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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 influence of cell membrane pore size on the permeability of biological macromolecules and to solve the key technical problems in molecular biology experiments of vascular cells. Methods Two types of cell co-culture cups with 0.4 μm and 1.0 μm pore size were used as the research objects. The vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) of rats were implanted On the inner and outer sides of the co-cultured cup bottom PET film. The experiment was divided into EC / VSMC co-culture application of low shear stress group, PET uninoculated cells quiescent group, PET film unilateral seeding cell quiescent group and PET membrane bilateral seeding cell quiescent group. The contents of platelet-derived growth factor (BB) and platelet-derived growth factor (BB) in ECs and VSMCs of low shear stress group were detected by ELISA. After stimulated by recombinant PDGF-BB (rPDGF-BB) The changes of intracellular signaling molecules such as p-ERK1 / 2 and p-Akt and nuclear matrix protein Lamin A were observed. Results PDGF-BB concentration in VSMCs in 0.4μm co-culture vessel was significantly higher than that in ECs treated with 0.5 Pa laminar flow low shear stress for 12 h. RPDGF-BB upregulated the expression of p-ERK1 / 2 and p-Akt, and down-regulated the expression of Lamin A, in cultured cells after 0.4μm and 1.0μm PET membranes were not inoculated. RpDGF-BB up-regulated the expression of p-ERK1 / 2 and p-Akt in contralateral cells and down-regulated the expression of Lamin A when monolayers were seeded on the outside of PET film. RPDGF-BB could only affect the expression of p-ERK1 / 2, p-Akt and Lamin A in the ipsilateral cells of 0.4μm PET film, but had no effect on the contralateral cells; while the 1.0μm PET film There was no significant difference in the expression of p-ERK1 / 2, p-Akt and Lamin A on both sides of the cells. Conclusion Both porous PET materials themselves allow the permeation of biological macromolecules, while cell seeding can affect the penetration of porous PET films into biological macromolecules. 0.4μm pore PET membrane on both sides of the cell inoculation, the permeability of biological macromolecules significantly weakened, closer to in vivo conditions.