同种异体骨髓间充质干细胞移植治疗创伤性脑损伤的遗传安全性评估

Genetic safety evaluation of allogeneic bone marrow mesenchymal stem cells in hosts following traumatic brain injury

目的初步探讨同种异体骨髓间充质干细胞(BMSCs)移植治疗创伤性脑损伤(TBI)的遗传安全性。方法(1)体内实验:分离提取并传代培养雄性SD大鼠的BMSCs。将微量药物注射套管植入并固定于12只雌性SD大鼠左侧侧脑室3 d后,用气压精密打击器对大鼠右侧大脑皮层进行打击以制备成中度TBI模型。分别于造模后4 h、3 d、6 d、9 d、12 d时经套管对TBI大鼠进行单次/多次BMSCs输注(2.5×105个/次,总体积10μL),并分别取造模后48 h、72 h、10 d和14 d时TBI大鼠(每时间点3只)的脑组织制备成石蜡标本,应用免疫荧光染色检测小胶质细胞激活情况,应用RNAscope?技术检测星形胶质细胞、神经元、小胶质细胞与移植的BMSCs的共定位情况,以观察同种异体BMSCs移植至TBI宿主体内后是否与宿主脑内细胞发生整合现象。(2)体外实验:先将冻存复苏的小胶质细胞株BV2用绿色荧光蛋白(GFP)阳性慢病毒颗粒转染,再将pHrodo RED探针预标记的BMSCs与经脂多糖预处理的BV2细胞分别按一定比例(BV2∶BMSCs=1∶1、1∶2、2∶1)进行共培养,36 h、72 h后于共聚焦荧光倒置显微镜下观察2种细胞间是否发生吞噬现象,以观察小胶质细胞对BMSCs的具体作用形式。结果(1)体内实验:造模后48 h、72 h、10 d和14 d时,TBI大鼠的脑组织石蜡切片中均未发现移植的BMSCs与星形胶质细胞、神经元存在共定位现象,但在造模后10、14 d时,TBI大鼠的小胶质细胞明显被激活并迁移至左侧侧脑室及脉络丛附近,且与移植的BMSCs发生共定位现象。(2)体外实验:不同比例的BV2细胞与BMSCs共培养36 h、72 h后均发生吞噬现象。结论同种异体BMSCs移植后并未与TBI宿主的星形胶质细胞、神经元发生整合现象,但其可被小胶质细胞吞噬,表明同种异体BMSCs移植治疗TBI具有遗传安全性。

Objective To investigate the genetic safety of allogeneic bone marrow mesenchymal stem cells(BMSCs)transplantation in traumatic brain injury(TBI).Methods(1)In vivo experiment:BMSCs from male SD rats were isolated and cultured.Moderate TBI models were prepared by implanting and fixing micro-drug injection cannula into the left ventricle of 12 female SD rats,and 3 d after that,striking the right cerebral cortex of the rats with pneumatic precision percussion device was performed.Four h,and 3,6,9,and 12 d after modeling,TBI rats were given a single/multiple BMSCs infusion(2.5×105/time,total volume 10μL)by cannula;48 and 72 h,and 10 and 14 d after modeling,brain tissues of TBI rats(3 at each time point)were prepared into paraffin specimens.Immunofluorescent staining was used to detect the microglia activation,and RNAscope®technology was used to detect the co-localization of astrocytes,neurons,microglia and transplanted BMSCs to observe whether the allogeneic BMSCs were integrated with the host brain cells after transplantation into TBI host.(2)In vitro experiment:the frozen and revived microglial cell line BV2 was transfected with green fluorescent protein(GFP)-positive lentiviral particles,and then,BMSCs prelabeled with pHrodo RED probe and BV2 cells pretreated with lipopolysaccharide were co-cultured in a certain ratio(BV2:BMSCs=1:1,1:2,2:1);after 36 and 72 h of co-culture,the phagocytosis between the 2 kinds of cells was observed under confocal fluorescence inverted microscope to observe the specific action forms of microglia on BMSCs.Results(1)In vivo experiment:48 and 72 h,and 10 and 14 d after modeling,no colocalization of transplanted BMSCs with astrocytes or neurons was found in paraffin sections of brain tissue in TBI rats;however,10 and 14 d after modeling,microglia in TBI rats were obviously activated and migrated to the left lateral ventricle and choroid plexus,and co-localization of microglia with transplanted BMSCs was observed.(2)In vitro experiment:phagocytosis occurred after co-culture of BV2 cells at different proportions with BMSCs for 36 and 72 h.Conclusion After transplantation,allogeneic BMSCs do not integrate with astrocytes or neurons of the TBI host,but they could be phagocytosed by microglia,indicating that allogeneic BMSCs transplantation for TBI is genetically safe.