Study On Construction And Identification Of The Recombinant Lentivirus Vector Expressing Shrna Of Rat Vsmc Stat3

Objective To investigate the primary culture method of rat thoracic aorta vascular smooth muscle cell (VSMC), analyze cell growth and biological characteristics and provide test material for the research of the proliferation of vascular diseases. To construct short hairpin RNA (shRNA) recombinant lentivirus vectors selectively silencing STAT3 gene. Methods The culture of rat thoracic aorta VSMC was done by tissue-piece inoculation. The cultured cells were observed and identified by morphology and immunohistochemistry with phase contrast microscope and immunohistochemical staining respectively. The cell viability was determined with Typan blue. Four oligonucleotides targeting of STAT3 gene were synthesized and cloned into lentivirus vector PLKO.1. The recombinant vector PLKO.1-STAT3-shRNA, packaging vector (pvsv-g) and envelope vector (pax-2) were cotransfected into the 293T cells by using lipofectamine reagent. Harvest media from cells and filter the lentivirus through a 0.45μm filter to remove the cells. The viral titer was checked by green fluorescent protein (GFP). After infected with the recombinant lentivirus, the protein expression of STAT3 in rat thoracic aorta VSMC was detected by Western blot. The gene silencing efficiency was measured by Western blotting. Results 85% inoculated tissue pieces survived. Primary cultured cells could be subcultured after 14 days and successfully passaged for more than 8 times, without noticeable changes in morphology and growth characteristics. The purity of the sixth passage VSMC was more than 97%. The cell viability was more than 94% by trypan blue. The cultured VSMC showed the typical "peak and valley" morphological appearance under microscope. Immunohistochemical staining with antibody against SM-α-actin demonstrated these cells were positive. Four shRNA expression vectors were correct by restricted endonuclease analysis and partial nucleotide sequencing. PLKO.1-STAT3-shRNA knocked down the expression of STAT3 protein dramatically by Western blotting. Conclusion The method of tissue-piece inoculation culture of rat thoracic aorta VSMC is simple, economic and efficient. It provides an ideal cell model for the research of the proliferation of vascular diseases. The recombinant lentivirus vectors expressing shRNA of STAT3 were constructed successfully. The results of this study lay the foundation for further studying on the role of JAK/ STAT3 signaling pathway in the proliferation of vascular diseases.