摘要目的观察猴视网膜下腔植入人胚眼视网膜色素上皮(RPE)后不同时段的眼底、荧光血管造影和组织学的改变。方法12～24周的人胚眼色素上皮片和酶解后的浓缩色素上皮细胞悬液经一步法或二步法植入猴眼视网膜下腔。结果猴眼发生排异反应的时间为2～6月，猴眼黄斑区发生排异反应的比例高于黄斑周围区。结论异种RPE视网膜下腔的移植在无免疫抑制剂的条件下，只能短期存活。

分类号R774

Immunorejection induced by patch transplants of human fetal retinal pigment epithelium in monkey’s subretinal spaces

Sheng Yaohua，Li Zengqi，Hu Yiqian

（Department of Ophthalmology，Xin Hua Hospital，the Second Medical University，Shanghai 200092）

AbstractObjectiveTo observe the fundus，angiographic and histological changes of monkey eyes in which human fetal retinal pigment epithelium（RPE） cells were transplanted into the subretinal spaces．MethodsSmall patches of RPE monolayer were dissected from human fetal eyes aged from 12～24 weeks and cultured in regular culture dishes；routine vitrectomy was performed and loaded patches was injected into the subretinal spaces of monkey eyes after producing a bleb detachment of the neural retina．The fundus and angiographic changes were followed up weekly in the first month post-operation and then monthly．The eyes in which we found remarkable immunorejection and those of 6 month post-operation were extracted．Photomicroscope and electromicroscope examination were taken．ResultsThese transplanted human fetal RPE patches could survive at least one month under the retina of monkey eyes and maintained intimate association with the host photoreceptors．Normal fluorescein angiogram could be seen．Remarkable immunorejection was initiated 12～24 weeks after transplantation in the monkey eyes．In fluorescein angiogram，marked hyperfluorescein was shown in the late phase．In histogram，large amount of monocytes infiltration could be found．The macular areas were more sensitive to the RPE xenografts than the other areas．ConclusionCultured human fetal RPE can be transplanted to the subretinal spaces of monkey eyes．The survival time of those xenografts is very limited if immuno-suppressive agents are not administered．How to keep the transplanted patches in monolayer status and their original orientation，and how to suppress the immunorejection are the key points in a successful RPE transplantation．

Key wordsretinal pigment epithelium xenoplastic transplantation immunorejection monkey human fetal

老年性黄斑变性(age-relatedmaculardegeneration，ARMD）是由于视网膜色素上皮(retinalpigmentepithelium，RPE)，Bruch’s膜和脉络膜毛细血管的渐进性病变而导致的光感受器疾病和视力下降。其中有相当一部分病例伴有视网膜下新生血管膜的形成，这些新生血管膜虽可经手术切除，但术后往往导致RPE的进一步的破坏^［1］。植入视网膜下腔的RPE细胞可启动修复机制，使部分光感受器外节再生^［2］。大鼠的同种RPE^［3］和人的异种RPE^［4］都可代偿、防止RCS鼠的遗传性光感受器的变性。同种RPE移植还可缓解Fischet-344鼠的老年性视网膜变化^［5］。因此在老年性黄斑变性手术中，同时植入RPE细胞应是一种合理的设想。此外RPE的移植也可能成为对于一些难治性眼底病在细胞和基因水平上治疗的新方法。国内外对于该课题的研究目前尚处于动物实验和临床前期阶段。

由于血－视网膜屏障的存在，视网膜下腔被认为是一个免疫豁免区^［6］。大鼠和兔的同种异体RPE移植无明显的排异反应发生^［7，8］。本文主要报道异种RPE移植的动物实验资料。我们将人胚眼的RPE片和RPE片经酶解后的浓缩细胞混悬液，经视网膜切开植入或注入至猴的视网膜下腔中。植入眼使用眼底摄影、荧光血管造影进行追踪，最后做组织切片检查。

1材料与方法

1．1RPE片的培养

组织材料取于12～24周的人胚眼，眼球在超净台中，沿睫状体扁平部剪开，剔除前部组织、玻璃体和视网膜。剩余眼杯在解剖显微镜下分成4块展平后，放入培养液中。再使用精细晶状体撕囊镊，将RPE撕离脉络膜毛细血管层。得到的RPE片放在含20%小牛血清和0.5%葡萄糖的培养液中，然后使用OT针头将其固定在培养皿的底部，并放入CO₂培养箱中，37℃恒温培养。培养液每周更换两次^［9］。

1．2植入方法

6个猕猴使用苯巴比妥(25mg／kg)做静脉麻醉。常规扩瞳后置入开睑器并做穹隆为基底的结膜瓣。采用二步法接受双眼RPE移植：按常规玻璃体切割术准备结膜切开和巩膜切口。先做局限性的玻璃体切割，切除与移植区视网膜相贴的玻璃体皮质。然后使用50μm直径的毛细玻璃管造成一局限性的视网膜脱离。最后将装有RPE片的直径为300μm的毛细管经原巩膜切口和视网膜裂洞进入视网膜下腔，植入RPE片。

猴眼术后1月内每周作常规荧光血管造影和眼底摄片。以后每月检查1次。

1．3组织学检查

视猴眼排异反应情况，在发生明确眼底表现后做眼球摘除。未发生反应者在第六个月获取组织学标本。摘除的眼球做角巩膜缘多点穿刺，浸泡在含2%戍二醛和1%多聚甲醛的磷酸缓冲液中至少48h。移植区使用1%锇酸作后固定，乙醇脱水和Epon包埋。切片作光镜和电镜检查。

2结果

人胚眼RPE片植入到猴眼黄斑及黄斑周围区共10眼，最长观察时间为6个月。黄斑区植入的6眼大多在3～4月后发生排异反应，而在黄斑周围区植入的4眼，其中3眼在观察期间未发生排异反应。

图1人胚眼RPE上皮片猴眼黄斑及黄斑周围区移植8周后。A．示两片色素上皮分别位于黄斑颞侧及颞上静脉间。靠近黄斑区的移植片，其表面有少许白色膜组织的复盖；B．在早期荧光血管造影中，二移植区都呈阻断荧光；C．在晚期荧光血管造影中，近黄斑的移植片呈荧光染色而远离黄斑区的移植片仍是阻断荧光；D．颞上侧移植片的组织切片示被移植的色素上皮呈单细胞排列。同时与宿主组织保持了良好的接触，且无明显的组织细胞浸润。Fig．1Eight weeks after human fetal RPE patches transplanted into macular and perimacular areas of monkey eyes．A．Fundus photograph of two RPE patches．One is just temporal to fovea，which is covered by white membrane；the other is along superior vascular arcade；B．In early fluorescein angiogram showing typical fluorescent blockade of two transplants；C．In late phase of angiogram，the transplant closed to fovea is stained，but the one located away from the fovea is remain unstained；D．Histogram of the patch located along superior vascular arcade．Transplanted cells maintain in monolayer and keep nice contact with host．Rejection was not discovered移植片同时植在黄斑及黄斑旁区8周后的荧光造影和组织学切片见图1。移植片边界清晰，局部平坦，但位于黄斑区的移植片似有薄膜复盖。早期荧光血管造影时两处都呈阻断荧光。至后期上方移植片仍呈阻断荧光，但黄斑区的植片已呈明显的荧光染色。组织学切片表示，移植片呈单细胞层，与光感受器外节保持良好的接触。脉络膜中无单核细胞积聚。RPE片植入猴眼24周的眼底照相、荧光血管造影及组织学切片见图2。移植片位于黄斑颞侧，剥离区可见一圆形脱色素区，移植片边界清晰，局部平坦，早期及后期荧光血管造影都无明显渗漏，移植片上可见正常网膜血管荧光。组织切片中提示植入片与宿主Bruch’s膜还有大量胶原组织的增生，但Bruch’s膜完整，局部无明显炎细胞浸润。移植片与光感受器外节对接良好。

图2 人胚眼RPE片猴眼黄斑区颞侧移植24周后。A．示移植片位于黄斑颞侧。圆形的脱色素区对应于局限性的视网膜脱离区。移植片表面有少许白色膜样组织复盖。B，C．早期、晚期荧光血管造影移植区都不出现明显的荧光染色。在移植片表面还能看到部分视网膜血管荧光。D．组织切片图中脉络膜和视网膜中都无明显的组织细胞浸润，宿主Bruch’s膜完好无损。移植片卷曲成管状，但与宿主Bruch’s膜和光感受器层保持了良好的接触。在移植片围成的腔隙中有大量的<