Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (7): 1199-1204.doi: 10.3969/j.issn.1673-8225.2011.07.013

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Retrovirus-mediated siRNA targeting transient receptor potential melastatin 7 gene suppresses activation of RBL-2H3 cells  

Lü Zhi-qiang, Wu Yi-mei, Jiang Shan-ping, Zhang Wei, Huang Lin-jie   

  1. Department of Respiratory Medicine, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China
  • Received:2010-09-23 Revised:2010-12-08 Online:2011-02-12 Published:2011-02-12
  • Contact: Jiang Shan-ping, Professor, Doctoral supervisor, Department of Respiratory Medicine, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China shanpingjiang @126.com
  • About author:Lü Zhi-qiang, Associate professor, Department of Respiratory Medicine, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China gzmeggiewoo@hotmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 81070017*; the Natural Science Foundation of Guangdong Province, No. 10151008901000078*; the Science and Technology Planning Project of Guangdong Province, No. 2009B030801093*; Medical Scientific Research Foundation of Guangdong Province, No. B2009071*

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Abstract:

BACKGROUND: Calcium ion plays an important role in the degranulation process for activated mast cells. Transient receptor potential melastatin 7 (TRPM7) is an important candidate channel for mast cells.
OBJECTIVE: To construct a recombinant retrovirus vector siRNA targeting rat TRPM7 gene and explore its influence on antigen-induced activation of RBL-2H3 cells.
METHODS: Three TRPM7-siRNA sequences and a negative sequence were designed and cloned into linearized pSuper-retro-neo-GFP vector. The above recombinants were transfected by lipofectamine 2000 into RBL-2H3 cells. The gene silencing efficacy of the 3 targets was evaluated by Western blot. The optimized pSuper-retro-neo-GFP-siTRMP7 and packaging plasmid were co-transfected into 293FT cells to produce retrovirus, which was applied to infect RBL-2H3 cells. The RNAi efficiency was confirmed by real-time PCR and Western blot. Measurement of β-hexosaminidase was performed before and after TRPM7-siRNA transfection to explore the changes on activation of RBL-2H3 cells.
RESULTS AND CONCLUSION: The gene silencing efficacy of siTRPM7-3 transfected group was highest among all Lipofectamine 2000 transfected groups (P < 0.05). Compared to the normal control group, TRPM7 expression of pSuper-retro-neo-GFP-siTRMP7-3 transfected group was significantly reduced both at mRNA and protein levels (P < 0.05). The results revealed that, down regulation of TRPM7 channel can suppress the antigen-induced activation of RBL-2H3 cells.

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