Effect of short hairpin RNA on growth and proliferation of human bone marrow mesenchymal stem cells

Jie CHENG; Zezhang TAO; Honggang DUAN; Bokui XIAO; Shiming CHEN

ABSTRACT

BACKGROUND:

Tetramethylpyrazine (TMP) can inhibit the expression of vascular endothelial growth factor (VEGF), but it is uncertain that TMP inhibit the growth and proliferation of HL-60 leukemic cells induced by VEGF.

OBJECTIVE:

To observe the effect of TMP on the proliferation of HL-60 leukemic cells induced by VEGF.

DESIGN:

Repetitive measurement and observation.

SETTING:

School of Medicine, Wuhan University of Science and Technology.MATERIALSThe experiment was carried out in the Molecular Biology Laboratory Center, School of Medicine, Wuhan University of Science and Technology from March to June in 2007. Human leukemic cell line HL-60 cells were purchased from Shanghai Institute of Cell Biology. TMP hydrochloride injection was produced by Wuxi Seventh Pharmaceutical Products Limited (Lot number011014), protamine sulfate injection was produced by Shanghai First Biochemical Pharmaceuticals (Batch number010302), and immunohistochemistry kit was purchased from Boster company.

METHODS:

①Human leukemic cell line HL-60 cells at log phase were used for the experiments. Cells were treated with 100 μg/L VEGF, and then TMP at final concentrations of 1.5, 15, 150 mg/L was added into culture medium. While the cells in medium without TMP were taken as blank control group, and the cells in medium with 20 mg/L protamine as positive control group. Meanwhile cells without treatment of VEGF were served as VEGF control group. After cells were incubated for 48 hours, the growth inhibiting rate of HL-60 cells was detected by MTT assay.②After HL-60 cells were treated with TMP at the final concentrations of 1.5, 15, 150 mg/L for 24 hours, the protein expression of VEGF in HL-60 cells was examined by SP immunohistochemistry.MAIN OUTCOME

MEASURES:

①Growth inhibiting rate of HL-60 cells.②Protein expression of VEGF.

RESULTS:

①Growth inhibiting rate of HL-60 cellsAfter HL-60 cells induced by VEGF were treated with 15 and 150 mg/L TMP, the absorbance value was significantly lower than that in VEGF control group (P < 0.05).②Protein expression of VEGFAfter HL-60 cells were treated with TMP for 24 hours, the protein expression of VEGF was down-regulated with increasing TMP concentration in a dependent manner. Significant differences were observed in the protein expression of VEGF between cells treated by TMP and the controls (P < 0.01).

CONCLUSION:

shRNA, by targeting hTERT mRNA, has no noticeable influence on growth and proliferation of hMSCs, and might be safe for the somatic cells which are normal but do not express hTERT.