鸟氨酸脱羧酶基因反义RNA对前列腺癌细胞生长的抑制作用

为研究鸟氨酸脱羧酶 (ODC)基因反义RNA对前列腺癌细胞的生长抑制作用 ,将表达ODC第 3外显子反义RNA的重组腺病毒rAd ODC Ex3as分别感染前列腺癌细胞株PC 3和LNCap .通过MTT法观察其对前列腺癌细胞增殖的影响 ,并确定不同细胞合适的感染滴度 ,再采用Western印迹和流式细胞术检测rAd ODC Ex3as对细胞中ODC表达的抑制作用、对细胞周期和凋亡的影响以及与CDK抑制物p2 1的关系 .实验显示 ,rAd ODC Ex3as分别以 5 0MOI、2 5MOI感染PC 3和LNCap细胞可明显抑制其生长增殖 ,而不引起细胞毒性作用 ;其对两种细胞中ODC表达的抑制作用分别为4 5 %和 5 9% .流式细胞DNA含量分析证实 ,rAd ODC Ex3as可引起PC 3和LNCap细胞周期G1期阻滞 ,但并未引起凋亡 .通过Western印迹发现 ,细胞中ODC表达的降低可诱导p2 1蛋白的过表达 .结果表明 ,rAd ODC Ex3as在体外能有效地干扰ODC基因的表达 ,并通过诱导p2 1的过表达使其细胞周期停于G1期 ,从而抑制前列腺癌细胞PC 3和LNCap的增殖 ,为其进一步基因治疗的研究打下基础 .     

FGF8在脊椎动物早期胚胎发育中的调控作用

成纤维细胞生长因子8 (fibroblast growth factor 8,FGF8)是成纤维细胞生长因子家族的成员之一,是一种组织发育过程中的重要分泌性调控信号分子,参与脊椎动物的多种组织器官的发生与发育.早期胚胎细胞通过表达FGF8在组织和器官发育、血管发生、血细胞生成、附肢发生和伤口愈合等方面发挥着重要作用.FGF8不但可以在细胞外通过胞内信号通路,而且也可以进入细胞内部发挥生物学功能.本文就FGF8在脊椎动物神经系统、内脏器官、肢体发育及不对称发育等组织、器官发育中的调控作用予以阐述.     

重组人成纤维细胞生长因子8b原核表达载体的构建和纯化研究

成纤维细胞生长因子8b(fibroblast growth factor, FGF8b)在生长因子中与内分泌癌的发生和发展相关联,是一个有潜在临床应用价值的候选分子,为了满足重组人FGF8b的研发需要,论文采用分子克隆的方法构建了以包涵体形式表达FGF8b的重组大肠杆菌,并初步摸索出包涵体蛋白的纯化和复性工艺条件,通过Western blot和MTT法鉴定了FGF8b的生物化学特征和促增殖活性,表明利用包涵体复性技术初步得到了具有活性的蛋白,为后续研发进程奠定了基础。     

成纤维细胞生长因子8(FGF8)研究进展

成纤维细胞生长因子8(fibroblast growth factor 8,FGF8)是成纤维细胞生长因子(FGFs)家族的成员之一。其在人胚胎时期多种组织内进行表达,对各种器官的形成中起着重要的作用。在正常成人体内,FGF8的表达水平受到严格的限制,然而在某些癌细胞或炎症部位中大量表达,特别是在激素类癌症的发生和发展中起着重要的作用。因此应用FGF8抗体治疗激素类癌症,为临床提供了新的治疗途径。     

疤痕组织中FGF变化的免疫组织化学观察

为探讨疤痕形成机理,我们对疤痕的不同部位分别行组织学和免疫组织化学观察,了解成纤维细胞生长因子（ＦＧＦ）在疤痕组织的分布特点。结果发现：同一部位的疤痕,其增生明显处的真皮内ＦＧＦ表达明显;疤痕边缘或萎缩部位的真皮内ＦＧＦ表达中等;正常真皮内ＦＧＦ阴性表达;ＦＧＦ阳性细胞较多,为成纤维细胞;增生明显处疤痕的中层有较多的ＦＧＦ阳性细胞。本文认为：疤痕形成与ＦＧＦ表达密切相关,增生性疤痕真皮的中心部位可能是组织增生最活跃的部位。     

miR-125b在前列腺癌1E8细胞运动转移中的作用及其分子机制的初步探讨

目的 研究 miR-125b在前列腺癌高低转移潜能细胞中的表达差异及其对高转移细胞株1E8细胞的运动转移中的作用和可能的分子机制.方法 realtime PCR法检测前列腺癌高低转移潜能配对细胞系中 miR-125b的表达差异.通过划痕实验及transwell实验观察1E8细胞及转染 miR-125b 抑制剂及其阴性对照后该细胞运动转移能力的变化.结果 realtime PCR结果显示高转移潜能1E8细胞中miR-125b表达水明显高于低转移潜能2B4细胞;下调miR-125b会减弱1E8细胞的运动转移能力.结论 miR-125b可促进前列腺癌细胞的运动转移能力.     

FGF21对能量代谢的调控

成纤维细胞生长因子21（fibroblast growth factor 21,FGF21）作为一种不依赖胰岛素的血糖调节因子,目前已被看做是治疗2型糖尿病的一个潜在的新型治疗因素.大量鼠类及灵长类动物模型的实验结果显示：FGF21可通过作用于脂肪组织及胰腺来降低血糖和甘油三酯含量,从而预防饮食诱导的肥胖及胰岛素抵抗.此外,FGF21也被证明可作为一种主要的内源性调控子,在禁食和酮症时起着关键的调控作用.然而,一些临床观察实验的结果表明,临床观察实验与动物模型实验之间虽然具有一定的相似性,但也存在很多不同,因而目前FGF21在人体中的生理学作用仍不明确.     

bFGF对乳腺癌细胞系MCF-7细胞增殖与凋亡的影响

目的观察碱性成纤维细胞生长因子（bFGF）对体外培养人乳腺癌MCF-7细胞增殖和凋亡的影响,初步探讨bFGF作用机制。方法在饥饿培养的MCF-7细胞中加入bFGF和PD98059处理,以MTT法、吖啶橙染色及流式细胞术观察细胞生长与凋亡情况;并用Western blot检测caspase-3蛋白含量。结果对照组细胞形态发生改变：核质固缩、有凋亡小体形成;细胞凋亡率较高;Western blot分析表明,caspase-3蛋白明显表达。bFGF处理后,细胞变饱满,凋亡现象减少;细胞增殖比明显增加;与对照组相比凋亡细胞比例下降,并诱导细胞进入S期;随着bFGF浓度增加,caspase-3蛋白表达水平降低,在一定范围内呈剂量依赖性。加入PD98059可抑制bFGF的这些作用。结论bFGF可以促进细胞增殖,加速人乳腺癌细胞系MCF-7细胞的细胞周期进程,抵抗无血清饥饿诱导的凋亡,其作用部分可能是通过Ras-Raf-ERK1/2途径介导的。     

FGF13通过ROS介导抑制乳腺癌细胞MCF7凋亡

[目的]探究成纤维细胞生长因子13(Fibroblast growth factor 13,FGF13)对乳腺癌细胞MCF7凋亡的调控作用。[方法]Western Blotting和免疫组织化学染色检测FGF13的表达;构建干扰和超表达FGF13的MCF7细胞株;TUNEL法和Western Blotting检测细胞凋亡;DCFH-DA荧光探针法检测细胞内活性氧(Reactive oxygen species, ROS)。[结果]与正常乳腺细胞相比,FGF13在乳腺癌MCF7细胞中表达升高(0.16±0.01 vs 3.89±0.23,P<0.001)。干扰FGF13后,MCF7细胞凋亡率升高(2.20%±0.10%vs 10.79%±0.69%,P<0.001),促凋亡蛋白Bax(0.99±0.02 vs 2.56±0.02)、Caspase 3(1.00±0.01 vs 1.70±0.01)和p53(0.98±0.01 vs 2.22±0.03)的表达上调(P<0.001),而抗凋亡蛋白Bcl-xl(0.99±0.01 vs 0.29±0.01)、Bcl-2(0....     

碱性成纤维细胞生长因子对胚胎神经细胞存活与生长的影响

本文利用大鼠胚胎中脑神经细胞作原代微团培养,培养物经不同浓度碱性成纤维细胞生长因子（ｂａｓｉｃ Ｆｉｂｒｏｂｌａｓｔ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｂＥＧＦ）,研究ｂＦＧＦ对细胞生长和分化的影响,并利用图象分析细胞形态的变化。结果表明ｂＥＧＦ可促进微团中的集落形成率明显增加,并显示量效应关系。图象显示ｂＦＧＦ可促进细胞神经突起增多,而且有丰富的神经纤维连结成网络状。认为ｂＥＧＦ能促进中脑细胞生长和分     

Overexpression of FGF9 in Prostate Epithelial Cells Augments Reactive Stroma Formation and Promotes Prostate Cancer Progression

Bone metastasis is the major cause of morbidity and mortality of prostate cancer (PCa). Fibroblast growth factor 9 (FGF9) has been reported to promote PCa bone metastasis. However, the mechanism by which overexpression of FGF9 promotes PCa progression and metastasis is still unknown. Herein, we report that transgenic mice forced to express FGF9 in prostate epithelial cells (F9TG) developed high grade prostatic intraepithelial neoplasia (PIN) in an expression level- and time-dependent manner. Moreover, FGF9/TRAMP bigenic mice (F9TRAMP) grew advanced PCa earlier and had higher frequencies of metastasis than TRAMP littermates. We observed tumor microenvironmental changes including hypercellularity and hyperproliferation in the stromal compartment of F9TG and F9TRAMP mice. Expression of TGFβ1, a key signaling molecule overexpressed in reactive stroma, was increased in F9TG and F9TRAMP prostates. Both in vivo and in vitro data indicated that FGF9 promoted TGFβ1 expression via increasing cJun-mediated signaling. Moreover, in silico analyses showed that the expression level of FGF9 was positively associated with expression of TGFβ1 and its downstream signaling molecules in human prostate cancers. Collectively, our data demonstrated that overexpressing FGF9 in PCa cells augmented the formation of reactive stroma and promoted PCa initiation and progression.     

Type 2 Fibroblast Growth Factor Receptor Signaling Preserves Stemness and Prevents Differentiation of Prostate Stem Cells from the Basal Compartment

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, which include basal, luminal, and neuroendocrine cells. Two types of P-SCs have been identified in both human and mouse adult prostates based on prostasphere or organoid cultures, cell lineage tracing, renal capsule implantation, and expression of luminal- and basal-specific proteins. The sphere-forming P-SCs are from the basal cell compartment that express P63, and are therefore designated as basal P-SCs (P-bSCs). Luminal P-SCs (P-lSCs) express luminal cytokeratins and Nkx3.1. Herein, we report that the type 2 FGF receptor (FGFR2) signaling axis is crucial for preserving stemness and preventing differentiation of P-bSCs. FGFR2 signaling mediated by FGFR substrate 2α (FRS2α) is indispensable for formation and maintenance of prostaspheres derived from P63⁺ P-bSCs. Ablation of Fgfr2 in P63⁺ cells in vitro causes the disintegration of prostaspheres. Ablation of Fgfr2 in vivo reduces the number of P63-expressing basal cells and enriches luminal cells. This suggests a basal stem cell-to-luminal cell differentiation. In addition, ablation of Fgfr2 in P63⁺ cells causes defective postnatal development of the prostate. Therefore, the data indicate that FGFR2 signaling is critical for preserving stemness and preventing differentiation of P-bSCs.     

Prostate Sphere-forming Stem Cells Are Derived from the P63-expressing Basal Compartment

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, including basal, luminal, and neuroendocrine cells. Multiple methods have been used to identify P-SCs in adult prostates. These include in vivo renal capsule implantation of a single epithelial cell with urogenital mesenchymal cells, in vitro prostasphere and organoid cultures, and lineage tracing with castration-resistant Nkx3.1 expression (CARN), in conjunction with expression of cell type-specific markers. Both organoid culture and CARN tracing show the existence of P-SCs in the luminal compartment. Although prostasphere cells predominantly express basal cell-specific cytokeratin and P63, the lineage of prostasphere-forming cells in the P-SC hierarchy remains to be determined. Using lineage tracing with P63^CreERT2, we show here that the sphere-forming P-SCs are P63-expressing cells and reside in the basal compartment. Therefore we designate them as basal P-SCs (P-bSCs). P-bSCs are capable of differentiating into AR⁺ and CK18⁺ organoid cells, but organoid cells cannot form spheres. We also report that prostaspheres contain quiescent stem cells. Therefore, the results show that P-bSCs represent stem cells that are early in the hierarchy of overall prostate tissue stem cells. Understanding the contribution of the two types of P-SCs to prostate development and prostate cancer stem cells and how to manipulate them may open new avenues for control of prostate cancer progression and relapse.     

Immunoreactive fibroblast growth factor (FGF) in a transplantable chondrosarcoma: inhibition of tumor growth by antibodies to FGF

Using a radioimmunoassay for bovine pituitary fibroblast growth factor (FGF), we have established the presence of the immunoreactive mitogen in extracts of a transplantable mouse chondrosarcoma. Both neutral and acidic extracts of the tumor contain an immunoreactive FGF (ir-FGF) that cross-reacts in a parallel and dose-dependent fashion in the radioimmunoassay. The ir-FGF is retained on heparin-Sepharose affinity columns and can be detected in the same molecular weight forms as rat pituitary FGF. Mice (C57/Bl) inoculated with the tumor (10 mg, im) show a decreased rate of tumor growth when passively immunized with the antiserum to FGF. The results establish the presence of FGF in this tumor and implicate its role in the etiology of its development.     

Transgenic expression of FGF8 and FGF10 induces transdifferentiation of pancreatic islet cells into hepatocytes and exocrine cells

FGF signaling is essential for normal development of pancreatic islets. To examine the effects of overexpressed FGF8 and FGF10 on pancreatic development, we generated FGF8- and FGF10-transgenic mice (Tg mice) under the control of the glucagon promoter. In FGF8-Tg mice, hepatocyte-like cells were observed in the periphery of pancreatic islets, but areas of alpha and beta cells did not decrease, whereas in FGF10-Tg mice, pancreatic ductal and acinar cells were found in islets, concomitantly with disturbed beta-cell differentiation. These results suggest that FGF8 and FGF10 play important roles in development of hepatocytes and exocrine cells, respectively, and explain the absence of FGF8 expression in normal islets and pancreatic hypoplasia in FGF10-deficient mice.     

成纤维细胞生长因子的特性与分子功能

成纤维细胞生长因子(FGFs)是一类蛋白质活性肽,在有机体内外显示广谱的生物学活性和功能,在生物发育学、生理学与临床药理学方面具有重要的作用和意义．对 FGFs 家族成员肽的特征特性,FGFs 的染色体定位、基因结构与功能,FGFs 的信号分子作用与促分裂素作用,FGFs 的作用受体、主动和被动调节与拮抗机理进行了综述．     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用