Sry基因监测腺病毒介导肝细胞生长因子修饰的骨髓间充质干细胞移植在大鼠烫伤创面愈合中的作用  

Study on evaluating Sry as an engrafting track of bone mesenchymal stem cells modified with adenoviral vector expressing human hepatocyte growth factor for healing of burn wounds

作　　者：他维玮[1] 哈小琴[2] 赵曼[1] 刘国军[1] 张尚弟[2] 

机构地区：[1]兰州大学第二医院检验科,730050 [2]兰州军区兰州总医院实验科、甘肃省干细胞与基因药物重点实验室

出　　处：《中国医药》2011年第12期1452-1455,共4页China Medicine

基　　金：全军医学科学技术研究“十一五”计划面上课题（06MB097）

摘　　要：目的制备地高辛标记的Sry探针，监测局部异体移植携带人肝细胞生长因子基因的重组腺病毒（Ad—HGF）修饰的骨髓间充质干细胞（BMSCs）在烫伤创面愈合中的作用。方法用雄性大鼠肝组织通过聚合酶链反应法克隆Sry基因，并将其构建至TEasyVector上，用地高辛标记法制备Sry基因原位杂交探针。密度梯度离心法体外分离培养雄性Wistar大鼠BMSCs，并转染Ad-HGF。复制16只背部深Ⅱ度烫伤雌性大鼠模型，于造模成功后，将Ad-HGF修饰的BMSCs一次性多点注射于创面真皮下。21d后，处死大鼠，取愈合创面新生组织和正常组织制作石蜡切片，原位杂交法检测组织切片中Sty基因的表达。结果①从8只雄性大鼠中成功克隆出Sry基因；②制备出地高辛标记的Sry探针；③16只雌性大鼠深Ⅱ度烧伤模型复制成功，用Ad-HGF修饰的BMSCs治疗后，原位杂交法证实全部雌鼠创面新生组织中有Sry表达，而正常组织中未检测到。结论外源移植的BMSCs参与了大鼠烫伤创面的修复，Sry基因可作为细胞追踪标记，为细胞治疗追踪技术及利用BMSCs治疗创伤的研究提供依据。Objective To prepare digoxin labeled cDNA probe of rat sex determining region of the Y （Sry） and to evaluate the effects of bone mesenchymal stem cells （BMSCs）with transfection of adenoviral vector expressing human hepatocyte growth factor （Ad-HGF） after heterogenous grafting on healing of burn wounds. Methods Sry gene from liver tissue of male rat was cloned by polymerase chain reaction method and constructed to T Easy Vector, prepared in situ hybridization cDNA probe of Sry labeled with Digoxin. BMSCs of male Wistar rat were isolated and cultured through density gradient centrifugation and prepared BMSCs with transfection of Ad-HGF. Sixteen female rats were subjected to boiling water for 12 seconds to produce partial skin burn（ pathologic grade 11 ）, then injected BMSCs modified with Ad-HGF into underplayed dermis of wound surface. The expressing of Sry gene in histological section through iN situ hybridization technique was detected. Results ① Sry gene from 8 male rats was successfully cloned ; ②Digoxin labeled cDNA probe of Sry was prepared; ③through in situ hybridization technique, the expression of Sty in cambium tissue of all the 16 female rats＇ wound surface was positive,while in the normal tissue it was negative. Conclusions Exogenous BMSCs participates repair of wound. Sry can be used as a cell trace maker. This experiment offers reference data for tracing technique of cell therapy and clinical application of BMSCs.

关 键 词：创伤 骨髓间充质干细胞 SRY基因 聚合酶链反应 DNA探针 

分 类 号：R641[医药卫生—外科学]