草甘膦致生殖细胞毒性效应及机制的初步研究

目的研究草甘膦对小鼠精母细胞(GC-2)毒性效应及机制。方法倒置显微镜和透射电镜观察草甘膦处理后GC-2细胞形态和超微结构;流式细胞仪检测草甘膦处理对GC-2细胞凋亡及细胞周期的影响;Western blot法检测凋亡及周期相关蛋白的表达水平。结果草甘膦能显著抑制GC-2细胞增殖,并导致细胞内质网和线粒体结构损伤;草甘膦对细胞增殖抑制主要与G1期周期阻滞和诱导GC-2细胞凋亡有关。蛋白质表达分析表明,cleaved-caspase3蛋白表达水平明显升高,提示草甘膦通过激活caspase3诱导GC-2细胞凋亡;同时,细胞周期负调控因子p21蛋白的表达上调,正调控因子Cyclin D1以及Cyclin E表达受到抑制,提示相关蛋白参与了细胞G1期阻滞。结论草甘膦对生殖细胞具有明显的毒性效应并与细胞周期和细胞凋亡调控相关。     

应用丙戊酸钠调节组蛋白乙酰化对肿瘤细胞周期相关蛋白的调控作用

目的 探讨应用组蛋白脱乙酰酶抑制剂丙戊酸钠(VPA)调节染色体组蛋白低乙酰化修饰水平对肿瘤细胞增殖周期相关蛋白Cyclin A、Cyclin DI、Cyclin E和P21waf/cipl的调控作用. 方法 应用0.75～4.00 mmol/ VPA干预肝癌细胞HepG2、胃癌细胞BGC-823、乳腺癌细胞MCF-7 48 h后,PI标记流式细胞术检测细胞周期;间接免疫荧光法分析Cyclin A、Cyclin D1、Cyclin E、P21waf/cip1蛋白表达;RT-PCR检测分析Cyclin A、Cyclin D1、Cyclin E、P21waf/cip1 mRNA表达. 结果 HepG2、BGC-823、MCF-7这3种细胞系培养48 h后,流式细胞术分析可见0.75～4.00 mmol/L、VPA实验组随药物浓度的增加而出现逐渐递增的细胞增殖周期G1期阻滞趋势.HepG2、BGC-823细胞Cyclin A蛋白及mRNA表达被明显下调;MCF-7细胞Cyclin A蛋白及mRNA表达在两个浓度组均未见明显变化;Cyclin D1蛋白及mRNA表达在3个细胞系均被明显下调;P21waf/cip1蛋白及mRNA表达在3个细胞均被明显上调;Cyclin E蛋白及mRNA表达则未见明显变化. 结论 应用VPA干预组蛋白乙酰化修饰可对HepG2、BGC-823、MCF-7细胞Cyc-lin D1、P21waf/cip1表达起明显的调控作用;对Cychn A的调控作用则随肿瘤细胞来源及表型的不同而有所差异,而对Cyclin E则无明显的调控作用.在VPA诱导肿瘤细胞增殖周期G1期阻滞过程中,下调CyclinD1和上调P21waf/cip1蛋白及mRNA表达可能是其共同作用途径.     

沉默PGRN基因对类风湿关节炎滑膜成纤维细胞增殖和凋亡的影响及机制研究

目的：探讨沉默颗粒蛋白前体（PGRN）基因对类风湿关节炎（RA）滑膜成纤维细胞增殖和凋亡的影响及其相关机制。方法：按照脂质体法将siRNA PGRN、siRNA NC转染至RA滑膜成纤维细胞MH7A，记为siRNA NC组、siRNA PGRN组，对照组（Control）未进行转染处理；将siRNA PGRN转染RA滑膜成纤维细胞MH7A后，加入丝裂原活化蛋白激酶（MAPK）信号通路激活剂Anisomycin，记为siRNA PGRN+Anisomycin组。采用qRT-PCR检测各组细胞PGRN mRNA表达水平；噻唑蓝（MTT）、克隆形成实验检测各组细胞增殖能力；流式细胞术检测各组细胞凋亡变化；Western blot检测各组细胞PGRN、细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原（PCNA）、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X(Bax)蛋白、MAPK通路蛋白表达。结果：与Control组、siRNA NC组相比，siRNA PGRN组内PGRN mRNA及蛋白表达、克隆形成率、存活率明显降低，凋亡率明显升高，Cyclin D1、PCNA、Bcl-...     

G6PI对阿霉素诱导类风湿关节炎关节滑膜细胞凋亡的保护作用

研究葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase,G6PI)对阿霉素(adriamycin,ADR)诱导类风湿性关节炎(rheumatoid arthritis RA)滑膜成纤维样细胞(fibroblast-like synoviocytes,FLS)凋亡的影响,探讨其作用机制。本研究从RA的关节滑膜组织中分离培养FLS,利用ADR建立细胞凋亡模型,通过流式细胞仪检测G6PI对细胞凋亡的影响,并通过western blot、Q-PCR检测细胞凋亡相关蛋白Bax、Bcl2、Caspase 3、Fas、Cyclin B1的表达;收集细胞培养上清,通过ELISA方法测定IL-1β和TNF-α含量。结果显示,G6PI(1μg/ml)和G6PI(10μg/ml)组RA-FLS的凋亡指数均显著低于ADR对照组;G6PI(1μg/ml)组与G6PI(10μg/ml)组间无显著差异。ADR凋亡模型中,Caspase 3、Fas和Bax表达明显上调,Cyclin B1下调;G6PI作用后,Caspase 3和Fas表达明显低于对照组,Cyclin B1表达则明显高于对照组。G6PI处理组的细胞培养上清中IL-1β和TNF-α含量均显著高于ADR对照组。研究表明G6PI可能通过Fas介导的Caspase通路来保护FLS免遭凋亡,同时促进RA-FLS分泌炎症因子IL-1β和TNF-α,从而参与RA的发生与发展。     

BDNF对诱导分化的SH-SY5Y细胞G_1期相关蛋白表达的影响

研究不同浓度的脑源性神经营养因子(BDNF)在体外诱导SH-SY5Y细胞分化时对G1期细胞周期相关蛋白表达的影响。采用全反式维甲酸(RA)和低(1ng/ml)、中(10ng/ml)、高(100ng/ml)三种不同浓度BDNF在无血清培养液中相继诱导SH-SY5Y细胞分化,形成均一的具有神经元形态的细胞。以实时定量RT-PCR法检测G1期细胞周期相关蛋白(包括cdk4,cdk6,cyclin D1,Cyclin E,p16和p27)的mRNA水平的表达变化。结果显示:不同浓度的BDNF处理后,Cdk4mRNA的表达水平均低于未分化细胞组(P<0.05);Cdk6和Cyclin D1的mRNA表达水平与RA组比较显著降低(P<0.05),Cyclin E和p16的mRNA表达水平仅在高浓度BDNF处理后明显降低,与未分化组比较P<0.05,p27的mRNA表达水平无显著变化。以上结果提示BD-NF在诱导SH-SY5Y细胞分化的同时无意促使其再进入细胞周期。同时,用RA预处理5d后改换BDNF培养SH-SY5Y细胞,可使细胞展现出成熟神经细胞的形态,是体外研究某些脑疾病的理想细胞模型。     

Caspase 3在PAB诱导宫颈癌HeLa细胞凋亡中的作用

目的研究Caspase 3在土荆皮酸诱导宫颈癌HeLa细胞凋亡中的作用。方法使用不同浓度的PAB处理He-La细胞，应用流式细胞仪检测细胞周期的分布，采用Western Blot方法检测HeLa细胞经PAB作用前后Bcl-2和Caspase3蛋白的表达变化。结果经PAB作用后，HeLa细胞出现剂量依赖性G2／M期阻滞伴随细胞凋亡，随药物浓度增加Bel-2蛋白表达逐渐下降，Caspase 3蛋白表达上调，Caspase 3活化百分率增高。结论PAB能通过调节Bcl-2蛋白的表达激活Caspase 3，从而诱导HeLa细胞发生凋亡。     

视黄酸依赖反义cyclin D1对HL-60细胞分化效应研究

目的 检测视黄酸(Retinoic acid，RA)依赖反义cyclin D1表达载体转染HL-60细胞后启动的诱导分化效应，并探讨其分子机制。方法 成功构建视黄酸依赖反义cyclin D1表达载体，以脂质体转染HL-60白血病细胞，经NBT还原实验、免疫荧光检测CD14表面抗原表达、RT-PCR以及Western blot等方法观测视黄酸处理后细胞分化效应的发生以及Rb基因的mRNA和蛋白表达水平的改变。结果 反义eyelin D1转染和未转染的HL-60细胞经RA处理后，NBT还原能力明显增强、CD14表面抗原表达显著增加，Rb基因的mRNA和蛋白表达水平均有所降低，其中，以反义cyclin D1转染细胞经RA处理后变化更为明显。结论 RA及其诱导的反义cyclin D1可协同诱导HL-60细胞分化成熟，下调Rb基因表达可能是其分子机制之一。     

EIF3D shRNA对乳腺癌细胞增殖、凋亡与侵袭能力的影响

目的探讨真核起始因子3D(EIF3D)shRNA对乳腺癌细胞增殖、凋亡与侵袭能力的影响。方法设计靶向EIF3D的shRNA序列,构建稳定沉默EIF3D的慢病毒载体,感染乳腺癌细胞MCF-7,同时设立阴性对照组与未处理组。qPCR和Western blot方法测定沉默效果,MTT方法测定感染后MCF-7细胞增殖能力变化,流式细胞术测定感染后MCF-7细胞凋亡率,Transwell侵袭实验检测感染后MCF-7细胞侵袭能力变化,Western blot方法测定感染后MCF-7细胞中Cyclin B1、基质金属蛋白酶-2(MMP-2)与Caspase-3的表达变化。结果感染EIF3D shRNA后,MCF-7细胞EIF3D的蛋白与mRNA表达均显著降低,提示稳定沉默EIF3D细胞株模型成功建立。沉默EIF3D能够显著抑制MCF-7细胞的增殖与侵袭能力,诱导细胞凋亡,下调Cyclin B1与MMP-2的蛋白表达,上调Caspase-3的表达(P0.01)。结论下调EIF3D能够抑制乳腺癌MCF-7细胞增殖与侵袭能力,并诱导细胞凋亡。     

淫羊藿苷对RA滑膜细胞凋亡及炎性因子表达的影响及机制研究

为探讨淫羊藿苷(icariin,ICA)对RA成纤维细胞样滑膜细胞(fibroblast-like synoviocytes,FLS)凋亡及炎性因子表达的影响,并探讨其可能的作用机制,体外分离及培养人RA FLS,用MTT和克隆细胞形成试验分别检测ICA对RA FLS增殖活性及克隆形成能力的影响,ELISA检测FLS分泌炎性因子TNF-α、IL-6的水平,流式细胞术检测FLS的凋亡情况,Western blotting检测ICA对RA FLS中细胞周期蛋白D1(Cyclin D1)、裂解的半胱氨酸蛋白酶3(Cleaved Caspase-3)、B淋巴细胞瘤2基因(B-cell lymphoma 2,Bcl-2)、Bcl-2相关蛋白(Bcl-2-associated X protein,Bax)及磷脂酰肌醇激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)信号通路相关蛋白表达的影响。结果显示,LPS诱导FLS后,ICA对RA FLS的增殖率及细胞克隆形成能力均呈浓度依赖性抑制效应(P0.05);ICA不同剂量组与LPS组相比,TNF-α、IL-6分泌水平明显降低(P0.05),细胞凋亡率明显增加(P0.05),Cyclin D1、Bcl-2、磷酸化PI3K(phospho-PI3K, p-PI3K)、磷酸化AKT(phospho-AKT, p-AKT)表达明显降低(P0.05),而Cleaved Caspase-3、Bax表达明显升高(P0.05)。提示ICA可降低人RA FLS的增殖能力,诱导其凋亡,并可降低其炎性因子的表达,这可能与抑制PI3K/AKT信号通路活化有关。     

人参皂苷Rg1抑制脂多糖诱导BV-2小胶质细胞增殖

目的探讨人参皂苷Rg1(Rg1)对脂多糖(LPS)诱导BV-2小胶质细胞中增殖细胞核抗原(PCNA)和细胞周期蛋白D1(cyclin D1)的调控作用及其对活化的BV-2小胶质细胞增殖的调控作用。方法体外培养BV-2小胶质细胞并分为空白对照组、LPS不同时间(1,3,6,8,12 h)处理组及Rg1不同浓度(10、20和50μmol/L)预处理组。采用Western blotting检测细胞增殖相关因子PCNA蛋白和cyclin D1蛋白的表达变化;RT-PCR检测细胞增殖相关因子PCNA和cyclin D1 mRNA的表达变化;免疫荧光染色法观察细胞内cyclin D1蛋白表达变化。采用流式细胞术检测细胞周期的变化。结果 LPS诱导BV-2小胶质细胞PCNA和cyclin D1mRNA和蛋白表达上调,并呈时间依赖性;不同浓度Rg1预处理下调LPS诱导的BV-2小胶质细胞PCNA和cyclin D1的mRNA和蛋白表达。结论Rg1通过下调LPS诱导的BV-2小胶质细胞PCNA和cyclin D1的表达水平来调控细胞周期进而抑制活化的小胶质细胞的增殖。     

Drosophila MAGE controls neural precursor proliferation in postembryonic neurogenesis

Necdin, a member of the MAGE family, is expressed abundantly in postmitotic neurons and is required for their differentiation and survival. In mammals, the MAGE family consists of more than 30 genes, whereas only one MAGE gene exists in the genome of nonmammalian vertebrates such as zebrafish and chicken. These nonmammalian MAGE genes are expressed in developing nervous system, and the primary structures of the encoded proteins resemble those of necdin-like MAGE proteins. Fruit fly Drosophila also carries a single necdin-like MAGE gene, which is highly expressed in neural stem cells (neuroblasts) during nervous system development. In the present study, we investigated the function of MAGE in Drosophila neurogenesis in vivo using an RNA interference (RNAi) -mediated gene knockdown system. Ubiquitous knockdown of Drosophila MAGE by double-stranded RNA injection into embryos was lethal at early stages of organogenesis. MAGE was then knocked down in developing mushroom bodies by RNAi-mediated gene silencing using the OK107-GAL4 driver. MAGE RNAi increased the population of proliferative neural precursors in larval mushroom bodies. At the pupal stage, RNAi-mediated MAGE knockdown led to a significant enlargement of the mushroom bodies as a result of increased neuronal population, presumably by accelerating the asymmetric division of neural stem cells. MAGE RNAi mushroom bodies of adult flies showed neurodegenerative changes such as vacuolation and nuclear DNA breaks, implying that supernumerary neurons undergo apoptosis during postpupal development. These results suggest that evolutionally conserved necdin-like MAGE is involved in both neural stem cell proliferation and neuronal survival during nervous system development.     

Smad3基因促进大鼠卵巢颗粒细胞增殖

目的 探讨Smad3基因对大鼠卵巢颗粒细胞增殖功能的影响.方法 21d SD雌性大鼠,腹腔注射孕马血清20IU/只,48h后收集卵巢颗粒细胞进行原代培养,用Smad3 基因特异的siRNA基因沉默后,采用免疫细胞化学法检测颗粒细胞中Smad3蛋白表达的变化,以判定基因沉默效率,Smad3基因沉默后用免疫细胞化学法检测...     

Expression of the G1-S modulators in hepatitis B virus-related hepatocellular carcinoma and dysplastic nodule: association of cyclin D1 and p53 proteins with the progression of hepatocellular carcinoma

Deranged expression of cell cycle modulators has been reported to contribute to the development and progression of hepatocellular carcinoma (HCC). However, their expression patterns remain poorly understood in hepatitis B virus (HBV)-related HCC, which constitutes about 65-70% of HCC in Korea. The aims of this study were to evaluate the expressions of G1-S modulators in HBV-related HCCs and dysplastic nodules (DNs), and to correlate with the histopathologic features of HCCs. Immunohistochemical expressions of cyclin D1, cyclin E, p53, p27, p21, p16, Rb, and PCNA proteins were investigated in 80 HCCs and 22 DNs. Cyclin D1 overexpression showed positive relationships with advanced tumor stage, poor differentiation, larger tumor size, microvascular invasion, intrahepatic meta-stasis, no tumor capsule formation, infiltrative growth, aberrant p53 expression, and high PCNA labeling index (LI) of HCC (p<0.05). Aberrant p53 expression showed positive relationship with poor differentiation of HCC (p<0.01). Expression of cyclin D1 or p53 was not observed in DNs. The p27 LI and p16 LI were lower in HCCs with intrahepatic metastasis (p<0.05). Cyclin D1 overexpression and aberrant p53 expression could be associated with the progression of HBV-related HCC, and might have a less crucial role in the DN-HCC sequence. In addition, elevated expression of p27 and p16 proteins might have inhibitory action to the intrahepatic metastasis of HBV-related HCC.     

Synergistic effects of AKAP95, Cyclin D1, Cyclin E1, and Cx43 in the development of rectal cancer

Objective: To explore the expression of A-kinase anchor protein 95 (AKAP95), Cyclin D1, Cyclin E1, and Connexin43 (Cx43) in rectal cancer tissues and assess the associations between each of the proteins and pathological parameters, as well as their inter-relationships. Methods: AKAP95, Cyclin D1, Cyclin E1, and Cx43 protein expression rates were evaluated by immunohistochemistry in 50 rectal cancer specimens and 16 pericarcinoma tissues. Results: The positive rates of AKAP95, Cyclin E1, and Cyclin D1 proteins were 54.00 vs. 18.75%, 62.00 vs. 6.25%, and 72.00 vs. 31.25% in rectal cancer specimens and pericarcinoma tissues, respectively, representing statistically significant differences (P < 0.05). The positive rate of Cx43 protein expression in rectal cancer tissues was 44.00% and 62.50% in pericarcinoma tissues, and the difference between them was not significant (P > 0.05). No significant associations were found between protein expression of AKAP95, Cyclin E1, Cyclin D1, and Cx43, and the degree of differentiation, histological type, and lymph node metastasis of rectal cancer (P > 0.05). However, significant correlations were obtained between the expression rates of AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43, respectively (P < 0.05). Conclusion: AKAP95, Cyclin E1, and Cyclin D1 protein expression rates were significantly higher in rectal cancer tissues compared with pericarcinoma samples, suggesting an association between these proteins and the development and progression of rectal cancer. In addition, the significant correlations between the proteins (AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43) indicate the possible synergistic effects of these factors in the development and progression of rectal cancer.     

Cyclin D1蛋白表达与卵巢癌转移相关性研究

目的:探讨细胞周期蛋白(CyclinD1)表达与卵巢癌转移的相关性和CyclinD1与P21WAF1、P53蛋白的关系。方法:应用蛋白免疫印迹技术(Westernblot)检测61例人卵巢癌组织及7例卵巢非癌组织中CyclinD1、P53及P21WAF1蛋白的表达。结果:卵巢癌组织中CyclinD1和P53蛋白表达率均明显高于卵巢非癌组织(P<0.05);P21WAF1蛋白表达卵巢癌组织低于卵巢非癌组织(P>0.05)。CyclinD1与P53及P21WAF1蛋白表达与卵巢癌组织类型无关(P>0.05)。P21WAF1蛋白表达与卵巢癌的临床分期相关(P<0.05),CyclinD1、P53蛋白表达与卵巢癌的临床分期无关(P>0.05)。CyclinD1、P53及P21WAF1蛋白表达与卵巢癌转移相关(P<0.05)。结论:CyclinD1蛋白表达与卵巢癌肿瘤转移相关,CyclinD1与P21WAF1、P53蛋白在卵巢癌发生、发展中可能有一定作用,但不起协同作用。     

热处理延缓维甲酸诱导P19细胞的生长及向神经元分化

研究热环境对P19细胞生长和RA诱导的向神经元分化的影响,为探讨热环境刺激对神经系统发育的影响提供参考。成熟的P19细胞在热环境的条件下培养1h后,消化接种至培养皿中正常培养,于不同时间点取样分析细胞数目。热环境处理P19细胞后用维甲酸诱导分化4d,随后接种至培养皿中,参照神经元的原代培养方法培养12d,用神经元抗体免疫鉴定,计数不同培养时间NSE、NF阳性神经元在总体细胞中的比例。结果发现接受热处理的P19细胞贴壁不良,生长始终缓慢,说明热环境刺激阻止细胞生长分裂。P19细胞经RA诱导分化后出现类似神经细胞的突起和纤维网络,而热环境导致P19细胞向神经元分化明显迟缓,NF阳性细胞比例增加。