人嗜铬细胞微囊化处理对大鼠异种移植的免疫隔离作用

摘要

背景:课题组前期在建立海藻酸钠-多聚赖氨酸-海藻酸钠微囊包裹活细胞制备技术的基础上,已证明微囊化嗜铬细胞有良好的镇痛效果,而该微囊包被材料的免疫隔离作用尚需明确.目的:观察海藻酸钠-多聚赖氨酸-海藻酸钠微囊化嗜铬细胞移植到大鼠眼前房和足胝部的免疫排斥反应,评价微囊化技术的免疫隔离作用.设计:随机对照动物实验.单位:华中科技大学同济医学院附属同济医院麻醉学教研室.材料:选用雌性 SD大鼠48只,鼠龄3个月,由华中科技大学同济医学院实验动物部提供.实验过程中对动物处置符合动物伦理学标准.实验所用海藻酸钠、多聚赖氨酸为美国Sigma公司产品,微囊发生器为德国赠送.方法:实验于2002-09/2003-09在华中科技大学同济医学院附属同济医院麻醉学实验室完成.①取6名脑死亡健康成人的肾上腺髓质,经分离、消化、培养后,制备成人嗜铬细胞悬液.供者家属对实验知情同意,实验方案通过医院伦理委员会批准.采用海藻酸钠-聚赖氨酸-海藻酸钠法制作空微囊和微囊化细胞.②48只大鼠被随机分为3组:人嗜铬细胞移植组、空微囊移植组、微囊化人嗜铬细胞移植组,每组分眼前房和足胝部两个部位进行移植,每个部位8只.人嗜铬细胞移植组分别将2×1010 L-1细胞悬液注入大鼠右眼前房和左足胝部.空微囊移植组和微囊化人嗜铬细胞组分别吸取空微囊(100个微囊)或ME-HCC(100个微囊,每个微囊包裹400～500个细胞)注入大鼠右眼前房和左足胝部.主要观察指标:于移植术后第7天采用ELISA法测定血清白细胞介素2水平.采用激光散射比浊仪测定血清IgG和IgM水平.移植术后第28天取大鼠右侧眼球及左侧足组织作常规切片,苏木精-伊红染色,40倍光镜下观察组织形态.结果:大鼠48只均进入结果分析.①血清白细胞介素2,IgG,IgM水平:空微囊移植组和微囊化人嗜铬细胞移植组均低于人嗜铬细胞移植组,差异有显著性意义(t=8.544～21.64,P < 0.01).②大鼠眼前房和足胝部组织形态:人嗜铬细胞移植组大鼠的眼前房内和足胝部可见大量淋巴细胞和中性粒细胞浸润.空微囊移植组和微囊化人嗜铬细胞移植组大鼠眼前房和足胝部仅见少量淋巴细胞和中性粒细胞.结论:海藻酸钠-多聚赖氨酸-海藻酸钠微囊化所产生的良好生物相容性及其机械稳定性,使之有效地发挥了免疫排斥隔离作用.%BACKGROUND: Based on previous technique prepared for encapsulating living cells with alginate-polysine- alginate (APA) microcapsules, it has been confirmed that microencapsulated chromaffin cells have good analgesic effects. The immunoisolated effects of such microcapsule materials need to be evaluated. OBJECTIVE: This study aimed to investigate the immunological rejections of APA microencapsulated chromaffin cells transplanted into rat anterior chamber of eyes and tendon of feet, and to evaluate the immunoisolated effect of microencapsulation.DESIGN: A randomized controlled animal experiment. SETTING: Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Forty-eight female SD rats, with the age of 3 months, were provided by the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology. The protocol was carried out in accordance with ethical guidelines for the use and care of animals. Alginate and polylysine used in the experiment were the products of Sigma Company, USA. Microcapsule generator was gifted by Germany. METHODS: This study was performed at the Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology from September 2002 to September 2003. Suprarenal medulla was taken from 6 healthy adult cadavers of brain death. After isolated, digested and cultured, suprarenal medulla was prepared into chromaffin cell suspension. Written informed consents were obtained from the family members of donors, and the protocol was given approval by the Ethics Committee of the hospital. Empty microcapsules and microencapsulated cells were prepared by APA. The 48 rats were randomly divided into the human chromaffin cell (HCC) group, the empty microcapsule group and the microencapsulated HCC (ME-HCC) group. In each group, there were two transplanted regions of anterior chamber of eyes and tendon of feet, with 8 rats used for each region. Each rat in the HCC group was perfused 2×1010 L-1 cell suspension into the anterior chamber of eyes and tendon of feet. Those in the empty microcapsule group and the ME-HCC group were perfused 100 empty capsules and ME-HCCs (100 microcapsules, 400-500 HCCs per microcapsule) into the same regions, respectively. MAIN OUTCOME MEASURES: On day 7 after transplantation, serum interleukin (IL)-2 level was determined by ELISA. Serum IgG and IgM levels were determined with a laser turbidimeter. On day 28 after transplantation, rat right eyeball and left feet were harvested, routinely sliced and stained by haematoxylin-eosin (HE). Histo-morphological structure was observed under a 40×light microscope. RESULTS: Forty-eight rats were included in the final analysis. Serum IL-2, IgG and IgM levels were significantly lower in the empty microcapsule group and ME-HCC group than in the HCC group (t=8.544-21.64, P < 0.01). A lot of lymphocyte and neutrophile infiltration could be found in the anterior chamber of eyes and tendon of feet of rats in the HCC group, but a little seen in that of the empty microcapsule group and ME-HCC group. CONCLUSION: APA microencapsulation has an effective immunoisolated effect on immunological rejection due to its good biocompatibility and mechanical stability.