骨形态发生蛋白7在前列腺癌骨转移中的意义

目的 研究骨形态发生蛋白7(BMP7)与前列腺癌骨转移的关系.方法 采用逆转录-聚合酶链反应(RT-PCR)法检测人前列腺癌PC-3、乳腺癌细胞MDA-MB-231、肾癌细胞KETR-3、膀胱癌细胞T24、胃癌细胞MGC-803中BMP7 mRNA表达.免疫组织化学PV法检测45例有骨转移,42例无骨转移前列腺癌患者前列腺癌组织的BMP7蛋白表达.结果 RT-PCR结果显示,BMP7相对吸光度均值在前列腺癌PC-3细胞(9.59×10-1±1.67×10-2)和乳腺癌细胞(9.50×10-1±3.89×10-2)显著增高;与胃癌细胞MGC-803(2.93×10-1±1.33×10-2)、膀胱癌细胞T24(2.96×10-1±1.21×10-2)、肾癌细胞KETR-3(3.26×10-1±1.53×10-2)比较,差异有统计学意义(P＜0.05).免疫组织化学结果显示,骨转移前列腺癌组BMP7表达吸光度均值(74.80±5.76)明显高于无骨转移前列腺癌组(65.94±1.76),两组差异有统计学意义(P＜0.05).结论 BMP7在嗜骨性转移的前列腺癌细胞中,以及有转移的前列腺癌组织中的高表达,表明BMP7与前列腺癌骨转移有关.     

骨形态发生蛋白-7在前列腺癌组织中的表达及意义

目的 研究骨形态发生蛋白-7(BMP-7)在前列腺癌(PCa)组织中的表达及意义.方法 经病理检查确诊PCa组织标本87例.患者平均年龄66(59～78)岁,术前或穿刺前血清总PSA(t-PSA)平均45.7(2.4～138.2)ng/ml.Gleason评分≤6分37例,7分18例,≥8分32例.临床分期:I期(T1a N0 M0)+Ⅱ期(T1b N0M0,T1cN0M0,T2N0M0)20例,Ⅲ期(T3N0M0)20例.Ⅳ期(T4N0M0,TxN1M0,TxN0M1)47例,其中TxN0M1 45例.根据核素骨扫描或正电子发射计算机体层成像CT检查结果分为:PCa无骨转移42例,PCa伴骨转移45例.以30例BPH组织标本为对照,患者平均年龄68(57～88)岁.免疫组织化学PV法检测BMP-7在PCa组织和BPH组织中的表达,统计学分析2组及PCa组内表达差异,PCa组织中BMP-7与血清t-PSA相关性采用Pearson相关性分析. 结果BPH组织中BMP-7表达吸光度A值为70.55±5.41,PCa组织中为70.47±6.18,2者比较差异无统计学意义(P>0.05).PCa无骨转移组BMP-7表达吸光度A值为65.94±1.76,伴有骨转移组为74.80±5.76,2者比较差异有统计学意义(P<0.05).Gleason评分≤6分者吸光度A值为65.96±1.56,7分者为65.83±2.75,≥8分者为78.06±1.39.≤6分和7分者分别与≥8分相比,BMP-7表达A值差异有统计学意义(P<0.05).临床分期编组中Ⅰ期+Ⅱ期BMP-7表达A值为65.86±1.72,Ⅲ期为65.87±1.85,Ⅳ期为74.49±5.83,Ⅰ期+Ⅱ期和Ⅲ期分别与Ⅳ期相比,差异有统计学意义(P<0.05).PCa组织中BMP-7表达A值与血清t-PSA值呈正相关关系(r=0.77,P<0.05).结论 病理Gleason高评分、临床分期、已发生骨转移的PCa组织中高表达BMP-7,BMP-7表达水平与血清t-PSA具有正相关性,提示BMP-7可能是促进PCa细胞发生骨转移的重要细胞因子之一.     

Epigenetic regulation of human bone morphogenetic protein 6 gene expression in prostate cancer.

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional molecules that regulate bone induction and organ development. Among BMPs, BMP-6 has been shown to be overexpressed in prostate cancer and is speculated to be associated with bone-forming skeletal metastasis. We investigated the regulatory mechanism of the BMP-6 gene expression in prostate cancer cell lines DU-145, LNCaP, PC-3, and PC-3M with regard to the methylation status of the CpG island in the 5' flanking region of the human BMP-6 gene. By sequence-specific analysis of methylated cytosines, we show here that the methylation status of the CpG loci around the Sp1 site of the BMP-6 promoter is related to its steady-state expression and an alternative splicing of messenger RNA (mRNA) in prostate cancer cell lines. Furthermore, a study of clinical cases of benign and malignant prostate lesion by in situ hybridization showed that BMP-6 expression was high at both primary and secondary sites in cases of advanced cancer with metastasis. Demethylation of the CpG loci around the Spl binding site was shown in cases with high BMP-6 expression by sequencing analysis of the methylated cytosine from paraffin-embedded materials. Our results suggested that during cancer progression, besides inactivation of tumor suppressor genes by hypermethylation, activation of certain genes like BMP-6 by selective demethylation was a common epigenetic event giving a variable character to the invading and metastasizing cancer cells.     

Downregulation of the expression of bone morphogenetic protein 7 in experimental pyelonephritis

Bone morphogenetic protein 7 (BMP 7) is a member of the transforming growth factor (TGF) beta superfamily and is involved in regeneration, repair, and development of specific tissues, for example kidney, gut, lens, and skeleton. BMP 7 has emerged as a renotrophic factor and experimental studies have shown its protective role against fibrotic processes. Tubulointerstitial changes are present in the pyelonephritic kidney which progresses to fibrosis. Renal fibrosis may lead to significant morbidity in the form of hypertension, proteinuria, and loss of renal function. The objective of this study was to investigate BMP 7 expression in experimental acute and chronic pyelonephritis models. Eighteen Wistar rats were injected with 0.1 mL solution containing E. coli ATCC 25922 10¹⁰ cfu mL^–1 into left renal medullae. Six rats were used as a sham group and were given 0.1 mL 0.9% NaCl. Pyelonephritic rats were sacrificed 24 h (group I, n=6), 1 week (group II, n=6), and 6 weeks (group III, n=6) after E. coli injection. Serum creatinine levels were analyzed. Renal tissues were studied histopathologically by use of hematoxylin and eosin and scored for diagnosis of pyelonephritis. BMP 7 expression was studied semiquantitatively by immunohistochemical staining. Acute (group I) and chronic (group II and group III) pyelonephritic histopathological changes were observed in experimental pyelonephritic groups. A gradual decrease in BMP 7 expression was observed in the tubulointerstitial and tubular area of the pyelonephritic kidneys, mildest in the acute pyelonephritic group and most severe in the chronic pyelonephritic 6th week group. A statistically significant difference was observed between tubulointerstitial BMP 7 expression by groups I and III (P=0.017) and by groups III and IV (P=0.000). Tubular BMP 7 expression was statistically significantly different between groups II and IV (P=0.009) and between groups III and IV (P=0.002). The data imply that BMP 7 has a major role in chronic pyelonephritis. Tubulointerstitial and tubular BMP 7 expression also had a significant negative correlation with fibrosis, tubular, atrophy, and vascular changes. Serum creatinine levels of the study group were all normal. We conclude that the decrease in renal BMP 7 expression in experimental chronic pyelonephritis is one of the factors responsible for fibrotic changes in persistent renal damage.     

Expression of bone morphogenetic protein messenger RNAs by normal rat and human prostate and prostate cancer cells

Human prostate cancer cells are known to produce several growth regulatory factors, including transforming growth factor β (TGFβ) and heparin-binding fibroblast growth factors (FGFs), which may play as-yet-undefined roles in prostate gland morphogenesis, as well as in prostate cancer cell behavior. Recently, a family of proteins in the extended TGFβ family, the bone morphogenetic proteins (BMPs), has been identified which stimulates bone formation in vivo, and in which, the proteins are likely involved in a variety of morphogenetic processes during embryogenesis. These powerful morphogenetic factors are capable of redirecting muscle mesenchyme cells to differentiate along the lines of bone tissue. We examined a number of well-characterized rat and human prostate cancer cell lines for the expression of BMP 2, 3, 4, and 6 messenger RNA. Poly(A + )-RNA was isolated from normal human and rat ventral prostate, from the rat prostate adenocarcinoma PAIII tumor and cultured cells derived from it, and from human prostate cancer cell lines PC-3, LNCaP, and DU-145. BMP mRNA levels were measured using BMP 2, 3, 4 and Vgr-1 (BMP 6) cDNA probes. Both normal and neoplastic prostate tissue expressed these BMP mRNAs, although the level of expression varied from tumor to tumor. Normal human prostate expressed BMP 4 mRNA predominantly, as did the human prostate cancers PC-3 and DU-145. PC-3 also expressed BMP 2 mRNA and BMP 3 mRNA in large amounts. Normal rat ventral prostate expressed all these BMP mRNAs, but the rat prostate adenocarcinoma PAIII expressed predominantly BMP 3 mRNA. The reason that different BMPs are expressed in varying amounts by these normal and neoplastic cells is unknown. However, if these BMPs are expressed in biologically active form, they could be responsible for important effects on normal prostate growth and morphogenesis, on neoplastic prostate cell behavior, and could even contribute to the capacity of prostatic cancer cells to stimulate new bone formation at metastatic tumor sites in bone. © 1994 Wiley-Liss, Inc.     

Targeting the HGF/SF receptor c-met using a hammerhead ribozyme transgene reduces in vitro invasion and migration in prostate cancer cells

BACKGROUND: Hepatocyte growth factor scatter factor (HGF/SF) elicits a number of biological activities including invasion and migration through activation of its tyrosine kinase receptor c-Met. Over expression of c-Met has been implicated in prostate cancer development and progression. This study examined the effect of a ribozyme transgene, designed to inhibit human c-Met expression, and its impact on in vitro invasion and migration in prostate cancer. METHODS: A transgene (Met 560) consisting of U1 snRNA, hammerhead ribozyme, and antisense was cloned into a modified pZeoU1-EcoSpe vector and transfected into DU-145 cells. The effect of HGF/SF was tested on prostate cancer cells whose expression of c-Met had been blocked by way of a ribozyme transgene. RESULTS: Met 560 stable transfectants (DU-145(+/+)) manifested a complete loss of c-Met expression at mRNA and protein levels. In contrast, control plasmid (DU-145(+/-)) and wild-type DU-145 cells (DU-145(-/-)) had similar levels of c-Met expression. HGF/SF significantly increased the in vitro invasiveness (mean 47.71 +/- SE 7.75; P < 0.01 vs. control 24.14 +/- 1.34), and migration (mean 48.44 +/- SE 3.51; P < 0.01 vs. control 22.95 +/- 1.47) of DU-145(-/-) cells, respectively. Similarly, HGF/SF also increased the invasion (62.33 +/- 6.34; P < 0.001 vs. control 24.5 +/- 2.35) and migration (46.14 +/- 2.26; P < 0.01 vs. control 21.82 +/- 1.62) of DU-145(+/-) cells. In contrast, DU-145(+/+) cells had lost its response to HGF/SF induced invasion (22.33 +/- 2.08; P > 0.05 vs. control 23.5 +/- 2.11) and migration (24.12 +/- 0.86; P > 0.05 vs. control 23.27 +/- 0.81). CONCLUSIONS: Targeting the HGF/SF receptor by way of a hammerhead ribozyme encoding antisense to c-Met, is an effective method to reduce the invasive or migration potential in prostate cancer, and may have important therapeutic implications.     

Influence of simultaneous targeting of the bone morphogenetic protein pathway and RANK/RANKL axis in osteolytic prostate cancer lesion in bone

Metastasis to bone is the leading cause of morbidity and mortality in advanced prostate cancer patients. Considering the complex reciprocal interactions between the tumor cells and the bone microenvironment, there is increasing interest in developing combination therapies targeting both the tumor growth and the bone microenvironment. In this study, we investigated the effect of simultaneous blockade of BMP pathway and RANK/RANKL axis in an osteolytic prostate cancer lesion in bone. We used a retroviral vector encoding noggin (RetroNoggin) to antagonize the effect of BMPs and RANK:Fc, which is a recombinant RANKL antagonist was used to inhibit RANK/RANKL axis. The tumor growth and bone loss were evaluated using plain radiographs, hind limb tumor measurements, micro PET/CT ((18)FDG and (18)F-fluoride tracer), and histology. Tibias implanted with PC-3 cells developed pure osteolytic lesions at 2-weeks with progressive increase in cortical bone destruction at successive time points. Tibias implanted with PC-3 cells over expressing noggin (RetroNoggin) resulted in reduced tumor size and decreased bone loss compared to the implanted tibias in untreated control animals. RANK:Fc administration inhibited the formation of osteoclasts, delayed the development of osteolytic lesions, decreased bone loss and reduced tumor size in tibias implanted with PC-3 cells. The combination therapy with RANK:Fc and noggin over expression effectively delayed the radiographic development of osteolytic lesions, and decreased the bone loss and tumor burden compared to implanted tibias treated with noggin over expression alone. Furthermore, the animals treated with the combination strategy exhibited decreased bone loss (micro CT) and lower tumor burden (FDG micro PET) compared to animals treated with RANK:Fc alone. Combined blockade of RANK/RANKL axis and BMP pathway resulted in reduced tumor burden and decreased bone loss compared to inhibition of either individual pathway alone in osteolytic prostate cancer lesion in bone. These results suggest that simultaneous targeting of tumor cells and osteoclasts may be the most effective method of inhibiting the progression of established osteolytic metastatic lesions in vivo.     

转染人骨形态发生蛋白在兔骨髓间充质干细胞中的表达

目的采用基因转移技术将人骨形态发生蛋白7(hBMP-7)基因转染兔骨髓间充质干细胞(BMSc),检测外源基因的表达.方法常规分子生物学技术构建hBMP-7逆转录病毒载体,制备含目的基因的重组逆转录病毒液,感染兔骨髓间充质干细胞,使用原位杂交以及免疫组织化学的方法检测hBMP-7在BMSc中的表达.结果原位杂交和免疫组化检测经hBMP-7基因转染的BMSc中出现阳性结果,未转染的BMSc中未见阳性结果出现.结论采用逆转录病毒介导的方法可以将hBMP-7转染至BMSc中,并有外源性基因的表达.     

HGF/c-Met信号系统与前列腺癌

原癌基因c-Met是前列腺癌基因治疗研究的理想靶点,HGF/c-Met信号系统的研究历史已久。本文就HGF/c-Met信号系统在前列腺癌的研究作一综述。     

姜黄素对前列腺癌细胞核转录因子抑制蛋白表达的影响

目的观察姜黄素对前列腺癌细胞核转录因子抑制蛋白(IkBα)表达的影响,探讨姜黄素抑制前列腺癌细胞增殖的作用机制。方法分别用10、25、50、75和100μmol/L 浓度的姜黄素对雄激素依赖性及雄激素非依赖性前列腺癌细胞株 LNCaP 和 PC3进行干预,5、12和24 h 后采用噻唑蓝(MTT)比色法观察细胞增殖情况;采用流式细胞术测定24 h 后细胞周期变化;5 h 后 Western 印迹法检测细胞中 IkBα的表达。结果姜黄素显著抑制 LNCaP 及 PC3细胞的生长,呈剂量和时间依赖性;姜黄素将两种前列腺癌细胞阻滞于 G_2、M 期[LNCaP 与 PC3细胞,空白对照分别为(11.4±1.3)%与(17.3±1.7)%,100μmol/L 姜黄素作用后分别为(27.3±2.8)%与(33.4±4.0)%],从而诱导肿瘤细胞凋亡;姜黄素作用于 LNCaP 细胞后,细胞中 IkBα表达无变化(F=0.129,P>0.05);但作用于 PC3细胞后,细胞中 IkBα的表达明显增强,呈现出显著的剂量依赖性(F=31.618,P<0.05)。结论姜黄素通过活化 IkBα在 PC3细胞中的表达发挥抑制 PC3细胞增殖的作用。对于 LNCaP 细胞,姜黄素可能通过抗氧化、抑制细胞内代谢产物形成等方式抑制 LNCaP 细胞增殖。     

Human bone morphogenetic protein 7 transfected nucleus pulposus cells delay the degeneration of intervertebral disc in dogs

The main reason for intervertebral disc (IVD) degeneration is the decrease in the quantity and activity of IVD cells with subsequent reduction of the extracellular matrix (ECM). In this study, we investigated a cell‐based repair strategy by injecting nucleus pulposus cells (NPCs) transduced with human bone morphogenetic protein (hBMP7) by adeno‐associated virus‐2 into the canine degenerative IVD to determine whether NPCs expressing hBMP7 could delay the degeneration of the IVD. Fourteen canines received annular punctures to induce disc degeneration. Eight weeks later, saline (group A), allogeneic NPCs (group B), or allogeneic NPCs transduced with hBMP7 (group C) were injected into the degenerative discs. Twelve weeks after the injection, MRI scan showed that the degeneration process of groups C was slower and less severe compared with that of groups B and C. The IVD stability in group C was superior to that in groups A and B in left‐right bending and rotation. HE, safranin‐O staining, and ELISA indicated that the degenerative degree of the IVD in group C was significantly milder than that in groups A and B. The study demonstrated that the implantation of NPCs‐hBMP7 could effectively maintained the structural integrity, ECM, and biomechanical properties of the canine degenerated discs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1311–1322, 2017.

     

HGF／SF、TGF－β1、NO在动脉硬化闭塞症发病中的意义

目的探讨肝细胞生长因子/散因子(HGF/SF)、转化生长因子β     

大肠癌细胞增殖中HGF/SF作用的观察

目的研究肝细胞生长因子/离散因子(HGF/SF)在诱导大肠癌细胞增殖中的作用。方法采用W estern B lot方法,检测HGF的受体c-met在受检大肠癌细胞株Caco-2,Colo3 2 0中的表达;观察Caco-2,Colo3 2 0中HGF/SF活化p 4 2/p 4 4MAPK和p 3 8MAPK的动态变化;应用[3H]-TdR,MTT方法观察p 4 2/p 4 4MAPK和p 3 8MAPK传导通路阻滞剂PD 9 8 0 5 9和SB 2 0 3 5 8 0对HGF/SF诱导的大肠癌细胞增殖的抑制作用。结果(1)c-met在Caco-2和Colo3 2 0中有表达。(2)HGF/SF激活p 4 2/p 4 4MAPK,p 3 8MAPK:2 0 ng/mL的HGF/SF处理细胞,p 4 2/p 4 4MAPK磷酸化在1 0m in达高峰(2.2 8±0.0 1);p 3 8MAPK变化与之相似(2.2 5±0.0 1)。(3)HGF/SF诱导大肠癌细胞的DNA合成增加依赖于p 4 2/p 4 4MAPK的激活,在2 4 h时点分别以2 0 ng/m lHGF/SF,不同浓度(1μmol/L,5μmol/L,1 0μmol/L)的PD 9 8 0 5 9和SB 2 0 3 5 8 0处理细胞,HGF/SF使胸腺啶吸收增加(P<0.0 1);PD 9 8 0 5 9以浓度依赖性抑制胸腺啶的吸收(P<0.0 1)。(4)HGF促进Caco-2细胞的增殖,而PD 9 8 0 5 9对这种增殖有抑制作用。结论HGF激活大肠癌细胞Caco-2和Colo3 2 0中p 4 2/p 4 4MAPK和p 3 8MAPK;p 4 2/p 4 4MAPK参与HGF/SF诱导的大肠癌细胞Caco-2有丝分裂;HGF促进大肠癌细胞Caco-2增殖;HGF/SF和p 4 2/p 4 4MAPK在大肠癌细胞中发挥作用可能有细胞选择性。     

骨缺损对脂肪源干细胞骨形态发生蛋白信号通路相关分子的影响

目的探讨脂肪源干细胞(ADSCs)是否存在骨形态发生蛋白(BMP)信号通路,以及骨缺损对大鼠内源性ADSCs中BMP信号通路分子表达的影响。 方法取30只Wistar大鼠随机分为空白对照组（A组5只）和实验组（B组25只）。A组不做处理,直接取腹股沟脂肪垫进行ADSCs培养。B组制造骨缺损模型,分别于造模后1、3、7、14、21d取腹股沟脂肪组织进行ADSCs培养,每一时间点取5只大鼠。原代培养7d,提取ADSCs总RNA,采用实时定量逆转录聚合酶链反应技术检测BMP受体(BMPR)和Smads的mRNA表达变化。实验数据用95％可信区间表示。 结果实验组骨缺损后1、3、7、14、21 d BMPR1a表达量均较对照组明显增加,差异有统计学意义(P＜0.05);骨缺损后7d表达量(2.532,2.552)增加达高峰。实验组骨缺损后1、3、7、14 d BMPRIb表达量较对照组明显增加,差异有统计学意义(P＜0.05);骨缺损后14 d表达量(6.628,6.648)增加达高峰。实验组骨缺损3、7、14、21 dBMPR2、Smad5、Smad8表达量较对照组明显增加,差异均有统计学意义(p＜0.05);骨缺损后14dBMPR2、Smad5表达量(3.538,3.658;8.055,8.149)增加达高峰,骨缺损后3 d Smad8表达量(3.657,3.759)增加达高峰。实验组骨缺损后7、14、21 d Smad1表达量较对照组明显增加,差异均有统计学意义(P＜0.05);骨缺损后7d表达量(3.641,3.771)增加达高峰。 结论 ADSCs表达BMPR,可能存在BMP信号通路,骨缺损可引起大鼠ADSCs中BMP信号通路相关分子的表达增加。     

Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro

Bone morphogenic proteins (BMPs) are growth factors important for skeletal development and bone growth. Noggin, one of the soluble BMP antagonists, regulates the action of BMPs on mesenchymal precursor cells, partially through a feedback type of inhibition. In this study, we constructed a novel BMP2/7 'fusion gene' that encodes both BMP2 and BMP7 genes in tandem by a linker. Polymerase chain reaction (PCR) and Western blotting showed that the BMP2/7 fusion gene construct led to the production of BMP2/7 heterodimers in A549 'producer' cells. When applied to C2C12 myoblastic cells, BMP2/7 heterodimers increased alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression (markers of osteoblastic differentiation) more effectively than either BMP2 or BMP7 homodimers. Moreover, this heterodimer induced significantly lower levels of Noggin expression in C2C12 cells than respective homodimers at similar doses. The addition of Noggin did not affect the heterodimer's activities in increasing osteoblastic differentiation in C2C12 cells. In contrast, BMP2 and BMP7 homodimers were largely inhibited by Noggin. Our finding suggests that the 'fusion gene' construct led to the production of bioactive BMP2/7 heterodimers, which were not antagonized by Noggin as effectively as it to BMP homodimers. The weaker Noggin antagonism on BMP heterodimers compared to homodimers may contribute to increased osteogenic potency of heterodimers in vitro and in vivo.