ABSTRACT
Aim To study the relationship between WntlOb and bone morphogenetic protein-9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs), and the molecular mechanisms underlying this process. Methods PCR, Western blot and histochemical staining were used to detect the effect of BMP9 on WntlOb and the effect of WntlOb on BMP9-induced osteogenic differentiation in MSCs. Meanwhile, real-time PCR, Western blot, oil red O staining, and flow cytometry assay were used to analyze the potential mechanism of Wnt10b affecting the function of BMP9. Results Wnt10b could be detected in C3H10T1/2, C2C12, MEFs and MC3T3-E1 cells. BMP9 up-regulated the expression of WntlOb in C3H10T1/2 cells. WntlOb enhanced the capability of BMP9 to increase the level of OCN and mineralization in C3H10T1/2 cells, and silencing Wnt10b attenuated these effects of BMP9. Wnt10b exhibited no substantial effect on cell cycle affected by BMP9, but it enhanced the effect of BMP9 on inducing phosphorylation of Smadl/5/8. While silencing Wnt10b attenuated this effect of BMP9. In addition, Wnt10b inhibited BMP9-induced adipogenic differentiation in C3H10T1/2 cells, and silencing Wnt10b promoted this effect of BMP9. Conclusions Wnt10b can promote BMP9-induced osteogenic differentiation of MSCs, which may be mediated through enhancing BMP/Smad signaling and reducing adipogenic differentiation.
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Abstract Split-hand/split-foot malformation (SHFM), also known as ectrodactyly is a rare genetic disorder. It is a clinically and genetically heterogeneous group of limb malformations characterized by absence/hypoplasia and/or median cleft of hands and/or feet. To date, seven genes underlying SHFM have been identified. This study described four consanguineous families (A-D) segregating SHFM in an autosomal recessive manner. Linkage in the families was established to chromosome 12p11.1-q13.13 harboring WNT10B gene. Sequence analysis identified a novel homozygous nonsense variant (p.Gln154*) in exon 4 of the WNT10B gene in two families (A and B). In the other two families (C and D), a previously reported variant (c.300_306dupAGGGCGG; p.Leu103Argfs*53) was detected. This study further expands the spectrum of the sequence variants reported in the WNT10B gene, which result in the split hand/foot malformation.
ABSTRACT
Objective To establish the GIOP model and extract BMSCs from the rat model.We aim to in-vesitigatethe effect ofrhPTH(1-34)for inhibiting β-catenin ubiquitination when combining with Micro-CT and bio-logical technology.We also investigate the influence of rhPTH(1-34)on the GIOP.Methods Female SPF emale rats wererandomly divided into normal control group,methylprednisolone group(model group),methylpredniso-lone+saline group(blankcontrol group)and methylprednisolone+rhPTH(1-34)group(test group). The proximal femoral cancellous bone was examined by Micro-CTand histopathological Staining. The expression of Wnt10b and β-catenin protein were detected. By comparing with inducedBMP-2,BMSCs were treated withrhPTH(1-34)and stained with ALP and alizarin red.Results(1)In Micro-CT,BV/TV,Tb.Th and Tb/N decreased,whereas Tb/sp increased in the test group comparedwith model group(P<0.05).ROI three-dimensional reconstruction of trabecu-lar bone in test group showed local bone repair;(2)Wnt10b and β-cateninexpression increased in the test group compared with the model model(P<0.05),indicating that rhPTH(1-34)can enhance the transcriptional activity of β-catenin(P<0.05)and promote the expression of Wnt10b andβ-catenin(P<0.05).Conclusion The inter-vention with rhPTH(1-34)can prevent GIOP by regulating the Wnt/β-catenin signaling pathway and inhibiting GIOP progress,which can improve the microstructure of bone.
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Objective To study the expression and clinical significance of Wnt10b gene in tissue samples of Hirschsprung disease (HD).Methods Sixty samples of stenotic intestines and 60 samples of normal intestines were collected from 60 patients with sporadic HD who were admitted to the Shengjing Hospital of China Medical University from 2003 to 2010.Tissue samples were stained by hematoxylin and eosin (HE),and then the expression of Wnt10b gene in the tissues was detected by immunohistochemical staining.The mRNA and protein expressions of Wnt10b in HD were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot.All data were analyzed by two independent sample t test.Results The results of HE staining confirmed HD in the 60 patients,and the samples met the requirement of the study.The expressions of Wnt10b were strong positive in the plasma of intramuscular and submucosal cells in stenotic intestines,while weak positive or negative in normal intestines.The results of immunohistochemical staining showed that the percentages of positive proteins in the stenotic intestines and the normal intestines were 0.061％ ± 0.014％ and 0.006％ ± 0.005％,respectively,with a significant difference (t =2.955,P ＜ 0.05).The results of qRT-PCR showed that the relative quantification of mRNA of Wnt10b was 23.5 ± 1.6 in the stenotic intestines,which was significantly higher than 13.1 ± 1.7 in the normal intestines (t =1.687,P ＜0.05).High mRNA expressions of Wnt10b were observed in 45 patients with HD,and low mRNA expressions of Wnt10b were observed in 15 patients.The results of Western blot showed that the relative protein expression of Wnt10b was 35.2 ± 2.3 in the stenotic intestines,which was significantly higher than 19.1 ± 1.3 in the normal intestines (t =2.046,P ＜ 0.05).High protein expressions of Wnt10b were observed in 43 patients,and low protein expressions of Wnt10b were observed in 17 patients.Conclusion There is a close relationship between the abnormal mRNA and protein expressions of Wnt10b in the intestines,which might play a role in the pathogensis congenital malformation of the alimentary tract.
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Objective To establish a rat skin organ culture model to study the initiation of hair follicle morphogenesis, and the dynamic expression of Wnt10b/β-catenin in the developing hair follicle. Methods The dorsal skins of SD rat at embryos 14-17 (E14-E17) were cultured on a gelatin sponge-supported tissue culture system at the air/liquid interface of DMEM with 10 % fetal bovine serum (FBS) for 3-6 days, some of which removed from El5 were cultured in DMEM with FBS of different concentrations. HE staining and fluoroimmunoassay were adopted. Results The model we established allowed skin tissues isolated from E14-E15 to develop in a manner that was histologically and temporarily similar to the process in vivo. However, the developing hair follicle ceased to continue when their morphogenetic process reached the forth stage, and the concentration of FBS did not show any significant effect on the development of hair follicle. Expression of Wnt10b/β-catenin was induced in culture, as it also occurred in vivo,but grew weak till it disappeared in culture for 6 days, which was accompanied by development halt of hair follicle. Conclusions A rat skin organ culture model is established in which the morphogenesis of hair follicle takes place in a manner similar to in vivo, and Wnt10b/β-catenin probably has a close connection with the early morphogenesis of hair follicle.