大鼠肺微血管内皮细胞通透系数检测的方法

摘要

Objective To explore an optimal method for the measurement of pulmonary microvascular endothelial cell (PMVEC) permeability coefficient. Methods A monolayer of rat PMVEC model was constructed by culturing a cell suspension on transwell filter or polycarbonate filter membrane. After the state of confluence of cells was affirmed with epithelial volt-ohmmeter or inverted microscope, the permeability coefficient was determined by means of transendothelial electrical resistance (TER), fluorescein isothiocyanate-dextran (Pd), and permeation of Hanks solution (Lp) across monolayers. Meanwhile,changes in PMVEC permeability expressed by the ratio of the observed value and the original value were observed after lipopolysaccharide (LPS) challenge for 0. 5 hour or 2 hours. Results The cells reached the state of confluence as observed under inverted microscope on the third day post-seeding, and the TER and Pd at this time-point were [(39.45 ± 3.96) Ω · cm2] and [(8. 52 ± 0. 50) × 10-6cm/s], respectively. After PMVEC were seeded on transwell filters, the TER increased steadily in a time-dependent manner after seeding of PMVEC, reaching the summit at the fourth day post-seeding [(49.84 ± 3. 93)Ω· cm2].Under the natural state, the TER, Pd and Lp of confluent PMVEC monolayers were (49. 84±3.93) Ω · cm2, (6.15 ± 0. 63) × 10-6 cm/s and (6. 80± 0. 62) × 10- 7 cm · s-1 · cm H2O-1, respectively.After PMVEC monolayers were challenged with 10 mg/L LPS for both 0. 5 hour and 2 hours, there was significant decrease in the permeability coefficient as measured by TER (0.87±0. 03, 0. 45±0. 04 vs. 1.00±0. 08, respectively, both P＜ 0. 05), and an increase in the permeability coefficient measured by Pd (1.33±0. 11, 2.43 ± 0. 14 vs. 1.00 ± 0. 10, respectively, both P＜ 0. 05) and the permeability coefficient measured by Lp (1.30± 0.07, 2. 38 ± 0. 15 vs. 1.00 ± 0. 11, respectively, both P ＜ 0. 05) when compared with the normal group. Conclusion Three methods, namely TER, Pd and Lp are available to use to assess PMVEC permeability coefficient. The combination of an inverted microscope, TER and Pd enhances the accuracy in determining PMVEC permeability coefficient, and it provides an experimental technique for studying the pathogenesis of acute lung injury in vitro.%目的 探讨肺微血管内皮细胞(PMVEC)通透系数检测的实验方法。方法 分别在trans well 小室和聚碳酸酯纤维膜上构建大鼠PMVEC单层模型，用电阻抗仪和倒置显微镜观察到细胞单层汇合后，分别应用电阻抗仪监测跨内皮细胞电阻(TER)，用异硫氰酸荧光素(FITC)-葡聚糖法检测通透系数(Pd)，用Hanks平衡液法检测通透系数（Lp）;同时观察脂多糖(LPS)刺激0.5h和2h后的PMVEC通透性变化，以测量值与其相应静息状态值的比值表示。结果 倒置显微镜下观察到细胞接种后3d时已汇合成单层时TER值为(39.45±3.96)Ω．cm2，Pd值为(8.52±0.50)×10-6 cm/s；随细胞接种时间增加，TER值稳定升高，接种后4d达高峰[(49.84±3.93)Ω．cm2]。静息状态下汇合成PMVEC单层的TER值、Pd值和LP值分别为(49.84±3.93) Ω·cm2、(6.15±0.63)×10-6 cm/s和(6.80±0.62)×10-7 cm．s-l．cm H2O-l。与未刺激组比较，10 mg/L LPS刺激PMVEC 0．5 h和2h后，TER法测定的通透性均下降（0.87±0.03、0.45±0.04比1．00±0.08），Pd法和Lp法测定的通透性均增加(Pd:1.33±0.11、2.43±0.14比1．00±0.10;Lp:1.30±0.07、2.38±0.15比1．00±0.11)，差异均有统计学意义（均P＜0.05）。结论 TER法、Pd法和Lp法均能有效检测PMVEC通透系数，倒置显微镜联合TER和Pd法所测值更加精确，从而为体外研究急性肺损伤发病机制提供实验方法。