static condition,vascular cells were cultured on the plate（with no cell on PET membrane）,on the outer side of PET membrane,and on the both sides of PET membrane,respectively.Then the recombinants PDGF-BB（rPDGF-BB） were added on the different sides of PET membrane.Western blotting was used to detect the change in expressions of p-ERK1/2,p-Akt and Lamin after cells were stimulated by rPGDF-BB.Results After low shear stress subjection for 12 h,the concentration of PDGF-BB in the medium from VSMC-side was significantly higher than that from EC-side.rPDGF-BB passed through 0.4 μm and 1.0 μm pores on the PET membrane and modulated expressions of p-ERK1/2,p-Akt and Lamin A in cells cultured on the opposite side of PET membrane and cells cultured on the plate separately.When cells were cultured on the both sides of PET membrane,rPDGF-BB only stimulated cells cultured on the same side of 0.4 μm pores on PET membrane,but had no specific effect on cells cultured on the opposite side.Conclusions PET membrane with both 0.4 μm and 1.0 μm pores was permeable to PDGF-BB,and cells cultured on the membrane could affect the permeability.The efficiency of PDGF-BB passing through 0.4 μm pores was significantly repressed with cells cultured on the both sides,which was more similar to that in vivo.