双重荧光标记法检测人视网膜色素上皮细胞吞噬的功能

Examination of phagocytic function in human retinal pigment epithelial cells by double fluorescent vital assay

目的:检测人视网膜色素上皮(humanretinalpig-mentepithelium,HRPE)细胞特异性及非特异性吞噬动力学,比较二者的不同之处。方法:用双重荧光标记法检测HRPE细胞吞噬动力学,即分别用红色染料硫氰酸罗达明(sulforho-damine,SR)、绿色荧光染料异硫氰酸酯荧光素(FITC)标记HRPE细胞和视杆细胞外节膜盘(rodoutersegments,ROS)。用1×1010个/L的FITC-ROS及自发绿色荧光的乳胶微球(leatexbeads,LB)于37℃孵育培养的正常HRPE细胞,在孵育的不同时间(5min￣48h)去除孵育物,终止吞噬反应。用配有特殊三通广谱吸收波长,长工作距离镜头的荧光显微镜实时,活体观察并记录结合及吞噬的数量。用扫描电镜及激光共聚焦扫描显微镜证明HRPE细胞对LB及ROS结合与吞噬。结果:孵育0.25h时ROS已结合于HRPE细胞表面,0.5h时可见极少数的ROS被HRPE细胞摄入细胞内;之后的孵育过程中被结合及被吞噬的ROS数目不断增加,至孵育18h对ROS的结合达到饱和,但摄入过程继续进行,至孵育24h,对ROS的吞噬达到饱和。当HRPE细胞与LB孵育时,至1.5h结合方启动,6h摄入开始;在观察的48h内被结合及被吞噬的LB数目随时间延长而呈线性增加。结论:HRPE细胞的特异性及非特异性吞噬动力学明显不同,HRPE细胞对ROS的结合及吞噬比其对LB的结合及吞噬发生得更早,而且结合及吞入过程均具有时间饱和性。

AIM： To investigate the kinetics in specific and non- specificphagocytosis of human retinal pigment epithelium （HRPE） cells, and to reveal the difference between them. METHODS： Double fluorescent vital assay was used to examine the kinetics. The HRPE cells and rod outer segments （ROS） were stained by sulforhodamine （SR） and FITC. The cultured HRPE cells were incubated with 1 ×10^10/L ROS or latex beads （LB） at 37℃, and then were rinsed at different incubation times to terminate the phagocysis. The kinetics of phagocytosis was measured vitally by fluorescent microscope which equipped with triple bands and broad wave-longs excitation/emition and long work-distance camera lens, together with CCDSPOT RTKE system. The binding and ingestion of ROS or LB were proved by scanning and transmission electron microscope, together with con-focal microscope. RESULTS： The kinetics study showed that the binding of ROS happened at 0.25h of incubation, and the ingestion of ROS happened at 0.5h; the amount of bound and ingested ROS increased with time proceeding and saturated at 18h and 24h, respectively. When HRPE cells incubated with LB, the bound and ingestion prolonged to 90min and 6h, respectively. The amount of bound and ingested LB increased with time proceeding during all the incubation time without saturation. CONCLUSION： The time that triggers binding and ingestion of ROS in HRPE cells are earlier than that in non- specific phagorytosis. There are saturation time of binding and ingestion in specific phagocytosis of HRPE cells, but not in nonspecific phagocytosis.