白内障囊外摘除联合人工晶体植入术的成败及并发症的多少，在很大程度上受人工晶体（IOL）本身因素的影响，其中生物相容性问题受到许多国内外学者的关注。为了寻求一种生物相容性好的IOL，本课题对聚甲基丙烯酸甲酯(PMMA)材料的IOL进行表面修饰，得到不同表面修饰的IOL。通过体内长期植入实验和体外细胞培养技术，观察修饰IOL的生物相容性。

1. 第一部分 氟-肝素表面修饰人工晶体的实验研究

目前临床上最常用硬质IOL材料是PMMA，它植入眼内引起一系列的炎症反应，免疫排斥反应，导致临床上并发症的出现，影响术后远期视力的恢复。为了减少IOL植入术后的并发症，必须提高PMMA IOL的生物相容性。本实验采用载能离子束联合低温等离子体技术对PMMA材料的IOL进行表面氟离子处理联合肝素表面修饰，得到氟－肝素表面双重修饰的IOL。体外实验已证实修饰后IOL的生物相容性得到提高，为过渡于临床研究，选用接近于人类的非人灵长类动物猕猴进行IOL植入的长期实验，并选用Pharmacia肝素修饰的人工晶体（HSM IOL）作对照。

20只猕猴随机分为5组：正常组，2只猕猴，不进行手术；对照组1，5只猕猴，10只眼，行晶状体囊外摘除联合后房型人工晶体植入术(ECCE+IOL)，植入未修饰的PMMA IOL；对照组2，3只猕猴，6只眼，施行ECCE+IOL，植入HSM IOL；实验组1， 5只猕猴，10只眼，施行ECCE+IOL，植入氟-肝素修饰的3.5×10¹⁴ions／cm² IOL；实验组2， 5只猕猴，10只眼，施行ECCE+ IOL，植入氟-肝素修饰的6.0×10¹⁴ions／cm² IOL。于术后15、30、60、90、180、360天在全麻下行裂隙灯眼前节检查，观察角膜水肿、前房渗出、后囊混浊形成情况；检眼镜检查眼底；修兹氏压陷式眼压计测量眼压。于术后不同时期透明角膜行前房穿刺抽取房水，作房水细胞计数；使用ELISA方法检测房水中sIL-2R的水平；放射免疫方法检测房水中TNF-a 的含量。使用免疫组化方法对术后180天眼内不同组织进行IL-2Ra 的蛋白检测；原位杂交方法对术后180天眼内不同组织进行TNF-a 和IL-2 mRNA的基因表达检测。利用计算计图像分析，对术后360天人工晶体表面粘附细胞进行分析。并对术后180天、360天的眼球进行扫描电镜、透射电镜、光镜的观察。实验结果使用SAS软件包进行秩和检验和方差分析。

结果如下：

1．角膜水肿：术后15天最明显，随时间延长而减轻乃至消失，对照组与实验组无差异。

2．前房渗出：术后15天最严重，以后逐渐减轻，术后90天前房渗出吸收，恢复正常。术后30天实验组1前房渗出明显低于对照组，具有统计学意义。

3．后囊混浊：术后30天开始，90天明显。术后90天、180天实验组后囊混浊的程度轻于对照组，术后360天实验组1后囊混浊的程度最轻。

4．并发症：IOL脱入玻璃体、瞳孔区色素堆积、瞳孔夹持、虹膜脱出、前房出血、眼球萎缩，与手术创伤有关。

5．房水细胞计数：术后15天最高，90天基本恢复正常。术后15天，对照组1明显高于其它各组，术后30天，实验组房水细胞计数低于对照组1，具有统计学意义。

6．sIL-2R和TNF-a ：房水sIL-2R的水平于术后15天增高，30天达高峰，90天恢复正常水平。术后30天对照组1房水sIL-2R的水平最高。房水TNF-a 的含量，术后15天达高峰，60天接近正常。术后15天实验组1房水中TNF-a 的含量低于其它各组。

7．免疫组化：正常眼角膜上皮细胞IL-2Ra表达弱阳性，虹膜基质有阳性表达，睫状体无色素上皮细胞表达阳性，视网膜的内外颗粒层弱阳性表达，神经节细胞层阳性表达。

8．原位杂交：

TNF-α：角膜上皮细胞阳性，虹膜基质表达阳性，睫状体的无色素上皮细胞弱阳性，视网膜内外颗粒层阳性，神经节细胞层的血管内皮细胞弱阳性表达。

IL-2：角膜上皮细胞弱阳性，虹膜基质、血管内皮细胞阳性，睫状体的无色素上皮细胞弱阳性，视网膜的内外颗粒层表达阳性，神经节细胞层的血管内皮细胞弱阳性表达。

9．人工晶体表面粘附细胞：术后180天、360天，光镜检查人工晶体表面光学部粘附细胞较少，周边粘附细胞较多，对照组1粘附细胞多于实验组。计算机分析粘附细胞的面积：细胞面积最大为巨细胞620.06μm²，粘附细胞最多为巨噬细胞，其面积平均99.99μm²。扫描电镜下，实验组人工晶体表面附着细颗粒状的蛋白膜，对照组表面附着纤维网状及颗粒样蛋白膜。

10．透射电镜：术后180天，角膜结构正常，虹膜基质疏松，睫状体与视网膜结构正常，各组间无差异。术后360天，虹膜基质疏松，胶原稀疏，其它组织结构正常。

11．光镜：后囊混浊的病理变化有纤维内障、Elschnig小体、Soemmerring＇s环。术后180天，对照组后囊混浊明显严重于实验组，其它病理变化如虹膜基质水肿，视网膜偶见轻度黄斑囊样水肿，实验组与对照组无差异。术后360天，对照组1的后囊混浊明显严重于实验组。术后360天还可见虹膜基质萎缩，其它组织的结构恢复正常，各组无差异。

12．正常猕猴角膜的大小几乎等同于人眼角膜，而眼球各个径线均小于人眼。眼内组织结构及白内障的病理变化基本于人类相同。

使用高等动物最理想的动物模型猕猴，研究IOL植入术后的远期疗效，分析氟-肝素修饰的IOL植入猕猴眼内比PMMA IOL引起眼的炎症反应、免疫反应轻，人工晶体表面粘附细胞少，后囊混浊形成的程度轻，具有较好的生物相容性。与Pharmacia HSM IOL相比，氟-肝素修饰IOL植入眼内引起后囊混浊的程度轻，具有显著性差异，其它临床和实验指标均无统计学意义。总之，氟-肝素表面双重修饰的IOL，其生物相容性良好，可试用于临床。

2. 第二部分 钛和碳表面修饰人工晶体的体外实验

使用等离子体技术对IOL进行不同材料的表面修饰，以寻求生物相容性更好的IOL。实验按IOL表面不同的修饰分为Ⅰ组-Ti、Ⅱ组-C (150ev)、Ⅲ组-C (800ev)、Ⅳ组-CH₄⁺ (150ev)和Ⅴ组-PMMA。进行体外细胞培养，观察血小板、粒细胞、巨噬细胞在不同修饰IOL表面粘附情况，粒细胞释放H₂O₂水平的检测及成纤维细胞在IOL表面的生长。结果表明：血小板粘附实验－Ⅰ、Ⅱ、Ⅲ组与Ⅴ组相比差异显著，修饰表面血小板粘附的数量较少。粒细胞实验－Ⅰ、Ⅱ、Ⅲ组IOL表面粘附粒细胞的数量少于Ⅴ组；粒细胞释放H₂O₂的水平，Ⅴ组明显高于Ⅰ、Ⅱ、Ⅲ、Ⅳ组。巨噬细胞粘附实验－巨噬细胞粘附于IOL表面的数量，Ⅰ、Ⅱ、Ⅲ、Ⅳ组少于Ⅴ组，并且Ⅰ组与Ⅱ、Ⅲ、Ⅳ组相比具有统计学意义。成纤维细胞生长实验－Ⅰ、Ⅲ组IOL表面成纤维细胞生长良好，与Ⅴ组比较差异明显。由此初步认为，体外细胞培养实验结果显示，Ti修饰的IOL比其它几组修饰的IOL生物相容性好，其次C (800ev)修饰的人工晶体也具有较好的生物相容性。若观察植入生物材料的长期生物相容性，必须进入下一个体内实验阶段。

关键词：　 氟-肝素修饰 PMMA IOL HSM IOL 生物相容性 表面粘附细胞 后囊混浊 房水 sIL-2R和TNF-a 病理变化 猕猴 钛和碳修饰 血小板 粒细胞 巨噬细胞 成纤维细胞 实验研究

0. The experimental study on manifold
   new-types surface modified IOLs

1. ABSTRACT

Success or failure and complications of extracapsular cataract extraction(ECCE) with implantation of a posterior chamber intraocular lens(IOL) depend on IOL itself to a great extent. The biocompatibility of IOLs has been studied by many authors. In order to get a better biocompatibility polymethylmethacrylate (PMMA) IOL surfaces were modified and observed in vivo and vitro.

　

0. PartⅠ The experimental study on F-heparin surface modified IOLs

At present PMMA is still the most common IOL material used. PMMA IOLs led to some degree of inflammatory response, foreign body reaction and complications. In our former study the technique of carrier energy ion beam combined with low temperature and low pressure plasma was used to modify the surfaces of PMMA IOLs with F ions and the heparin solution. In vitro, it was confirmed that the F-heparin modified IOLs(FHM IOLs) possessed better biocompatibility than that of PMMA IOLs. In this study the nonhuman primate animals-monkeys were chosen to implant FHM IOLs for one year to compare the results with PMMA IOLs. Pharmacia heparin surface modified (HSM) IOLs were also used as a control.

Twenty monkeys were used and classified into 5 groups. In normal group two monkeys (4 eyes) were not operated. Operations were done on all of the monkeys except in the normal group. In Control group 1, 10 eyes of 5 monkeys were implanted with PMMA IOLs. Control group 2, 6 eyes (3 monkeys) were implanted with HSM IOLs. Experimental group 1, 10 eyes(5 monkeys) were implanted with F(3.5×10¹⁴ions／cm²)-HM IOLs. Experimental group 2, 5 monkeys(10 eyes) were implanted F(6.0×10¹⁴ions／cm²)-HM IOLs. All of the eyes were examined by slitlamp microscope, ophthalmoscope and schiotz tonometer under general anesthesia at postoperative 15, 30, 60, 90, 180, 360 days. At different periods postoperatively aqueous from anterior chamber was aspirated to calculate cells and measure sIL-2R by ELISA and TNF-αby radioimmunoassay. The level of IL-2Rα protein was measured by immunohistochemistry, whereas the level of TNF-αand IL-2 mRNA expression was studied by the method of in situ hybridization. Cells adherence on the surface of IOLs were analyzed by computer. And the eye tissues were examined by scanning electron microscope(SEM), transmission electron microscope(TEM) and light microscope(LM). Statistical evaluation was performed by using rank sum test and analysis of variance.

The results were as follows:

1.Corneal edema: At 15 day postoperatively corneal edema became worse, but decreased gradually and disappeared. There was no significant difference between the control and the experimental groups.

2.Anterior chamber exudation: At 15 day postoperatively anterior chamber exudation became prominent, decreased gradually and absorbed at 90 day. At 30 day the severity of anterior chamber exudation in experimental groups was less than that in control groups.

3.Posterior capsule opacification (PCO): It occurred at 30 day and was obvious at 90 day. In experimental groups the degree of PCO was lower than that in the control groups at 90 day and 180 day. And in experimental group 1 the degree of PCO was lowest among the three groups at 360 day.

4.Complications: There were luxation of IOL into vitreous, very dense aggregation of pigments in pupil area, pupil capture, prolapse of iris, hyphema, atrophy of eyeball. These complications were associated with trauma during and after operation.

5.Cells in the aqueous: Cellular count was highest at 15 day and recovered at 90 day. At 30 day cells of control group 1 were much more than that of other groups.

6.sIL-2R and TNF-α: The level of sIL-2R in aqueous increased at 15 day, reached the highest level at 30 day and recovered at 90 day. At 30 day the level of sIL-2R of control group 1 in aqueous was the highest. The content of TNF-α in aqueous was the highest at 15 day and recovered at 60 day. At 15 day the content of TNF-α in experimental group 1 was lower than in the other groups.

7.Immunohistochemistry: The level of IL-2Rα protein expression was positive in normal corneal epithelium, stroma of iris, nonpigmented ciliary epithelium, inner and outer granular layers and nerve-ganglion cells layer in retina.

8.In situ hybridization: The level of both TNF-α mRNA expression and IL-2 mRNA expression was positive in normal corneal epithelium, stroma of iris, nonpigmented ciliary epithelium, inner and outer granular layers and nerve-ganglion cells layer in retina.

9.Cellular adhesion on the IOL surface: Cellular adhesion was less on the IOL center and more on the IOL border by LM at 180 day and 360 day postoperatively. The cells deposited on the IOL in control groups were more than that in experimental groups. By computer analysis the largest deposited cells on the IOLs were giant cells with the average area of 620.06μm². The most deposited cells on IOL were macrophages with the average area of 99.99μm². By SEM in the experimental groups on the surface of IOLs there was fine granular proteinic membrane, but in control groups there was fibrinous reticular proteinic membrane.

10.TEM: At 180 day the structure was normal in cornea, ciliary body and retina, but was loose in stroma of iris. At 360 day the structure was loose in stroma of iris and collagenous fibers, but other tissues were normal.

11.LM: The types of morphological changes of PCO included fibrosis-type, pearl-type and Soemmerring＇s ring . At 180 day the PCO of control groups was much obviously severe than that of experimental groups. There were also some pathological changes such as edema of iris stroma and macular cystoid edema. At 360 day the PCO of control group 1 was the most severe and iris stroma atrophy existed.

12.The size of cornea in normal monkeys was about the same as in human cornea and the ocular axes in normal monkeys were shorter than that in human eyes. The structure of ocular tissues and pathological changes in monkeys was almost the same as in human.

We studied the long-time effect of the IOL implantation by using higher mammal animal model. F-heparin modified IOLs induced lower inflammatory reaction, foreign-body reaction, less cellular deposits, less dense PCO than that of PMMA IOL and possessed a better biocompatibility. Comparing with HSM IOLs, F-heparin modified IOLs showed less PCO.

According to the above mentioned fact, F-heparin modified IOL may be used for clinic trial due to its better biocompatibility.

　

1. PartⅡ In vitro experiment of Ti and C surface modified IOLs

In order to find out IOLs with much better biocompatibility different kinds of materials were used to modify surface of IOLs with the technique of low pressure plasma. There were 5 groups according to different modification. Group Ⅰ-Ti, group Ⅱ-C (150ev), group Ⅲ-C (800ev), group Ⅳ-CH₄⁺(150ev) and group Ⅴ-PMMA. In vitro the cultured platelets, granulocytes, macrophages, fibroblasts were observed to adhere and grow on the IOLs. The level of H₂O₂ released by granulocytes was measured. The results were as following:

Key words: F-heparin modification, PMMA IOLs, HSM IOLs, biocompatibility, adhered cells, PCO, aqueous, sIL-2R and TNF-a , pathological changes, monkeys, Ti and C modification, platelets, granulocytes, macrophages, fibroblasts, experimental study

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