[Construction of ADAM28 shRNA interference vector and its inhibitory effect on human periodontal ligament stem cells].

PURPOSE: To investigate the inhibition of ADAM28 shRNA interfering vector on ADAM28 gene expression in human periodontal ligament stem cells(HPDLSC), and provide experimental evidence for gene therapy against congenital hypoplasia of tooth root(CHTR) disease.

METHODS

Four pairs of shRNA specific interfering fragments were designed, synthesized, and connected with pGPU6/GFP/Neo vector. ADAM28 shRNA interfering vector was constructed and identified, and transfected into HPDLSC for 48 h, and then the inhibition efficiency was detected by real-time fluorescence quantitative RT-PCR（qRT-PCR）and Western blot. Statistical significance was assessed by SPSS 21.0 software package.

RESULTS

Enzyme digestion and sequencing identification demonstrated that the double strands shRNA was correctly inserted into the expression vector pGPU6/GFP/Neo, and the recombinant interfering vector was successfully constructed and highly transfected into HPDLSC. QRT-PCR and Western blot showed that pGPU6/GFP/Neo-ADAM28-shRNA1-4 had significant inhibition efficiency, and shRNA1 had the highest inhibition efficiency. There were significant differences between ADAM28-shRNA1-4 group and non-transfection group, negative control group, respectively(P<0.05).

CONCLUSIONS

ADAM28 shRNA interference vector can effectively inhibit ADAM28 gene expression in HPDLSC.