γ-分泌酶抑制剂DAPT抑制Jurkat T细胞增殖

目的:探讨γ-分泌酶抑制剂DAPT在体外对人T淋巴细胞白血病Jurkat T细胞株增殖的抑制作用及其某些机制.方法:通过倒置显微镜和MTT法检测DAPT对Jurkat T细胞聚集的影响和对其增殖的抑制作用;利用碘化丙锭染色结合流式细胞术检测DAPT对Jurkat T细胞周期的影响;藉免疫印迹法检测DAPT对Jurkat T细胞周期蛋白ICBP90表达的影响.结果:DAPT能显著影响Jurkat T细胞的聚集,抑制Jurkat T细胞的增殖,使其细胞周期停滞于S期,且周期蛋白ICBP90的表达下调.结论:γ-分泌酶抑制剂DAPT能够抑制Jurkat T细胞的增殖和细胞周期的进行,其抑制作用可能与ICBP90蛋白表达的下调有关.     

2-ME抑制人子宫内膜癌细胞株增殖的研究

目的　探讨2-甲氧雌二醇(2-ME)对人子宫内膜癌细胞株KLE细胞体外增殖和凋亡的抑制作用.方法选用人子宫内膜癌细胞株KLE进行体外培养,实验组加入不同浓度2-ME的培养液,对照组不含2-ME.用四甲基偶氮唑蓝(MTT)比色法观察2-ME对人子宫内膜癌细胞株KLE增殖的抑制作用;药物作用后的克隆形成实验;电子显微镜(电镜)观察细胞形态变化;流式细胞仪(FCM)观察细胞的凋亡率及细胞周期的变化.结果2-ME浓度为10.0～50.0μM时,明显抑制KLE细胞的增殖(P<0.01),并具有时间依赖性和剂量依赖性.2-ME作用后G0/G1期细胞增加,并伴随G0/G1期细胞的增加,出现细胞凋亡峰和凋亡率的升高(P<0.05).电镜下观察到KLE细胞染色体边集、核固缩、凋亡小体.结论2-ME对人子宫内膜癌KLE细胞株增殖有抑制作用,并能促进其凋亡.     

新型TRK激酶小分子抑制剂的筛选与验证

原肌球蛋白相关激酶(Tropomyosin-Related Kinase,TRK)属于受体酪氨酸激酶家族,具有调节细胞增殖、分化、凋亡、代谢等作用.在多种肿瘤细胞中发现TRK激酶编码基因NTRK的融合现象,TRK蛋白过表达或激酶活性组成性激活促进了肿瘤的发生发展.以TRK激酶为靶标的小分子抑制剂正处于研发之中,如Entrectinib等.本研究以Entrectinib为阳性对照,从小分子化合物库中筛选得到Crizotinib、LY2874455等7个新颖的TRK激酶小分子抑制剂,结构计算提示Dovitinib等4个化合物均属于ATP竞争性抑制剂.在NTRK1基因融合的KM12细胞体外增殖实验中,全部的TRK激酶抑制剂均能抑制细胞增殖,且LY2874455最为显著.在细胞的联合用药实验中,发现Crizotinib与Entrectinib的联用具有协同作用.     

榄香烯乳对B16细胞株的诱导分化作用

观察榄香烯乳对Ｂ１６黑色素瘤细胞株是否具有诱导分化作用。通过ＭＴＴ显色法测定榄香烯乳在体外对Ｂ１６细胞株的ＩＣ５０,腹腔给药观察榄香烯乳对体内Ｂ１６细胞株皮下、肺转移结节的抑制作用。选取低于ＩＣ５０的药物浓度研究榄香烯乳作用后Ｂ１６细胞株超微结构和成瘤性的改变。榄香烯乳对体内外Ｂ１６细胞株均有抑制作用。药物作用后,瘤细胞生长抑制,增殖减慢,接触抑制恢复,成瘤性降低,电镜观察细胞恶性度下降,出现较为     

维药异常黑胆质成熟剂的抗肿瘤作用及其对细胞迁移的影响

探讨维药异常黑胆质成熟剂(ASM)抗肿瘤作用及其对肿瘤细胞迁移的影响.体外培养人肝癌Bel-7402细胞株、人乳腺癌BCAP 细胞株、人宫颈癌 Hela 细胞株、人胃癌BGC-823细胞株,采用四甲基偶氮唑蓝(tetrzolium-based colorimetric assay,MTT)法检测不同浓度ASM(1、2.5、5、10、15、20、50mg/mL)对人肝癌Bel-7402细胞株、人乳腺癌BCAP细胞株、人宫颈癌Hela细胞株、人胃癌BGC-823细胞株4种肿瘤细胞增殖的影响;选择人肝癌Bel-7402细胞株为研究对象,采用细胞划痕运动实验和扫描电镜技术,观察不同浓度ASM对肝癌细胞迁移的影响.MTT检测结果显示,ASM对人肝癌Bel-7402细胞株、人乳腺癌BCAP细胞株、人宫颈癌Hela细胞株、人胃癌BGC-823细胞株生长均有一定抑制作用,且在15～20mg/mL作用下最为明显,对不同肿瘤细胞株抑制作用不一;体外细胞划痕运动实验和扫描电镜结果显示,ASM对人肝癌Bel-7402细胞运动有一定抑制作用.由此推论,ASM对肿瘤细胞增殖和肿瘤细胞迁移有一定抑制作用.     

美洲大蠊提取物对肝星状细胞增殖能力的影响

目的:研究美洲大蠊提取物样品Ⅰ、Ⅱ、Ⅲ对体外培养大鼠肝星状细胞株(HSC-T6)增殖的影响。方法:用四甲基偶氮噻唑蓝(MTT)法检测不同浓度样品在不同培养时间作用下HSC-T6细胞的增殖能力。结果:与空白对照组比较,样品Ⅰ抑制了HSC-T6的增殖,且随时间的延长抑制作用加强,在一定的浓度范围内具有剂量依赖性,培养72 h时作用最强。样品Ⅱ与样品Ⅰ作用效果相似,但抑制作用略小于样品Ⅰ。样品Ⅲ在作用24 h时,有一定抑制作用,而在其他时间段基本没有抑制作用。结论:样品Ⅰ、Ⅱ、Ⅲ均能抑制HSC-T6的增殖,有可能成为防治肝纤维化的理想药。     

nm23-H1基因对乳腺癌细胞MCF-7S增殖及移动特性的影响

目的：探讨nm23—H1基因转染乳腺癌细胞后对其增殖和转移能力的影响。方法：用人nm23—H1基因与真核表达质粒pcDNA3．1(—)构建成重组表达质粒pcDNA3．1—NMHl，转染人乳腺癌细胞MCF—7S，经G418筛选4周后，筛选出稳定表达nm23—H1的细胞株，绘制其生长曲线，检测其增殖、移动能力，用SPSS统计软件处理实验数据，分析nm23—H1基因对乳腺癌细胞的作用。结果：与对照组相比，转染nm23—H1基因的MCF—7S细胞的对数生长期延长，细胞增殖速度减慢，移动距离缩短，克隆形成率降低。结论：nm23—H1基因在乳腺癌细胞的体外实验中有抑制癌细胞生长、增殖、转移能力的作用。     

酪氨酸激酶抑制剂在肿瘤治疗中的研究进展

恶性肿瘤正一直严重威胁着人类生命，尽管诊断和治疗水平有所进步，但很多肿瘤生存率一直很低。近年来随着科学的发展，我们对肿瘤的生物学特性有了更深一步的认识。人类蛋白酪氨酸激酶（PTKs）在肿瘤的发生发展过程中起着非常重要的作用，它已成为一种很有前景的肿瘤治疗新靶点。PTKs抑制剂研究已成为当今世界抗肿瘤研究的热点领域，这些药物在临床前期研究中已显示很好的抗肿瘤效应，一些正在临床上作为很有前景的抗癌药。特别是BCR-ABL酪氨酸激酶抑制剂imatinib（STI571）在治疗慢性髓细胞白血病显著成功使得科学家们更热心于投入这一领域的研究，目前，至少有30多种酪氨酸激酶抑制剂在肿瘤治疗的不同临床试验阶段，大约120多个临床试验正在世界范围内进行。在此对酪氨酸激酶在肿瘤中的作用及酪氨酸激酶抑制剂在肿瘤治疗中的研究进展作一综述。     

CML相关抗原诱导正常T细胞增殖的TCR Vα基因选用和克隆性研究

目的：了解慢性粒细胞白血病（CML）相关抗原体外诱导T细胞的TCR Vα亚家族限制性表达和克隆性增殖情况。方法：采用混合淋巴细胞和肿瘤细胞培养（MLTC）方法，利用CML细胞、K562细胞和bcr3-abl2多肽体外诱导脐血或正常人的T细胞增殖，用RT-PCR和基因扫描分析MLTC后T细胞的29个TCRVtx亚家族基因的互补决定区（CDR3），了解各Vd亚家族的限制性表达情况和T细胞克隆性增殖特点。结果：经CML抗原刺激后增殖的T细胞仅表达部分Vd谱系基因（1—14个亚家族），两例脐血均出现有Vα3亚家族T细胞克隆性增殖趋势；正常人外周血出现寡克隆增殖的Va5、Vα14亚家族T细胞。结论：CML相关抗原体外诱导脐血和正常人T细胞出现抗原相关的TCR Vα优势利用和克隆性增殖。     

人肝癌HepG2细胞异质性亚系的建立及生物学行为初探

目的：从人肝癌细胞株HepG2母系克隆分离异质性亚系，探讨肝癌异质性机理。方法：应用有限稀释法对培养人肝癌细胞株HepG2进行单细胞克隆分离异质性亚系，并应用细胞计数法、流式细胞仪检测其细胞周期和DNA含量，MTT法检测其对不同抗癌药物敏感性，同时应用裸鼠脾种植肝转移在体实验模型探讨其生物学行为的差异。结果：分离到HepG2—D3及HepG2—F4两个细胞亚系。HepG2—F4亚系细胞呈长梭形，体外增殖能力强，但脾脏内种植后不成瘤，不转移(0／10)；HepG2—D3亚系细胞呈多角形、多突起，脾脏内种植成瘤率高(10／10)，并在肝脏形成广泛转移(10／10)。结论：从人肝癌细胞株HepG2母系成功分离建立了两个异质性细胞亚系，两在形态学、增殖能力及其对不同抗癌药物敏感性、成瘤和转移能力方面均存在差异，该亚系可为进一步研究肝癌异质性尤其是转移的确切机制提供良好的素材。     

Activating mutations in ALK provide a therapeutic target in neuroblastoma

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.     

Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors.

Long-term potentiation (LTP) in the hippocampus is thought to contribute to memory formation. In the Ca1 region, LTP requires the NMDA (N-methyl-D-aspartate) receptor-dependent influx of Ca2+ and activation of serine and threonine protein kinases. Because of the high amount of protein tyrosine kinases in hippocampus and cerebellum, two regions implicated in learning and memory, we examined the possible additional requirement of tyrosine kinase activity in LTP. We first examined the specificity in brain of five inhibitors of tyrosine kinase and found that two of them, lavendustin A and genistein, showed substantially greater specificity for tyrosine kinase from hippocampus than for three serine-threonine kinases: protein kinase A, protein kinase C, and Ca2+/calmodulin kinase II. Lavendustin A and genistein selectively blocked the induction of LTP when applied in the bath or injected into the postsynaptic cell. By contrast, the inhibitors had no effect on the established LTP, on normal synaptic transmission, or on the neurotransmitter actions attributable to the actions of protein kinase A or protein kinase C. These data suggest that tyrosine kinase activity could be required postsynaptically for long-term synaptic plasticity in the hippocampus. As Ca2+ calmodulin kinase II or protein kinase C seem also to be required, the tyrosine kinases could participate postsynaptically in a kinase network together with serine and threonine kinases.     

BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.     

Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases.     

Tyrosine kinase receptor indistinguishable from the c-met protein

Growth factor receptors with protein tyrosine kinase activity are central to the control of proliferation of both normal and malignant cells. Using anti-phosphotyrosine antibodies, we have previously identified a transmembrane glycoprotein with abnormally high protein tyrosine kinase activity in a human gastric tumour cell line (GTL-16). Electrophoresis under non-reducing conditions revealed that this kinase (relative molecular mass 145,000 (145 K)) is disulphide-linked to a 50K chain in an alpha beta-complex of 190K (p190). From its novel two-chain structure, we deduced that p190 was the prototype of a new class of tyrosine kinase receptors. We now show that p190 is indistinguishable from the protein encoded by the c-met proto-oncogene and that the alpha beta-subunit structure is conserved in other human cell lines. We also show that the high level of p190 found in the GTL-16 cell line is accompanied by amplification and overexpression of c-met. This provides the first example of a functional alteration of c-met in a human tumour cell line.     

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ～600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.     

Corticosterone activates Erk1/2 mitogen-activated protein kinase in primary hippocampal cells through rapid nongenomic mechanism

Nongenomic effects of glucocorticoids (GC) in various cell types have been well documented, but it still remains unknown whether the mechanism also works in hippocampus which is a crucial target of glucocorticoids in neural system during physiological and/or pathophysiological processes. We present here that corticosterone (B) could rapidly activate Erk1/2 mitogen-activated protein kinase (MAPK) in primarily cultured hippocampal cells within minutes, with a bell-shaped time dependent curve which peaked at 15min and then went down to normal level in 30 min. This activation was blocked by protein kinase C (PKC) inhibitor (Go6976), G protein inhibitor (GDPβs), and MEK(MAPK/extracellular signal-regulated kinase kinase) inhibitor(PD98059), but not by protein kinase A (PKA) inbibitor (H89), tyrosine kinase inhibitor (genistein), and glucocorticoid receptor ( GR ) antagonist (RU38486). Thus, the rapid activation of Erk1/2 MAPK in primary hippocampal cells induced by B was likely mediated by a G protein coupled receptor (GPCR) pathway with involvement of PKC, which belonged to the nongenomic rather than genomic mechanism of GC' s effects.     

溶液的酸度AG与pH的关系探究

荷兰科学家HenkVanLubeck提出了在溶液中应用[H+]和[OH-]比值的对数即AG来表示其酸度。关于AG,本文从四个方面论述它的优点及其与pH的关系,并介绍有关AG的计算。     

Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.     

Effects of pigment glands and gossypol on somatic cell culture of upland cotton (Gossypium hirsutum L.)

The effects of pigment glands and gossypol on the somatic cell culture of upland cotton were studied, using the materials as follows: three pairs of glanded and glandless upland cotton near isogenic lines, TM-1, and Coker 312. The results showed that the pigment glands and gossypol contents in the explants had great inhibiting effect on the induction and growth of callus in somatic cell culture of upland cotton, and the induction rate of callus and the single callus weight of glandless cotton were much higher than those of their glanded near isogenic lines. It was easier to obtain regeneration plants from glandless cotton than from their glanded near isogenic lines. There was a significant inverse correlation between the gossypol contents in the explants and callus induction rate, with the correlation coefficient of -0.84. The vitro gossypol in the medium had some inhibiting effect on the induction and growth of callus, especially for the glandless cotton. However, a certain concentration of vitro gossypol in the medium (0.1 mg/L) was an aid to the steadiness growth of callus in glandless cotton somatic cell culture, with a high rate of embryogenic cells which was in favor of plant regeneration, and it was also relatively easy to obtain regeneration plants when they were transferred into differentiation medium with 0.1 mg/L of vitro gossypol, even for some cultivars which are difficult in somatic cell culture. In addition, the gossypol content and its variation in the seedlings and callus during culture of Coker 312 were discussed, as well as the relationship between gossypol variation in the explants and its somatic cell culture. The probability of vitro gossypol used in cotton somatic cell culture for the improvement of somatic cell culture was suggested.