AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Cloning of Rabbit Bone Morphogenetic Protein 15 and Its Expression During in vitro Maturation of Rabbit Oocytes

Partial cDNA sequence of rabbit BMP15 was cloned by RT-PCR from rabbit ovaries, showing a similarity of 83%-90% with the BMP15 nucleotide sequences in humans, mice, ovine, sheep, cows and pigs. The expression of BMP15 in rabbit cumulus-oocyte complexs during oocytes in vitro maturation （IVM） was measured by fluorescent quantitative RT-PCR method. BMP 15 was expressed at low levels in immature oocytes and increased to the highest level at 16h of IVM, which coincides with the time of cumulus cell expansion, then declined slowly under IVM cultivation. The expression pattern of BMP 15 suggested that it might be important in cumulus expansion in rabbits.

The biological function of type I receptors of bone morphogenetic protein in bone

Bone morphogenetic proteins （BMPs） have multiple roles in skeletal development, homeostasis and regeneration. BMPs signal via type I and type II serine/threonine kinase receptors （BMPRI and BMPRII）. In recent decades, genetic studies in humans and mice have demonstrated that perturbations in BMP signaling via BMPRI resulted in various diseases in bone, cartilage, and muscles. In this review, we focus on all three types of BMPRI, which consist of activin-like kinase 2 （ALK2, also called type IA activin receptor）, activin- llke kinase 3 （ALK3, also called BMPRIA）, and activin-like kinase 6 （ALK6, also called BMPRIB）. The research areas covered include the current progress regarding the roles of these receptors during myogenesis, chondrogenesis, and osteogenesis. Understanding the physiological and pathological functions of these receptors at the cellular and molecular levels will advance drug development and tissue regeneration for treating musculoskeletal diseases and bone defects in the future.

Influence of bone morphogenetic protein type IA receptor conditional knockout in lens on expression of bone morphogenetic protein 4 in lens

AIM: To investigate the influence of bone morphogenetic protein type IA receptor [BMPR-IA(ALK3)] conditional knockout in lens on expression of bone morphogenetic protein 4(BMP4) in lens during the development of the vertebrate eye.METHODS: Cre-positive mice were mated with Crenegative mice to generate 50% Cre-positive(conditional knockout, CKO) 4 embryos, 8 eyes and 50% Cre-negative offspring(wild type, WT) 4 embryos, 8 eyes. The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm.Removal of paraffin wax and dehydrating for sections,and then the procedure of in situ hybridization was processed, BMP4 MK1784-m(BOSTER) was used, and observed the expression of BMP4 in the lens in experimental group and control group. We selected SPSS11.0 software for statistical analysis, P【0.05 showed that the difference was statistically significant.· RESULTS: Four embryos of each genotype were examined, totally we had 8 embryos, 16 eyes. We got the uniform outcomes in all the embryos. We found ALK3 was required during lens growing, but was not essential for the formation of lens. We observed that the expression of BMP4 in the lens was significantly reduced in all 8 ALK3 CKO lens, BMP4 expression was normal in all the 8 WT lens, P 【0.01. This phenomenon became increasingly visible in accordance with embryo development. The most apparent alteration was present at stage E15.5.CONCLUSION: ALK3 is essential for lens growth. The influence of ALK3 on the expression of BMP4 is present during the development of mice lens.

Bone Morphogenetic Protein 15 as a Candidate Gene for Prolificacy of Jining Grey Goat

On the basis of the ovine bone morphogenetic protein 15(BMP15)gene,two pairs of primers(PI and P2)were designed to amplify exons 1 and 2 of the BMP15 gene in five randomly selected does of both Angora and Jining Grey goats.The sequences of BMP15 exon 1(P1 amplification)of Angora and Jining Grey goats were identical.There was a 3-nucleotide(CTT)insertion in positions 268 to 270 of goat BMP 15 exon1 compared with that of sheep(GenBank accession number AF236078),which caused a leucine insertion in the 12th position of amino acid sequence.Sequence length of goat BMP 15 exon 2(P2 amplification)was identical with that of sheep(AF236079),but there were seven nucleotide and four amino acid changes between goat and sheep.The nucleotide in the 963rd position of BMP15 exon 2 was A for Angora goat and sheep,and G for Jining Grey goat.Based on this A963G mutation,primer pair P3 was designed to detect single nucleotide polymorphism of BMP15 exon 2 in breeds of high prolificacy(Jining Grey),moderate prolificacy(Boer)and low prolificacy(Angora and Inner Mongolia Cashmere)by polymerase chain reactionsingle strand conformation polymorphism(PCR-SSCP).Three genotypes(AA,AG and GG)were detected in Jining Grey goats,two genotypes(AG and GG)in Boer,and only the AA genotype in Angora and Inner Mongolia Cashmere goats.Sequencing revealed one mutation(A963G)in genotype GG compared with genotype AA,and this mutation resulted in an amino acid change of serine→glycine(S300G).In Jining Grey goats,frequencies of AA,AG and GG genotypes were 0.008,0.059 and 0.933,respectively.Genotypic distributions of the BMP 15 gene were significantly different(P<0.05 or P<0.001)between Jining Grey and Boer,Angora,and Inner Mongolia Cashmere goats.In Jining Grey goats,the does with the GG genotype had 0.71(P<0.05)or 1.57(P<0.05)additional kids than did those with AG or AA genotypes,and does with the AG genotype had 0.86(P<0.05)more kids than did those with the AA genotype.These results tentatively indicate that the BMP15 gene is either a major gene that affects prolificacy in Jining Grey goats,or may be a molecular marker in close linkage with such a gene.

Expression of Vascular Endothelial Growth Factor in Bone Morphogenetic Protein-2 Induced Osteogenesis

An experimental model of femoral muscular pouch in 20 mice was adopted. The expression of VEGF was examined by in situ hybridization method and immunohistochemical method in bone morphogenetic protein- 2 induced osteogenesis . The experimental results demonstrated that the expression signals of VEGF mRNA and VEGF appeared in cytoplasm during condensation of mesenehymal cell. As the mesenchymal cells differentiated into precartilage, the expression signals decreased in mesenehymal cells, but increased in chondrocytes and kept getting denser in the process of cartilage maturity. The peak expression of VEGF mRNA and VEGF in the experimental group appeared on the 14 th day, accompanied by numerons hypertrophic chondrocytes. When mature cartilage calcified and neu, bone trabecula formed, the expression of VEGF mRNA and VEGF decreased in chondrocytes, but still expressed moderately in the osteoblasts and osteocytes. Signals of VEGF mRNA and VEGF can not be detected in the control groups.

Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases

The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.

Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

To construct the recombinant adeno-associated virus （rAAV） vector with human bone morphogenetic protein 7 （BMP7） and observe the BMP7 mRNA expression in vitro, BMP7 CDS sequence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombinant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated, the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×10^11 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

Establishment and identification of fibroblast clones expressing human bone morphogenetic protein 2

Objective:To establish fibroblasts stably expressing human bone morphogenetic protein 2(hBMP2). Methods:Eukaryonic expression vector(pcDNA3.1-B2) was transduced into NIH3T3 cells using SofastTM, a new generation cationic polymer gene transfection reagent. The positive cell clones were selected with G418. The stable transfection and expression of BMP2 in the NIH3T3 cells were determined by RT-PCR and immunohistochemical stain. Results: BMP2 mRNA was transcripted and expressed in the transfected NIH3T3 cells. Conclusion: With positive compound transfection, outside human BMP2 gene can be successfully transducted into NIH3T3 cells, which is the key step to induce periodontal cells to osseous phenotypes.

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole(Cynoglossus semilaevis)

BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp,which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR（qRT-PCR）. The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages（egg, larva, juvenile and fingerling stages）.The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch（dph）; then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to ＋109. The predicted transcription factor binding sites（E-box binding factors, zinc finger transcription factor, etc.） in this region might participate in the transcriptional regulation of the bmp2 gene.

Molecular Cloning and Sequence Analysis of FullLength cDNA Encoding Human Bone Morphogenetic Protein—7

Objective:To clone the full-length human bane morphogenetic protein-7 (BMP-7 ) gene and analyse its sequence, to aid in investigation of its function and structure. Methods : Total RNA was isolated from Chinese fetal kidney by the acid gmnidinium thiocyanate phenol-chloroform method. Two overlapping segments of human BMP- 1 cDNA were obtained by reverse transcription (RT)-PCR. Following application, the two segments were ligated to each other and subcloned into POEM-T easy vector to form PEGM-T easy/hBMP-7 recombinant plasmid. Sanger dideoxy chain-termination method was used to sequence the cDNA. Results. There was 750 bp fragment obtained RT-PCR using #2 primer from 5' end of BMP-7 gene (PCR by using # 2 and # 1) ,and 540 bp fragment from 3' end was generated by KT-PCR using # 4 primer (PCR using # 3 and # 4). Full-length cDNA encoding BMP-7 was obtained by religation of two segments. When compared with hBMP-7 sequence in Gene bank (XM30619) ,our full-length BMP-7 cDNA has a G instead of a T at nucleotide 862. This change results in valine substituting for phenylalanine in the protein. Conclusion. This is the first time that BMP-7 cDNA was successfully cloned from Chinese fetal kidney. BMP-7 cDNA plays an important role in healing injuries of the osteo-articular system. This makes BMP-7 is an attractive target far various clinical applications.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Expression of mature peptide of human bone morphogenetic protein-2 in Escherichia coli

To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expression vectorpDH in which foreign gene was controlled by PRPL promoters. E. coli DH5a transformed with recombinant plasmid pDHB2m wasinduced at 42℃to express the target protein. The expressed product was partially purified and refolded, and then implanted intorat thigh muscles to assay its bone inductive activity. Results: After induction, a protein band on SDS-PAGE gel with an apparentmol. wt. of 13kD was observed to anticipate in the strain carrying pDHB2m, but not in the control. The expressed hBMP-2m accounted for 45%-60% of the total bacterial protein. The expressed product existed in a form of inclusion body. After partially purified and refolded, rhBMP-2m could induce the formation of cartilage and bone tissue heterotopically. Conclusion: The maturepeptide of human bone morphogenetic protein-2 has ben successfully expressed in E. coli and the product has ectopic bone inductive activity.

血清骨形态发生蛋白9与生长分化因子15水平在脓毒症诊断及预后评估中的价值

目的:探讨血清骨形态发生蛋白9(BMP9)与生长分化因子15(GDF15)表达在脓毒症诊断和预后评估中的价值。方法:收集脓毒症患者30例作为脓毒症组,非脓毒症重症患者31例为非脓毒症组,同期体检的健康成人23例为对照组。追踪随访脓毒症组患者28d转归,分为存活组16例和死亡组14例。分析比较3组一般资料、临床指标及血清BMP9与GDF15水平。采用Logistic回归分析脓毒症发病及预后的危险因素。通过受试者工作特征曲线(ROC)的曲线下面积(AUC)评估血BMP9及GDF15在脓毒症诊断及预后预测中的价值。结果:与非脓毒症组和对照组比较,脓毒症组血清BMP9表达明显增高(P均<0.05)。与对照组比较,脓毒症组血清GDF15表达明显增高(P<0.05)。脓毒症死亡组患者血清BMP9、GDF15水平显著高于存活组(P均<0.05)。Logistic回归示血BMP9、C反应蛋白(CRP)、序贯器官衰竭评分(SOFA)是脓毒症发病的独立危险因素,GDF15是脓毒症患者死亡的独立影响因素。BMP9联合CRP诊断脓毒症的AUC为0.845,特异性83.9%,灵敏度73.3%,诊断效能与序贯器官衰竭评分相当(AUC 0.809,特异性83.9%,灵敏度70.0%)。GDF15联合中性粒细胞与淋巴细胞的比值(NLR)对脓毒症患者预后预测的AUC为0.898,特异性80%,灵敏度100%,预测效能与急性生理与慢性健康状况评估(APACHE II)评分相当(AUC 0.687,特异性93.9%,灵敏度53.3%)。结论:脓毒症患者BMP9和GDF15表达明显增高,BMP9联合CRP对脓毒症诊断价值较高。GDF15联合NLR对脓毒症患者预后预测效能较高。

CO-TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS WITH HUMAN BMP2 AND VEGF165 GENES

Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.

EXPRESSION OF rhBMP-7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

GDF-9和BMP-15在PCOS卵泡发育及胰岛素抵抗中的研究进展

生长分化因子-9(growth differentiation factor-9,GDF-9)和骨形态生成蛋白-15(bone morphogenetic protein-15,BMP-15)是转化生长因子-β(transforming growth factor-β,TGF-β)超家族的组成部分,在卵巢中参与颗粒细胞和卵母细胞的增殖分化,影响卵母细胞的发育和质量,调节卵巢功能。GDF-9和BMP-15异常表达,影响卵泡发育和透明带结构,可能是导致多囊卵巢综合征(polycystic ovary syndrome,PCOS)患者不孕的重要环节。BMP-15激活Smad1/5/8信号通路,可能还与PCOS患者的胰岛素抵抗密切相关。但是,血清中GDF-9和BMP-15蛋白难以精确定量,血清GDF-9和BMP-15水平难以作为PCOS特异性诊断与治疗评估的标志。综述GDF-9和BMP-15在卵泡发育中的作用、PCOS患者卵巢GDF-9和BMP-15异常表达对卵泡发育及胰岛素抵抗的影响,探讨GDF-9和BMP-15在PCOS诊治中的潜在应用价值。

过表达lncRNA HAGLR促胫骨骨折大鼠骨髓间充质干细胞成骨分化的机制研究

目的研究骨质疏松(OP)-胫骨骨折(TF)大鼠长链非编码RNA同源盒D基因簇反义生长相关长链非编码RNA(lncRNA HAGLR)与其下游靶基因的表达情况,并探讨lncRNA HAGLR对大鼠骨髓间充质干细胞(MSC)成骨分化的作用与机制。方法30只SD雌性大鼠随机分为sham组、OP组、OP-TF组,每组10只。ELISA法检测大鼠血清碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(TRAP)的水平。对大鼠MSC细胞系R7500使用成骨分化诱导培养基进行诱导,并分为MSC组和成骨诱导组(Osteogenic-MSC组)。分别转染pcDNA-HAGLR、pcDNA-NC、miR-19a-3p的模拟物(miR-19a-3p mimic)、mimic的阴性对照(NC mimic)、miR-19a-3p的抑制剂(miR-19a-3p inhibitor)、miR-19a-3p inhibitor的阴性对照(NC inhibitor)至R7500后进行相应分组。双荧光素酶报告基因实验验证lncRNA HAGLR和miR-19a-3p以及骨形态发生蛋白2(BMP2)和miR-19a-3p的靶向关系。qRT-PCR检测各组lncRNA HAGLR和miR-19a-3p的表达。Western blot检测BMP2、ALP、胶原蛋白I(COL-I)、骨钙素(OCN)、骨桥蛋白(OPN)的表达。ALP染色和AR染色检测MSC的成骨分化能力。结果OP组和OP-TF组的血清ALP和TRAP水平均高于sham组,差异有统计学意义(P<0.05)。而OP组与sham组胫骨组织中lncRNA HAGLR、miR-19a-3p、BMP2的表达水平比较差异均无统计学意义(P>0.05),而OP-TF组胫骨组织中lncRNA HAGLR、BMP2的表达水平均明显低于sham组和OP组(P<0.05),OP-TF组胫骨组织中miR-19a-3p的表达水平高于sham组和OP组(P<0.05)。与MSC组相比,Osteogenic-MSC组的lncRNA HAGLR表达水平明显升高(P<0.05),而miR-19a-3p的表达降低(P<0.05)。双荧光素酶报告基因实验表明lncRNA HAGLR与miR-19a-3p具有靶向关系,miR-19a-3p与BMP2具有靶向关系。pcDNA-HAGLR组的miR-19a-3p的表达水平低于pcDNA-NC组(P<0.05)。miR-19a-3p mimic组与NC mimic组的lncRNA HAGLR的表达水平比较,差异无统计学意义(P>0.05)。与NC mimic组相比,miR-19a-3p mimic组BMP2的表达水平降低(P<0.05),miR-19a-3p表达水平升高(P<0.05)。pcDNA-HAGLR组细胞较pcDNA-NC组具有更强的成骨分化能力和更高的ALP活性(P<0.05)。miR-19a-3p inhibitor组细胞较NC inhibitor组具有更强的成骨分化能力和更高的ALP活性(P<0.05)。结论胫骨骨折大鼠lncRNA HAGLR和BMP2表达降低,miR-19a-3p表达增高。过表达lncRNA HAGLR通过靶向调控miR-19a-3p/BMP2轴促进大鼠MSC的成骨分化。

骨形态发生蛋白/Wnt信号通路调控成骨:揭示骨骼形成和重塑的分子机制

背景:成骨细胞是负责骨骼形成和重塑的主要细胞类型,其功能的正常发挥受到多种信号通路的精密调控,其中,骨形态发生蛋白和Wnt信号通路在成骨过程中起着关键作用。目的:综述了骨形态发生蛋白/Wnt信号通路在调控成骨细胞功能中的作用,并分析其在不同生理和病理条件下的变化,以期进一步揭示骨骼形成和重塑的分子机制。方法:以中文检索词“BMP信号通路,Wnt信号通路,成骨”及英文检索词“BMP signaling pathway,Wnt signaling pathway,osteogenesis”在中国知网、万方和PubMed数据库中检索研究原著,检索时限为各数据库建库至2023年6月的相关文献,最终筛选61篇文献进行分析总结。采用文献综述的方法,对骨形态发生蛋白/Wnt信号通路调控成骨作用的研究进行梳理和分析。结果与结论:①骨形态发生蛋白和Wnt信号通路在成骨细胞的分化、增殖和成熟过程中发挥重要作用。骨形态发生蛋白信号通路主要通过激活Smad蛋白来调控成骨相关基因的表达,Smad蛋白被激活后进入细胞核,调控与成骨相关的基因表达,与骨形态发生蛋白不同,Wnt信号通路主要依赖于β-catenin的激活来发挥生物学效应。②不同生理和病理状态下,骨形态发生蛋白/Wnt信号通路的调控作用会受到多种因素的影响。生长因子及激素及机械应力等会在一定程度上影响骨形态发生蛋白/Wnt信号通路的活性。③骨形态发生蛋白/Wnt信号通路在调控成骨过程中与其他信号通路相互作用,共同构成复杂的调控网络。④中药和天然化合物可以通过调控信号通路来促进骨骼健康,为中药治疗骨骼疾病提供了新的可能性。⑤未来研究可进一步探讨骨形态发生蛋白/Wnt信号通路与其他信号通路的相互作用以及其在不同生理和病理条件下的变化,解析此复杂网络中的关键节点和调控机制,为骨骼相关疾病的治疗提供更精确的靶点,也为揭示骨骼形成和重塑的分子机制提供新的思路。

小尾寒羊高繁殖力候选基因BMP15和GDF9的研究

以控制Belclare和Cambridge绵羊高繁殖力的骨形态发生蛋白 15 (bonemorphogeneticprotein 15 ,BMP15 )基因和生长分化因子 9(growthdifferentiationfactor 9,GDF9)基因为候选基因 ,采用PCR RFLP技术检测BMP15基因和GDF9基因在高繁殖力绵羊品种 (小尾寒羊、湖羊 )以及低繁殖力绵羊品种 (多赛特羊、特克塞尔羊、德国肉用美利奴羊 )中的单核苷酸多态性 ,同时研究这两个基因对小尾寒羊高繁殖力的影响。结果表明 :在 5个绵羊品种中都没有检测到GDF9基因的G8突变 (C→T) ,也没有检测到BMP15基因的B4突变 (G→T)。高繁殖力的小尾寒羊在BMP15基因编码序列第 718位碱基处发生了与Belclare绵羊和Cambridge绵羊相同的B2突变 (C→T) ,而其余 4个绵羊品种则没有发生这种突变。对于BMP15基因的B2突变 ,在小尾寒羊中检测到AA、AB两种基因型 ,A等位基因频率为 0 734,B等位基因频率为 0 2 6 6。小尾寒羊与其余 4个绵羊品种间B2突变基因型分布差异极显著 (P <0 0 0 1)。突变杂合基因型 (AB)小尾寒羊平均产羔数比野生纯合基因型 (AA)多 0 6 2只 (P <0 0 1)。研究结果表明 ,BMP15B2突变对小尾寒羊高繁殖力影响作用十分明显 。