原子力显微镜观测小分子化合物J2对大鼠角膜移植术后内皮细胞膜表面的影响

The observation of the effects of a micromolecular compound J2 on rat corneal allograft using atomic force microscopy

目的探讨应用原子力显微镜（AFM）观察大鼠角膜移植术后的内皮细胞，观测小分子J2化合物对角膜内皮细胞超微结构的影响。方法队列研究方法。将30只取右眼行穿透性角膜移植术后的实验大鼠编号后计算机随机抽号分为2组，每组15只。A组为J2药实验组，B组为安慰剂对照组。每组于术后第5、10、15、20、25天，分别计算机选号随机取下3只大鼠的角膜植片，经苏木素-伊红染色及AFM样品固定。两组AFM观测数据平均粗糙度（Ra）、平均峰值高度（Rp）、平均低谷高度（Rv）采用单因素方差分析。结果A组植片平均存活时间为（33．12±6．80）d，B组为（18．87±4．19）d，两组比较差异有统计学意义（F=47．7449，P=0．000）。AFM观测发现两组样品在植片存活第5天均显示为较规则的六边形结构，直径约15μm，可见细胞表面的凹凸不平结构。随着时间延长，两组样品出现不同改变，术后第20天：B组样品的细胞结构无法辨认，凹凸不平的表面分辨困难，而A组的细胞仍然保持结构清晰。Ra、Rp和Rv在同期比较中，两组结果存在差异有统计学意义。术后第5天，A组：Ra（97．64±31．58）rim，Rp（297．79±25．19）nm，Rv（545．55±25．83）nm；B组：Ra（112．61±34．29）nm，Rp（265．06±24．17）nm，Rv（544．41±21．78）nm（Fa=30．9416，P=0．0000；Fp=263．6018，P=0．0000；Pv=1．2013，P=0．2735）。术后第10天，A组：Ra（102．98±32．98）nm，Rp（711．38±21．94）nm，Rv（639．89±22．58）Ilm；B组：Ra（222．85±31．28）nm，Rp（111．22±20．35）nm，Rv（746．49±23．17）nm（Fa=2086．4535，P=0．0000；Fp=53768．4676，P=0．0000；Pv=3257．3178，P=0．0000）。术后第15天，A组：Ra（87．44±34．97）nm，Rp（344．18±21．09）nm，Rv（482．61±22．27）nm；B组：Ra（197．64±35．72）am，Rp（510．76±24．98）nm，Rv（545．62±23．17）nm（Fa=1458．1057，P=0．0000；Fp=7788．6963，P=0．0000；Pv=1153．2860，P=0．0000）。术后第20天，A组：Ra（85．85±32．53）nm，Rp（348．69±21．26）nm，Rv（367．65±23．12）nm；B组：Ra（201．36±34．12）nm，Rp（788．58±20．34）nm，Rv（563．33±21．01）nm（Fa=1801．1215，P=0．0000；Fp=67057．9516，P=0．0000；Pv=11770．2195，P=0．0000）。术后第25天，A组：Ra（104．97±32．47）nm，Rp（395．05±20．38）nm，Rv（396．17±21．59）am；B组：Ra（43．85±31．28）nat，Rp（249．88±20．79）nm，Rv（154．88±22．37）nm（Fa=551．4134，P=0．0000；Fp=7458．9255，P=0．0000；Pv：18070．5189，P=0．0000）。结论角膜移植术后，A、B两组的角膜内皮细胞应用AFM观测可发现在Ra、Rp及Rv比较上存在差异，小分子化合物J2可延迟角膜移植发生排斥反应的时间。

Objective To study the cell membrane of corneal endothelium with a micromolecular compound J2 in corneal allograft of rat using atomic force microscopy （AFM）. Methods Cohort study.Subjects were divided into two groups： group A（ n = 15 ） ： experimental group; group B （n = 15 ） ： placebo control group. At the fifth, tenth, fifteen, twentieth, twenty-fifth day after penetrating keratoplasty, the donor implant was separated from receipt bed, one part of which was stained by HE and the others fixed into AFM sample. Amplitude and height images were obtained in the tapping mode with a scan rate of 2 Hz and an integral gain of 0. 3 to 0. 5. Statistical analysis was performed using single-factor analysis of variance and P value was calculated. Results The average transplant survival time in group A was （33.12 ± 6. 80） d, and those in group B was （18.87 ± 4. 19）d. There were significant difference between two group （F = 47. 7449, P = 0. 00 ）. There were obvious differences on ultrastructure measured by AFM between two groups. At the fifth day after penetrating keratoplasty, regular hexagonal structure of corneal endothelium was observed by AFM in both two group. The diameter of corneal endothelium was about 15 μm, uneven microstructure of cell could be found. The time being, different changes were arose in two group： a clear microstructure could be found in group A, however the microstructure of cell could not be recognized in group B. One way analysis of variance showed that significant differences on parameters （ Ra, Rp and Rv） were found between two groups （ P 〈 0. 05 ）. At the fifth day after penetrating keratoplasty, group A ： Ra （97.64 ±31.58）nm, Rp（297.79 ±25. 19）nm, Rv（545.55 ±25.83）nm; group B： Ra（ll2. 61 _±34. 29） nm, Rp（265.06 ±24. 17） nm, Rv（544. 41 ±21.78） nm（Fa =30. 9416,P =0. 0000; Fp = 263. 6018 ,P = 0. 0000; Pv = 1. 2013, P =0. 2735）. At the tenth day after penetrating keratoplasty, group A：Ra（ 102. 98 ± 32. 98）nm, Rp（711.38 ±21.94）nm, Rv（639. 89 ±22. 58）nm; group B：Ra （222. 85 ±31.28） nm, Rp （ 111.22± 20. 35 ） nm, Rv （ 746. 49 ± 23. 17 ） nm （ Fa = 2086. 4535, P = 0. 0000 ; Fp = 53768. 4676, P = 0. 0000; Pv =3257. 3178, P = 0. 0000）. At the fifteenth day after penetrating keratoplasty, group A： Ra （87.44 ±34. 97）nm, Rp（344. 18 ±21.09）nm, Rv（482. 61 ±22. 27） nm; group B：Ra（ 197.64 ±35.72） nm, Rp（510. 76 ±24. 98）nm, Rv（545.62 ±23.17） nm（Fa = 1458. 1057,P = 0. 0000; Fp =7788. 6963, P = 0. 0000 ;Pv = 1153. 2860, P = 0. 0000 ）. At the twentieth day after penetrating keratoplasty, group A ： Ra （85.85 ±32. 53）nm, Rp（348.69 ±21.26）nm, Rv（367.65 ±23.12）nm; group B： Ra （201.36 ±34. 12） nm, Rp （788.58 ± 20. 34） nm, Rv （563.33 ± 21.01） nm（Fa = 1801. 1215,P = 0.0000;Fp = 67 057. 9516, P = 0. 0000 ; Fv = 11 770. 2195, P = 0. 01300 ） . At the twenty-fifth day after penetrating keratoplasty, group A：Ra （ 104. 97 ± 32. 47 ） nm, Rp （ 395.05 ± 20. 38 ） nm, Rv （ 396. 17 ± 21.59 ） nm; group B ： Ra（43.85 ± 31.28 ） nm, Rp （ 249. 88 ± 20. 79 ） nm, Rv （ 154. 88 ± 22. 37 ） nm （ Fa = 551. 4134, P=0.0000; Fp =7458.9255, P = 0.0000; Pv = 18 070.5189, P = 0.0000）. Condusions The morphology and ultrastrueture of corneal endothelium in group A with J2 were different from group B by observation with AFM. J2 was an effect mieromolecular in prevention of corneal allograft rejection.