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

doi:10.3969/j.issn.2095-4344.2016.07.005

Chinese Journal of Tissue Engineering Research ›› 2016, Vol. 20 ›› Issue (7): 947-956.doi: 10.3969/j.issn.2095-4344.2016.07.005

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

Cao Jin, Fu Pei-rong, Fang Jing, Yang Jian-kun, Wei Hua-wei, Li Si-yuan, Gao Feng, Xi Yong-ming

Department of Spine Surgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China

Received:2015-11-24 Online:2016-02-12 Published:2016-02-12
Contact: Xi Yong-ming, M.D., Associate professor, Chief physician, Department of Spine Surgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
About author:Cao Jin, Studying for master’s degree, Department of Spine Surgery, Affiliated Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
Supported by:
the National Natural Science Foundation of China, No. 81470104

Abstract

Abstract:

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 mRNA (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 BamHI and EcoRI 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 293T 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 293T cells with a viral titer of 1-5 ×108 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 mRNA 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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Cao Jin, Fu Pei-rong, Fang Jing, Yang Jian-kun, Wei Hua-wei, Li Si-yuan, Gao Feng, Xi Yong-ming. Constructing and identifying a lentiviral vector of RNA interference targeting matrix metalloproteinases-3 gene in human degenerative nucleus pulposus cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(7): 947-956.

Figures/Tables 6

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

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