摘要
目的:分析壳聚糖-DNA超微颗粒在关节内的转基因效应。方法:实验于2005-09/2006-06在上海交通大学医学院健康科学研究所骨科细胞与分子生物学实验室完成。实验材料:①模型制备:采用切断内侧副韧带,切除内侧半月板的方法制备骨关节炎兔模型。②基因产品:白细胞介素(interleukin,IL)1Ra基因、IL-10基因。实验分组:15只新西兰兔按随机数字表法分为3组:①空载体对照组(n=3),造模后5d两侧膝关节关节腔注射400μL壳聚糖-PcDNA3.1溶液,共3次,每48h1次。②IL-1Ra基因治疗组和IL-10基因治疗组,每组6只,造模后5d对照侧膝关节关节腔分别注射20μg裸DNA(PcDNA3.1-IL-1Ra或PcDNA3.1-IL-10),实验侧膝关节关节腔注射400μL壳聚糖-DNA超微颗粒(含20μgIL-1Ra或IL-10),注射次数及间隔时间同空载体对照组。实验评估:①采用酶联免疫吸附分析及免疫组织化学检测IL-1Ra和IL-10基因的表达和分布。②苏木精-伊红染色和甲苯胺蓝染色观察骨关节炎软骨组织学变化。结果:纳入新西兰兔15只,均进入结果分析。①IL-1Ra和IL-10基因在关节滑液中的表达:空载体对照组及IL-1Ra基因治疗组对照侧膝关节滑液中未检测到IL-1Ra表达,实验侧于第1次基因注射后7,14d检测到IL-1Ra表达。IL-10基因治疗组对照侧和实验侧均未检测到IL-10表达。②IL-1Ra基因在兔膝关节的分布:IL-1Ra基因治疗组兔软骨表层和中间层部分细胞内表达IL-1Ra,至少持续到第1次基因注射后14d。在滑膜组织中未观察到明显的IL-1Ra表达。③兔骨关节炎软骨组织学变化:空载体对照组呈早期骨性关节炎的典型性改变。苏木精-伊红染色显示软骨细胞坏死,蛋白多糖甲苯胺蓝染色不均一,软骨表层和中间层大部分区域失染,软骨细胞簇聚区域其周围深染。IL-1Ra基因治疗组在软骨损坏方面明显减轻,甲苯胺蓝部分失染。结论:①壳聚糖-DNA超微颗粒的转染效率与基因产品有关。②将IL-1Ra用关节腔直接注射壳聚糖-DNA超微颗粒的方法直接转移入关节腔能一定程度上减轻骨性关节炎的进程。
AIM： To analyze the transgenic effect of chitosan-DNA nanoparticles in knee joints.
METHODS： The experiment was accomplished in the Laboratory of Cell and Molecular Biology, Department of Orthopedics, Institute of Health Science, Medical College of Shanghai Jiao Tong University between September 2005 and June 2006. The operation model of osteoarthritis （OA） was established with excision of the medial collateral ligament plus medial meniscectomy. Gene products： interleukin-1Ra （IL-1Ra）, IL 10. Fifteen white New Zealand rabbits were used in this study and then divided into three groups randomly. （1）Rabbits in Group 1 （placebo, n =3） received three consecutive intra-articular injections at 48-hour intervals of chitosan-DNA nanoparticles （400 μL） containing PcDNA3.1（2） Rabbits in Group 2 （control, n =6）： The controlled knee received injections of 400 μL chitosan-DNA nanoparticles containing 20 μg PcDNA3.1-plasmid （chitosan-1L-1Ra）, while the lesioned knee was injected with the same volume of sodium sulfate buffer containing 20 μg PcDNA3.1-1L-1Ra plasmid. （3）Rabbits in Group 3 （experiment, n =6）： The controlled knee received injections of 400 μL chitosan-DNA nanoparticles containing 20 μg PcDNA3.1-1L-10 （chitosan-1L-10）, while the lesioned knee was injected with the same volume of sodium sulfate buffer containing 20 μg PcDNA3.1-1L-10 plasmid. All injections were given 5 days post-surgery. The injection times and intervals were identical with those of Group 1. Both the expression and distribution of IL-1Ra and IL-10 in synovial fluid were tested through enzyme-linked immunosorbent assay and immunohistochemical analysis. Hematoxylin-eosin staining and toluidine blue staining ware applied for the histological examination of OA cartilage.
RESULTS： All the fifteen New Zealand rabbits were analyzed in the result. （1）Expression of IL-1Ra and IL-10 in synovial fluid： A detectable level of human IL-1Ra was found in the lesioned rabbit knees at 7, 14 days after first injection. On the contrary, no clear expression of IL-1Ra was detected in the controlled knee joint synovial fluid in Group 2 and placebo group. However, human IL-10 was not detectable in all of the rabbit knees in Group 3.（2）Distribution of IL-1 Ra in rabbit chondrocyte： A small group of chondrocytes in a few areas of the superficial and middle zones were found to stain positive for human IL-1Ra. And the expression of foreign gene would last at least 14 days after first injection. The synovial membrane specimens of the rabbit knees given chitosan-1L-1Ra nanoparticle injection were negative for human IL-1Ra. （3）Histological change of OA cartilage： Specimens from the placebo group showed morphologic characteristic change of early OA. Hematoxylin-eosin staining showed chondrocyte death, and toluidine blue staining for proteoglycan was distributed unevenly, the areas in the superficial and intermediate layers mostly destained and only the areas around cell clusters still darkly stained. Treatment of joints with chitosan-1L-1Ra nanoparticles had less severe lesions of cartilage compared with the control. Toluidine blue staining showed partially destaining.
CONCLUSION： （1）The transfection efficiency of chitosan-DNA nanoparticles is closely related to the gene product. （2）A reduction degree of OA severity is also noted in the histologic cartilage lesions of the group that receive the chitosan-1L-1Ra injection.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第9期1613-1616,1623,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
上海市自然科学基金资助(04ZR14064)
武警浙江总队医院横向合作项目(0536J1A181)~~
