首页期刊介绍编委会投稿指南期刊订阅广告合作留言板联系我们English
李杰,谢燕燕,王馨,王霖虹,和红霞,孙庆云,晏亚辉,闫振宇.人凝血因子 IX 基因在小鼠脂肪干细胞中的稳定表达[J].中国实验动物学报,2020,28(4):532~538.
人凝血因子 IX 基因在小鼠脂肪干细胞中的稳定表达
Stable expression of human coagulation factor IX in mouse adipose-derived stem cells
投稿时间:2020-02-08  
DOI:10. 3969 / j.issn.1005-4847. 2020. 04. 014
中文关键词:  目的基因  重组腺病毒  脂肪间充质干细胞
英文关键词:target gene  recombinant adenovirus  adipose-derived stem cell(ADSC)
基金项目:
作者单位E-mail
李杰 华北理工大学附属医院血液科,河北 唐山 063000 hblljj2017@ 163.com 
谢燕燕 华北理工大学附属医院血液科,河北 唐山 063000 15081920603@ 163.com 
王馨 华北理工大学附属医院血液科,河北 唐山 063000  
王霖虹 华北理工大学附属医院血液科,河北 唐山 063000  
和红霞 华北理工大学附属医院血液科,河北 唐山 063000  
孙庆云 华北理工大学附属医院血液科,河北 唐山 063000  
晏亚辉 华北理工大学附属医院血液科,河北 唐山 063000  
闫振宇 华北理工大学附属医院血液科,河北 唐山 063000 hbyzy2011@ 163.com 
摘要点击次数: 144
全文下载次数: 38
中文摘要:
      目的 将携带人凝血因子 IX(hFIX)基因(F9)的重组腺病毒转染 C57BL/ 6 小鼠脂肪间充质干细胞 (adipose-derived stem cell,ADSC),经过传代培养后,观察 F9 是否在 ADSC 中稳定表达,探索 ADSC 是否可作为血友病基因治疗的载体细胞。 方法 取 C57BL/ 6 小鼠腹股沟区脂肪,依据组织块悬浮法分离培养小鼠 ADSC 并进行传代培养,应用携带 hFIX 基因并含 GFP 荧光标记的重组腺病毒转染第 3 代 ADSC,转染后再次进行传代培养至第 4、 5 代细胞。荧光显微镜下观察细胞携带荧光数量,RT-PCR 检测 F9 基因表达,ELISA 法检测细胞上清液及 Western Blot 检测细胞内携带目的基因表达蛋白情况。 结果 (1)重组腺病毒转染后荧光显微镜下可见荧光表达,细胞传代后仍具有荧光。 (2)RT-PCR 结果显示:四组 ADSC 均可表达内参 GAPDH 基因片段,A、B、C 组可检测到目的基因 F9 的表达,D 组未检测到 F9 的表达。 (3) ELISA 法检测凝血因子 IX 抗原( hFIX:Ag) 结果显示:A 组( 81. 62 ± 8. 82)ng / mL、B 组(52. 50 ± 3. 25)ng / mL、C 组(47. 41 ± 4. 00)ng / mL 明显高于 D 组检测值(0. 76 ± 0. 44)ng / mL,差 异具有显著性(P< 0. 05)。 (4)Western Blot 法检测四组细胞内 hFIX 蛋白表达情况,结果显示 A 组蛋白表达灰度值 (0. 68 ± 0. 10)、B 组(0. 49 ± 0. 15)、C 组(0. 18 ± 0. 05)明显高于 D 组蛋白表达灰度值(0. 02 ± 0. 01),差异具有显 著性(P < 0. 05)。 结论 重组腺病毒转染 ADSC 后,经过传代后培养,仍可表达较高的 hFIX 活性,可以作为血友病 基因治疗的载体细胞。
英文摘要:
      Objective To observe whether hFIX is stably expressed in adipose-derived stem cells (ADSCs) and to explore whether ADSCs can be used as a vector cell line for hemophilia gene therapy following transformation by recombinant adenoviruses carrying human coagulation factor IX ( hFIX) gene, F9. Methods ADSCs were isolated from C57BL/ 6 mice and cultured by tissue mass suspension. The third generation of ADSCs was transfected by recombinant adenovirus carrying F9 and GFP fluorescence marker. After transfection, the cells were subcultured again to the 4th and 5th passages. The level of fluorescence expressed by the cells was observed by fluorescence microscopy, the expression of F9 was detected by RT-PCR, and the protein expression of hFIX was detected by ELISA and western blotting. Results Fluorescence was observed after recombinant adenoviral transfer, and was still identified after cell passaging. RT-PCR revealed that all four groups of ADSCs could express internal reference GAPDH, but there was no expression of the target gene F9 in group A, and the expression of F9 could be detected in group D. Detection of hFIX:Ag by ELISA showed that the detection values of groups A (81. 62 ± 8. 82) ng / mL, B (52. 50 ± 3. 25) ng / mL, and C (47. 41 ± 4. 00) ng / mL were significantly higher than that of group D (0. 76 ± 0. 44) ng / mL, and there was a significant difference between the two groups (P< 0. 05). hFIX expression in the four groups of ADSCs was detected by western blotting. The result showed that the gray value of histone expression in groups A (0. 68 ± 0. 10), B (0. 49 ± 0. 15) and C was significantly higher than that in group D (0. 02 ± 0. 01), and the difference was statistically significant (P< 0. 05), while the gray value of histone expression in group A was significantly higher than that in group D (P< 0. 05). Conclusions The recombinant adenoviruses were transformed into ADSCs and cultured after passaging, and could still express high hFIX activity, thus demonstrating their potential as vector cells for hemophilia gene therapy.
查看全文  查看/发表评论  下载PDF阅读器
关闭
您是第 1770016 位访问者
版权所有:中国实验动物学会 主管单位:中国科学技术协会 主办单位:中国实验动物学会    中国医学科学院医学实验动物研究所
地  址: 北京市朝阳区潘家园南里5号 邮编:100021 电话:010-67779337 E-mail:bjb2@cnilas.org
本系统由北京勤云科技发展有限公司设计
微信关注二维码