Effect Of Silencing TGF-β1Expression By Small Interfering RNA On Ossification Of The Ligamentum Flavum In Mice

BackgroundOLF is a very common disease in clinical medicine; its cause is unknown, many factors are included in its pathogenesis, such as age, race, genes or growth factors and stress change. The growth factors about the formation of bone, the super family of transforming growth factors, play a very important role in the pathogenesis. The international organization confirmed that TGF-β1, BMP-2are involved in the different periods of ligament. However, there are few reports in the world about the correlation between TGF-β1and BMP-2gene expression, and their role in the process of OLF.This study starts from the etiology, form pathology and biochemical process of spinal ligament ossification, target TGF-β1gene in yellow ligament cells of mouse by siRNA interference technology, and observe the effect of osteogenic cells of yellow ligament in mice after silencing TGF-β1, to find an efficient and unique method to prevent OLF, thus provides a new way of thinking for clinical treatment of such disease.ObjectiveTo study the effect of TGF-β1on ossification of ligamentum flavum, RNAi technique was used to inhibit the expression of TGF-β1gene on fibroblast of mice ligamentum flavum which have been induced into ossification.MethodsFibroblasts of mice ligamentum flavum were cultivated in vitro and rhBMP-2was used to induce the ossification of fibroblast of mice ligamentum flavum. After ossification, those osteoblasts were cultivated and photographed with morphological observation, and were identified with alkaline phosphatase staining and alizarin red staining of calcified nodules after have being cultivated15and25days respectively. Eukaryotic expression vector (siRNA-pSilencer2.0U6-TGFβ1) carried red fluorescent protein was constructed to transfect the osteoblasts; after that, the expression of TGF-β1and BMP-2in osteoblasts were detected by immunofluorescence technique before and after transfection; the expression change of TGF-β1mRNA in osteoblasts was determined by Real-time PCR; Western blot was used to measured the expression change of protein of TGF-β1and BMP-2in osteoblasts; ELISA (enzyme linked immunosorbent assay) was used to determine the expression change of ALP and OC (osteocalcin) in osteoblasts.ResultsAfter ossification had been induced successfully, ligamentum flavum cell morphological observation showed that alkaline phosphatase staining and alizarin redstaining of calcified nodules were both positive, and those cells had typical osteoblast biology. After transfected by siRNA-pSilencer2.0U6-TGFβ1in72hours, immunofluorescence detection displayed decline in the fluorescence intensity of TGF-β1and BMP-2; compared with the negative control and bland control, Real-time PCR showed that the expression of the TGF-β1mRNA in experiment group decreased significantly by41.94%and47.82%, respectively (P<0.01); western blot showed that the ratio of TGF-β1/β-action in experiment group decreased remarkably by35.88%and44.75%, respectively (P<0.01) and BMP-2/β-action decreased significantly by81.79%,86.06%, respectively (P<0.01); ELISA displayed that ALP in experiment group decreased notably by24.14%,32.30%, respectively (P<0.01) and OC decreased remarkably by17.01%,21.63%, respectively (P<0.01).Conclusion1. Eukaryotic expression vector (siRNA-pSilencer2.0U6-TGFβ1) could effectively inhibit the expression of TGF-β1directly and inhibit the expression of endogenous BMP-2indirectly in osteoblasts, 2. rhBMP-2could induce yellow ligament cells to osteoblasts, and inhibit the expression of TGF-β1gene can delay its progress in the ossification...