中华神经科杂志
中華神經科雜誌
중화신경과잡지
Chinese Journal of Neurology
2013年
4期
257-264
,共8页
任瑞芳%黄良国%蒋国红%黄名璐%施迎兵
任瑞芳%黃良國%蔣國紅%黃名璐%施迎兵
임서방%황량국%장국홍%황명로%시영병
脑源性神经营养因子%重组,遗传%骨髓移植%间质干细胞移植%慢病毒属%脑出血%大鼠
腦源性神經營養因子%重組,遺傳%骨髓移植%間質榦細胞移植%慢病毒屬%腦齣血%大鼠
뇌원성신경영양인자%중조,유전%골수이식%간질간세포이식%만병독속%뇌출혈%대서
Brain-derived neurotrophic factor%Recombination,genetic%Bone marrow transplantation%Mesenchymal stem cell transplantation%Lentivirus%Cerebral hemorrhage%Rats
目的 观察脑源性神经营养因子(BDNF)基因重组慢病毒转染骨髓间质干细胞(MSCs)移植治疗大鼠脑出血的疗效.方法 分离培养大鼠MSCs；将带有BDNF的慢病毒载体转染MSCs;RT-PCR、蛋白质印迹检测转基因MSCs BDNF基因及蛋白水平表达.制备大鼠脑出血模型,采用抽签法随机分为磷酸盐缓冲液(PBS)组、MSCs组、空病毒转染骨髓问质干细胞(MSCs-EGFP)组、脑源性神经营养因子基因重组慢病毒转染骨髓间质干细胞(MSCs-EGFP-BDNF)组,每组15只.72 h后细胞移植,记录各组细胞移植后7、14、21 d神经功能缺损改善程度；免疫荧光双标检测MSCs脑内迁移和分化情况.结果 MSCs-EGFP-BDNF组BDNF基因及蛋白水平表达明显高于MSCs组及MSCs-EGFP组；与PBS组(7 d:2.0±0.4,14 d:1.7 ±0.2,21 d:1.3±0.2)相比,MSCs组(7 d:1.6±0.2,14 d:1.2 ±0.3,21 d:0.8±0.2)、MSCs-EGFP组(7 d:1.6±0.3,14 d:1.1 ±0.2,21 d:0.8 ±0.3)及MSCs-EGFP-BDNF组(7 d:1.2±0.3,14 d:0.6±0.1,21 d:0.2 ±0.2)大鼠神经功能评分均有不同程度改善(F=6.667、18.417、20.882,均P＜0.05),其中MSCs-EGFP-BDNF组改善最为显著；免疫荧光双标显示MSCs-EGFP-BDNF组胶原纤维酸性蛋白、神经元特异性核蛋白、环核苷酸磷酸二酯酶阳性率明显高于MSCs组及MSCs-EGFP组,而MSCs组与MSCs-EGFP组比较差异无统计学意义.结论 BDNF基因重组慢病毒修饰的MSCs基因及蛋白水平表达均增高；修饰的MSCs移植后可迁移至脑出血灶周围存活并分化表达神经细胞标志物,促进脑出血后神经功能修复.
目的 觀察腦源性神經營養因子(BDNF)基因重組慢病毒轉染骨髓間質榦細胞(MSCs)移植治療大鼠腦齣血的療效.方法 分離培養大鼠MSCs；將帶有BDNF的慢病毒載體轉染MSCs;RT-PCR、蛋白質印跡檢測轉基因MSCs BDNF基因及蛋白水平錶達.製備大鼠腦齣血模型,採用抽籤法隨機分為燐痠鹽緩遲液(PBS)組、MSCs組、空病毒轉染骨髓問質榦細胞(MSCs-EGFP)組、腦源性神經營養因子基因重組慢病毒轉染骨髓間質榦細胞(MSCs-EGFP-BDNF)組,每組15隻.72 h後細胞移植,記錄各組細胞移植後7、14、21 d神經功能缺損改善程度；免疫熒光雙標檢測MSCs腦內遷移和分化情況.結果 MSCs-EGFP-BDNF組BDNF基因及蛋白水平錶達明顯高于MSCs組及MSCs-EGFP組；與PBS組(7 d:2.0±0.4,14 d:1.7 ±0.2,21 d:1.3±0.2)相比,MSCs組(7 d:1.6±0.2,14 d:1.2 ±0.3,21 d:0.8±0.2)、MSCs-EGFP組(7 d:1.6±0.3,14 d:1.1 ±0.2,21 d:0.8 ±0.3)及MSCs-EGFP-BDNF組(7 d:1.2±0.3,14 d:0.6±0.1,21 d:0.2 ±0.2)大鼠神經功能評分均有不同程度改善(F=6.667、18.417、20.882,均P＜0.05),其中MSCs-EGFP-BDNF組改善最為顯著；免疫熒光雙標顯示MSCs-EGFP-BDNF組膠原纖維痠性蛋白、神經元特異性覈蛋白、環覈苷痠燐痠二酯酶暘性率明顯高于MSCs組及MSCs-EGFP組,而MSCs組與MSCs-EGFP組比較差異無統計學意義.結論 BDNF基因重組慢病毒脩飾的MSCs基因及蛋白水平錶達均增高；脩飾的MSCs移植後可遷移至腦齣血竈週圍存活併分化錶達神經細胞標誌物,促進腦齣血後神經功能脩複.
목적 관찰뇌원성신경영양인자(BDNF)기인중조만병독전염골수간질간세포(MSCs)이식치료대서뇌출혈적료효.방법 분리배양대서MSCs；장대유BDNF적만병독재체전염MSCs;RT-PCR、단백질인적검측전기인MSCs BDNF기인급단백수평표체.제비대서뇌출혈모형,채용추첨법수궤분위린산염완충액(PBS)조、MSCs조、공병독전염골수문질간세포(MSCs-EGFP)조、뇌원성신경영양인자기인중조만병독전염골수간질간세포(MSCs-EGFP-BDNF)조,매조15지.72 h후세포이식,기록각조세포이식후7、14、21 d신경공능결손개선정도；면역형광쌍표검측MSCs뇌내천이화분화정황.결과 MSCs-EGFP-BDNF조BDNF기인급단백수평표체명현고우MSCs조급MSCs-EGFP조；여PBS조(7 d:2.0±0.4,14 d:1.7 ±0.2,21 d:1.3±0.2)상비,MSCs조(7 d:1.6±0.2,14 d:1.2 ±0.3,21 d:0.8±0.2)、MSCs-EGFP조(7 d:1.6±0.3,14 d:1.1 ±0.2,21 d:0.8 ±0.3)급MSCs-EGFP-BDNF조(7 d:1.2±0.3,14 d:0.6±0.1,21 d:0.2 ±0.2)대서신경공능평분균유불동정도개선(F=6.667、18.417、20.882,균P＜0.05),기중MSCs-EGFP-BDNF조개선최위현저；면역형광쌍표현시MSCs-EGFP-BDNF조효원섬유산성단백、신경원특이성핵단백、배핵감산린산이지매양성솔명현고우MSCs조급MSCs-EGFP조,이MSCs조여MSCs-EGFP조비교차이무통계학의의.결론 BDNF기인중조만병독수식적MSCs기인급단백수평표체균증고；수식적MSCs이식후가천이지뇌출혈조주위존활병분화표체신경세포표지물,촉진뇌출혈후신경공능수복.
Objective To observe the curative effect of transplantation of mesenchymal stem cells (MSCs) transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor (BDNF) gene on intracerebral hemorrhage in rats.Methods MSCs were isolated from the rat bone marrow,cultured and transfected by recombinant lentiviral vectors carrying BDNF gene.Intracerebral hemorrhagic models were constructed and randomly divided into 4 groups:phosphate buffered saline transplanted (PBS) group,MSCs group,mesenchymal stem cells transfected with empty lentiviral vectors transplanted (MSCs-EGFP) group and mesenchymal stem cells transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor gene transplanted (MSCs-EGFP-BDNF) group.PBS and MSCs were transplanted according to the groups 72 hours after the establishment of models.The improvements of the neurological function were recorded of each group 7 d,14 d,and 21 d after the transplantation.Double labeling immunofluorescent staining were used to detect the migration and the differentiation of transplanted MSCs.Results MSCs-EGFP-BDNF group had significant higher levels of BDNF gene and protein expression than MSCs group and MSCs-EGFP group.All MSCs transplanted groups (MSCs groups:7 d:1.6 ±0.2,14 d:1.2 ±0.3,21 d:0.8 ±0.2; MSCs-EGFP groups:7 d:1.6 ±0.3,14 d:1.1 ±0.2,21 d:0.8 ±0.3; MSCs-EGFP-BDNFgroup:7 d:1.2 ±0.3,14 d:0.6 ±0.1,21 d:0.2±0.2) had more improvements in the neural function (F=6.667,18.417,20.882,all P ＜0.05) than PBS group(7 d:2.0 ±0.4,14 d:1.7 ±0.2,21 d:1.3 ±0.2),and MSCs-EGFP-BDNF group had the most significant improvement.With double labeling immunofluorescent staining,the MSCs-EGFP-BDNF group had significantly higher positive rates of glial fibrilary acidicprotein,neuron specific nuclear protein,2',3 '-cyclic nucleotide 3'-phosphodiesterase than MSCs group and MSCs-EGFP group,while there was no significant differences between the latter two.Conclusions The expression levels of gene and protein are higher for the MSCs modified with recombinant lentiviral vectors carrying BDNF gene.The modified MSCs can migrate to the perihematomal brain issue of intracerebral hemorrhage,express the characteristic molecules of neurons and improve the neural function after intracerebral hemorrhage.
