低渗刺激下晶状体上皮细胞HLE B-3调节性容积回缩机制的研究

背景调节性容积回缩（RVD）现象在生物界各种细胞中普遍存在，它不仅与细胞容积的稳定性相关，还参与多种细胞的生理功能和病理过程，但迄今为止，关于晶状体上皮细胞（LECs）的RVD机制研究仍较少。目的观察低渗刺激下人LECs系HLE B-3细胞株RVD的过程，并探讨其可能的作用机制及影响因素。方法应用氢离子缓冲剂（HEPES）将林格液分别配成质量分数45％、35％、25％和15％的低渗液（实际渗透压分别为165、195、225和255mOsm／kg），用NaOH将pH值调至7．4或6．8。HLE B-3在含质量分数10％胎牛血清（FBS）的DMEM／F12培养基中进行体外培养，待细胞贴壁后稳定生长时，将HLEB一3细胞置于特制的培养板内，分别用不同质量分数的低渗林格液灌流培养，观察其对细胞RVD的影响。以45％低渗林格液刺激细胞作为对照组，通过Cl^-、K^＋通道阻断剂干预，分别用无HCO3^＋、无Ca^2＋、无Cl^-或高K^＋林格液灌流改变细胞外液离子成分或酸化溶液pH，在Image-Pro Plus6．0图像处理软件控制下实现时滞拍摄固定视野内的活细胞图像，分析细胞RVD的动态变化。结果在低渗刺激下细胞容积膨胀，随后即出现了RVD过程。45％低渗林格液刺激细胞产生RVD为（59．1±7．8）％，随着林格液渗透压的逐渐降低，细胞容积逐渐膨胀，细胞的RVD能力逐渐增强，细胞最大膨胀容积与RVD存在s形正相关（r=0．990，P〈0．05），当细胞最大膨胀容积在115％～135％时，RVD对其变化敏感。10mmol／L K^＋通道阻断剂四乙基氯化铵（TEA）、5mmol／LBaCl2、100μmol／L5-硝基-2（3-苯丙胺），苯甲酸（NPPB）和相同浓度的氯Cl^-通道阻断剂二异硫氰基芪-2，2^＋-二磺酸（DIDS）可分别使RVD减少（16．5±1．6）％、（14．7±2．28）％（P〈0．01）。当细胞外液为高K^＋及无Cl-的环境时，RVD分别减少（5．8±1．6）％和（2．7±0．8）％，差异有统计学意义（P〈0．01），而在无Ca^2＋条件下，RVD几乎消失。细胞外液为pH6．8时，仅能暂时延缓细胞的RVD过程，而非明显的抑制，尤其是在容积回缩的前10min内，pH6．8的45％低渗林格液刺激时细胞的RVD速率为（0．86±0．24）％／min，明显低于pH7．4的45％低渗林格液刺激时的RVD速率（3．24±0．84）％／min，两组间差异有统计学意义（P〈0．05）。结论低渗刺激下人LECs可产生RVD，细胞内一定量的ca“存在是RVD激活的前提，而K^＋和Cl^-是HLE B-3 RVD过程的关键性离子，Cl^-、K^＋离子通道则是与细胞容积变化相关性Cl^-、K^＋外流的一个重要途径，同时低渗液的酸化也可以延缓RvD的过程。

Background It is widely appreciated that many animal cells rely on the mechanism of regulatory volume decrease (RVD) after swell under the hypotonic environment,which involved in some processes of cellular physiology.But the RVD of lens epithelial cells(LECs) still is being further researched.Objective Present study was to clarify the possible mechanisms and influencing factors in the RVD of LECs.Methods Human LECs line (HLE B-3)were cultured and passaged in DMEM/F12 containing 10％ fetal bovine serum(FBS),and before volume measurement,cells were stuck to the base of a perfusion chamber,Ringer solution osmolality was decreased from 15％Hypo to 45％ Hypo,and the cells stimulated by 45％ Hypo Ringer solution were used as the control group.Some experiments were performed in the presence of high extracellular K+ concentration,chloride or potassium channel inhibitor,experiments were also carried out in the nominal absence of Ca2+,Cl-or HCO-3 to test the effect of a decrease in intracellular concentration of these ions on the cell volume response.The volume changes of living cells were measured by lag-time microphotograph acquisition and analysis system (IPP6.0).Results Time course of cell volume change after hypotonic shock in HLE B-3 cells was observed.The cell swelling was followed by a gradual volume recovery,indicating the presence of RVD was influenced by the hypotonic stress.Under the stimulation of 45 ％Hypo Ringer solution,the rates of RVD were (59.1 ±7.8)％.RVD was correlated positively to the maximum cell volume (r =0.99,P＜0.05)in S shape,and RVD changes were sensitive to alter maximum cell volume in the range of 115％-135％.RVD reduced to (16.5 ± 1.6) ％,(14.7 ± 2.3) ％,respectively after acted by potassium channel inhibitor,TEA(10 mmol/L)and BaCl2(5 mmol/L)as well as chloride channel inhibitor,NPPB(100 μmol/L)and DIDS(100 μmol/L),with significant differences in comparison with the control group(all P＜0.01).RVD decreased by(5.8±1.6)％ and(2.7±0.8)％ in high concentration of K+ in extracellular fluid and the absence of Cl-(P＜0.01).RVD was significantly inhibited under the absence of Ca2+.When the 45％ Ringer solution was pH6.8,the process of RVD delayed.The rate of RVD in the first ten minutes was (0.86±0.24)％/min,showing a significant decline in comparison with (3.24±0.84) ％ / min of pH 7.4 (P ＜0.05).Conclusions HLE B-3 have RVD ability under the hypotonic stress stimulation.A certain intracellular Ca2+ concentration is the premise of RVD activation,and Cl efflux and K+ efflux are the key mechanism of RVD of HLE B-3.Acidic environment of hypotonic solution delays the occurrence of RVD.