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

作　　者：赛佳明[1] 马学晓[1] 邱晨生 陈伯华[1] 胡有谷[1] 

机构地区：[1]青岛大学附属医院脊柱外科,山东省青岛市266003

出　　处：《中国脊柱脊髓杂志》2015年第12期1090-1094,共5页Chinese Journal of Spine and Spinal Cord

基　　金：国家自然科学基金资助项目(编号:81171758)

摘　　要：目的 ：研究慢病毒载体GV115介导Caspase-3 si RNA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 si RNA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 si RNA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。Objectives： To explore the biological effects of GV115-Caspase3 siRNA on human intervertebral disc nucleus pulposus（h IDNP） cells. Methods： The h IDNP cells were collected from 12 patients with traumatic vertebral burst fracture（22-36 years old）. The h IDNP cells were isolated and subcultured by using tissue culture technique. The cells of second generation were grouped into GV115-Caspase3 si RNA group, GV115 group and control group. The h IDNP cells were transfected by GV115-Caspase3 siRNA and detected by the immunofluence method. The viability of h IDNP cells was detected by MTT method. The synthesis of collagen type Ⅱ and glycosaminoglycan were detected by Western-Blot and Antonopulos methods. Results： The h IDNP cells were successfully isolated and cultured. After one week, the h IDNP 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 si RNA 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 h IDNP cells, enhance the cells ′ viability and promote the synthesis of extracellular matrix.

关 键 词：髓核细胞 慢病毒 CASPASE-3 RNA干扰 基因治疗 

分 类 号：R681.5[医药卫生—骨科学] Q813[医药卫生—外科学]