中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
4期
316-321
,共6页
晶状体上皮细胞%调节性容积回缩%低渗刺激%细胞离子通道
晶狀體上皮細胞%調節性容積迴縮%低滲刺激%細胞離子通道
정상체상피세포%조절성용적회축%저삼자격%세포리자통도
Lens epithelial cells%Regulatory volume decrease%Hypotonic stress stimulation%Cellular ion channel
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255 mOsm/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HLE B-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Ca2+、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 Plus 6.0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.10 mmol/L K+通道阻断剂四乙基氯化铵(TEA)、5 mmol/L BaCl2、100 μmol/L 5-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Ca2+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10 min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Ca2+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.
揹景 調節性容積迴縮(RVD)現象在生物界各種細胞中普遍存在,它不僅與細胞容積的穩定性相關,還參與多種細胞的生理功能和病理過程,但迄今為止,關于晶狀體上皮細胞(LECs)的RVD機製研究仍較少. 目的 觀察低滲刺激下人LECs繫HLE B-3細胞株RVD的過程,併探討其可能的作用機製及影響因素. 方法 應用氫離子緩遲劑(HEPES)將林格液分彆配成質量分數45％、35％、25％和15％的低滲液(實際滲透壓分彆為165、195、225和255 mOsm/kg),用NaOH將pH值調至7.4或6.8.HLE B-3在含質量分數10％胎牛血清(FBS)的DMEM/F12培養基中進行體外培養,待細胞貼壁後穩定生長時,將HLE B-3細胞置于特製的培養闆內,分彆用不同質量分數的低滲林格液灌流培養,觀察其對細胞RVD的影響.以45％低滲林格液刺激細胞作為對照組,通過Cl-、K+通道阻斷劑榦預,分彆用無HCO3、無Ca2+、無Cl-或高K+林格液灌流改變細胞外液離子成分或痠化溶液pH,在Image-Pro Plus 6.0圖像處理軟件控製下實現時滯拍攝固定視野內的活細胞圖像,分析細胞RVD的動態變化.結果 在低滲刺激下細胞容積膨脹,隨後即齣現瞭RVD過程.45％低滲林格液刺激細胞產生RVD為(59.1±7.8)％,隨著林格液滲透壓的逐漸降低,細胞容積逐漸膨脹,細胞的RVD能力逐漸增彊,細胞最大膨脹容積與RVD存在S形正相關(r=0.990,P＜0.05),噹細胞最大膨脹容積在115％ ～ 135％時,RVD對其變化敏感.10 mmol/L K+通道阻斷劑四乙基氯化銨(TEA)、5 mmol/L BaCl2、100 μmol/L 5-硝基-2(3-苯丙胺),苯甲痠(NPPB)和相同濃度的氯Cl-通道阻斷劑二異硫氰基芪-2,2’-二磺痠(DIDS)可分彆使RVD減少(16.5±1.6)％、(14.7±2.28)％(P＜0.01).噹細胞外液為高K+及無Cl-的環境時,RVD分彆減少(5.8±1.6)％和(2.7±0.8)％,差異有統計學意義(P＜0.01),而在無Ca2+條件下,RVD幾乎消失.細胞外液為pH6.8時,僅能暫時延緩細胞的RVD過程,而非明顯的抑製,尤其是在容積迴縮的前10 min內,pH6.8的45％低滲林格液刺激時細胞的RVD速率為(0.86±0.24)％/min,明顯低于pH7.4的45％低滲林格液刺激時的RVD速率(3.24±0.84) ％/min,兩組間差異有統計學意義(P＜0.05). 結論 低滲刺激下人LECs可產生RVD,細胞內一定量的Ca2+存在是RVD激活的前提,而K+和Cl-是HLE B-3 RVD過程的關鍵性離子,Cl、K+離子通道則是與細胞容積變化相關性Cl-、K+外流的一箇重要途徑,同時低滲液的痠化也可以延緩RVD的過程.
배경 조절성용적회축(RVD)현상재생물계각충세포중보편존재,타불부여세포용적적은정성상관,환삼여다충세포적생리공능화병리과정,단흘금위지,관우정상체상피세포(LECs)적RVD궤제연구잉교소. 목적 관찰저삼자격하인LECs계HLE B-3세포주RVD적과정,병탐토기가능적작용궤제급영향인소. 방법 응용경리자완충제(HEPES)장림격액분별배성질량분수45％、35％、25％화15％적저삼액(실제삼투압분별위165、195、225화255 mOsm/kg),용NaOH장pH치조지7.4혹6.8.HLE B-3재함질량분수10％태우혈청(FBS)적DMEM/F12배양기중진행체외배양,대세포첩벽후은정생장시,장HLE B-3세포치우특제적배양판내,분별용불동질량분수적저삼림격액관류배양,관찰기대세포RVD적영향.이45％저삼림격액자격세포작위대조조,통과Cl-、K+통도조단제간예,분별용무HCO3、무Ca2+、무Cl-혹고K+림격액관류개변세포외액리자성분혹산화용액pH,재Image-Pro Plus 6.0도상처리연건공제하실현시체박섭고정시야내적활세포도상,분석세포RVD적동태변화.결과 재저삼자격하세포용적팽창,수후즉출현료RVD과정.45％저삼림격액자격세포산생RVD위(59.1±7.8)％,수착림격액삼투압적축점강저,세포용적축점팽창,세포적RVD능력축점증강,세포최대팽창용적여RVD존재S형정상관(r=0.990,P＜0.05),당세포최대팽창용적재115％ ～ 135％시,RVD대기변화민감.10 mmol/L K+통도조단제사을기록화안(TEA)、5 mmol/L BaCl2、100 μmol/L 5-초기-2(3-분병알),분갑산(NPPB)화상동농도적록Cl-통도조단제이이류청기기-2,2’-이광산(DIDS)가분별사RVD감소(16.5±1.6)％、(14.7±2.28)％(P＜0.01).당세포외액위고K+급무Cl-적배경시,RVD분별감소(5.8±1.6)％화(2.7±0.8)％,차이유통계학의의(P＜0.01),이재무Ca2+조건하,RVD궤호소실.세포외액위pH6.8시,부능잠시연완세포적RVD과정,이비명현적억제,우기시재용적회축적전10 min내,pH6.8적45％저삼림격액자격시세포적RVD속솔위(0.86±0.24)％/min,명현저우pH7.4적45％저삼림격액자격시적RVD속솔(3.24±0.84) ％/min,량조간차이유통계학의의(P＜0.05). 결론 저삼자격하인LECs가산생RVD,세포내일정량적Ca2+존재시RVD격활적전제,이K+화Cl-시HLE B-3 RVD과정적관건성리자,Cl、K+리자통도칙시여세포용적변화상관성Cl-、K+외류적일개중요도경,동시저삼액적산화야가이연완RVD적과정.
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.
