| Cleidocranial dysplasia(CCD)is an autosomal dominant hereditary disease characterized by skeletal dysplasia and caused by heterozygous mutations in Runt-related transcription factor 2(RUNX2).Typical clinical manifestations include large fontanelles,hypoplastic or absent clavicles,anomalies of eruption,and supernumerary teeth,and osteoporosis and fractures have been documented in a handful of cases.Bone marrow stromal cells(BMSCs)possess the multipotential properties and are essential for maintaining bone homeostasis.Studies have shown that RUNX2 haploinsufficiency will change the biological properties of bone marrow stromal cells in CCD patients(BMSCs-CCD),its intrinsic mechanism remains to be explored.Previous studies have demonstrated that RUNX2 directly repressed endogenous micro RNA-31(mi R-31)expression in rat BMSCs,but it has not been confirmed in human BMSCs.The aim of this study was to analyze the changes in the biological characteristics of BMSCs-CCD.Moreover,the effect of mi R-31 on the osteogenesis of BMSCs-CCD and its possible mechanism were discussed.Part ? Isolation,culture and biological characterization of bone marrow stromal cells of CCD patientObjective: To isolate and culture bone marrow stromal cells from CCD patients(BMSCs-CCD)and normal control(BMSCs),and to analyze the differences of the biological characteristics between them.Methods: BMSCs were isolated and cultured by cancellous bone adherent culture.Real-time quantitative PCR analysis was performed to detect the expression of RUNX2;CCK-8,flow cytometry and Brd U cell proliferation assay for cell proliferation capacity;Alkaline phosphatase staining,alizarin red staining and Western blot analysis was performed after osteogenic induction for cell osteogenic differentiation Ability;Crystal violet staining for clone formation ability;Detection of pluripotency transcription factors for stemness;Detection of senescence-related proteins and ?-gal senescence staining experiments for senescence characters;Cellular immunofluorescence for comparing the expression of BMI1.Results:(1)The growth rate of BMSCs-CCD was slower than that of BMSCs,the percentage of BMSCs-CCD in the S/G2 phase was lesser and the Brd U positive rate of BMSCs-CCD were lower.(2)Comparing 3 cases of CCD patients with normal peers,the m RNA level and protein level of RUNX2 in BMSCs-CCD were significantly reduced.The expression of RUNX2,Osteopontin(OPN)and Osteocalcin(OCN)was lower in BMSCs-CCD relative to BMSCs after osteogenic induction,the area of alkaline phosphatase staining positive and alizarin red-positive mineralized areas of BMSCs-CCD was decreased.(3)Fewer colony-forming units were observed in BMSCs-CCD than that in BMSCs,and The expression levels of OCT4,NANOG and SOX2 were clearly reduced in BMSCs-CCD relative to BMSCs at the protein level.More SA-?-gal positive cells were observed in BMSCs-CCD,the expression levels of the senescence related markers p16,p21 were higher in BMSCs-CCD,and less BMI1 nuclear localization was indicated in BMSCs-CCD than that in BMSCs.Conclusions: The proliferation,osteogenic differentiation potential and stemness of BMSCs-CCD decreased,whereas the senescence increased compared to that of BMSCs.Changes in the biological properties of BMSCs-CCD may lead to decreased osteogenesis capacity,which in turn affects the stability of the skeletal system of CCD patients and is associated with osteoporosis.The decreased expression of BMI1 which is essential for the regulation of stem cell self-renewal and cellular senescence,may be responsible for the senescence related characteristics of BMSCs-CCD.Part ? Mechanism of micro RNA-31 in regulating osteogenic differentiation and senescence of BMSCs in CCD patientsObjective: To analyze the effect of micro RNA-31(mi R-31)on osteogenic differentiation and senescence of BMSCs in CCD patients,and to explore the mechanism of RUNX2 haploinsufficiency affecting the expression of mi R-31.Methods: Real-time PCR was used to detect the differential expression of mi R-31 in BMSCs-CCD and BMSCs;The small interfering RNA was used to knock down RUNX2(si-RUNX2)in BMSCs to detect the expression of mi R-31 after knockdown;According to prediction tools,the mi R-31 luciferase reporter,which contained the binding site,was constructed.The expression vectors of mutant and wild-type mi R-31 were transfected with si-RUNX2 in 293 T cells to detect the difference in luciferase activity;Lentiviruses expressing mi R-31 inhibitor were generated to knock down mi R-31 in BMSCs-CCD,and alkaline phosphatase staining,alizarin red staining and Western blot analysis were used for osteogenic differentiation ability;The ?-gal senescence staining assay was used for senescence after knockdown.And the expressions of senescence markers were compared.Results:(1)The expression of mi R-31 is higher in BMSCs-CCD compared to BMSCs.(2)Down-regulation of RUNX2 resulted in mi R-31 up-regulation in BMSCs.The luciferase activity was enhanced by interfering with RUNX2,while the enhancement effect was abolished by mutations in the binding site.(3)Interfering with mi R-31 could promote osteogenic differentiation of BMSCs-CCD.(4)At the same time,mi R-31 knockdown led to decreased numbers of SA-?-gal-positive cells,and decreased expression of p16 and p21 with significantly increased Bmi1 level.Conclusions: RUNX2 regulated the transcriptional control of mi R-31 by directly binding to the promotor region of mi R-31 in human.RUNX2 haploinsufficiency leads to the up-regulation of mi R-31 transcriptionally in BMSCs-CCD.Mi R-31 was critically involved in osteogenesis-associated intrinsic mechanisms of BMSCs-CCD.,and may induce aging-related properties of BMSCs-CCD by regulating BMI1.Part ? in vivo study of mi R-31 in BMSCs-CCDObjective: To explore whether BMSCs modified by mi R-31 knocking-down lentivirus could obtain enhanced osteogenesis ability in vivo.Methods: Lev-GFP or Lev-mi R-31 inhibitor-treated BMSCs-CCD as well as the vacuity contrast groups were attached to HA/?-TCP biomaterial,respectively,which was implemented in the nude mice subcutaneously.Eight weeks after implantation,the specimens were harvested and subjected to H&E staining,Masson trichrome and immunohistochemical to detect the mineralized tissues and the expression of osterix(OSX)and OCN.Results:(1)Eight weeks after implantation,the mineralized tissues were significantly increased in Lev-mi R-31 inhibitor compared to Lev-GFP and Sham.The bone formation of Lev-mi R-31 inhibitor treated group was better than that of the BMSCs group.(2)The osteogenic factors both during the early stage and late stage were higher in Lev-mi R-31 inhibitor than that in Lev-GFP and Sham.Conclusions: Interfering with mi R-31 could improve the osteogenesis ability of BMSCs-CCD in vivo and that the application of mi R-31-modified BMSCs-CCD might have implications for treating bone disorders of CCD patients in the future. |
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