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.

Improved Osteointegration of Ti-6Al-4V-implants of Different Surface Texture by the Use of Bone Morphogenetic Protein-3 (BMP-3)

Half of altogether 60 cylindrical implant devices made of titanium-aluminum-vanadium alloy （ Ti-6Al-4V） were plusna-sprayed with a hydroxyapatite-couting and the other half had a corundum blasted porous surface. 15 implants of each group of the titanium test buplants were coated with 230 μg porcine, high-purified BMP- 3-precipitute per implant. In each case a BMP- 3-couted and an uncoated control-device were implanted into the femoral part of the putellofemoral joint of the right and left leg of 30 adult giant rabbits. Histomorphological and histomorphometrical we found in both groups with BMP- 3-coated test devices an improved osteointegrution. Stutistical evaluation using the t-test for matched samples showed 5 weeks after surgery a significant higher volume of tony formed bone of the BMP- 3-coated corundum- blasted or hydroxyapathe- coated Ti- 6Al- 4 V test devices compared to the non-couted controls of the same t）pe （p 〈 0.01, t-test for matched samples）. In both implant groups with BMP-couting a synergetic effect was verifiable although the bone ongrowth in the hydroxyaputite coated implants was more extensive than in the corundum blasted implants. Light microscopy demonstrated osteointegrution without connective tissue membrane around the surface of the implants. Our results indicate that composite metal implants,as used in endoprosthetics and implantology , are suitable carriers for BMP- 3 and im proved fixation of the implants can be achieved. The hydroxyapatite surface is superior to the corundum-blasted surface with regards to the observed parameters because of its pronounced bioactivity and its osteoconductive characteristics.

Effects of bone morphogenetic protein-4 on spatial memory and cholinergic expression in the dentate gyrus after fornix-fimbria transection in rats

BACKGROUND： Previous experiments have confirmed bone morphogenetic proteins （BMPs） upregulate cholinergic expression in neurons isolated from the embryonic rat hippocampus and cerebral cortex. Therefore, BMPs could be useful for treating Alzheimer＇s disease and other neurodegenerative diseases. OBJECTIVE： BMP-4 was infused into the hippocampal dentate gyrus of fomix-fimbria transected rats to test the effects of BMP-4 on cholinergic expression in dentate gyrus neurons, and to observe changes in spatial memory behavior. DESIGN： A randomized controlled animal experiment. SETTING： Department of Neurosurgery and Laboratory for Cell Biology, Institute of Geriatrics, General Hospital of Chinese PLA. MATERIALS： Twenty-seven healthy adult male Sprague Dawley （SD） rats, weighing 250-300 g, were provided by the Laboratory Animal Center of the General Hospital of Chinese PLA. Reagents： BMP-4 （B-2680, Sigma Company） and choline acetyl transferase （CHAT） antibody （AB5042, Chemicon Company） were used in this study. Equipments： a rat stereotaxic instrument （type： SN-2N, Narushige Group, Japan） and Image-prog-plus image analysis software （Media Cybernetics company, USA） were used in this study. The protocol was carried out in accordance with ethical guidelines for the use and care of animals. METHODS： This experiment was performed in the Institute of Geriatrics, General Hospital of Chinese PLA between July 2004 and March 2005. Rats were randomly divided into 4 groups： Alzheimer＇s disease group （n = 7）, normal control group （n = 5）, BMP-4-Alzheimer＇s disease group （n = 8）, and model group （n = 7）. In the Alzheimer＇s disease group, the left hippocampal fomix-fimbria of rats was transected to mimic Alzheimer＇s disease symptoms. In the BMP-4-Alzheimer＇s disease group, 1 μt L BMP-4 （10 mg/L） was perfused into the left dentate gyrus with a microinjector at 1 μ L/min. In the model group, 1 μ L saline was perfused into the same position by the same method. Twenty-eight days after injection, Morris water maze test was performed in all rats to test spatial memory. Time-to-platform and swim-path length were recorded. Immunohistochemical staining of cholinergic neurons was performed on brain sections containing dentate gyrus. The area covered by ChAT-positive cells was analyzed using an Image-prog-plus image analysis software. MAIN OUTCOME MEASURES： Area covered by ChAT-positive cells in the dentate gyrus. Time-to-platform and swim path-length. RESULTS： Twenty-seven rats were included in the final analysis. In the Alzheimer＇s disease group, the area covered by ChAT-positive cells was significantly smaller compared with the normal control group （F = 76.03, P 〈 0.01）. The area covered by ChAT-positive cells was significantly larger in the BMP-4- Alzheimer＇s disease group than in the model group （F = 35.17, P 〈 0.05）, but significantly smaller than in the normal control group （F = 40.17, P 〈 0.05）. Time-to-platform and swim-path length were significantly longer in the Alzheimer＇s disease group than in the normal control group （F =24.62 and 631.58, respectively, both P 〈 0.05）. Time-to-platform and swim-path length were significantly shorter in the BMP4-Alzheimer＇s disease group compared with the model group （F= 22.06 and 606.89, respectively P 〈 0.05）. CONCLUSION： Injection of BMP-4 into the dentate gyrus of Alzheimer＇s disease model rats alleviates central cholinergic system injury and concomitantly improves spatial memory.

Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells

Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.

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.

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1,Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

Human bone morphogenetic protein-2 gene transfer induces human mesenchymal stem cell proliferation and differentiation in vitro

Objective: To identify eukaryotic expression vector of human bone morphogenetic protein 2 pcDNA3/BMP2, verify its expression in transfected human mesenchymal stem cells (hMSCs) and the effect on hMSCs differentiation. Methods: The BMP2 gene was cloned into a eukaryotic expression vector pcDNA3. Transfected the recombinant into hMSCs by liposome. Immunnohistochemistry and in situ hybridization methods were used to identify the expression of BMP2 mRNA and protein; ALP and Von Kossa stains were performed to identify the BMP2 gene differentiated effect on the hMSCs. Results: The pcDNA3/BMP2 fragments were as large as theory. BMP2 mRNA and protein were expressed and synthesized both in 48 h and 4 weeks after transfection, the ALP and Ca deposit exhibition, which marked the osteogenic lineage of hMSCs, were enhanced and sped. Conclusion: Transfection of pcDNA3/BMP2 is able to provide transient and persistent expression in hMSCs, and promote the MSCs differentiation to osteogenic lineage.

AB004. Regulation of retinal angiogenesis and vascular permeability by bone morphogenetic protein signaling

The bone morphogenetic protein(BMP)family of proteins has a multitude of roles throughout the body.It plays important roles in development and in the adult vascular endothelium,by modulating the angiogenic response.The endothelial-specific receptor BMP receptor Alk1 is of particular importance in the proper remodeling of the vasculature and its ligand BMP9 has been shown to be a potent inhibitor of neovascularization.Dysregulated BMP signaling has been linked to multiple vascular diseases and can lead to the abnormal angiogenesis.We therefore investigated the role of BMP9/Alk1 signaling in retinal angiogenesis,and its therapeutic implications for vascular pathologies of the eye.

糖尿病视网膜病变患者血清BMP4、Netrin-4水平与病情分期及预后的关系研究

目的分析糖尿病视网膜病变(DR)患者血清骨形态发生蛋白4(BMP4)、神经轴突导向因子4(Netrin-4)水平,并研究其与患者病情分期及预后的关系。方法选取2019年10月至2022年11月该院收治的186例2型糖尿病患者作为研究对象,依据是否发生视网膜病变分为DR组(108例)和非DR组(78例),同期选取该院100例体检健康者作为对照组。依据DR患者病情分期将DR患者分为Ⅰ期(19例),Ⅱ期(14例),Ⅲ期(22例),Ⅳ期(23例),Ⅴ期(18例),Ⅵ期(12例),依据DR患者治疗后的视力残疾情况分为预后良好组(72例)及预后不良组(36例)。采用酶联免疫吸附试验检测血清BMP4、Netrin-4、空腹血糖(FPG)、糖化血红蛋白(HbA1c)、收缩压(SBP)、舒张压(DBP)、甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平,分析不同病情分期DR患者血清BMP4、Netrin-4水平。采用多因素Logistic回归分析DR发生的影响因素,分析不同预后DR患者血清BMP4、Netrin-4水平,采用受试者工作特征(ROC)曲线分析DR患者预后不良的诊断价值。结果DR组FPG、HbA1c、SBP、DBP、TG、TC、BMP4、Netrin-4水平高于对照组及非DR组,HDL-C水平低于对照组及DR组(P<0.05)。不同病情分期DR患者血清BMP4、Netrin-4水平比较,差异有统计学意义(P<0.05)。HbA1c、BMP4及Netrin-4水平是DR发生的影响因素(P<0.05)。预后不良组血清BMP4、Netrin-4水平高于预后良好组(P<0.05)。血清BMP4、Netrin-4水平联合诊断DR患者预后不良的效能优于各指标单独诊断。结论DR患者血清BMP4、Netrin-4水平升高,可辅助评估患者病情分期,联合诊断患者预后不良的效果较好。

基于TGF-β通路考察BMP4/FBN1对脊髓室管膜瘤增殖侵袭性的影响

目的基于TGF-β通路考察BMP4/FBN1对脊髓室管膜瘤(SCE)增殖侵袭性的影响及其相关机制。方法收集SCE患者肿瘤组织,同时收集正常组织作为对照,分为对照组、病例组。利用SCE患者肿瘤组织培养肿瘤细胞,并用携带FBN1 shRNA,BMP4 shRNA的慢病毒基因敲除FBN1或BMP4蛋白的表达。通过qRT-PCR和Western Blot检测肿瘤组织样本中FBN1和BMP4基因和蛋白表达水平。在BMP4低表达和FBN1低表达肿瘤细胞中通过qRT-PCR检测TGF-β基因表达水平;同时通过CCK-8细胞增殖实验与Transwell侵袭实验进一步检测敲低FBN1或BMP4蛋白的表达对肿瘤细胞增殖和侵袭能力的影响。结果与对照组相比,病例组BMP4和FBN1基因表达量升高(分别为P<0.05,P<0.01),蛋白表达量也升高(P<0.01);敲低FBN或BMP4蛋白不仅可下调SCE细胞中TGF-β基因的表达(分别为P<0.05、P<0.01),同时抑制了SCE细胞的增殖与侵袭能力(分别为P<0.05、P<0.01)。结论本研究基于TGF-β通路,考察关键分子BMP4/FBN1对SCE增殖侵袭性的影响,提示FBN1、BMP4与TGF-β之间可能存在相互作用,为临床治疗罕见病SCE提供可用的理论基础。

AN IMMUNOCYTOCHEMICAL STUDY OF BONE MORPHOGENETIC PROTEIN IN EXPERIMENTAL FRACTURE HEALING OF THE RABBIT MANDIBLE

A monoclonal antibody raised against bone morphogenetic protein (BMP-McAb) has been used to demonstrate the presence of bone morphogenetic protein(BMP) in experimental fracture healing. Rabbit mandibles were fractured using standardized methods and left to heal for 3, 7, 14, 21 and 24 d, respectively. The avidin-biotin complex (ABC) method demonstrated an accumulation of positively stained primitive mesenchymal cells at the fracture site in the hematoma stage of bone repair. These cells appeared to undergo differentiation into positively-stained chondroblasts and osteoblasts during the phase of callus formation. Undifferentiated mesenchymal cells showed a high positive reactivity in the early post-fracture stages but a much lower reactivity during the remodelling phase.The results of our study suggest that bone inductive processes are accompanied by the presence of BMP in osteoprogenitor cells during fracture healing of the mandible and that BMP may play a significant role in osteogenesis during bone healing.

Influence of bone morphogenetic protein on articular cartilage regeneration following periosteal grafting

Background: Autologous periosteal grafting is used as treatment for articular cartilage defect. Objective: To study the effect of bone morphogenetic protein (BMP) on articular cartilage regeneration following periosteal grafting. Methods: 16 healthy 15 week-old New Zealand white rabbits of both sexes (32 knees) were randomly divided into experimental group (group A) and control group (group B). A4.0 mmdiameter full-thickness articular cartilage defect was created in the femoral intercondylar fossa in all rabbits. Following this, a4.0 mmdiameter section of the periosteum was harvested from the anteromedial part of the upper tibial bone. In group A (eight rabbits, 16 knees), the cartilage defect was covered with periosteum, into which 20 μg BMP and 20% Pluronic were injected. In group B (eight rabbits, 16 knees), the cartilage defect was covered with periosteum, into which the same dosage of 0.9% NS (Normal saline) and 20% Pluronic were injected. All rabbits were sacrificed at 4, 8, and 12 weeks postoperatively, the cartilage defect areas were examined macroscopically and microscopically, and the morphology of the chondrocytes and collagen fibers were examined by scanning electron microscopy. Results: The filling of the defects with regenerated tissue was observed in both the group. The most notable improvement was that the cartilage regeneration in group A was obviously superior to that in group B, with the total histological score in group A significantly higher. Conclusion: BMP is an effective factor that could promote regeneration of articular cartilage and lead to successful cartilaginous resurfacing following periosteal

动脉阻塞性疾病患者血浆骨形态发生蛋白-4的表达变化及其与炎症和血管损伤的相关性

目的:骨形态发生蛋白-4(bone morphogenentic protein-4,BMP4)在动脉粥样硬化(atherosclerosis,AS)的病理过程中具有重要调节作用,但相关的临床研究较少。本研究拟观察以AS为主要病理特点的动脉阻塞性疾病(arterial occlusive disease,ACD)患者血浆BMP4的表达情况,并分析血浆中BMP4与炎症因子和血管损伤标志物之间的相关性。方法:共招募38名诊断为ACD的患者(ACD组)和38名体检志愿者(对照组),抽取ACD组患者术前和对照组体检时的静脉血,比较2组血常规指标的差异。采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测血浆中BMP4、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-1β、IL-10及血管内皮钙黏蛋白(vascular endothelial cadherin,VE-cadherin)的表达变化,并进一步分析BMP4与以上各指标之间的相关性。结果:与对照组相比,ACD组患者血常规结果表现为中性粒细胞-淋巴细胞比值[neutrophil to lymphocyte ratio,NLR;1.63(1.26,1.91)vs 3.43(2.16,6.61)]和血小板-淋巴细胞比值[platelet to lymphocyte ratio,PLR;6.37(5.26,7.74)vs 15.79(7.97,20.53)]升高、淋巴细胞-单核细胞比值[lymphocyte to monocyte ratio,LMR;5.67(4.41,7.14)vs3.43(2.07,3.74)]下降(均P<0.05);ACD组患者血浆BMP4[581.26(389.85,735.64)pg/mL vs 653.97(510.95,890.43)pg/mL]、TNF-α[254.16(182.96,340.70)pg/mLvs293.29(238.90,383.44)pg/mL]及内皮标志物VE-cadherin[1.54(1.08,2.13)ng/mL vs 1.85(1.30,2.54)ng/mL]的水平均显著升高,而抗炎因子IL-10的水平显著下降[175.89(118.39,219.25)pg/mLvs135.92(95.80,178.04)pg/mL](均P<0.05)。2组间促炎因子IL-1β的差异无统计学意义[300.39(205.39,403.56)pg/mL vs 378.46(243.20,448.69)pg/mL;P=0.09]。相关分析结果表明:血浆BMP4水平与促炎因子IL-1β(r=0.35)、TNF-α(r=0.31)以及内皮标志物VE-cadherin(r=0.47)呈正相关,与抗炎因子IL-10呈负相关(r=-0.37;均P<0.01)。结论:ACD患者血浆BMP4的水平升高,且与患者的炎症水平和血管损伤程度具有相关性。

BMP-4与17β-雌二醇对MBA-1细胞护骨素表达的影响

目的探讨骨形态生成蛋白-4(BMP-4)与17β-雌二醇对小鼠骨髓基质细胞株MBA-1细胞护骨素表达的影响。方法用BMP-4及17β-雌二醇干预MBA-1细胞后,用RT-PCR和Western印迹检测护骨素表达的变化。结果在无BMP-4或17β-雌二醇干预的情况下,MBA-1细胞在自身分化成熟过程中伴有护骨素的表达增加;经BMP-4与17β-雌二醇干预后可上调MBA-1细胞护骨素表达增加。结论BMP-4与17β-雌二醇可上调MBA-1细胞护骨素表达,从而诱导MBA-1细胞向成骨细胞方向分化。

BMP-4对甲状腺乳头状癌细胞生物学行为的影响及其分子机制

目的观察骨形态发生蛋白4(BMP-4)对甲状腺乳头状癌(PTC)细胞增殖、凋亡和侵袭能力的影响,并探讨其与Smad1信号通路的关系。方法常规培养PTC细胞系B-CPAP、TPC-1、K1细胞,采用qPCR法检测BMP-4 mRNA表达水平。取BMP-4 mRNA表达量较低的B-CPAP细胞,将其分为阴性对照组和过表达组,分别感染阴性片段、过表达BMP-4重组慢病毒;取BMP-4 mRNA表达量较高的TPC-1细胞,将其分为阴性对照组和干扰组,分别感染无效干扰片段和干扰BMP-4重组慢病毒。采用MTT法检测各组细胞增殖能力,流式细胞仪检测细胞凋亡情况,Corning实验检测细胞侵袭能力,qPCR法及Western blotting法检测E-Cadherin、Vimentin、Smad1 mRNA及蛋白表达。结果B-CPAP细胞过表达组细胞增殖能力较阴性对照组增加(P<0.01),细胞凋亡及侵袭能力无明显变化(P均>0.05),E-Cadherin、Vimentin、Smad1 mRNA及蛋白表达无明显变化(P均>0.05)。TPC-1细胞干扰组细胞增殖能力较阴性对照组下降,细胞凋亡增加,细胞侵袭能力下降,Vimentin蛋白表达降低(P<0.05或<0.01),E-Cadherin、Smad1蛋白表达无明显变化(P均>0.05)。结论BMP-4可促进PTC细胞增殖、抑制其凋亡,增强其侵袭转移能力,其机制与诱导上皮间质转化有关;BMP-4促进PTC侵袭转移不是通过Smad1信号通路发挥作用的。

前列腺癌组织中BMP-4的表达及临床意义

目的探讨骨形态发生蛋白-4(bone morphogenetic protein-4,BMP-4)在前列腺癌组织中的表达,分析其表达下调对前列腺癌细胞增殖和侵袭的影响。方法采用免疫组化En Vision法检测58例前列腺癌及40例前列腺良性增生(benign prostatic hyperplasia,BPH)组织中BMP-4蛋白的表达;利用Western blot法检测前列腺癌细胞(LNcap、PC-3、Du145)和BPH细胞中BMP-4蛋白的表达;将BMP-4 siRNA和对照siRNA分别转染LNcap,分为未处理组、对照siRNA组和BMP-4 siRNA组,采用Western blot法检测三组LNcap细胞中BMP-4、MMP-2和MMP-9的表达量;利用CCK8分别检测三组细胞的增殖能力;采用Tanswell小室实验检测三组细胞的侵袭能力。结果前列腺癌组织中BMP-4蛋白阳性率高于BPH组织(P <0. 05),BMP-4蛋白在前列腺癌细胞中的表达量高于BPH细胞。BMP-4 siRNA组细胞中BMP-4蛋白表达低于未处理组和对照siRNA组; BMP-4 siRNA组细胞增殖能力及细胞侵袭能力均低于未处理组和对照siRNA组。BMP-4 siRNA组细胞中MMP-2和MMP-9蛋白表达量低于未处理组和对照siRNA组。结论 BMP-4高表达于前列腺癌组织中,与前列腺癌细胞增殖和侵袭相关,有望成为前列腺癌潜在的治疗靶点。

BMP4、HIF-1α在上皮性卵巢癌中的相关性研究

目的:检测上皮性卵巢肿瘤组织中及体外不同缺氧浓度下卵巢癌细胞株SKOV-3细胞中骨形态发生蛋白4(bone morphogenetic protein4, BMP4)、缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)的表达,分析探讨BMP4、HIF-1α在上皮性卵巢癌转移、侵袭中的作用及与卵巢上皮癌预后的关系。方法:回顾性分析2014年1月-2017年12月在南通市第一人民医院诊治且资料完整的卵巢癌患者82例。采用免疫组织化学方法检测卵巢肿瘤组织BMP4、HIF-1α的表达。利用二氯化钴(CoCl2)构建缺氧模型,按实验要求培养SKOV-3细胞,置于0、50、100、150、200μmol/L含CoCl2的DMEM培养基中培养。其中将用0μmol/L CoCl2培养基的细胞设为常氧对照组,其余均为缺氧组。培养8 h,提取蛋白,用Western Blot检测,定量分析不同缺氧浓度下卵巢癌细胞株SKOV-3中BMP4、HIF-1α的表达情况。结果:BMP4、HIF-1α在正常卵巢中不表达,其阳性表达率随卵巢肿瘤恶性程度、临床分期、病理分级升高依次递增。体外不同缺氧状态下,随着CoCl2浓度的增高,卵巢癌细胞株SKOV-3中HIF-1α、BMP4的蛋白水平表达均逐渐增强(均P<0.05)。结论:BMP4、HIF-1α的表达与卵巢肿瘤发生发展、浸润转移密切相关,缺氧微环境可促进上皮性卵巢癌侵袭和转移。

BMP-4和Ki-67在泌乳素腺瘤中的表达及临床意义

目的探讨骨形态生成蛋白4(BMP-4)和细胞核增殖相关抗原(Ki-67)在泌乳素(PRL)腺瘤中的表达及与腺瘤侵袭性的关系.方法应用免疫组织化学和原位杂交技术检测 35例PRL腺瘤及8例正常垂体组织中BMP-4mRNA、BMP-4和Ki-67蛋白的表达水平,并对三者的表达水平的关系进行统计分析.结果 8例正常垂体组织的BMP-4和Ki-67表达均阴性;35例PRL腺瘤中,BMP-4mRNA、BMP-4表达水平与PRL腺瘤的侵袭性呈正相关 (r=0.885,P＜0.01);Ki-67在2～3级与0～1级的表达水平有显著性差异(P＜0.05),但 BMP-4mRNA、BMP-4的表达较Ki-67更具灵敏性(P＜0.05).结论 BMP-4在PRL腺瘤中呈特异性高表达,且与PRL腺瘤侵袭性呈正相关.

异氟醚通过BMP4/Smad信号通路对自发性高血压大鼠脑损伤的作用机制研究

目的:探究异氟醚通过调节骨发生形态蛋白4(BMP4)及其下游Smad蛋白对自发性高血压大鼠(SHR)脑损伤的作用机制。方法:采用随机数字表法将48只SHR分为模型组、异氟醚组、阳性药物组、异氟醚+LDN193189组,健康大鼠作为健康对照组,每组12只。各组大鼠分别放入氧气箱中,健康对照组、模型组、阳性药物组大鼠通入特殊气体(30%O_(2)、70%N_(2))1 h,异氟醚组、异氟醚+LDN193189组通入混杂2%异氟醚的特殊气体1 h,通过麻醉气体检测仪检测异氟醚含量,确保异氟醚浓度始终保持在2%。通气结束后,异氟醚+LDN193189组大鼠腹腔内注射BMP4/Smad通路抑制剂溶液20μL(LDN193189,5 mg/kg),阳性药物组大鼠腹腔内注射缬沙坦溶液20μL(10 mg/kg),健康对照组、模型组、异氟醚组大鼠腹腔内注射等量的生理盐水。氧气箱通气实验及注射每日1次,持续10 d。无创血压检测仪检测尾动脉收缩压变化;神经学行为评分评价行为功能;脑含水量检测脑水肿程度;原位末端转移酶标记法(TUNEL)染色和尼氏染色观察海马组织病理学变化及神经元损伤情况;荧光定量聚合酶链式反应(PCR)及蛋白免疫印迹(Western Blot)法检测海马组织BMP4、Smad2 mRNA及蛋白表达水平。结果:与模型组比较,异氟醚组、阳性药物组、异氟醚+LDN193189组大鼠海马神经细胞凋亡减少,尼氏小体增多,血压、神经学评分、脑组织含水量显著降低,BMP4、Smad2 mRNA及蛋白表达量显著升高(P<0.05);与异氟醚组比较,异氟醚+LDN193189组大鼠神经细胞凋亡增多,尼氏小体急剧减少,血压、神经学评分、脑组织含水量显著升高,BMP4、Smad2 mRNA及蛋白表达量显著降低(P<0.05);异氟醚组与阳性药物组各项数据差异均无统计学意义(P>0.05)。结论:异氟醚可通过促进BMP4/Smad信号通路减轻SHR脑损伤。

骨成型蛋白-4参与糖尿病心肌病“高糖记忆”现象的机制

目的 研究骨成型蛋白-4(BMP4)在糖尿病心肌病(DCM)“高糖记忆”中的作用。方法 体外实验:原代培养C57BL/6小鼠乳鼠心肌细胞,随机分为正常浓度葡萄糖组(Normal glucose,NG)、高浓度葡萄糖组(High glucose,HG)、“高糖记忆”组(HGNG),分别采用NG(5.6 mM)、HG(33 mM)、HG培养4 d+NG培养4 d,每组均培养8 d。培养结束后采用Western Blotting(WB)法检测凋亡蛋白Caspase-3及BMP4在DCM心肌细胞中的表达情况。以BMPs受体抑制剂DMH1作用于DCM“高糖记忆”模型细胞,观察Caspase-3及BMP4表达变化。体内实验:注射链脲佐菌素(50 mg/kg)连续5 d建立糖尿病C57BL/6小鼠模型,每日给予二甲双胍(Metformin,MET,320 mg/kg)灌胃治疗,连续给药8周,血糖仪检测血糖、WB法检测caspase-3及BMP4在小鼠心脏组织中表达情况。结果 在HG组中,HG可明显诱导心肌细胞BMP4蛋白表达,与NG组比较差异显著(P<0.01);DCM“高糖记忆”模型(HGNG组)心肌细胞BMP4蛋白表达显著升高,与NG组比较差异显著(P<0.01),与HG组比较无统计学差异(P>0.05)。DMH1可明显抑制DCM“高糖记忆”模型细胞中BMP4及Caspase-3蛋白表达(P<0.01)。MET可降低糖尿病小鼠血糖及心体比指数,抑制Caspase-3蛋白表达(P<0.01),但并不抑制BMP4蛋白表达。结论 BMP4是产生DCM“高糖记忆”现象的机制之一,BMPs受体抑制剂DMH1改善DCM“高糖记忆”引起的心肌细胞损伤。