2,3-吲哚醌诱导人神经母细胞瘤SH-SY5Y细胞凋亡及周期阻滞

背景与目的:2,3-吲哚醌(isatin,ISA)是存在于哺乳动物体液及组织中的一种天然物质,已发现其对肿瘤细胞的生长有抑制作用,但具体作用机制尚不明。本研究以人神经母细胞瘤细胞SH-SY5Y为靶细胞,观察ISA对SH-SY5Y细胞的作用及其机制。方法:采用荧光染色、流式细胞术(flow cytometry,FCM)及Western blot等方法检测不同浓度ISA(0、100、200、400μmol/L)诱导SH-SY5Y细胞凋亡及细胞周期阻滞的作用机制。结果:400μmol/L ISA作用48h后,在荧光显微镜下观察到SH-SY5Y细胞出现核固缩、DNA断裂等典型的凋亡形态学改变。Western blot结果显示,随ISA浓度的增加,Bcl-2蛋白表达下降、Bax蛋白表达无明显改变,Bcl-2/Bax下降。100、200、400μmol/L ISA处理SH-SY5Y细胞48h,活化Caspase-3的表达率分别为19.28%、25.88%、33.43%,明显高于对照组(P<0.05)。Western blot进一步检测到其下游底物Caspase激活的脱氧核糖核酸酶抑制剂(inhibitor of caspase-activated DNase,ICAD)被降解。细胞周期分析表明,经100、200、400μmol/L ISA处理48h后,G1期SH-SY5Y细胞数明显增加,呈明显的G1期阻滞;磷酸化的ERK蛋白及Cyclin D1(CDK1)蛋白表达显著减少(P<0.05)。结论:ISA能明显诱导SH-SY5Y细胞凋亡和细胞周期G1期阻滞,该作用可能与Bcl-2/Bax降低、Caspase-3激活,以及下调磷酸化ERK和细胞周期因子CDK1表达有关。     

mTOR通路对神经母细胞瘤SH-SY5 Y细胞自噬作用的影响

目的：研究雷帕霉素靶蛋白（ mammalian target of rapamycin,mTOR）通路对神经母细胞瘤SH-SY5Y细胞自体吞噬作用的影响。方法：应用不同浓度的雷帕霉素作用于神经母细胞瘤细胞,采用实时荧光定量PCR检测雷帕霉素作用前后神经母细胞瘤细胞株的mTOR、LC3及Beclin 1 mRNA表达情况。结果：雷帕霉素能抑制mTOR表达且呈现量效依赖关系,不同浓度的雷帕霉素分别对SH-SY5Y细胞作用72h后,mTOR mR-NA的表达随浓度增加逐渐下降。而LC3及Beclin 1 mRNA的表达随浓度增加逐渐上升,神经母细胞瘤中mTOR与LC3及Beclin 1的表达呈负相关。结论：雷帕霉素通过抑制mTOR信号通路促进神经母细胞瘤的自体吞噬作用,动态观察mTOR可作为神经母细胞瘤治疗效果监测的指标之一。     

Midkine对神经母细胞瘤TNB1细胞生长与分化的影响

目的:通过对中期因子(midkine,MK)在神经母细胞瘤TNB1细胞中的生物学作用进行研究,揭示该因子在肿瘤细胞生长和分化中相关分子机制.方法:1)利用神经母细胞瘤临床患者数据库,明确MK的表达与神经母细胞瘤患者生存率之间的关系.在体外细胞实验中,利用携带MKshRNA载体的慢病毒体外感染神经母细胞瘤TNB1细胞,分...     

9-顺维甲酸联合细胞毒性药物对SH-SY5Y神经母细胞系增殖及诱导凋亡的影响

目的：探讨9-顺维甲酸联合细胞毒性药物对SH—SY5Y神经母细胞系增殖及诱导凋亡方面的作用，为9-顺式维甲酸治疗神经母细胞瘤可能有效方案的设计提供实验依据．方法：1）采用MTT比色法测定不同单药组和双药组在不同时间及不同浓度下SH—SY5Y细胞的抑制率；2）采用流式细胞仪测定不同单药组和双药组在不同时间及不同浓度下SH—SY5Y细胞的凋亡指数。结果：1）DDP＋RA双药组中，低剂量组在24和48h以及高剂量组在48h细胞抑制率较相应DDP单药组高（P〈0．01）；UP-16＋RA双药组中，低、高剂量组在24h细胞抑制率较相应LIP—16单药组高（P〈0．01）；DDP＋RA和UP—16＋RA双药组（除了低剂量DDP＋RA组在24h）细胞抑制率均高于RA组（500ng／孔）（P〈0．01）.2）单药组和双药组细胞凋亡指数均高于对照组（P〈0．01）；双药组细胞凋亡指数均高于相应单药组（P〈0．01），结论：细胞毒性药物与RA组合用药细胞抑制率和早期细胞凋亡指数较单药高，提示细胞毒性药物与RA在抑制细胞生长和诱导凋亡方面表现很好的协同作用.     

神经母细胞瘤增殖凋亡的研究

目的　本实验通过研究神经母细胞瘤的增殖、凋亡的失衡 ,结合神经母细胞瘤的分化 ,以探讨增殖、凋亡的改变与神经母细胞瘤预后的关系。方法　分别取正常对照组、良性肿瘤组、恶性肿瘤组 3组组织制作石蜡切片 ,运用免疫组织化学法检测增殖细胞 ,计算增殖细胞占细胞百分比 ,求得增殖指数 (PI)。运用原位DNA末端标记法 ,检测凋亡细胞 ,计算凋亡细胞占细胞百分比 ,求得凋亡指数 (AI)。结合神经母细胞瘤的组织学分类 ,了解增殖指数、凋亡指数与神经母细胞瘤预后情况。结果　预后好的组织与预后差的组织 2组的PI相比P <0 .0 1。预后好的组织与预后差的组织两组的AI相比P <0 .0 1。Ⅲ期或Ⅳ期死亡与健在分别PI相比较P <0 0 1。Ⅲ期或Ⅳ期死亡与健在分别AI相比较P <0 0 1。结论　PCNA指数越高 ,神经母细胞瘤预后越差。凋亡细胞和凋亡小体越多 ,凋亡指数越高 ,神经母细胞瘤预后越好。联合检测神经母细胞瘤的增殖指数、凋亡指数结合凋亡小体可提高对神经母细胞瘤诊断及预后判断的准确性 ,并对治疗指导有重要价值     

吴茱萸碱对神经母细胞瘤SK-N-SH细胞增殖、迁移及侵袭的影响

目的：探讨吴茱萸碱（Evo）是否通过调控lncRNA LINC00858 表达调控神经母细胞瘤SK-N-SH 细胞的增殖、迁移及侵袭。方法：在体外以3、6、12 μmol/L Evo 处理人神经母细胞瘤SK-N-SH 细胞,利用RNA干扰技术分别将si-NC、si-LINC00858转染至SK-N-SH 细胞,将pcDNA、pcDNA-LINC00858 转染至SK-N-SH 细胞并经12 μmol/L Evo 处理,实验分为对照组、Evo 低剂量组、Evo 中剂量组、Evo 高剂量组、si-NC 组、si-LINC00858 组、Evo+pcDNA 组、Evo+pcDNA-LINC00858 组。采用qPCR法检测各组细胞LINC00858 的表达量,MTT、Transwell 实验分别检测细胞的增殖、迁移、侵袭能力,WB 法检测细胞中cyclinD1、MMP-2、 MMP-9和p21 蛋白的表达。结果：与对照组相比,Evo低、中、高剂量组SK-N-SH 细胞中LINC00858 表达均显著降低（均P<0.05）,细胞增殖抑制率显著升高、迁移及侵袭细胞数显著减少（均P<0.01）,cyclinD1、MMP-2、MMP-9 蛋白表达降低、p21 蛋白表达升高（均P<0.01）。与si-NC 组相比,si-LINC00858 组细胞的增殖抑制率、迁移和侵袭细胞数及相关蛋白表达变化同Evo 低、中、高剂量组。与Evo+pcDNA 组相比,Evo+pcDNA-LINC00858 组细胞的增殖抑制率显著降低、迁移及侵袭细胞数均显著增多（均P<0.01）,cyclinD1、MMP-2、MMP-9蛋白表达升高、p21蛋白表达降低（均P<0.05）。结论：Evo 通过下调LINC00858 表达抑制神经母细胞瘤SK-N-SH细胞的增殖、迁移及侵袭。     

神经母细胞瘤增殖、凋亡与休眠消退的相关性研究

目的：本实验通过研究神经母细胞瘤的增殖、凋亡的改变，结合神经母细胞瘤的分化，以探讨增殖、凋亡的改变与神经母细胞瘤休眠消退的关系。方法：肿瘤休眠消退组：神经母细胞瘤休眠消退6例，男4例，女2例。取组织制作成石蜡切片，运用免疫组织化学方法检测增殖细胞，计算增殖细胞占细胞百分比，求得增殖指数（PI）。运用原位DNA末端标记法，检测凋亡细胞，计算凋亡细胞占细胞百分比，求得凋亡指数（AI）。结果：休眠消退病例中5例凋亡指数大于9％，5例增殖指数大于13％，6例增殖率减凋亡率均小于4％。结论：增殖指数小于13％，凋亡指数大于9％，特别是增殖指数减凋亡指数小于4％，神经母细胞瘤休眠消退的可能性大。     

人参皂苷Rh2对神经母细胞瘤SH-SY5Y的增殖、凋亡、迁移及侵袭的影响

目的:针对神经母细胞瘤（neuroblastoma,NB）早期易转移的特性,研究人参皂苷Rh2（ginsenoside Rh2）对神经母细胞瘤的增殖、凋亡、迁移及侵袭的影响。方法:用不同（5、10、20、40、80 μg/ml）浓度人参皂苷Rh2来干预神经母细胞瘤细胞系SH-SY5Y。Cell counting kit-8(CCK-8)法、细胞划痕试验、Transwell小室模型检测细胞增殖、迁移及侵袭能力;蛋白质印迹法（Western-blot,WB）试验检测不同浓度下神经母细胞瘤细胞内基质金属蛋白酶2（matrix metalloproteinase2,MMP2）、Bax、Bcl-2、β-catenin蛋白的表达。结果:人参皂苷Rh2可以时间-浓度依赖性地抑制神经母细胞瘤增殖、迁移及侵袭,降低迁移相关蛋白MMP2的表达,上调了促凋亡蛋白Bax的表达水平同时降低抗凋亡蛋白Bcl-2的表达。人参皂苷Rh2处理的神经母细胞瘤内β-catenin水平降低。结论:人参皂苷Rh2可以在体外抑制神经母细胞瘤的增殖,下调MMP2蛋白抑制肿瘤细胞迁移及侵袭,通过wnt通路抑制细胞生长。     

神经母细胞瘤增殖、凋亡与休眠消退的相关性研究

目的　本实验通过研究神经母细胞瘤的增殖、凋亡的改变 ,结合神经母细胞瘤的分化 ,以探讨增殖、凋亡的改变与神经母细胞瘤休眠消退的关系。方法　肿瘤休眠消退组 :神经母细胞瘤休眠消退 6例 ,男 4例 ,女 2例。取组织制作成石蜡切片 ,运用免疫组织化学方法检测增殖细胞 ,计算增殖细胞占细胞百分比 ,求得增殖指数 (PI)。运用原位DNA末端标记法 ,检测凋亡细胞 ,计算凋亡细胞占细胞百分比 ,求得凋亡指数 (AI)。结果　休眠消退病例中 5例凋亡指数大于 9% ,5例增殖指数大于 13 % ,6例增殖率减凋亡率均小于 4%。结论　增殖指数小于 13 % ,凋亡指数大于 9% ,特别是增殖指数减凋亡指数小于 4% ,神经母细胞瘤休眠消退的可能性大。     

PBDE-47对人神经母细胞瘤SH-SY5Y细胞致突变作用

背景与目的:探讨2,2',4,4'-四溴联苯醚(2,2',4,4'-tetrabromodiphenyl ethers,PBDE-47)对人神经母细胞瘤SH-SY5Y细胞的致突变作用.材料与方法:设空白对照组、溶剂对照组、3个不同BDE-47剂量的试验组(1 μg、2 μg、4 μg/ml)和阳性对照组(MMC),共6组.SH-SY5Y细胞分别暴露于各组不同受试物24 h后,采用胞质分裂阻断法测定微核率、核浆桥率. 结果:PBDE-47可诱导SH-SY5Y细胞核分裂指数呈剂量依赖性下降,微核细胞率和核浆桥率呈剂量依赖性增加;2 μg/ml和4 μg/ml的染毒剂量可引起SH-SY5Y细胞核分裂指数明显降低(P＜0.05)以及微核细胞率和核浆桥率的明显增加(P＜0.05),而仅在4 μg/ml PBDE-47染毒组发现微核率明显增加(P＜0.05).结论:PBDE-47可诱导SH-SY5Y细胞微核和核浆桥的形成,具有致突变作用.     

Proliferation and Differentiation of Myelodysplastic CD34+ Cells

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders of hematopoiesis involving hyperproliferative and ineffective hematopoiesis associated with morphologic evidence of marrow cell dysplasia resulting in refractory cytopenia(s), and an increased risk of transformation into acute myeloblastic leukemia (AML). The administration of colony-stimulating factor(s) (CSFs) to patients with MDS increased blood neutrophil concentrations, in most patients, and was also expected to be beneficial and to prevent infections. However, the progression to AML during the treatment with CSFs was suspected in some patients. Therefore, extensive in vitro studies were expected to lead to the establishment of criteria for selection of patients who are likely to benefit from CSF's as well as to establish the overall value of the different types of CSFs therapy. For this purpose, in vitro colony assays provide an excellent tool for investigating the biologic characteristics of MDS progenitor cells. However, conditions of the culture must be such that each progenitor can express its full potential for proliferation and differentiation.

Because of the above, MDS progenitor cells cannot be used because they carry an impairment in proliferation and differentiation. To address this problem, one needs to know how many cells are being handled and the maximum numbers of colonies and clusters expected. CD34, a stem cell phe-notype, is at present one of the best markers of progenitor cells, and can be used for purposes of purification. Using a defined number of CD34⁺ cells, it was feasible to make direct investigations on MDS progenitor cells. In this review the properties of MDS progenitor cells are described, in association with proliferation and differentiation, with special emphasis on the phenotypic subpopula-tions of MDS CD34⁺ cells.     

丙戊酸促进神经母细胞瘤细胞自噬性死亡

目的探讨丙戊酸(VPA)对神经母细胞瘤细胞自噬性死亡的调控。方法用含不同浓度的VPA培养基培养SH-SY5Y细胞,应用MTT法检测细胞的存活率,乳酸脱氢酶(LDH)检测细胞损伤,单丹磺酰尸胺(MDC)染色评价细胞质内酸性自噬泡的形成,Western blotting法检测巨自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的比值及Beclin-1的表达。结果与对照组比较,VPA孵育48 h后,SH-SY5Y细胞存活率明显降低,且具有剂量依赖性(P<0.05);SH-SY5Y细胞培养基中LDH明显增高,且具有剂量依赖性(P<0.05);VPA诱导细胞内LC3-Ⅱ/LC3-Ⅰ的比值及Beclin-1的表达水平明显上调(P<0.05)。结论 VPA诱导神经母细胞瘤细胞细胞自噬性死亡,为神经母细胞瘤的治疗提供新的手段。     

Induction of Erythroid Differentiation by 5-Fluorouracil in K562 Leukemia Cells

K562 cell line, established from a patient in the blast crisis of chronic myeloid leukemia, expresses high levels of c-myc and bcr/abl gene products. Exposure of K562 cells to 5-fluorouracil (5-FU) resulted in a marked benzidine-positive erythroid differentiation with concomitant reduction in cell proliferation. No change in c-myc mRNA or protein levels occurred during 96 h of drug treatment. In contrast, a biphasic change of p210 ^bcr/abl and the abl -associated kinase activities was observed upon treatment with 5-FU. The change in p210 ^bcr/abl expression may be mediated at the translational level, since the steady-state level and the enzymatic activity of p210 ^bcr/abl are reduced, whereas bcr/abl mRNA levels are unaltered. The results are consistent with the existence of pleiotropic differentiation pathways in K562 cells.     

Effect of Human Interferons on Morphological Differentiation and Suppression of N-myc Gene Expression in Human Neuroblastoma Cells

The activity of human interferons (HuIFNs) to induce morphological changes and the suppression of N- myc gene expression on human neuroblastoma cells (GOTO and KP-N-RT) was evaluated. Morphological differentiation, characterized as the extension and bifurcation of neurites, the formation of multinucleated giant cells and the formation of neurite networks, was induced by treatment with recombinant HuIFN-γ (rHuIFN-γ and also with natural HuIFN-γ on human neuroblastoma cells (GOTO and KP-N-RT). But recombinant HuIFN-αA and recombinant HuIFN-βdid not induce any changes. The rHuIFN-β and rHuIFN-γ inhibited the growth of GOTO and KP-N-RT cells more strongly than the rHuIFN-αA did. The expression of N- myc gene was suppressed in GOTO cells treated with rHuIFN-γ. The suppressive effect of rHuIFN-γ was dependent on the duration of the treatment. However, rHuIFN-αA and rHuIFN-β did not suppress N- myc gene expression. Moreover, both morphological differentiation and the supprcssive effect on N- myc gene expression by rHuIFN-γ were inhibited in the presence of cycloheximide. These results suggest that the morphological changes and N- myc gene expression in neuroblastoma cells are closely related. Furthermore, this decreased N- myc gene expression during the morphological differentiation may be related to the proteins induced by HuIFN-γ.     

RORα高表达对人胃癌MGC803细胞增殖与迁移侵袭的影响

目的 观察RORα高表达对MGC803细胞增殖与迁移侵袭的影响。方法 构建高表达RORα人胃癌MGC803细胞。Real-time PCR或RT-PCR和Western blot检测RORα、MMP-9与TIMP3 mRNA和蛋白表达。MTT、流式细胞术、迁移和侵袭实验检测RORα高表达对MGC803细胞增殖、细胞周期、迁移和侵袭能力的影响。结果 RORα高表达MGC803细胞RORα mRNA和蛋白表达显著上调。在48、72与96 h,RORα高表达细胞的增殖活性明显低于对照组和空载体组（P<0.05）。RORα高表达G2/M期细胞明显高于对照组和空载体组（P<0.05）。高表达组细胞的迁移距离较对照组与空载体组明显减少（P<0.05）。高表达组穿膜细胞数较对照组与空载体组明显减少（P<0.05）。RORα高表达可明显下调MMP-9和上调TIMP3mRNA与蛋白。结论 成功构建RORα高表达MGC803细胞,RORα高表达可抑制MGC803细胞增殖与迁移侵袭,将细胞阻滞于G2/M期,其机制可能与下调MMP-9和上调TIMP3有关。     

The Effects of Purified Artemia Extract Proteins on Proliferation,Differentiation and Apoptosis of Human Leukemic HL-60 Cells

There has been an increment in the number of studies focused on marine bioactive materials. Many peptides and other biomaterials with anticancer potential have been extracted from various marine animals. Artemia extracts have found uses in sun-light protection cosmetics and anti-aging products. However, contents of biochemical compounds in Artemia spp. and molecular mechanisms of have not been clearly studied in leukemic cells in vitro. In this work, we isolated and purified proteins of Artemia Urmiana. Six clear fractions (A-F) observed on DEAE-cellulose chromatography were assayed for effects on cell growth, differentiation and apoptosis using the human leukemic HL-60 cell line. Cell proliferation analysis by MTT and BrdU assays indicated that did not affect cells, growth. Cells treated with crude extract and fractions A, B and C, but not E and F (up to 100 μg/mL), exhibited increase of cell growth in a dose dependent manner. Stimulatory effects of fraction D were observed at concentrations of 10 μg/ml and above. In nitro blue tetrazolium (NBT) reduction assays, treatment with 100 μg/mL of fraction E or F for 96 hr increased the fraction of differentiated cells up to 14.8 ± 3.56% and 16.5 ± 2.08% respectively. Combination of those fractions with retinoic acid had significant synergistic effects on the differentiation of cells (56.8 ± 3.7% and 67.4 ± 4.2%, p≤0.01). Annexin-V FITC staining for apoptosis and flow cytometric assays indicated induction of apoptosis by fractions E and F up to 23.8 and 31.8% of cells.     

过表达RbAp46基因对白血病细胞系U937增殖与分化的影响

目的 探讨过表达的RbAp46基因对白血病细胞系U937生物学特性的影响。方法 运用电穿孔介导的转基因技术, 在U937中建立稳定表达外源RbAp46基因的细胞株。用锥虫蓝拒染法判定细胞活力,细胞计数判定细胞生长速率。将处在对数生长期U937/RbAp46和U937/CMV各以2×105/ml浓度接种,加入TPA 50ng/ml,同时以培养液作为空白对照,培养72 h后收集细胞,流式细胞术测定细胞表面分化抗原CD11b的表达,RT-PCR分析p21WAF1/CIP1mRNA的表达,流式细胞术检测细胞周期分布情况。结果 转染RbAp46 的U937细胞的生长速率要比对照组低1倍左右。无论TPA诱导与否,RbAp46都能增加U937细胞表面CD11b的表达。相对于对照组,正常条件下,RbAp46转染的细胞p21WAF1/CIP1mRNA水平没有明显增加,细胞在G1期分布增加,但是经TPA诱导后,p21WAF1/CIP1mRNA水平的明显增加,细胞阻滞在G1期。结论 过表达的外源RbAp46基因能抑制U937的增殖,并为细胞分化准备了条件,使细胞更易于启动分化,p21WAF1/CIP1可能参与该过程。     

Fibulin-5 is a Prognostic Marker that Contributes to Proliferation and Invasion of Human Glioma Cells

Fibulin-5 has recently been considered as a potential tumor suppressor in human cancers. Several studieshave shown that it is down-regulated in a variety of tumor types and inhibits tumor growth and metastasis. Thisstudy was aimed to investigate the clinical significance of fibulin-5 in glioma and its role in cell proliferationand invasion. We found that the expression of fibulin-5 in glioma tissues was significantly lower than those innormal brain (NB) tissues. Negative expression was significantly correlated with advanced clinical stage (gradeIII+IV) . Furthermore, Fibulin-5 negative expression was correlated with a shorter overall survival of gliomapatients. Multivariate Cox repression analysis indicated that fibulin-5 was an independent factor for predictingoverall survival of glioma patients. Overexpression obviously inhibited cell proliferation in U251 and U87cells. Furthermore, it significantly reduced the number of migrating and invading glioma cells. In conclusion,impaired expression of fibulin-5 is correlated with the advanced tumor stage in glioma. Otherwise, Fibulin-5 isan independent prognostic marker for predicting overall survival of glioma patients. Mechanistically, it mayfunction as a tumor suppressor via inhibiting cell proliferation and invasion in gliomas.     

桂皮酸对NB4细胞增殖和分化作用的实验研究

目的探讨桂皮酸对NB4细胞的增殖和分化的作用.方法采用光镜观察形态变化,流式细胞术检测CD11b和CD33.结果桂皮酸作用NB4细胞后,可使NB4细胞增殖明显受抑,诱导NB4细胞向终未细胞分化;实验组与对照组的分化率差异明显(P＜0.01);CD11b表达升高,CD33表达下降(P＜0.05).结论桂皮酸对NB4细胞具有抑制增殖和诱导分化的作用.