慢病毒载体GV115介导Caspase-3 siRNA转染人椎间盘髓核细胞的生物学效应

Biological effects of GV115-Caspase3 siRNA on human intervertebral disc nucleus pulpous cells

中文关键词:  髓核细胞  慢病毒  Caspase-3  RNA干扰  基因治疗

英文关键词:Nucleus pulposus cell  Lentivirus  Caspase-3  RNAi  Gene therapy

中文摘要:

  【摘要】 目的：研究慢病毒载体GV115介导Caspase-3 siRNA转染人椎间盘髓核细胞的生物学效应。方法：采集12例外伤致脊柱爆裂性骨折患者（22~36岁）术中切除的椎间盘髓核组织，采用组织块法分离培养髓核细胞并传代。取第2代髓核细胞，分为GV115-Caspase3 siRNA组、GV115组和对照组，各组12个细胞培养孔。在荧光显微镜下观察计数阳性髓核细胞，计算GV115-Caspase3 siRNA对人椎间盘髓核细胞的转染效率；采用免疫荧光法检测三组髓核细胞中Caspase-3表达；采用MTT法检测三组髓核细胞活性；采用Western-Blot法和Antonopulos法分别检测三组髓核细胞的Ⅱ型胶原和蛋白多糖含量。结果：椎间盘髓核细胞被成功分离培养，培养1周后细胞达到80%融合，进行传代培养。转染后1周（85.6±1.3）％的髓核细胞可被GV115-Caspase3 siRNA转染而表达绿色荧光素。GV115-Caspase3 siRNA组Caspase-3免疫荧光阳性细胞率[（19.4±3.2）%]较GV115组[（84.3±9.2）%]和对照组[（83.9±8.7）%]明显减少（P＜0.05），OD值（1.56±0.21）较GV115组（0.91±0.15）和对照组（0.92±0.17）高（P＜0.05），Ⅱ型胶原免疫印迹染色强度（1.32±0.09）较GV115组（0.81±0.05）和对照组（0.79±0.04）高（P＜0.05），蛋白多糖含量（0.56±0.09） 较GV115组（0.35±0.06）和对照组（0.34±0.05）高（P＜0.05）。结论：GV115-Caspase3 siRNA可高效转染人椎间盘髓核细胞，增强髓核细胞的生物活性，并促进细胞外基质的合成。

英文摘要:

  【Abstract】 Objectives: To explore the biological effects of GV115-Caspase3 siRNA on human intervertebral disc nucleus pulposus(hIDNP) cells. Methods: The hIDNP cells were collected from 12 patients with traumatic vertebral burst fracture(22-36 years old). The hIDNP cells were isolated and subcultured by using tissue culture technique. The cells of second generation were grouped into GV115-Caspase3 siRNA group, GV115 group and control group. The hIDNP cells were transfected by GV115-Caspase3 siRNA and detected by the immunofluence method. The viability of hIDNP cells was detected by MTT method. The synthesis of collagen type Ⅱ and glycosaminoglycan were detected by Western-Blot and Antonopulos methods. Results: The hIDNP cells were successfully isolated and cultured. After one week, the hIDNP cells reached 80% confluence, which were subcultured and transfected by GV115-Caspase3 siRNA, and the percentage of positive cells was (85.6±1.3)％ under a fluorescence microscope. The Caspase-3 expression in GV115-Caspase3 siRNA group[(19.4±3.2)%] was less than that in the GV115 group[(84.3±9.2)%] or the control group[(83.9±8.7)%], the difference was statistically significant(P＜0.05). The OD value of GV115-Caspase3 siRNA group(1.56±0.21) was higher than that of GV115 group(0.91±0.15) or control group(0.92±0.17), the difference was statistically significant(P＜0.05). The collagen type Ⅱ immunoblotting staining intensity of GV115-Caspase3 siRNA group(1.32±0.09) was higher than that of GV115 group(0.81±0.05) or control group(0.79±0.04). The glycosaminoglycan content of GV115-Caspase3 siRNA group(0.56±0.09) was higher than that of GV115 group(0.35±0.06) or control group(0.34±0.05), the difference was statistically significant. Conclusions: GV115-Caspase3 siRNA can efficiently transfect the hIDNP cells, enhance the cells′ viability and promote the synthesis of extracellular matrix.