摘要
目的构建Bcl-2基因修饰慢病毒质粒,建立稳定表达Bcl-2基因的骨髓间充质干细胞系,探讨Bcl-2基因修饰的骨髓闸充质干细胞治疗ALF的可行性。方法 PCR合成Bcl-2基因片段并插入GV287空载体后扩增进行慢病毒包装,使用Bcl-2慢病毒液转染大鼠骨髓间充质干细胞,使用单纯未处理骨髓间充质干细胞,移植急性肝损伤模型大鼠,观察移植后大鼠存活情况及其肝脏病理改变。结果得到纯化的骨髓间充质干细胞细胞株;成功分离Bcl-2基因DNA片段并构建Bcl-2基因重组慢病毒质粒,并成功转染到骨髓间充质干细胞中,经荧光显微镜检测GFP荧光及免疫印迹蛋白检测,转染后细胞可高表达Bcl-2基因,转染后细胞状态较好,可进行移植实验;模型鼠经过尾静脉移植骨髓间充质于细胞后,共聚焦显微镜下观察肝脏冰冻切片病理,骨髓间充质干细胞,可以定植到损伤肝脏组织。结论初步证实,骨髓间充质干细胞能够在急性肝损伤大鼠肝脏归巢,并且能发挥一定程度治疗作用。
Objective To construct Bcl-2 gene-modified lent ivirus, establish stable expression of Bcl-2 gene in bone marrow mesenchymal stem cell lines,and discuss the feasibility of bone marrow Bcl-2 gene-modified mesenchymal stem cells therapy in acute liver failure （ALF ） to the problems of liver apoptosis and regeneration. Methods Bcl-2 gene fragment synthesized by polymerase chain reaction （PCR） was inserted into GV287 empty vector to be amplified and packaged in lentivirus. Bcl-2 lentivirus were transfected into rat bone marrow mesenchymal stem cells. Then bone marrow mesenchymal stem cells were transplanted into rats with acute liver in ju ry , and survival rates and liver pathology were observed after transplantation. Results The purified bone marrow mesenchymal stem cells lines were obtained. Bcl-2 gene fragments were separated for construction of Bcl-2 gene lentivirus, which was transfected into bone marrow mesenchymal stem cells successfully. Through detecting by green fluorescent protein （GFP） fluorescence and western b lo t, transfected cells were in good condition with high expression of Bcl-2. Rat models were transplanted with bone marrow mesenchymal stem cells through tail vein, and PKH 26 fluorescence-labeled bone marrow mesenchymal stem cells can be detected in damaged liver tissue by liver pathology frozen section after transplantation. Conclusion We preliminarily confirm that bone marrow mesenchymal stem cells can home to liver tissue in rats with acute liver injury with certain therapeutic effects.
出处
《肝脏》
2017年第1期27-31,共5页
Chinese Hepatology
基金
黑龙江省自然科学基金项目(D201106)面上项目
