The Effect Of Periostin Expression Up-regulation On The Biological Behaviors Of BMSCs In Ovariectomized Rats

Postmenopausal Osteoporosis(PMO)is a common and frequently-occurring disease in the process of human aging,which is mainly due to ovarian function declines after menopause in women,and estrogen secretion is reduced,causing bone metabolic abnormalities,severe bone loss caused by the disease.Chronic periodontitis is a common oral complication of PMO.Relevant studies have proved that the level of estrogen in the body not only affects the bone metabolism of the whole body,but also directly affects the function and metabolism of the periodontal area.For many years,estrogen replacement therapy has been the preferred treatment for PMO,which can significantly improve women's quality of life,especially in inhibiting bone resorption,but its long-term use can increase the risk of breast and reproductive system tumors.Studies have shown that mesenchymal stem cells(BMSCs)in the bone marrow of jawbone can enter the periodontal tissue through vascular channels,differentiating into periodontal precursor cells and participating in the regeneration of periodontal tissue.Studies on bone marrow mesenchymal stem cells(PMO-BMSCs)and inflammatory periodontal membrane stem cells(P-PDLSCs)in postmenopausal osteoporosis have confirmed that their osteogenic differentiation ability is significantly decreased and apoptosis is increased during the induction of osteogenesis,which may be an important cause of osteoporosis and periodontal bone tissue defects.Mamalis et al.found that estrogen and its receptors(ER)were involved in periodontal tissue metabolism and functional alterations mainly by regulating the expression of Periostin.Periostin also protects cells from hypoxic-induced cell damage and inhibits apoptosis by mediating integrin-dependent cell proliferation,adhesion,and movement,according to numerous studies.However,whether Periostin expression up-regulation could improve the biological function and local microenvironment of periodontal,and survival rate of BMSCs has not been reported.For this purpose,this study intends to use the lentiviral vector system to transfer Periostin targeted genes to PMO-BMSCs,and preliminarily explore the regulating effect of Periostin on the physiological functions of PMO-BMSCs by up-regulating the expression of Periostin,so as to provide more theoretical basis for the treatment of periodontal disease accompanied by postmenopausal osteoporosis.contents: 1.Establishment and evaluation of rat osteoporosis modelFemale SD rats were randomly divided into two groups.Rats in the castrated group(OVX group)were treated with modified single back incision to remove bilateral ovaries to establish a rat osteoporosis model,while those in the SHAM group were treated with the same surgical approach,but only the same amount of adipose tissue around the ovaries was removed.The body weight of the two groups of rats was recorded every week after the operation.Three months after the modeling,Three months after the modeling,1% of pentobarbital sodium was overdosed by intraperitoneal injection of anesthetic to kill the rats,and the femur was taken for Micro-CT scanning.The success of the modeling was evaluated after 3d reconstruction,and the related bone morphological parameters were analyzed.2.Isolation,culture and identification of BMSCsAfter successful modeling,the bone marrow of the two groups of rats was extracted under aseptic conditions,and the bone marrow mesenchymal stem cells were isolated and cultured using the whole bone marrow adherent method,followed by flow cytometry for the detection of immune markers on the surface of stem cells,and osteogenic and adipogenic induction to identify the differentiation characteristics of stem cells.3.BMSCs(OVX-Virus)transfected by recombinant lentivirus(pGC-Fu-POSTN)BMSCs from OVX group were transfected by recombinant lentivirus containing enhanced green fluorescent protein(EGFP)and Periostin gene,and the MOI and optimal infection conditions were determined by preliminary experiments.BMSCs of the third generation OVX group were transfected officially according to the results of preliminary experiments.About 72 hours after infection,when the abundance of fluorescence expression was high,the infection efficiency was observed by fluorescence microscope,and follow-up experiments(OVX-Virus group)were conducted.4.Periostin up-regulation on the biological behaviors of OVX-BMSCsThe OVX group and the SHAM group cells of third generation were used as the control groups,and the OVX-Virus group cells on the fourth day of Virus transfection were used as the experimental group.After osteoblastic induction,ALP staining and alizarin red staining were used to identify the osteoblastic ability of the three groups.The proliferation ability of the three groups was detected by CCK8 assay.Cell cycle and apoptosis were detected by flow cytometry.Total RNA was extracted and real-time quantitative PCR(RT-PCR)was used to detect the genes related to osteogenesis and angiogenesis.Total intracellular proteins were extracted,and the contents of osteogenic and angiogenic proteins in the three groups were determined by Western Blot.Results:1.After the establishment of the rat model,weekly weighing records were made for the two groups of rats,indicating that the weight growth trend of castrated rats was significantly higher than that of the sham operation group;Three months after the modeling,rats were sacrificed to death under anaesthetic overdose and their femurs were taken for micro-ct scanning.The scanning results showed that the number and density of cancellous bone trabeculae in OVX group were significantly reduced,and the interosseous space in cancellous bone was widened.The analysis of bone morphological parameters showed that there were significant differences in bone volume fraction and thickness of bone trabeculae between the two groups.All the above results indicated that the osteoporosis model of female castrated rats was successfully established.2.BMSCs were isolated and cultured by whole bone marrow adherent method.After subculture,morphological observation under the microscope showed that the morphology of bone marrow mesenchymal stem cells was gradually uniform,with triangular or polygonal forms and a "vortex" arrangement.The detection results of stem cell surface markers in flow cytometry showed that the extracted cells had high expression of CD29 and CD90,and little or no expression of CD34 and CD45,which were consistent with the immunological characteristics of bone marrow mesenchymal stem cells.Calcium nodules and lipid droplets were observed after induction of osteogenesis and adipogenesis.The above results indicated that BMSCs were successfully extracted.3.According to lentivirus infection conditions and MOI selection principles,the MOI of BMSCs used in this study was determined to be 70 based on the pre-experiment results.The infection condition was conventional medium +Polybrene,and the culture was resumed after 8h transfection.After infection for about 72 h,the expression of green fluorescence of enhanced green fluorescent protein(EGFP)was observed by fluorescence microscope,and the infection efficiency was about 80%,and the cells grew well,indicating that the lentivirus carrying the target gene was successfully transfected into the host cells,which could be used for subsequent experiments.4.After virus transfection,alkaline phosphatase activity was enhanced,staining was deepened,and the number of calcified nodules stained with alizarin red increased compared with OVX group,and after the quantitative analysis of alizarin red,the difference was statistically significant;The proliferation rate of BMSCs in OVX group was lower than that in sham operation group,and was increased by Periostin.Cell cycle detection by flow cytometry showed that the proportion of S-phase BMSCs in OVX-Virus group was significantly higher than that in OVX group(P<0.05).The apoptosis rate of OVX-Virus group BMSCs was significantly lower than that of OVX group(P<0.05).Osteogenic genes ALP,BSP,COL1,Periostin were detected by RT-PCR,and were up-regulated after virus transfection(P<0.05).Western Blot analysis for osteogenic proteins also demonstrated an increase in ALP,BSP,COL1,Periostin expression(P<0.05).Conclusion:1.A rat model of osteoporosis was established by resection of bilateral ovaries through a modified single incision on the back,and body weight monitoring and micro-ct scanning after surgery 3 months confirmed the successful establishment of the postmenopausal osteoporosis model.2.BMSCs from OVX group were transfected with recombinant lentivirus containing enhanced green fluorescent protein(EGFP)and rat Periostin gene,and green fluorescence was successfully expressed by fluorescence microscopy,indicating that the lentivirus carrying the target gene was successfully transfected into host cells.3.Periostin expression up-regulation could improve osteogenic differentiation and proliferation of BMSCs,and inhibit apoptosis.