Expression profile of microRNA secreted by rat condylar chondrocytes under tensile stress / 中华口腔医学杂志

Objective: To preliminarily explore the mechanism of tensile stress regulating endochondral osteogenesis of condyle by analyzing the expression profiles of significantly different microRNAs (miRNAs) in exosomes of rat mandibular condylar chondrocytes (MCC) under quiescent and cyclic tensile strain (CTS) conditions. Methods: Rat condylar chondrocytes were cultured under static and CTS conditions respectively (10 SD rats, male, 2 weeks old), and exosomes were extracted. The two groups of exosomes were named as control group and CTS group respectively. The differential expression miRNAs were screened by high-throughput sequencing. Bioinformatics analysis and prediction of target genes related to osteogenesis were performed by TargetScan and miRanda website. Results: The exosomes of rat condylar chondrocytes cultured under tensile stress showed a "double concave disc" monolayer membrane structure, the expression of CD9 and CD81 were positive, and the particle size distribution accorded with the characteristics of exosomes, which was consistent with that of static cultured rat condylar chondrocytes. A total of 85 miRNAs with significantly different expression were detected by high-throughput sequencing (P<0.05). The main biological processes and molecular functions of differential miRNAs were biological processes and protein binding, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathway enrichment analysis showed that there was significant enrichment in mammalian target of rapamycin (mTOR) signal pathway. The candidate target genes of miR-199a-5p include bone morphogenetic protein 3 (BMP3), endothelin converting enzyme 1, and miR-186-5p may target Smad8 and BMP3 to exert osteogenesis-related functions. Conclusions: Compared with static state, tensile stress stimulation can change the expression of miRNAs such as miR-199a-5p, miR-186-5p in the exocrine body of rat condylar chondrocytes, which can be considered as a mean to regulate the application potential of the exosomes.

Effect of BMP-2 and -3 overexpression on osteogenic and chondrogenic differentiation of prenatal mouse intervertebral disc cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein (BMP)-2 and -3 over expressions on chondrogenesis and osteogenesis of prenatal mouse intervertebral disc cells and provide experimental evidences for the application of BMPs in the therapy of disc diseases.</p><p><b>METHODS</b>The prenatal mouse intervertebral disc cells were infected with a recombinant adenovirus expressing BMP-2 and BMP-3 for 5-7 days, and the expressions of collagen type I (Col I), collagen type II (Col II), aggrecan, osteocalcin, osteoprotegerin and osteopontin mRNAs were detected with RT-PCR. The expression of cartilage matrix was evaluated with toluidine blue staining, and alkaline phosphatase (ALP) activity was detected with ALP reading and ALP staining.</p><p><b>RESULTS</b>BMP-2 and -3 overexpression did not enhance chondrogenesis and osteogenesis of annulus fibrosus (AF) cells or cause significant increases in the expressions of Col I, Col II or aggrecan mRNA in nucleus pulposus (NP) cells. Adenovirus-mediated overexpression of BMP-2 and BMP-3, however, promoted osteogenesis of NP cells and significantly increased the expression of osteocalcin mRNA; the overexpression of BMP-2, but not BMP-3, enhanced the mRNA expressions of osteoprotegerin and osteopontin. Toluidine blue staining demonstrated that BMP-2 and BMP-3 overexpression did not obviously affect the secretion of cartilage matrix. In NP cells, BMP-2 and -3 overexpression significantly enhanced ALP activity, which was not observed in AF cells.</p><p><b>CONCLUSION</b>Adenovirus-mediated BMP-2 and -3 overexpression can promote the osteogenic differentiation of NP cells but can not affect osteogenesis of AF cells or chondrogenesis of NP cells.</p>

Effect of human bone morphogenetic protein 2, 3, 6, and 12 on osteosarcoma cell line UMR106 / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of human bone morphogenetic protein (hBMPs) 2/3/6 and 12 on osteosarcoma cell UMR106.@*METHODS@#Adenovirus-BMP2/3/6 and 12 (AdBMP2/3/6 and12) were used to treat the cell line. Their proliferation, apoptosis, and transmigration were detected by Trypan blue exclusion test, TdT-mediated biotinylated-dUTP nick end labeling (TUNEL), acridine orange-ethidium bromide (AO/EB) double fluorescent dye staining, and transwell-room test, respectively. The alkaline phosphatase (ALP) activity was detected to reflect the differentiation of tumors.@*RESULTS@#Compared with the control groups, the cell survival rate of the experimental groups treated with AdBMP2/3/6 and 12 showed a significant time-dependent decrease (P<0.01). The apoptosis indexes were increased significantly (P<0.01) and the results from TUNEL and AO/EB method were consistent. The cell numbers of transmembrane significantly decreased at 24,48, and 72 h (P<0.01). AdBMP2/3/6 and 12 treatment enhanced the activity of ALP activity from day 3 and this effect might still be observed up to day 9 of the treatment (P<0.01).@*CONCLUSION@#hBMPs2/3/6 and 12 can inhibit the proliferation and transmigration, and induce their apoptosis and differentiation in osteosarcoma cell line UMR106.

Differential expression of Bmp2, Bmp4 and Bmp3 in embryonic development of mouse anterior and posterior palate / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The palate is differently regulated and developed along the anterior-posterior axis. The Bmp signal pathway plays a crucial role in palatogenesis. Conditioned-inactivation of Bmp type I receptor Alk2 or Alk3 in the neural crest or craniofacial region leads to palatal cleft in mice. However, how different Bmp members are involved in palatogenesis remains to be elucidated. In the present study, mRNA expression patterns of Bmp2, Bmp3 and Bmp4 in the developing anterior and posterior palates were examined and compared, focusing on the fusion stage.</p><p><b>METHODS</b>To detect the expression of Bmp mRNA, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagital and coronal sections of mice head from E13 to E18.</p><p><b>RESULTS</b>The expression of these Bmps were developmentally regulated in the anterior and posterior palates prior to, during and after palatal fusion. During palatal fusion, Bmp4 expression shifted from the anterior to the posterior palate, Bmp2 was highly expressed in both the anterior and posterior palates in this process, whereas Bmp3 was only localized in the posterior palate. They showed generally non-overlapping pattern in their expression domains. Thereafter, their expression was detected in both the anterior and posterior palates regulating osteogenesis and myogenesis respectively.</p><p><b>CONCLUSIONS</b>Bmp signalling is involved in palatogenesis in multiple stages and has multiple roles in regulating anterior and posterior palatal development. Disturbances of Bmp signalling during palatogenesis might be a possible mechanism of cleft palate.</p>

Reciprocal action between BMP-2 and BMP-3 in cultured fibroblast in vitro / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To explore reciprocal action between BMP-2 (bone morphogenetic protein-2) and BMP-3 for better understanding of the mechanism of BMP during bone fracture union.</p><p><b>METHODS</b>rhBMP-2 was added into the cultured fibroblasts with the concentration of 1,200 ng/ml. The expression of BMP-3 in fibroblasts was detected by immunohistochemistry. Eukaryotic expression vector pcDNA3-BMP-3 was transfected into the fibroblasts. After the effective expression of BMP-3 was identified, BMP-2 was also detected by immunohistochemistry in BMP-3 expression cells. The fibroblasts transfected with empty vector pcDNA3 were used as the control.</p><p><b>RESULTS</b>Exogenous rhBMP-2 could promote the expression of BMP-3 in fibroblasts. BMP-3 also could be detected in these cells.</p><p><b>CONCLUSIONS</b>BMP-2 and BMP-3 could reciprocally adjust the expression in fibroblasts.</p>