p16基因甲基化的芯片定量检测

p16基因的失活与多种肿瘤相关,但p16基因缺失率较低,突变更为罕见,p16基因启动子区CpG岛甲基化与其蛋白表达密切相关．DNA甲基化已成为目前研究的热点,现有的技术包括：Southernblot法、限制性内切酶-PCR法、DNA测序法、甲基化特异性PCR(MSP)、     

纳米TiO2光催化氧化对胃癌细胞周期的影响

以胃癌细胞系BGC-823为研究对象,采用倒置相差显微镜观察纳米TiO2光催化氧化前后BGC-823细胞形态的变化,通过流式细胞仪考察其对BGC-823细胞凋亡率及细胞周期的影响。结果表明,纳米TiO2光催化氧化使BGC-823细胞呈受激坏死或凋亡晚期继发性坏死状态;导致BGC-823细胞周期阻滞于G2/M期并诱导细胞凋亡,从而抑制其DNA合成及增殖分化进程。当TiO2比负载量为9.8×10-7g/cm2、光照时间为40 min时,G2/M期阻滞较为显著,细胞凋亡率达24.34%。     

四氢吡啶并[4,3-d]二氢嘧啶酮衍生物的合成及其初步抗癌活性测定

为寻找新型抗癌药物的先导化合物,设计合成了16种未见文献报道的四氢吡啶并[4,3-d]二氢嘧啶酮衍生物2a～2p,结构经1H NMR,IR,MS,元素分析确证.初步的生物活性测试结果表明,在100μg/mL浓度下,目标化合物对白血病K562细胞的增殖表现出显著的抑制活性,对卵巢癌HO-8910和肝癌SMMC-7721细胞系的增殖也表现出一定的抑制活性.     

新型吡咯类氨基酸缀合物的合成与抗肿瘤活性测定

以苯甲醛或3,4-二甲氧基苯甲醛为原料经Wittig-Horner反应、Van Leusen吡咯合成法、水解等多步反应,设计并合成了16个未见文献报道的吡咯类氨基酸缀合物7a~7p.其结构经1H NMR,13C NMR及HRMS确证.采用噻唑蓝(MTT)法测试了目标化合物对MCF-7,MGC80-3,Hep G2,CT-26及HUVEC细胞的增殖抑制作用.结果表明,几乎所有化合物对人体正常细胞HUVEC无明显抑制作用,化合物7a~7h对Hep G2细胞株有一定的抑制活性,化合物7i~7p对MCF-7及MGC80-3较强的抑制活性.其中化合物7o~7p对MCF-7细胞株的抑制活性最强.     

鹿茸寡肽的制备及其促成骨细胞的增殖作用

从梅花鹿鹿茸中提取了天然鹿茸总多肽(VATP). 在进一步分离纯化的过程中, 筛选出具有促进成骨细胞增殖的高活性肽组分(VAP-B), 并通过制备型HPLC对其纯化, 得到了分子量分布约为200~600的小肽活性组分(VAP-B2). 细胞周期分析结果表明, 鹿茸肽VAP-B2组分促进了人成骨肉瘤细胞OS-732的周期转化, 使其细胞周期S期细胞指数明显增加, 即表现为S期DNA含量明显提高. 碱性磷酸酶(ALP)活性检测结果表明, 随着鹿茸肽VAP-B2剂量的增加, ALP的活性明显增加. 这与成骨细胞增殖分化及成熟过程中, 细胞的周期性变化和骨形成标志物碱性磷酸酶水平的明显增高相符.     

细胞代谢组学用于木犀草素抑制MCF-7细胞的机制研究

基于气相色谱质谱联用(GC-MS)技术将代谢组学的方法结合细胞周期的实验,研究木犀草素作用于MCF-7细胞的作用机理。细胞活性实验验证,木犀草素对MCF-7细胞有抑制作用,GC-TOF/MS对加药细胞和未加药细胞代谢物进行指纹图谱分析,并进一步应用偏最小二乘判别分析(OPLS-DA)对代谢组学数据进行多维统计分析。结合木犀草素将细胞周期抑制在S期(Synthesis),推测木犀草素通过阻碍核酸代谢中的磷酸戊糖途径抑制MCF-7细胞的增殖。     

非病毒性基因载体的合成研究

基因治疗是一种有效的治疗先天性遗传性疾病以及后天获得性疾病的手段。它通过激发细胞的生物活性或者抑制细胞非正常的功能来治疗或者预防疾病的发生,例如细胞的基因紊乱,细胞的无序增殖。目前基因治疗所面临的问题是缺乏有效的基因递送载体。基因载体主要分为病毒性基因载体和非病毒性基因载体。与病毒性基因载体相比,非病毒性基因载体具有毒性小、安全性高、易于制备、能够荷载分子量大的DNA等优点。本文综述了非病毒性基因载体的合成研究进展。     

反义c-Ha-ras寡聚核苷酸对转化细胞的生长抑制及其p21蛋白合成的抑制

本文合成了两个15聚核苷酸AS-1和AS-2,前者与c-Ha-ras mRNA的前三个密码子及其上游的核蛋白体结合点附近的单链区互补,后者与细胞核内未成熟c-Ha-rag RNA第一内含子3′端及第二外显子5′端区域互补,它们可分别阻断c-Ha-ras mRNA的翻译和拼接过程,从而降低c-Ha-ras癌基因的表达水平,抑制了转化细胞的增殖。这种抑制作用随浓度的增加(从4μmol/L至10μmol/L)而增加,加入AS-1或AS-2 12h后抑制作用达到高峰,以后逐渐下降。用ELISA方法测定转化细胞中c-Ha-ras产物p21蛋白水平,结果显示增殖受抑制细胞p21水平较对照细胞降低30％左右。     

细胞代谢组学用于羽扇豆醇干预人乳腺癌细胞MCF-7的机理探究

应用基于气相色谱-质谱联用（GC-MS）的代谢组学方法结合细胞周期实验,研究羽扇豆醇体外抑制人乳腺癌细胞MCF-7增殖的作用机理。代谢组学的研究结果表明：通过正交偏最小方差判别分析（OPLS-DA）可以很好地区分羽扇豆醇作用的MCF-7细胞代谢谱与对照组细胞代谢谱,模型参数为：R²Ycum=0.988,Q²Ycum=0.964。VIP（variable importance in the projection）值大于1的差异代谢物进一步用t检验进行单位分析,选择t<0.05（VIP>1）的代谢物作为羽扇豆醇作用组的生物标志物,得到琥珀酸、磷酸、亮氨酸、异亮氨酸等11种代谢差异物。结合羽扇豆醇将细胞周期抑制在G1期这一现象,推测羽扇豆醇可能是主要抑制了三羧酸循环中的琥珀酰辅酶A的生成和底物磷酸化生成ATP的反应来抑制MCF-7细胞的增殖。本实验从代谢组学角度为乳腺癌抗肿瘤机制提供新的线索。     

青蒿二烯功能模块与酵母底盘的适配性

设计构建人工酿酒酵母细胞合成青蒿二烯的关键是使外源功能模块与底盘细胞适配,本文通过对外源功能模块中的载体、蛋白表达和启动子进行优化,以提高功能模块与底盘细胞的适配性. 使用着丝粒载体和附加型载体构建了2种青蒿二烯功能模块,在过表达甲羟戊酸（MEV）途径中关键基因（截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMGR及法尼基焦磷酸合酶基因ERG20）的2种酵母底盘中进行适配,得到适配性较好的人工合成细胞,其产量为11.2 mg/L;将青蒿二烯合酶基因（ADS）与ERG20进行融合构建融合蛋白功能模块,在选定底盘中适配性进一步提高,青蒿二烯的产量提升至17.5 mg/L;采用不同强度的启动子（TDH3p,TEF1p和PGK1p）对融合蛋白功能模块进行调控,最终得到功能模块与底盘间适配关系更好的人工合成细胞,其产量提升到71.8 mg/L.     

Expression of Cdk4, Cyclin D1 and Rb in exogenous wild type P16 gene transfected bladder cancer cell line

Cell cycle control plays a key role in the growth of normal mammalian cells. One of the fundamental abnormalities in human tumors is dysregulated cell cycle control. It is generally accepted that active Cdk4–Cyclin D1 complexes help cells to pass through the R point, a point of no return, after which cells become committed to a new round of replication. Accumulated evidences indicate that Cdk4 is the ‘primary sensor’ for driving cells through the R point and it is widely known that P16INK4a can arrest cells in G1.But how can the expression of exogenous P16INK4 gene affect the activity of Cdk4–Cyclin D1 remains unclear. In this study, using exogenous wild type P16 gene and antibodies for P16, Cdk4, Cyclin D1 and Rb proteins, we examined the expression of exogenous wild type P16 gene and the changes of cell cycle regulatory genes-Cdk4, Cyclin D1 and Rb in human bladder cancer cells. The cell growth analysis revealed that the proliferation of P16 gene transfected cancer cells was inhibited after the transfection of exogenous wild P16 gene. The immunocytochemical results indicated that after the transfection of exogenous wild type P16 gene, the expression of Cdk4, Cyclin D1 and Rb were negative in the nuclei while the expression of P16 significantly increased in the nuclei and the cytoplasm. Our results suggest that the transfection of exogenous wild P16 gene induces the inhibition of proliferation of the bladder cancer cells and the increasing expression P16 inhibits the expression of Cdk4, Cyclin D1 and Rb in nuclei, which results in the cell cycle being inhibited at G0/G1 phase. As a consequence, exogenous P16 has negative effects on the malignant proliferation of bladder cancer cells and it may be considered as target for potential anti-cancer drugs.     

Synthesis and Biological Evaluation of Spirocyclic Chromane Derivatives as a Potential Treatment of Prostate Cancer

As a significant co-activator involved in cell cycle and cell growth, differentiation and development, p300/CBP has shown extraordinary potential target in cancer therapy. Herein we designed new compounds from the lead compound A-485 based on molecular dynamic simulations. A series of new spirocyclic chroman derivatives was prepared, characterized and proven to be a potential treatment of prostate cancer. The most potent compound B16 inhibited the proliferation of enzalutamide-resistant 22Rv1 cells with an IC₅₀ value of 96 nM. Furthermore, compounds B16–P2 displayed favorable overall pharmacokinetic profiles, and better tumor growth inhibition than A-485 in an in vivo xenograft model.     

The co-transfection of p16(INK4a) and p14(ARF) genes into human lung cancer cell line A549 and the effects on cell growth and chemosensitivity

Two functionally and structurally different proteins, p16(INK4a) and p14(ARF), encoded by the gene INK4a/ARF located at 9p21 are cyclin-dependent kinase (cdk) inhibitors and important cell cycle regulators. More and more evidences have been accumulated to show that the exogenous p16(INK4a) or p14(ARF) can inhibit the cell growth and/or induce the apoptosis. But it is still unclear if they can play positive role when combine with the conventional chemotherapy in cancer treatment. Here we show that cationic liposome-mediated gene transfection of INK4a/ARF into lung cancer cell line A549, in which the INK4a/ARF locus was lost, suppressed the growth and induced apoptosis. When treated with five different chemotherapy drugs with different mechanism after the transfection, A549 got an increased chemosensitivity for adriamycin and cisplatin and an unchanged result for topotecan, taxol or vinorelbine. The results indicated that cell cycle redistribution and increased apoptosis index after transfection might be the main explanation for the enhanced chemosensitivity. The combination of gene therapy with conventional chemotherapy is not always better than single chemotherapy. This trial will be of benefit to the treatment of lung cancer when combine the conventional chemotherapy and gene therapy in the future.     

Synergistic Growth-Suppressive Effects of Quercetin and Cisplatin on HepG2 Human Hepatocellular Carcinoma Cells

Quercetin, a natural flavonoid, exhibits anticancer effects. The aim of this study is to determine whether the combination of quercetin with cisplatin, a conventional chemotherapeutic drug, would have synergistic suppressive effects on hepatocellular carcinoma (HCC) cells. To this end, HepG2 cells were exposed to quercetin (50 μM) or cisplatin (10 μM) alone or combination of both and cell proliferation and apoptosis were investigated. Our data revealed that the combination of quercetin and cisplatin was significantly (P?<?0.05) effective in inducing growth suppression and apoptosis in HepG2 cells, when compared with single agent treatment. Quercetin combined with cisplatin modulated the expression of numerous genes involved in cell cycle progression and apoptosis. Treatment with quercetin rather than cisplatin resulted in a marked elevation of p16 expression in HepG2 cells. Targeted reduction of p16 using RNA interference technology partially reversed quercetin-induced cell cycle G1 arrest and apoptosis in HepG2 cells. In conclusion, quercetin has suppressive activity against HCC cells through p16-mediated cell cycle arrest and apoptosis and its combination with cisplatin yielded synergistic inhibitory effects in suppressing cell growth and inducing apoptosis.     

p41-Arc, a regulatory subunit of Arp2/3 complex, can induce premature senescence in the absence of p53 and Rb

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the tumor suppressor proteins, such as Rb or p53. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-β-galactosidase (SA-β-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. p41-Arc has been known to be a putative regulatory component of the mammalian Arp2/3 complex, which is required for the formation of branched networks of actin filaments at the cell cortex. In this study, we demonstrate that p41-Arc can induce senescent phenotypes when it is overexpressed in human tumor cell line, SaOs-2, which is deficient in p53 and Rb tumor suppressor genes, implying that p41 can induce senescence in a p53-independent way. p41-Arc overexpression causes a change in actin filaments, accumulating actin filaments in nuclei. Therefore, these results imply that a change in actin filament can trigger an intrinsic senescence program in the absence of p53 and Rb tumor suppressor genes.     

The function of p27KIP1 during tumor development

Timely cell cycle regulation is conducted by sequential activation of a family of serine-threonine kinases called cycle dependent kinases (CDKs). Tight CDK regulation involves cyclin dependent kinase inhibitors (CKIs) which ensure the correct timing of CDK activation in different phases of the cell cycle. One CKI of importance is p27^KIP1. The regulation and cellular localization of p27^KIP1 can result in biologically contradicting roles when found in the nucleus or cytoplasm of both normal and tumor cells. The p27^KIP1 protein is mainly regulated by proteasomal degradation and its downregulation is often correlated with poor prognosis in several types of human cancers. The protein can also be functionally inactivated by cytoplasmic localization or by phosphorylation. The p27^KIP1 protein is an unconventional tumor suppressor because mutation of its gene is extremely rare in tumors, implying the normal function of the protein is deranged during tumor development. While the tumor suppressor function is mediated by p27^KIP1''s inhibitory interactions with the cyclin/CDK complexes, its oncogenic function is cyclin/CDK independent, and in many cases correlates with cytoplasmic localization. Here we review the basic features and novel aspects of the p27^KIP1 protein, which displays genetically separable tumor suppressing and oncogenic functions.     

Gene expression profiling of tumor-associated macrophages after exposure to single-dose irradiation

Radiotherapy (RT) is a common cancer treatment approach that accounts for nearly 50% of patient treatment; however, tumor relapse after radiotherapy is still a major issue. To study the crucial role of tumor-associated macrophages (TAMs) in the regulation of tumor progression post-RT, microarray experiments comparing TAM gene expression profiles between unirradiated and irradiated tumors were conducted to discover possible roles of TAMs in initiation or contribution to tumor recurrence following RT, taking into account the relationships among gene expression, tumor microenvironment, and immunology. A single dose of 25 Gy was given to TRAMP C-1 prostate tumors established in C57/B6 mice. CD11b-positive macrophages were extracted from the tumors at one, two and three weeks post-RT. Gene ontology (GO) term analysis using the DAVID database revealed that genes that were differentially expressed at one and two weeks after irradiation were associated with biological processes such as morphogenesis of a branching structure, tube development, and cell proliferation. Analysis using Short Time-Series Expression Miner (STEM) revealed the temporal gene expression profiles and identified 13 significant patterns in four main groups of profiles. The genes in the upregulated temporal profile have diverse functions involved in the intracellular signaling cascade, cell proliferation, and cytokine-mediated signaling pathway. We show that tumor irradiation with a single 25-Gy dose can initiate a time-series of differentially expressed genes in TAMs, which are associated with the immune response, DNA repair, cell cycle arrest, and apoptosis. Our study helps to improve our understanding of the function of the group of genes whose expression changes temporally in an irradiated tumor microenvironment.     

Inactivation patterns of p16/INK4A in oral squamous cell carcinomas

The p16/INK4A is one of the major target genes in carcinogenesis and its inactivation has frequently been reported in other types of tumors. The purpose of this study is to evaluate inactivation patterns of p16/INK4A in oral squamous cell carcinoma. Six different oral cancer cell lines, SCC-4, SCC-9, SCC-15, SCC-25, KB, and SNUDH- 379 were examined for inactivation of p16/INK4A genes. In the analysis of p16/INK4A gene inactivation, PCR amplification, direct sequencing, and methylation-specific PCR methods were adopted for evaluation of homozygous deletion, point mutation, and promoter hypermethylation, respectively. Homozygous deletion was detected in SCC-25 and SCC-9. SCC-15 showed hypermethylated promoter region within p16/INK4A gene. It is suggestive in the present study that inactivation patterns of p16/INK4A were mainly homozygous deletion, promoter methylation rather than point mutation in oral squamous cancer cell lines, so treatment modalities of oral squamous cell carcinoma should be focused on these types of inactivation.