转录调节因子Ets-1在大鼠血管平滑肌细胞增殖与凋亡中的作用

目的： 研究转录调节因子Ets-1对大鼠血管平滑肌细胞增殖与凋亡的影响及其可能的机制。 方法： 用定量细胞DNA片段的方法观察大鼠血管平滑肌细胞增殖与凋亡。用Western印迹法检测磷酸化视网膜母细胞瘤（RB-P）蛋白表达。 结果： Ets-1抑制大鼠血管平滑肌细胞凋亡。反义P21WAF1/CIP1能够阻断Ets-1的抗凋亡作用，并抑制Ets-1诱导的平滑肌细胞增殖。Ets-1能够上调RB-P蛋白表达，反义P21WAF1/CIP1可以阻断Ets-1诱导的RB-P蛋白表达。 结论： 在大鼠血管平滑肌细胞中，Ets-1通过P21WAF1/CIP1旁路发挥抑制凋亡和促进增殖作用。     

肿瘤相关抗原基因OVA66特异的RNA干扰对肿瘤细胞生物学特性的影响

构建OVA66基因的特异小干扰RNA表达载体,转染HeLa细胞,分析对该基因表达和肿瘤生物学特性的影响。以RT-PCR、Westernblot方法检测OVA66基因在多种肿瘤细胞系的表达谱。利用计算机辅助设计和合成针对OVA66的特异小干扰RNA的DNA片段,定向克隆于特定的干扰载体(pSUPER)。利用脂质体法将重组载体转染于OVA66高表达细胞系HeLa,筛选得到shRNA-OVA66稳定表达细胞。采用Real-timePCR检测目的基因的表达;FACS、Westernblot方法分析目的蛋白的表达。MTT、3H-TdR掺入等方法检测干扰OVA66基因表达后对HeLa细胞增殖和凋亡的影响。双酶切鉴定得到针对OVA66基因特异的shRNA表达载体(pSUPER-shRNA-OVA66),并经序列分析证实。经检测pSUPER-shRNA-OVA66稳定表达的HeLa细胞(shRNA-OVA66)中目的基因mRNA水平降低;OVA66蛋白表达受到抑制,表明设计的靶片段对目的基因有显著的抑制效果。并显示shRNA-OVA66细胞DNA合成下降;细胞周期分析表明阻断OVA66基因表达可抑制shRNA-OVA66细胞增殖能力,并引发细胞凋亡。OVA66特异的shRNA表达载体构建成功,阻断OVA66基因表达可抑制细胞增殖,促使细胞凋亡,为OVA66蛋白肿瘤生物学功能与肿瘤发生、发展的研究奠定基础。     

p27^Kipl反义寡核苷酸对B淋巴瘤细胞WEHI—231细胞周期的影响

目的 探讨p27^Kipl在抗原受体介导的B淋巴瘤细胞WEHI-231细胞周期停止信号中的作用。方法 用抗IgM抗体诱导WEHI-231细胞细胞周期停止，用合成的p27^Kipl反义寡核苷酸抑制p27^Kipl基因的表达，采用流式细胞仪，分析细胞核的DNA含量和细胞周期的变化，用体外激酶实验检测Cdk2的活性，Western blot检测Rb蛋白的磷酸化水平。结果 合成的p27^Kipl反义寡核苷酸能阻断抗IgM抗体诱导的p27^Kipl基因表达的上调，用p27^Kipl反义寡核苷酸处理WEHI-231细胞，可恢复抗原受体交联引起的周期素依赖性激酶Cdk2活性的降低，以及Rb蛋白磷酸化水平的下降，并使细胞周期恢复运转。结论 p27^Kipl可能在抗原受体信号介导的WEHI-231细胞的细胞周期停止中发挥主要作用。     

Cyr61促进RA滑膜细胞增殖及炎症因子调控研究

目的:采用类风湿性关节炎(RA)患者滑膜细胞的体外培养,研究基质蛋白Cyr61在RA滑膜细胞增殖中的作用及其机制。方法:通过Real-time PCR、Western blot和免疫组化检测RA病人的滑膜组织和细胞中Cyr61的表达情况;用3H-TdR掺入法检测滑膜液(SF)对滑膜细胞增殖的影响;用ELISA方法检测RA患者滑膜液中Cyr61蛋白的水平。结果:RA病人的滑膜组织和细胞中高表达Cyr61;SF能刺激滑膜细胞发生明显增殖;且RA患者滑膜液中含高浓度的Cyr61蛋白。用SiRNA干扰技术抑制滑膜细胞中Cyr61基因表达,再加入滑膜液后,则滑膜细胞增殖明显降低。同时,将SF与anti-Cyr61抗体共同孵育后再刺激滑膜细胞,FLS也不再发生明显增殖。进一步研究滑膜液中与上调Cyr61表达有关的炎症细胞因子,发现SF中IFNγ-和TNFα-具有上调Cyr61蛋白表达的作用。结论:Cyr61蛋白是促进滑膜细胞增殖的重要调控基因;RA患者滑膜液中含有高浓度的炎症因子IFNγ-和TNFα-,通过上调Cyr61蛋白表达而促进滑膜细胞增殖,可能是促进RA病理性滑膜增生的重要因素之一。     

mdx小鼠骨骼肌组织成肌、成脂、成骨基因的特异性表达

背景：干细胞移植是治疗肌营养不良症的有效方法之一,但移植的干细胞在病理骨骼肌中成肌表达较低。 目的：通过比较mdx小鼠和C57小鼠的骨骼肌形态及成肌、成脂、成骨基因表达的差异,探讨mdx小鼠骨骼肌病理改变的可能机制。 方法：取mdx小鼠与C57小鼠的骨骼肌组织行冰冻切片,苏木精-伊红染色和Vonkossa染色观察两种小鼠肌肉组织的形态特征;提取mdx小鼠和C57小鼠骨骼肌组织总RNA,real-time PCR检测成肌、成脂、成骨相关基因的表达。 结果与结论：mdx小鼠骨骼肌有肌纤维坏死和再生,伴有轻度脂肪、纤维结缔组织增生,Vonkossa染色可见钙结节沉积,而C57小鼠的骨骼肌细胞形态清晰,核位于细胞周边。与C57小鼠比较,mdx小鼠肌肉组织成骨、成脂基因表达有不同程度的上调(P < 0.05),而成肌基因表达下调(P< 0.05)。dystrophin基因缺失及成肌基因表达下调、成骨和成脂基因上调是造成mdx小鼠肌肉组织变性坏死的原因。     

反义核酸抑制miR-20、miR-106表达对293T细胞增殖的影响

目的:通过反义核酸技术抑制miR-20、miR-106的表达,检测对293T细胞增殖的影响。方法:应用特异性针对miR-20、miR-106的反义核酸转染293T细胞,用形态学、流式细胞技术研究反义核酸对细胞增殖的抑制作用,用定量PCR和ELISA研究miRNA和相关靶基因的表达。结果:研究发现反义核酸可有效抑制miR-20和miR-106的表达,并明显抑制293T细胞的增殖;研究还表明反义miR-106可明显上调抑癌基因Rb的表达。结论:反义核酸通过抑制miRNA表达,上调抑癌基因Rb的表达,能明显抑制293T细胞增殖。     

肌特异性miRNA在肌细胞分化过程及肌营养不良中的表达

目的:观察肌特异性miRNA(miR-1,-133a和-206)在肌细胞增殖和分化过程中的变化,探讨肌特异性miRNA与肌营养不良的关系。方法:培养小鼠成肌细胞C_2C_(12)并诱导分化成熟,应用实时荧光定量PCR(q-PCR)分别检测C_2C_(12)在增殖期和分化期(1、3、5 d)miR-1、-133a、-206的变化;免疫组织化学法筛选dystrophin缺失型营养不良病例(DMD),q-PCR检测miR-1、-133a、-206在DMD肌组织中的表达情况。结果:增殖期C_2C_(12)细胞的miR-1、-133a、-206表达量较低,分化期3者表达均升高,并随着成肌细胞分化时间的延长,miRNAs表达显著升高,其中miR-1表达升高最明显;筛选的10例DMD标本均存在不同程度的dystrophin蛋白表达减弱或缺失,与非特异性改变肌组织对比,miR-1、-133a、-206表达均升高,其中miR-206在DMD)患者肌组织的表达水平显著升高。结论:miR-1、-133a、-206均能促进成肌细胞的分化,其中miR-206在肌营养不良的病变过程中可能发挥重要作用。     

microRNA-224在弥漫大B细胞淋巴瘤的表达及作用

目的研究miR-224在弥漫大B细胞淋巴瘤(DLBCL)患者中的表达与临床病理特征的关系,观察miR-224对弥漫大B淋巴瘤细胞系OCI-LY10增殖、侵袭能力的影响。方法收集86例DLBCL组织,并以22份无肿瘤细胞侵犯的正常淋巴结组织标本为对照,采用real-time PCR方法检测miR-224的相对表达水平,分析miR-224表达水平与患者临床病理特征的关系。对弥漫大B淋巴瘤细胞系OCI-LYl0进行miR-224反义寡核苷酸(ASO)转染并培养,MTT法检测细胞增殖能力,Transwell小室检测细胞侵袭能力,real-time PCR方法与Western blot方法检测细胞中bcl-2的表达水平。结果DLBCL患者淋巴瘤中miR-224表达水平显著低于正常淋巴结组织(P0.05),miR-224在DLBCL患者中的表达与患者年龄、性别、疾病分期均无相关性(P0.05)。miR-224ASO明显升高OCI-LYl0细胞的增殖侵袭能力,显著升高bcl-2蛋白与m RNA表达水平(P0.05)。结论miR-224抑制OCI-LY10细胞增殖侵袭可能与下调bcl-2蛋白表达相关。     

siRNA沉默MIF基因抑制大细胞肺癌H460细胞增殖

目的: 研究小干扰RNA(siRNA)阻断巨噬细胞抑制因子(MIF)基因表达对大细胞肺癌细胞株H460细胞增殖的抑制效应并探讨其抑瘤机制。方法: 在体外培养的H460细胞中,采用免疫荧光法观测siRNA转染效率,Western blotting检测MIF蛋白的表达,MTT法和平板克隆形成实验检测 MIF siRNA对H460细胞活性和增殖的抑制作用,Hoechst染色出现后荧光显微镜观测转染后细胞的形态学变化,流式细胞仪观测细胞凋亡。结果: 与阴性对照组相比,siRNA能有效阻断MIF蛋白的表达,显著抑制H460细胞活性及增殖能力(P<0.05);Hoechst染色可见转染MIF siRNA后的H460细胞出现典型的凋亡形态学改变;流式细胞术结果显示转染MIF siRNA1和MIF siRNA2后细胞凋亡较阴性对照组显著增加 。结论: MIF在大细胞肺癌的发生发展过程中发挥着重要作用,siRNA阻断MIF蛋白表达,可抑制H460细胞增殖,提示其极有可能成为肺癌的新治疗靶点。     

Yap通过长链非编码RNA MALAT1调控肝癌细胞增殖

目的观察Yap对肝癌细胞增殖的影响及可能机制。方法应用Western blot与q RT-PCR方法检测Yap与MALAT1蛋白在肝癌组织及癌旁正常组织中的表达,应用CCK-8法检测si Yap后肝癌细胞增殖活力的改变以及MALAT1的表达变化,过表达MALAT1检测si Yap后肝癌细胞增殖活力的改变。结果 Yap与MALAT1蛋白在肝癌组织中表达水平均高于癌旁正常组织,沉默Yap基因的表达后肝癌细胞增殖活力受到抑制,MALAT1的表达水平减少,过表达MALAT1后阻断Yap表达,Yap对肝癌细胞增殖抑制作用消失。结论 Yap参与肝癌细胞增殖可能与调控MALAT1相关。     

Expression of transforming growth factor-beta 1 and its relation to endomysial fibrosis in progressive muscular dystrophy.

Progressive muscular dystrophy is characterized by muscle fiber necrosis, regeneration, and endomysial fibrosis. Although absence of dystrophin has been known as the cause of muscle fiber degeneration, pathogenesis of interstitial fibrosis is still unknown. Transforming growth factor-beta 1 (TGF-beta 1) induces accumulation of extracellular matrix in various diseases, such as liver cirrhosis and interstitial pneumonitis. To investigate its function on the pathogenesis of progressive muscular dystrophy, it was necessary to determine the degree of TGF-beta 1 expression and the site of TGF-beta 1 immunoreactivity. In Duchenne muscular dystrophy and most of Becker muscular dystrophy, high TGF-beta 1 immunoreactivity expressed on muscle fibers and extracellular space. In other myopathies with endomysial fibrosis, however, TGF-beta 1 was seldom observed. We also examined the immunoreactivity of the latent TGF-beta binding protein, which is bound to the TGF-beta precursors. In all Duchenne muscular dystrophy and half of Becker muscular dystrophy cases, high latent TGF-beta 1 binding protein immunoreactivity was seen, but in other myopathies its immunoreactivity was seldom seen on muscle fibers or extracellular space. Therefore TGF-beta 1 may play an important role in synthesis and accumulation of extracellular matrix in progressive muscular dystrophy.     

Increased expression of decorin during the regeneration stage of mdx mouse

Satellite cells exist in postnatal muscle tissue and constitute the main source of muscle precursor cells for growth and repair. These cells carry out important roles for skeletal muscle formation postnatally during growth of muscle mass as well as damage-induced regenerative processes. Muscle regeneration supports muscle function in aging and has a role in the functional impairment caused by progressive neuromuscular diseases. Major substances controlling this process are growth factors and extracellular matrix. Myostatin, a member of TGF-β family, was mainly expressed in muscle tissue. Decorin, a member of the small leucine-rich proteoglycan gene family, is composed of a core protein and a dermatan/chondroitin sulfate chain. Recent studies have shown that decorin enhanced the proliferation and differentiation of myogenic cells by suppressing myostatin activity. Thus, decorin appears to be a new molecule in the myostatin signaling pathway and a promising target for treatment of progressive neuromuscular diseases. Therefore, in this study, we examined the localization of decorin as well as myostatin in a muscular dystrophy model in mdx mice and B10 Scott Snells mice as a control to elucidate the differences between decorin and myostatin messages as well as protein distribution. This study revealed increased expression of decorin protein as well as mRNA at the regenerative stage of mdx mice compared to early stages, while only weak expression of decorin was detected in the control mice. Our study contributes to identifying the relationship between decorin and myostatin as well as the development of a therapeutic strategy for progressive neuromuscular diseases.     

Interference with myostatin/ActRIIB signaling as a therapeutic strategy for Duchenne muscular dystrophy

Since the discovery of the myostatin/ActRIIB signaling pathway 15 years ago, numerous strategies were developed to block its inhibitory function during skeletal muscle growth. Accumulating evidence demonstrates that abrogation of myostatin/ActRIIB signaling ameliorates pathology and function of dystrophic muscle in animal models for Duchenne muscular dystrophy (DMD). Therapeutic trials in healthy man and muscular dystrophy patients suggest feasibility of blockade strategies for potential clinical use. However, many key questions on the effect of myostatin/ActRIIB blockade remain unresolved; such as the underlying molecular mechanism that triggers muscle growth, the effect on muscle regeneration and adult muscle stem cell regulation and whether it causes long term metabolic alterations. Current therapeutic strategies aim to systemically abrogate myostatin/ActRIIB signaling. Although this ensures widespread effect on musculature, it also interferes with ActRIIB signaling in other tissues than skeletal muscle, thereby risking adverse effects. This review discusses current knowledge on myostatin/ActRIIB signaling and its potential value as a therapeutic target for DMD.     

Analysis of motor control and kinesthetic perception in patients with progressive muscular dystrophy

The purpose of this study was to examine motor control and kinesthetic perception of upper extremity in patients with Duchenne progressive muscular dystrophy. Nine normal subjects and nine subjects with muscular dystrophy performed a pursuit tracking task with step wave target by means of isometric contraction, and simultaneously estimated magnitude of muscular tension during tracking behavior. The results were as follows: (1) the muscular electrical activity measured from the EMGs was directly proportional to the muscular tension for both the normal and the muscular dystrophy groups, (2) the speed of step response for the muscular dystrophy group was slower than that for the normal group, and (3) the exponent of power function for the muscular dystrophy group tended to be smaller than that for the normal group. These results were discussed in terms of the stage of disability in progressive muscular dystrophy.     

Elimination of myostatin does not combat muscular dystrophy in dy mice but increases postnatal lethality

Myostatin is a TGF-beta family member and a negative regulator of skeletal muscle growth. It has been proposed that reduction or elimination of myostatin could be a treatment for degenerative muscle diseases such as muscular dystrophy. Laminin-deficient congenital muscular dystrophy is one of the most severe forms of muscular dystrophy. To test the possibility of ameliorating the dystrophic phenotype in laminin deficiency by eliminating myostatin, we crossed dy(W) laminin alpha2-deficient and myostatin null mice. The resulting double-deficient dy(W)/dy(W);Mstn(-/-) mice had a severe clinical phenotype similar to that of dy(W)/dy(W) mice, even though muscle regeneration was increased. Degeneration and inflammation of muscle were not alleviated. The pre-weaning mortality of dy(W)/dy(W);Mstn(-/-) mice was increased compared to dy(W)/dy(W), most likely due to significantly less brown and white fat in the absence of myostatin, and postweaning mortality was not significantly improved. These results show that eliminating myostatin in laminin-deficiency promotes muscle formation, but at the expense of fat formation, and does not reduce muscle pathology. Any future therapy based on myostatin may have undesirable side effects.     

Distinct roles for telethonin N‐versus C‐terminus in sarcomere assembly and maintenance

The N‐terminus of telethonin forms a unique structure linking two titin N‐termini at the Z‐disc. While a specific role for the C‐terminus has not been established, several studies indicate it may have a regulatory function. Using a morpholino approach in Xenopus, we show that telethonin knockdown leads to embryonic paralysis, myocyte defects, and sarcomeric disruption. These myopathic defects can be rescued by expressing full‐length telethonin mRNA in morpholino background, indicating that telethonin is required for myofibrillogenesis. However, a construct missing C‐terminal residues is incapable of rescuing motility or sarcomere assembly in cultured myocytes. We, therefore, tested two additional constructs: one where four C‐terminal phosphorylatable residues were mutated to alanines and another where terminal residues were randomly replaced. Data from these experiments support that the telethonin C‐terminus is required for assembly, but in a context‐dependent manner, indicating that factors and forces present in vivo can compensate for C‐terminal truncation or mutation. Developmental Dynamics 239:1124–1135, 2010. © 2010 Wiley‐Liss, Inc.     

Non-sarcolemmal muscular dystrophies

The muscular dystrophies are characterised by progressive muscle weakness and wasting. Pathologically the hallmarks are muscle fibre degeneration and fibrosis. Several recessive forms of muscular dystrophy are caused by defects in proteins localised to the sarcolemma. However, it is now apparent that others are due to defects in a wide range of proteins including those which are either nuclear-related (Emery-Dreifuss type muscular dystrophies, oculopharyngeal muscular dystrophy), enzymatic (limb-girdle muscular dystrophy 2A, myotonic dystrophy) or sarcomeric (limb-girdle muscular dystrophies 1A and 2G). Although the clinical and molecular basis of these disorders is heterogeneous all display myopathic morphological features. These include variation in fibre size, an increase in internal nuclei, and some myofibrillar distortion. Degeneration and fibrosis occur, but usually not to the same extent as in muscular dystrophies associated with sarcolemmal protein defects. This review outlines the genetic basis of these "non-sarcolemmal" forms of dystrophy and discusses current ideas on their pathogenesis.     

Functional requirements for fukutin-related protein in the Golgi apparatus

Two forms of congenital muscular dystrophy (CMD), Fukuyama CMD and CMD type 1C (MDC1C) are caused by mutations in the genes encoding two putative glycosyltransferases, fukutin and fukutin-related protein (FKRP). Additionally, mutations in the FKRP gene also cause limb-girdle muscular dystrophy type 2I (LGMD2I), a considerably milder allelic variant than MDC1C. All of these diseases are associated with secondary changes in muscle alpha-dystroglycan expression. To elucidate the function of FKRP and fukutin and examine the effects of MDC1C patient mutations, we have determined the mechanism for the subcellular location of each protein. FKRP and fukutin are targeted to the medial-Golgi apparatus through their N-termini and transmembrane domains. Overexpression of FKRP in CHO cells alters the post-translational processing of alpha- and beta-dystroglycan inhibiting maturation of the two isoforms. Mutations in the DxD motif in the putative active site of the protein or in the Golgi-targeting sequence, which cause FKRP to be inefficiently trafficked to the Golgi apparatus, did not alter dystroglycan processing in vitro. The P448L mutation in FKRP that causes congenital muscular dystrophy changes a conserved amino acid resulting in the mislocalization of the mutant protein in the cell that is unable to alter dystroglycan processing. Our data show that FKRP and fukutin are Golgi-resident proteins and that FKRP is required for the post-translational modification of dystroglycan. Aberrant processing of dystroglycan caused by a mislocalized FKRP mutant could be a novel mechanism that causes congenital muscular dystrophy.