微量注射不同载体转基因至损伤肌腱的效率、分布和组织反应

Efficiency and distribution of gene delivery to the injured tendons by microinjection and tissue reactions caused by vectors

目的探索微量注射法以不同转基因载体转基因至损伤肌腱的转基因效率、分布和组织反应。方法用微量注射器将10μl携带增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）报告基因的pCMV—EGFP、pCAGGS—EGFP、AAV2-EGFP和AdS-EGFP载体直接注射入鸡的损伤并缝合的趾深屈肌腱。在术后第3，7，14，21天分别取肌腱，进行冰冻切片后在荧光显微镜下观察肌腱内的EGFP表达量及分布，并将肌腱切片作HE染色后观察各不同载体在肌腱中引起的炎症反应。结果荧光显微镜下观测发现，注射4种转基因载体的肌腱，转染3d后即可见肌腱内有增强绿色荧光蛋白表达；在第7天时表达最明显；第14天时，各组表达增强绿色荧光蛋白表达量下降；21d时很少有表达增强绿色荧光蛋白的细胞。用微量注射器在腱段两点注射能保证近损伤肌腱内转染细胞均匀分布。注射AAV2-EGFP和AdS-EGFP的肌腱的增强绿色荧光蛋白明显多于质粒载体的增强绿色荧光蛋白，而AAV2-EGFP和Ad5-EGFP两组间无明显差异。HE染色发现质粒载体和AdS—EGFP对肌腱的组织反应较重，可见较多炎症细胞浸润，以淋巴细胞和中性粒细胞为主。注射AAV2-EGFP的肌腱炎症反应较轻。结论用微量注射器在腱段两点注射转基因载体能转染近损伤处的整个肌腱段的细胞。在研究的4种基因治疗方法中，AAV2和AdS在损伤肌腱中的转基因效率最强，在转基因后第7天表达最明显，而且AAV2引起的肌腱组织反应最轻。提示AAV2载体比AdS和质粒载体更有利于作为转基因治疗肌腱损伤的载体，微量注射载体是转基因至损伤肌腱的合适方法。

Objective To investigate efficiency and distribution of gene delivery to the injured tendons by microinjection and tissue reactions caused by different vectors. Methods By using a mi- croinjection technique, 10μl of pCMV-EGFP, pCAGGS-EGFP, AAV2-EGFP and Ad5-EGFP harboring enhanced green fluorescence protein （EGFP） gene were respectively injected to two sites of the proximal stump of 48 transected digital flexor tendons in 18 chickens. At days 3, 7, 14 and 21, the tendons were harvested for observing distribution and expression of EGFP under a fluorescence microscope by using fro- zen tissue sections. The tendon sections were also stained with hematoxylin and eosin to examine inflam- mation caused by these vectors. The other 24 normal flexor tendons were served as controls. Results Compared with normal tendon tissues, the EGFP expression was observed in tendons at days 3, 7, 14 and 21 after injection. The EGFP expression was observed at day 3 and reached peak at day 7 for all vectors. The EGFP expression was decreased at day 14 but seldom seen at day 21. EGFP was distributed evenly in the injected tendon segment adjacent to the cut level. The EGFP expression in the tendons injected with AAV2-EGFP and Ad5-EGFP was higher than that with pCMV-ECFP and pCAGGS-EGFP injection, with insignificant statistical difference upon the EGFP expression between AAV2-EGFP and AdS-EGFP vectors. Tissue reactions of the tendons caused by the liposome-plasmid vector （including pCMV-EGFP and pCAGGS-EGFP） were the most prominent among all vectors. Infiltration of inflammatory cells, chiefly lymphocytes and neutrophilic granulocytes, were found. The tendons injected with AAV2 vectors presented gentle inflammatory reactions. Conclusions Microinjection to two sites of each tendon stump delivers the transgene to the entire tendon segment adjacent to the cut. Gene delivery by the AAV2 and AdS vectors has the highest efficiency among four vectors tested, when expression level peaks at day 7 after injection and AAV2 vector causes the slightest tissue reactions in the tendons, indicating that the AAV2 vector is a promising gene delivery vector and microinjection is practical for tendon gene therapy.