| Objectives:1.To explore the dose and mechanism of apoptosis of vertebral growth plate chondrocytes induced by tumor necrosis factor(TNF-α)and aldosterone in vitro.2.To establish an in situ drug delivery system that can be injected into the intervertebral disc and simulate the release process of candidate drugs to determine drug concentration,sterilization and encapsulation.3.To establish an animal model of implantable in situ drug delivery system containing candidate drugs to induce apoptosis of chondrocytes in unilateral vertebral growth plate and to explore its effect and verify its mechanism.The study tends to provide a theoretical and scientific basis for exploring a new treatment method for early-onset scoliosis(Early Onset Scoliosis,EOS).Methods:1.Flow cytometry and TUNEL staining were used to detect the apoptosis of chondrocytes in vertebral growth plate of a 2-month pigs induced by 0-90ug/ml TNF-α.qPCR and Western blotting were used to determine whether it could induce cell scorch and necrosis at the same time.Similarly,to detect the effect of chondrocyte apoptosis induced by 10-7M to 10-3M aldosterone.The mechanism of aldosterone action was predicted by network pharmacology and verified by WB and qPCR at the cell level in vitro.2.An implantable in situ drug delivery system suitable for intervertebral drug delivery was constructed.The release concentrations of TNF-αand aldosterone in the drug delivery system were screened by ELISA method and liquid mass spectrometry,and the drugs delivery system were sterilized and encapsulated.3.To establish an animal model of apoptosis of chondrocytes in unilateral vertebral growth plate of young pigs induced by an implantable drug delivery system loaded with aldosterone,to explore and verify the imaging,histology,apoptosis and mechanism.Results:1.TNF-α induced apoptosis of chondrocytes in vertebral growth plate at the concentration of 15-90ug/ml,but also induced cell death and cell necrosis.The release process of Poloxamer/NaCl gel encapsulated with TNF-α was simulated in vitro shows that the drug was almost not released effectively(drug loading concentration:1 ug/ml;release concentration:less than 100pg/ml,release rate:0.01%).2.Aldosterone significantly induced chondrocyte apoptosis in the range of 10-7M to 103M by inhibiting the activity of PI3K/Akt,and the effect was time-dependent and dose-dependent.The drug release process of the aldosterone-encapsulated in situ drug delivery system lasted for 3 days,and the drug release concentration was 2 orders of magnitude lower than that of the drug loading concentration(drug loading concentration:10-4M;release concentration:1.35 × 10-6M,release rate:3.6%).3.In situ injectable drug delivery system containing 10-3M aldosterone could induce apoptosis of chondrocytes in unilateral vertebral growth plate of young pigs by inhibiting PI3K/Akt signal pathway.Morphologically,the arrangement of chondrocytes in vertebral growth plate was disordered and the height of growth plate was decreased.TUNEL staining showed an apoptotic area centered on the injection range.At the same time,this procedure will not cause intervertebral disc degeneration and interbody fusion.Conclusions:1.Aldosterone can inhibit the apoptosis of chondrocytes induced by PI3K/Akt and it is an alternative drug to interfere with vertebral growth,2.Poloxamer 407/NaCl hydrogel is a kind of in situ drug delivery system which meets the requirements of surgical operation.It can adjust the concentration of Poloxamer 407 or NaCl to regulate the entanglement degree between micelles of drug loading system,and adapt to the different needs of drug molecular weight,operation time and release rate.3.The in situ drug delivery system containing aldosterone is a implant which can be used to interfere with the growth of vertebral body.The intervertebral injection does not cause intervertebral disc degeneration or spinal fusion,so it can be used as a new treatment method of EOS further study. |
PDF Full Text Request