中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2009年
8期
841-845
,共5页
刘勇%陈二涛%冯东福%潘栋超%汪洋
劉勇%陳二濤%馮東福%潘棟超%汪洋
류용%진이도%풍동복%반동초%왕양
脑源性神经营养因子%干细胞%转染%视网膜
腦源性神經營養因子%榦細胞%轉染%視網膜
뇌원성신경영양인자%간세포%전염%시망막
Brain-derived neurotrophic factor%Stem cell%Transfection%Retina
目的 探讨移植hBDNF-GFP基因转染神经干细胞后对视神经损伤大鼠视网膜BDNF表达的影响.方法 ①78只SD大鼠随机分为正常对照组(n=6)、视神经夹伤组(n=72).视神经夹伤组动物行右侧视神经夹伤后随机平均分为三个亚组:分别于玻璃体腔内注射0.01 mol/L PBS(PBS组),GFP基因转染神经干细胞(GFP-NSCs组)和hBDNF-GFP基因转染神经干细胞(hBDNF-GFP-NSCs组).各组动物单纯暴露左侧视神经作为假损伤组.分别于移植后3 d,7 d,14 d和28 d处死动物取视网膜,ELISA法检测各组视网膜中BDNF含量.②另取6只视神经损伤大鼠移植hBDNF-GFP-NSCs,分别于2周、4周和8周各处死2只大鼠,取眼球做冰冻切片观察移植细胞存活、迁移情况.③35只sD大鼠随机分为4组:正常对照组(n=5)、右眼视神经损伤NSCs治疗组(n=10)、GFP-NSCs治疗组(n=10)及hBDNF-GFP-NSCs治疗组(n=10).④各组于术后4周和8周分别处死5只大鼠,West-era-blot法检测视网膜组织中BDNF表达.结果 ①ELISA结果显示,正常对照组BDNF含量与假损伤组间比较差异无统计学意义(P>0.05),移植手术后第3天时,三个损伤亚组视网膜匀浆上清中hBDNF含量明显增加(与假损伤组比较P<0.05),而三组间差异无统计学意义(P>0.05);7 d时,GFP-NSCs组与假损伤组比较差异具有统计学意义(P<0.05),而PBS组、hBDNF-GFP-NSCs组与假损伤组比较差异无统计学意义(P>0.05),PBS组与GFP-NSCs组间及hBDNF-GFP-NSCs组与GFP-NSCs组间差异具有统计学意义(P<0.05);移植第14天和28天时,PBS组和GFP-NSCs组中视网膜BDNF含量降低,而hBDNF-GFP-NSCs组中BDNF含量与其他三组比较明显升高,差异有统计学意义(P<0.05).②冰冻切片示移植后,hBDNF-GFP-NSCs可在宿主视网膜内存活,并逐渐迁移至视网膜各层.③Western-blot检测显示,各组内第4周和第8周BDNF表达差异无统计学意义,移植hBDNF-GFP-NSCs组视网膜组织中BDNF表达明显高于于其他各组.结论 hBDNF-GFP基因转染神经干细胞移植后可在宿主视网膜长期存活,且可以稳定高表达BDNF.
目的 探討移植hBDNF-GFP基因轉染神經榦細胞後對視神經損傷大鼠視網膜BDNF錶達的影響.方法 ①78隻SD大鼠隨機分為正常對照組(n=6)、視神經夾傷組(n=72).視神經夾傷組動物行右側視神經夾傷後隨機平均分為三箇亞組:分彆于玻璃體腔內註射0.01 mol/L PBS(PBS組),GFP基因轉染神經榦細胞(GFP-NSCs組)和hBDNF-GFP基因轉染神經榦細胞(hBDNF-GFP-NSCs組).各組動物單純暴露左側視神經作為假損傷組.分彆于移植後3 d,7 d,14 d和28 d處死動物取視網膜,ELISA法檢測各組視網膜中BDNF含量.②另取6隻視神經損傷大鼠移植hBDNF-GFP-NSCs,分彆于2週、4週和8週各處死2隻大鼠,取眼毬做冰凍切片觀察移植細胞存活、遷移情況.③35隻sD大鼠隨機分為4組:正常對照組(n=5)、右眼視神經損傷NSCs治療組(n=10)、GFP-NSCs治療組(n=10)及hBDNF-GFP-NSCs治療組(n=10).④各組于術後4週和8週分彆處死5隻大鼠,West-era-blot法檢測視網膜組織中BDNF錶達.結果 ①ELISA結果顯示,正常對照組BDNF含量與假損傷組間比較差異無統計學意義(P>0.05),移植手術後第3天時,三箇損傷亞組視網膜勻漿上清中hBDNF含量明顯增加(與假損傷組比較P<0.05),而三組間差異無統計學意義(P>0.05);7 d時,GFP-NSCs組與假損傷組比較差異具有統計學意義(P<0.05),而PBS組、hBDNF-GFP-NSCs組與假損傷組比較差異無統計學意義(P>0.05),PBS組與GFP-NSCs組間及hBDNF-GFP-NSCs組與GFP-NSCs組間差異具有統計學意義(P<0.05);移植第14天和28天時,PBS組和GFP-NSCs組中視網膜BDNF含量降低,而hBDNF-GFP-NSCs組中BDNF含量與其他三組比較明顯升高,差異有統計學意義(P<0.05).②冰凍切片示移植後,hBDNF-GFP-NSCs可在宿主視網膜內存活,併逐漸遷移至視網膜各層.③Western-blot檢測顯示,各組內第4週和第8週BDNF錶達差異無統計學意義,移植hBDNF-GFP-NSCs組視網膜組織中BDNF錶達明顯高于于其他各組.結論 hBDNF-GFP基因轉染神經榦細胞移植後可在宿主視網膜長期存活,且可以穩定高錶達BDNF.
목적 탐토이식hBDNF-GFP기인전염신경간세포후대시신경손상대서시망막BDNF표체적영향.방법 ①78지SD대서수궤분위정상대조조(n=6)、시신경협상조(n=72).시신경협상조동물행우측시신경협상후수궤평균분위삼개아조:분별우파리체강내주사0.01 mol/L PBS(PBS조),GFP기인전염신경간세포(GFP-NSCs조)화hBDNF-GFP기인전염신경간세포(hBDNF-GFP-NSCs조).각조동물단순폭로좌측시신경작위가손상조.분별우이식후3 d,7 d,14 d화28 d처사동물취시망막,ELISA법검측각조시망막중BDNF함량.②령취6지시신경손상대서이식hBDNF-GFP-NSCs,분별우2주、4주화8주각처사2지대서,취안구주빙동절편관찰이식세포존활、천이정황.③35지sD대서수궤분위4조:정상대조조(n=5)、우안시신경손상NSCs치료조(n=10)、GFP-NSCs치료조(n=10)급hBDNF-GFP-NSCs치료조(n=10).④각조우술후4주화8주분별처사5지대서,West-era-blot법검측시망막조직중BDNF표체.결과 ①ELISA결과현시,정상대조조BDNF함량여가손상조간비교차이무통계학의의(P>0.05),이식수술후제3천시,삼개손상아조시망막균장상청중hBDNF함량명현증가(여가손상조비교P<0.05),이삼조간차이무통계학의의(P>0.05);7 d시,GFP-NSCs조여가손상조비교차이구유통계학의의(P<0.05),이PBS조、hBDNF-GFP-NSCs조여가손상조비교차이무통계학의의(P>0.05),PBS조여GFP-NSCs조간급hBDNF-GFP-NSCs조여GFP-NSCs조간차이구유통계학의의(P<0.05);이식제14천화28천시,PBS조화GFP-NSCs조중시망막BDNF함량강저,이hBDNF-GFP-NSCs조중BDNF함량여기타삼조비교명현승고,차이유통계학의의(P<0.05).②빙동절편시이식후,hBDNF-GFP-NSCs가재숙주시망막내존활,병축점천이지시망막각층.③Western-blot검측현시,각조내제4주화제8주BDNF표체차이무통계학의의,이식hBDNF-GFP-NSCs조시망막조직중BDNF표체명현고우우기타각조.결론 hBDNF-GFP기인전염신경간세포이식후가재숙주시망막장기존활,차가이은정고표체BDNF.
Objective To investigate the effects of human Rrain-derived neurotrophic factor-gamma fetopro-tein (hBDNF-GFP) gene-transfected neural stem cell (NSC) transplantation on BDNF expressions in the retina of rots after optic nerve (ON) crush injury. Method ①Seventy-eight Sprague-Dawley (SD) rats were randomly as-signed into a control group (n = 6) and ON crush group (n = 72). In the ON crush group, the right ON was crushed while the left NO was exposed as sham injury. Rats in the ON crush group were divided into three sub-groups: PBS group (intravitreons injection of 0.01 mol/L phosphate buffered solution); GFP group (intravitreous transplantation of GFP gene-transfected NSCs); and hBDNF-GFP group (intravitreous transplantation of hBDNF-GFP gene-transfected NSCs). Rats were sacrificed 3, 7, 14 and 28 days after transplantation, and BDNF expres-sions in retinal homogenates was detected by using enzyme-linked immunosorbent assay (ELISA). ②The hBDNF-GFP-NSCs were transplanted intravitreous into six rats after ON crush injury. Following this, two rats were sacri-riced 2, 4 and 8 weeks after transplantation. The survival and location of NSCs in host retina were observed by frozen section analysis. ③Adult SD rats were randomly divided into four groups: control group (n = 5); NSC group (NSC transplantation, n = 10); GFP-NSC group (GFP-NSC transplantation, n = 10); and hBDNF-GFP-NSC group (hBDNF-GFP-NSC transplantation, n = 10). Four and eight weeks after transplantation, five rats from every group were sacrificed. Western blot analysis was used to determine retinal BDNF expression. Results ① There was no significant difference in BDNF expression between the control group and sham-injury groups (P >0.05). Three days after NSC transplantation, BDNF expression increased significantly in the three injured sub-groups compared with the sham-injury group, (P < 0.05), whereas no significant inter-group differences in BDNF expressions among three injured sub-groups were observed (P > 0.05). Seven days after transplantation, there was a significant difference in BDNF expression between the GFP-NSC group and the sham-injury groups (P <0.05), whereas there were no significant differences in BDNF expressions among the PBS, hBDNF-GFP-NSC and sham-injury groups (P > 0.05). Fourteen and 28 days after transplantation, BDNF expressions decreased in the PBS group and the GFP-NSC groups, while BDNF expressions in the hBDNF-GFP-NSC group increased significant-ly compared with the other three groups (P < 0.05); ②Frozen section showed that transplanted hBDNF-GFP-NSCs could survive and gradually extended to all layers of the host retina. ③Westem blot revealed there were no differences in BDNF expressions between 4-week and 8-week intervals in the hBDNF-GFP-NSC group. Compared with other three groups, BDNF expressions in the retina increased significantly after hBDNF-GFP-NSC transplanta-tion. Conclusions The hBDNF-GFP gene-trausfected NSCs can survive in the host retina and BDNF expressions are stable at a high level.
