Objective    To construct the lentivi

ral vector of mouse p38MAPK gene RNAi and study its effect on p38MAPK expression and apoptosis of MC3T3-E1 cells.     Methods    Three complementary oligoDNA fragments of mouse p38MAPK gene were designed and synthesized. After phosphorylation and annealing

double-strand DNA was subcloned into HpaⅠ sites of the siRNA expression vector PLL3.7. The inserted sequence was identified after screening and restriction enzyme digestion. 293T cells were co-transfected with lentiviral vector and recombinant plasmids. Viral titer was determined by flow cytometry (FCM). Recombinant lentivirus vector system was used to transfect the MC3T3-E1 cells. mRNA expression levels of p38MAPK were analyzed by real time PCR. MC3T3-E1 cells were collected in 7 d after culture in high glucose. Cell apoptosis was detected by FCM. Protein level in p-p38MAPK was measured by Western blotting.     Results    The lentiviral vector containing shRNAs targeting p38MAPK gene was successfully constructed and transfected into MC3T3-E1 cells. Recombinant plasmids were named p38MAPK-shRNA1

p38MAPK-shRNA2

and p38MAPK-shRNA3

respectively. Their viral titer was 2.4×10

8

2.8×10

8

and 2.5×10

8

TU/ml

respectively. The expression of p38MAPK mRNA was 78.8%

84.3% and 60.2%

and was significantly lower in MC3T3-E1 cells of different p38MAPK-shRNA-transfected groups than in control group（P0.01）. Western blot analysis showed that the p-p38MAPK protein expression level was significantly higher in MC3T3-E1 cells of high glucose group and non-p38MAPK-transfected group than in those of control group (P0.01) and was significantly lower in MC3T3-E1 cells of high glucose group and non-p38MAPK-transfected group than in those of control group after p38MAPK-shRNA transfection (P0.01). FCM showed that the apoptosis rate of MC3T3-E1 cells was significantly higher in high glucose group than in control group (P0.01) and significantly lower in high glucose group than in p38MAPK-shRNA transfection group (P0.01).     Conclusion    The recombinant

lentivirus vector of shRNA targeting p38MAPK gene constructed by our group can effectively inhibit the expression of p38MAPK in MC3T3-E1 cells

thus decreasing their apoptosis induced by high glucose.