人基质金属蛋白酶3基因RNA慢病毒载体构建及在人退变髓核细胞中的鉴定（英文）

Constructing and identifying a lentiviral vector of RNA interference targeting matrix metalloproteinases-3 gene in human degenerative nucleus pulposus cells

背景：抑制椎间盘细胞外基质的降解可以延缓椎间盘退变的进程,基质金属蛋白酶3是降解Ⅱ型胶原和蛋白多糖细胞外基质成分的关键酶。目的：构建人基质金属蛋白酶3特异性shR NA重组慢病毒载体,感染人退变的髓核细胞,鉴定干扰效果。方法：根据基质金属蛋白酶3基因mR NA序列,设计4组靶向基质金属蛋白酶3基因的短发夹RNA序列,合成、退火形成双链DNA片段,与经过DNA内切酶Bam HⅠ、EcoRⅠ双酶切后的LV3载体连接转入感受态细胞,对长出的克隆进行菌落PCR鉴定,并对阳性克隆进行测序及比对分析。通过转染293T细胞对慢病毒进行包装和滴度测定。用慢病毒质粒载体感染人退变的髓核细胞,荧光显微镜观察感染率,在72和96 h使用实时PCR和Western blot检测干扰效果。结果与结论：（1）酶切和测序两种方法结果证明4种质粒载体的插入序列完全正确,慢病毒载体构建成功并获得相应的慢病毒,病毒悬液的滴度为（1-5）×10~8 TU/m L。（2）含有GCC AGG CTT TCC CAA GCA AAT干扰序列的干扰效率最高,72和96 h基质金属蛋白酶3基因表达水平干扰分别为98%和72%,96 h基质金属蛋白酶3蛋白水平干扰57.2%。（3）实验成功构建了靶向人基质金属蛋白酶3基因RNA干扰慢病毒载体,为进一步研究基质金属蛋白酶3功能和用慢病毒进行基因治疗奠定了基础。

BACKGROUND： Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3（MMP3） is considered as a key enzyme for degradation of extracellular matrix components such as type II collagen and aggrecan. OBJECTIVE： To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells. METHODS： According to the human MMP3 m RNA（NM_002422.4） sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by Bam HI and EcoR I enzymes, and then transfected into the competent cells. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293 T cells. Human degenerative nucleus pulposus cells were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours. RESULTS AND CONCLUSION： The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293 T cells with a viral titer of 1-5 ×10~8 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 m RNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfully constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.