课题背景：在利用病毒载体的基因治疗中可能发生诱发肿瘤、免疫源性等安全性问题，临床应用存在较大的障碍。聚乙烯亚胺是近年来兴起的新型非病毒基因载体，本实验利用其作为目的基因骨形态发生蛋白7DNA 的载体材料，通过体内转染的方式干预骨折的愈合过程，试图寻找到一条有效增强老年大鼠骨折修复能力的途径。

同行评价：实验利用非病毒载体聚乙烯亚胺体内转染骨形态发生蛋白7基因，发现其可促进老年大鼠骨折愈合。文章选题拟解决当前的科研难题，选题有意义，科研设计较合理，其结果为老年骨折的基因治疗提供了实验依据。

相关链接：聚乙烯亚胺是一种阳离子型聚合物载体，具有较高的基因转染效率。虽然实验证明聚乙烯亚胺自身已是一种高效的基因转入试剂，但给其加上靶向配体还能够增强它对某些细胞系的转染活性，特别适合进行体内转染。聚乙烯亚胺对细胞的毒性主要是由于其对细胞膜有较高的亲和黏附所致，但其毒性比阳离子脂质体的毒性小，且易于通过修饰降低毒性。

摘要
目的：为避免病毒载体带来的安全隐患，利用新型的非病毒载体聚乙烯亚胺体内转染骨形态发生蛋白7基因，观察其对老年大鼠骨折愈合过程的影响，以寻找能够有效促进老年骨折愈合的基因治疗方法。
方法：实验于2006-04/2007-04在解放军第二军医大学细胞生物学实验室和实验动物中心完成。①实验分组：取雄性SD大鼠24只，其中20月龄18只随机分为基因治疗组、生理盐水组和空白对照1组3组，4月龄6只为空白对照2组。②实验方法：所有大鼠建立股骨骨折模型，1周后基因治疗组骨折断端经皮注射聚乙烯亚胺和骨形态发生蛋白7基因转染试剂 250 μL，生理盐水组断端经皮注射相同体积生理盐水，其他2组不加处理。③观察指标：骨折第8周摄X射线片，然后取标本行组织病理切片、Ⅰ型胶原免疫组化染色，观察各组骨折愈合情况。
结果：24只大鼠均进入结果分析。骨折第8周4月龄大鼠骨折基本愈合，3组老年大鼠骨折均未完全愈合，但X射线片、苏木精-伊红染色和Ⅰ型胶原免疫组织化学染色结果显示，与生理盐水组和空白对照1组相比，基因治疗组骨痂明显增多、骨痂骨化过程提前，Ⅰ型胶原染色深、面积较大，说明愈合速度加快、愈合能力改善。
结论：20月龄老年大鼠骨折愈合过程明显延缓，聚乙烯亚胺介导骨形态发生蛋白7基因体内转染的方法可以增强老年大鼠骨折修复能力、促进老年大鼠骨折愈合。
关键词：基因转染；老年骨折； BMP-7；聚乙烯亚胺；组织构建

韩庆林，苟三怀，王良泽，康健.聚乙烯亚胺体内转染骨形态发生蛋白7基因修复老年大鼠的股骨骨折[J].中国组织工程研究与临床康复，2008，12(7):1205-1208 [www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1205(ps).pdf]

In vivo bone morphogenetic protein 7 gene transfection mediated by polyethyleneimine for femoral fracture healing in old rats

Abstract

AIM：To avoid the safety hidden danger by virus vector, non-virus vector polyethyleneimine (PEI) was transfected into bone morphogenetic protein 7 (BMP-7), and its influence on bone fracture healing of old rats was observed, so as to find a effective gene therapy for enhancing the fracture healing in old rats.
METHODS: The experiment was performed in the Cytobiology Laboratory and Experimental Animal Centre of Second Military Medicine University of Chinese PLA from April 2006 to April 2007. ①Twenty-four male SD rats were selected including 18 20-month-old rats, which were divided into gene therapy group, normal saline group and blank control group one (n=6), and 6 4-month-old rats as control group 2. ②Femoral fracture models were established in all rats. One week later, gene therapy group was injected with 250 μL PEI/pcDNA-BMP-7 plasmid complex into fracture site percutaneously, normal saline group injected with matching normal saline by the same way, while the other groups were not given treatment. ③At the 8th week, the healing results were checked through X rays, histological observation and type Ⅰ collagen immunohistochemical staining.
RESULTS: All 24 rats were involved in the result analysis. The 4-month-old rats acquired fracture healing 8 weeks after operation, while the other groups did not. But compared with the normal saline and control group 1, the gene therapy group had more callus around the fracture site and the ossification process was advanced with deep and large type Ⅰ collagen staining, which suggested the fracture was approaching totally healed.
CONCLUSION: Fracture healing is significantly delayded in 20-month-old rats. In vivo hBMP-7 gene transfection mediated by PEI effectively enhances the fracture healing of old rats.

Han QL, Gou SH, Wang LZ, Kang J.In vivo bone morphogenetic protein 7 gene transfection mediated by polyethyleneimine for femoral fracture healing in old rats.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(7):1205-1208(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1205(ps).pdf]