The Effects Of Wnt5a On Osteogenic And Adipogenic Differentiation Of Stem Cells From The Apical Papilla In Vitro

Objective: The stable stem cells from the apical papill(SCAP) with Wnt5 a overexpression and knockdown were established, detecting the related factor of osteogenic and adipogenic expression. To discuss the effects of Wnt5 a on osteogenic and adipogenic differentiation of SCAP in vitro, which would provide theoretic evidence and experimental support in Wnt5 a regulating biological effect of SCAP.Methods: SCAP were primarily cultured and isolated by enzyme digestion method and limited dilution technique. And were identified.The lentivirus with overexpressed Wnt5 a and knockdown Wnt5 a were built and stablly transfecting SCAP, The SCAP with overexpressed Wnt5 a and knockdown Wnt5 a were obtained, which was screened using puromycin. The m RNA and protein expression of Wnt5 a in SCAP were detected by q RT-PCR and Western Blot. The SCAP with overexpressed Wnt5 a and knockdown Wnt5 a as well as their relevant empty vector controls were osteoinduced, in vitro, respectively in 2 weeks, 3 weeks and 4 weeks. The total RNA and protein were extracted and the expression of ALP, BSP, DSPP, OCN were detected by q RT-PCR and Western Blot. The SCAP with overexpressed Wnt5 a and knockdown Wnt5 a as well as their relevant empty vector controls were induced into adipogenic differentiation in 2 weeks in vitro. The total RNA and total protein were extracted, and the expression of PPAR-γ were detected in q RT-PCR and Western Blot. The related data were statistically analyzed by software SPSS 19.0.Results: 1. Wnt5 a were stablly transfecting SCAP, which transfection rate was nearly 90%. Compared with its relevant empty vector controls, the overexpressed Wnt5 a group had a significantly increased Wnt5 a expression(P<0.05), the knockdown Wnt5 a group had significantly downregulated Wnt5 a expression(P<0.05).The stable SCAP with overexpressed and knockdown Wnt5 a were established. 2. SCAP mineralization factors related to osteognic:(1) ALP: At 2 weeks of osteognic induction, compared with empty vector controls, the expression of ALP m RNA decreased in overexpressed Wnt5 a group(P<0.05) and the expression of protein increased(P<0.05); The expression of ALP m RNA showed no obvious difference between knockdown Wnt5 a group and the empty vector control(P>0.05), the expression of protein decreased in knockdown Wnt5 a group(P<0.05). The expression of m RNA and proteinin overexpressed Wnt5 a group were higher than knockdown Wnt5 a group(P<0.05). At 3 weeks of osteognic induction, compared with empty vector controls, the expression of ALP m RNA and protein increased in overexpressed Wnt5 a group(P<0.05),decreased in knockdown Wnt5 a group(P<0.05); The expression of ALP m RNA and protein in overexpressed Wnt5 a group were higher than knockdown Wnt5 a group(P<0.05). At 4 weeks of osteognic induction, compared with empty vector controls, the expression of ALP m RNA increased in overexpressed Wnt5 a group(P<0.05),The expression of ALP protein showed no obvious difference between knockdown Wnt5 a group and the empty vector control(P>0.05), The expression of m RNA and proteindecreased in knockdown Wnt5 a group(P<0.05); The expression of ALP m RNA and protein in overexpressed Wnt5 a group were higher than knockdown Wnt5 a group(P<0.05).(2) BSP: At 2 weeks, 3 weeks and 4 weeks of osteognic induction, compared with empty vector controls, the expression of BSP m RNA and protein rised in overexpressed Wnt5 a group(P<0.05), and decreased in knockdown Wnt5 a group(P<0.05).The expression of BSP m RNA and protein in overexpressed Wnt5 a group was higher than knockdown Wnt5 a group(P<0.05).(3) DSPP: At 2 weeks of osteognic induction,compared with empty vector controls, the expression of DSPP m RNA and protein rised in overexpressed Wnt5 a group(P<0.05), the expression of m RNA decreased in knockdown Wnt5 a group(P<0.05), but the expression of protein in knockdown Wnt5 a group was not detected. The m RNA expression in overexpressed Wnt5 a group was higher than knockdown Wnt5 a group(P<0.05). At 3 weeks and 4 weeks of osteognic induction, compared with empty vector controls, the expression of DSPP m RNA and protein rised in overexpressed Wnt5 a group(P<0.05), and decreased in knockdown Wnt5a group(P<0.05).The expression of m RNA and protein in overexpressed Wnt5 a group was higher than knockdown Wnt5 a group(P<0.05).(4) OCN: At 2 weeks of osteognic induction, compared with empty vector controls, the expression of OCN m RNA and protein rised both in overexpressed and knockdown Wnt5 a group(P<0.05); The expression of OCN m RNA and protein in overexpressed Wnt5 a group were lower than knockdown Wnt5 a group(P<0.05). At 3 weeks and 4 weeks of osteognic induction, the expression of OCN m RNA and protein in overexpressed Wnt5 a group was higher than its empty vector controls(P<0.05), and knockdown Wnt5 a group was lower than its empty vector controls(P<0.05); The expression of OCN m RNA and protein in overexpressed Wnt5 a group was higher than knockdown Wnt5 a group(P<0.05). 3. PPAR-γ of SCAP factor related to adipogenic: compared with empty vector controls, the expression of PPAR-γ m RNA and protein rised in overexpressed Wnt5 a group(P<0.05), and decreased in knockdown Wnt5 a group(P<0.05).The expression of PPAR-γ m RNA and protein in overexpressed Wnt5 a group was higher than knockdown Wnt5 a group(P<0.05).Conclusion: 1. The SCAP with overexpressed and knockdown Wnt5 a were established by stable transfecting Wnt5 a lentivirus vector which transfection rate was nearly 90%. 2. Overexpressed Wnt5 a can promote the expressions of osteogenic factors ALP、BSP、DSPP、OCN in vitro, knockdown Wnt5 a can inhibit their expressions. The changes become obvious in 3 weeks 、4 weeks of osteognic induction. 3. Overexpressed Wnt5 a can promote the expression of adipogenic factor PPAR-γ in vitro, knockdown Wnt5 a can inhibit the expression. 4. Under the conditions of this study, we preliminary confirmed that Wnt5 a has promoting effect in regulating the osteogenic and adipogenic differentiation of SCAP,which would provide experimental support to the molecular mechanisms of root development, clinic treatment and tissue engineering.