| Backgroundosteoporosis is a kind of systemic metabolic bone disease which can reduce the bone density and bone mass of the whole body,destroy bone microstructure and increase bone brittleness due to various reasons,and thus prone to fracture.Osteoporosis can be divided into two categories:primary and secondary.Primary osteoporosis is divided into three types:postmenopausal osteoporosis(type Ⅰ),senile osteoporosis(Ⅱ)and idiopathic osteoporosis(including adolescent type),among which postmenopausal osteoporosis is caused by the decline of estrogen levels after menopause,which is very harmful to the life and health of elderly women.Currently,the treatment for osteoporosis mainly includes calcium supplement,vitamin D,bone resorption inhibitors,bone formation promoters,traditional Chinese medicine,etc,but all of them have their own limitations and cannot effectively treat osteoporosis for a long time.Therefore,it is very important to find new treatment methods.In addition,some mirnas can affect osteogenic differentiation of BMSC by affecting multiple signaling pathways,promoting bone remodeling and improving osteoporosis symptoms.Therefore,searching for mirnas that can affect osteogenic differentiation of BMSC is helpful to find new molecular targets for osteoporosis treatment.osteoporosis is a kind of systemic metabolic bone disease which can reduce the bone density and bone mass of the whole body,destroy bone microstructure and increase bone brittleness due to vaious reasons,and thus prone to fracture.Osteoporosis can be divided into two categories:primary and secondary.Primary osteoporosis is divided into three types:postmenopausal osteoporosis(typeⅠ),senile osteoporosis(Ⅱ)and idiopathic osteoporosis(including adolescent type),among which postmenopausal osteoporosis is caused by the decline of estrogen levels after menopause,which is very harmful to the life and health of elderly women.Currently,the treatment for osteoporosis mainly includes calcium supplement,vitamin D,bone resorption inhibitors,bone formation promoters,traditional Chinese medicine,etc.,but all of them have their own limitations and cannot effectively treat osteoporosis for a long time.Therefore,it is very important to find new treatment methods.In addition,some mirnas can affect osteogenic differentiation of BMSC by affecting multiple signaling pathways,promoting bone remodeling and improving osteoporosis symptoms.Therefore,searching for mirnas that can affect osteogenic differentiation of BMSC is helpful to find new molecular targets for osteoporosis treatment.Objective1.1.miR-18a-5p was overexpressed and inhibited in vitro,and osteoblastic markers were detected by various methods to clarify the effect of miR-18a-5p on osteogenic differentiation of BMSC.2.The targeting relationship between miR-18a-5p and notch2 was demonstrated by bioinformatics analysis and double luciferase reporting assay3.In vivo,miR-18a-5p was verified to promote osteogenic function,providing a new target and idea for miRNA treatment of osteoporosis.The primary BMSC cells of young rats were isolated and cultured in vitro to induce the differentiation of BMSC to osteogenic direction,and the expression changes of miR-18a-5p during osteogenic differentiation were detected.Methods1.Primary BMSC cells of rat femur were isolated and extracted at 4 weeks to induce osteogenic differentiation of BMSC in vitro,and osteogenic markers and expression levels of miR-18a-5p were detected to determine whether the expression of miR-18a-5p was correlated with osteogenic differentiation of BMSC.2.In vitro,the mimics and inhibitors of miR-18a-5p were transfected with BMSC to up-regulate and down-regulate the expression level of miR-18a-5p,and the osteoblastic differentiation of BMSC was detected by ARS staining,ALP staining,qRT-PCR,WB and other methods.To demonstrate the regulatory effect of miR-18a-5p on osteogenic differentiation of BMSC.3.Bioinformatics analysis method was used to predict and screen the mRNA of the target gene of miR-18a-5p,and the overexpression and inhibition levels of miR-18a-5p were respectively detected to determine the expression level of the target gene,and the regulatory relationship between the two was preliminatively determined,and the target relationship was verified by double luciferase reporter gene experiment and response experiment.4.A rat model of postmenopausal osteoporosis was established by bilateral ovariectomy,which was divided into two groups:bilateral ovariectomy group(OVX)and sham group(sham)without ovariectomy.The femurs of experimental animals were analyzed by micro-CT on BMD and bone histomorphometry to verify whether the postmenopausal osteoporosis model was successfully established.In order to verify the effect of miR-18a-5p on osteogenic differentiation in vivo,adenovirus containing miR-18a-5p and control reagent were injected into the right femoral bone marrow cavity in rats with postmenopausal osteoporosis.Results1.The results of in vitro culture of BMSC and qRT-RCR showed that compared with growth medium,the expression level of osteogenic markers of BMSC was significantly increased in osteogenic induction medium,and the expression level of miR-18a-5p was increased during osteogenic differentiation of BMSC.No significant difference was found in normal cell proliferation,suggesting that miR-18a-5p may regulate osteogenic differentiation of BMSC.2.After transfection of miR-18a-5p mimics and inhibitors with BMSC,detection results showed that overexpression of miR-18a-5p could promote osteogenic differentiation of BMSC,while inhibition of miR-18a-5p expression could inhibit osteogenic differentiation of BMSC.3.Bioinformatics analysis predicted that notch2 was a potential target gene of miR-18a-5p,and double luciferase reporting experiments confirmed that notch2 was a target gene of miR-18a-5p.4.The results of micro-CT animal experiments showed that the rat model of postmenopausal osteoporosis was successfully established,and miR-18a-5p could effectively improve the symptoms of postmenopausal osteoporosis compared with the control group..ConclusionBy cultivating BMSC in vitro and inducing it to osteogenic differentiation,we found that the expression level of miR-18a-5p significantly increased during the process of osteogenic differentiation of BMSC.Then,by expressing and inhibiting the expression level of miR-18a-5p,we found that overexpression of miR-18a-5p could promote osteogenic differentiation of BMSC.Inhibition of miR-18a-5p can inhibit osteogenic differentiation of BMSC,which proves that miR-18a-5p can promote osteogenic differentiation of BMSC.Then,the targeted inhibition relationship between miR-18a-5p and downstream target gene notch2 was predicted and verified.Finally,animal experiments further confirmed that miR-18a-5p can promote osteogenic differentiation in vivo,providing a new target and idea for the treatment of osteoporosis. |
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