| Currently, with the rapid development of the deer-feeding industry, venison appears inmany European countries as a dining table food. While deer antlers could enhance the bodyimmunity, resist the senility, and promote the wound healing and the bone reparation in thesense of traditional Chinese medicine (TCM), they are widely used for treatment andnourishing. As a unique appendix organ of mammals which could regenerate fully andperiodically, deer antlers are one excellent biological model for the study of organ repair andregeneration. The regeneration of deer antlers could be derived from antler-forming stemcells. The growth rate of antlers could reach to1—2cm/d, approximate that of cancer cells.But the rapid growth does not arouse any canceration, which could provide a new researchmodel for cancer.Galectin-1(Gal-1), a sugar-binding protein with the molecular weight of14kDa, isone type of animal lectins. It could be related to numerous biological processes, e.g. thenerve repair, angiogenesis and cartilage formation. The current research on Gal-1is mainlyfocused on the carcinogenesis and embryonic development process. Regeneration of deerantlers involve growth of nerves, blood vessels and cartilage, etc. The Gal-1content inantler-forming stem cells is different from that in normal cells, and even between differentantler-forming stem cells. Interestingly, the appearance of Gal-1in antler-forming stem cellsdoes not lead to any canceration, which is important for the study of the role played byGal-1in cancer and the deer antler regeneration.The antler-forming stem cells were selected as the experimental materials for thepreliminary investigation of the role Gal-1played in the formation of antler blood vessels,nerves and cartilage in vitro with the lentiviral vector mediated RNAi (RNA interference)technology, providing a support for the late research in vivo. The experiment was designedas follows: a high score RNAi target sequence of Gal-1was screened with the help of theon-line design software, general design principles and the reported literature and Blast in theNCBI database was applied to remove the off-target ones. An oligodeoxynucleotide ofshRNA (short hairpin RNA) was designed empirically and synthesized by a commercialcompany. The positive-and anti-sense strands of the synthesized shRNA were annealed invitro to form the double strands, which was then inserted into the vector plasmid handled bythe restriction enzyme to recombine. The recombined plasmid was preliminary identified byPCR and then sequenced. The recombined vector plasmid pLVTHM, packaging plasmid psPAX2and coated plasmid pMD2.G were purified by endotoxin-free plasmid kit andco-transfected into293t cells by Roche transfection reagents. The recombinant virus werecollected and concentrated at24h and48h respectively. The collected antler-formingperiosteal cells were primarrily cultured, infected by the concentrated viral solution andobserved72h after infection using an inverted fluorescence microscope.Result：two high score RNAi target sequences of Gal-1were selected in this study. Thepositive-and anti-sense strands of the synthesized shRNA were annealed, forming a smallfragment of nucleotide double strands. The fragment was inserted into the vector plasmidpLVTHM, which was verified by PCR and sequencing. The fluorescence response of theantler-forming periosteum cells was obvious. The results indicated that, the lentivirus vectormediated RNAi was successfully developed, abundant concentrated virus solution wasobtained and antler-forming periosteum stem cells with Gal-1gene silencing which could beinherited were obtained. This study could provide a support for the future study in vivo ofGal-1function. |
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