电场干预对血管平滑肌细胞血小板衍化生长因子受体分布的影响

Effect of electric field on the distribution of platelet derived growth factor receptor in vascular smooth muscle cells

目的:观察电场作用下血管平滑肌细胞膜血小板衍化生长因子受体表达的变化及其与细胞迁移生长的关系。方法:①实验于2003-08/2004-08在解放军第三军医大学附属西南医院心内科及烧伤实验室完成。选用健康Wistar大鼠120只。②按常规组织块贴壁法原代培养血管平滑肌细胞。③通过外加电场干预装置(基本结构包括:观测小室和细胞培养室、电极系统、直流电源。细胞培养室的中央部分是对细胞行为进行显微镜下直接观测的地方,即观测小室。是该装置的关键部分。与细胞培养室相连的电极采用琼脂-盐桥电极。电源系统为输出电压0.1～200V,输出电流0.01～10mA,连续可调的电压电流双稳直流电源),对体外培养的大鼠主动脉血管平滑肌细胞直流电场干预,间断显微细胞图像记录和分析,观察不同强度电场、不同作用时间下血管平滑肌细胞迁移和细胞形态的变化;采用免疫荧光染色方法检测电场干预后,与血管平滑肌细胞迁移密切相关的血小板衍化生长因子受体膜表达,观察血小板衍化生长因子受体表达变化与细胞生物行为变化的关系。④两组间差异比较采用t检验。结果:①在施加150mV/mm电场干预后,血小板衍化生长因子受体表达增加(P<0.05～0.01),部分细胞血小板衍化生长因子受体表达呈不对称分布,在细胞向阴极伸展的板状突起中有明显荧光着色。血小板衍化生长因子受体在血管平滑肌细胞的重新分布在电场干预30min后出现,在6h电场干预中仍持续存在。②在150mV/mm电场干预下,培养的血管平滑肌细胞有明显的电场趋化性,细胞向阴极迁移的距离明显长于无电场作用对照组,血管平滑肌细胞在电场作用下迁移速度亦明显加快[(13.84±0.79),(4.32±0.42)μm/h,P<0.01),其电场趋化性与血小板衍化生长因子受体的不对称分布相一致。结论:外加直流电场干预下,大鼠血管平滑肌细胞的血小板衍化生长因子受体表达上调并呈极性不对称分布,这一改变可能与血管平滑肌细胞形状发生改变和向电场阴极面定向迁移有关。

AIM： Tu observe the change of platelet derived growth factor receptor （PDGFR）, and its relation with the migration and growth of vascular smooth muscle cells （VSMCs） under electrical field. METHODS： ①The experiment was conducted at the Department of Cardiology anTI Burn Laboratory, Southwest Hospital, Third Military Medical Universily of Chinese PLA from August 2003 to August 2004. 120 healthy Wistar rats were selected.②VSMCs were cultured primarily with routine tissue block adhering wall method. ③Direet current electric fields was used to intervene VSMCs at aorta of rats euhured in vitro （basically composed of observing cabin, cell euhure, electrode system and direct power source： Central part of cell culture room was the place where direct ohservation was done on cell behavior under mieroseope, which was ohservational eahin. It was a key of this equipment. Eleetrode connected to cell culture room was using japanese isinglass-sah bridge electrode. Output electric pressure of the power supple system was 0.1-200 V, and the output erectric eurrent was 0.01-10 mA with a douhle stable direct power supply, which eouhl adjust electrie pressure and current.） by extra interventional assembly of electrical field. It was recorded and analyzed with discontinuous micro-cell image. The changes of migration and form of VSMCs were ohserved under electrical field at different intensity and different effect time. Expression of PDGFR closely related to migration of VSMCs was detected with immunofluorescent staining method after the intervention of electrical field. Relationship between the changes of the expression of PDGFR and the changes of bio-behavior was observed.④ Difference between the two groups was eompared with t-test. RESULTS：①Under 150mV/mm the direct current electric field, the expression of PDGFR increased （P 〈 0.05-0.01）, and part of PDGFR distributed asymmetriely on the membrane of VSMCs. There was marked fluorescent staining at the board-like apophysis extended towards cathode. The PDGFR appeared after the VSMCs redistributed at electrical field for 30 minutes, and still existed at the 6^th hour under electrical field. ②Under 150 mV/mm the direct eurrent electric field, the cultured VSMCs had signifieant chemotaxy towards electrical field. The distance of cellular migration towards cathode was longer distinctly higher than that in control group （no electrical field intervention）. The speed of migration of VSMCs was quickly clearly under electrical field intervention [（13.84±0.79）, （4.32±0.42）μm per hour. P 〈 0.01]. and its chemotaxy towards electrical field was coincidence with the asymmetrical distribution of PDGFR. CONCLUSION： The expression of PDGFR of VSMCs of rats increases and shows polar asymmetrical distribution under extra direct electrical field intervention. This ehange can be related with the changes of the form of VSMCs and its oriented migration towards the cathode of electrical field.