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1.
PTEN/PI3K/Akt信号通路对K562细胞凋亡调控的研究   总被引:2,自引:1,他引:2       下载免费PDF全文
 目的 探讨PTEN/PI3K/Akt信号传导通路对人慢性粒细胞白血病细胞系K562的增殖、凋亡调控的研究及可能的分子作用机制。 方法 将携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad-PTEN-GFP)及空载体(Ad-GFP)腺病毒,转染人慢性粒细胞白血病细胞系K562。通过MTT检测细胞生长曲线,流式细胞术检测细胞凋亡率和细胞增殖指数,同时用细胞光镜、电镜形态等方法检测细胞凋亡,荧光定量PCR(FQ-PCR)检测PTEN及凋亡相关基因Bcl-2、Bcl-xL、Bax mRNA水平变化,Western blot检测PTEN及Akt、p-Akt蛋白水平变化。 结果 与Ad-GFP组相比,Ad-PTEN-GFP 转染K562细胞后,细胞增殖受抑,增殖指数降低,凋亡率增加,p-Akt表达降低,抗凋亡相关基因Bcl-2、Bcl-xL mRNA表达降低,促凋亡基因Bax mRNA表达增加。 结论 过表达PTEN可能通过抑制PI3K/Akt通路抑制K562细胞系增殖,促进细胞凋亡。  相似文献   

2.
目的:观察逆转录病毒介导的双自杀基因对K562细胞的杀伤作用,探讨慢性粒细胞白血病的基因治疗方法。方法:通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,经G418筛选后大量培养产病毒的阳性克隆PA317/CD tk细胞株,收集病毒上清,浓缩后转染K562细胞,再次经G418筛选,获得稳定表达双自杀基因的K562/(:D tk细胞株。用RTl_PCR检测双自杀基因的表达。给予前体药物5-氟胞嘧啶(5-flourocytosine,5-FC)和/或无环乌苷(Ganciciovir,GCv)后MTT法测定转基因组及未转基因组K562细胞的存活率。结果:双自杀基因在K562细胞中可稳定表达,联合使用5-FC和GCv对细胞增殖的杀伤作用及旁杀伤效应高于单独使用5-FC或GCv。结论:逆转录病毒介导自杀基因可有效杀死K562细胞,双自杀基因较单一自杀基因具有更强的抗肿瘤作用。  相似文献   

3.
目的 研究ras p21蛋白在bcr-abl p210蛋白抗凋亡信号传导通路中的作用。方法 应用逆转录病毒载体pLXSN将反义N-ras1 cDNA转导到K562细胞系中后,通过足叶乙甙对细胞进行诱导凋亡。结果 转导反义N-ras1 cDNA的K562细胞内N-ras p21表达下降。反义N-ras转民细胞较对照细胞K562易发生凋亡,对药物的敏感性显著增高。结论 ras p21蛋白是bcr-abl p210蛋白介导抗凋亡信号的重要的下游蛋白,阻断该蛋白的功能有可能成为治疗人类慢性髓性白血病的1个选择点。  相似文献   

4.
木黄酮作用于K562细胞机制的初步研究   总被引:3,自引:0,他引:3  
目的:探讨酪氨酸激酶抑制剂木黄酮(Genistein)作用于慢性粒细胞性白血病(CML)的机制。方法:通过细胞增殖、活力检测、形态学观察、半固体集落培养及DNA凝胶电泳和流式细胞术,观察木黄酮对K562细胞生长的影响。结果:(1)经≥5mg/L木黄酮处理2d的K562细胞,增殖抑制率达到50%以上,且与作用剂量和时间呈正相关;形态渐趋向凋亡但体积涨大;(2)K562细胞集落抑制达50%时的木黄酮浓度为10mg/L,也与时间和剂量呈正相关;(3)K562细胞经10mg/L木黄酮处理4d,DNA凝胶电泳可见清晰的梯状条带;(4)K562细胞分别经2.5mg/L、5mg/L和10mg/L木黄酮处理1d和2d后,流式细胞术检测细胞凋亡率分别为0.62%、1.64%、2.71%和0.68%、4.09%、8.4%;2d后G2期细胞分别占总细胞数的17%、34.5%和79.5%,而G1期和S期细胞逐渐明显减少。结论:木黄酮对K562细胞具有显的抑制增殖作用,其机制与诱导凋亡有关。  相似文献   

5.
目的:探讨SHIP基因对人白血病细胞系K562细胞周期的调控作用.方法:用携带野生型SHIP基因及绿色荧光蛋白 (green fluorescent protein,GFP)基因的慢病毒及空载体慢病毒转染人白血病K562细胞.FCM检测转染效率、细胞凋亡率及细胞周期变化,实时荧光定量PCR(real-time fluorescent quantitative PCR,RFQ-PCR)检测SHIP mRNA水平的变化,Western 印迹法检测SHIP、cyclin D1、p21WAF1/CIPI、p27KIP1蛋白表达水平的变化.结果:与转染空载体组和未转染组相比,转染野生型SHIP基因的K562细胞增殖减慢,凋亡率明显上升(P<0.05);细胞周期显示,G0/G1期延长,G1期前出现亚二倍体的凋亡峰,S期和G2/M期比例降低;cyclin D1表达降低,p21WAF1/CIPI和p27KIP1表达增加.结论:转染野生型SHIP基因可使细胞周期阻滞在G0/G1期,抑制白血病细胞增殖,促进白血病细胞凋亡.  相似文献   

6.
 目的 探讨辛伐他汀(SV)联合阿糖胞苷(Ara-C)对K562 细胞增殖与凋亡的影响。方法 不同浓度SV和Ara-C单用或者联合处理K562细胞,对照组为K562细胞。药物作用24、48、72 h后收集细胞,分别观察各组细胞形态,采用MTT法检测不同组别细胞的生长抑制率,采用流式细胞术检测细胞早期凋亡率、细胞坏死比例。结果 SV联合Ara-C组与单药组相比细胞形态明显有核固缩现象,且可见凋亡小体形成,并且随着处理时间的增加,抑制率也增大。其中15 μmol/L SV联合20 μmol/L Ara-C的细胞抑制作用最为显著,72 h细胞抑制率为(72±1)%,明显高于15 μmol/L SV组的(45±2)%和20 μmol/L Ara-C组的(44±0)%(P<0.01),表现为协同抑制作用(24、48 h金氏Q值为1.24和1.19)。流式细胞术检测发现20、15和 10 μmol/L SV组K562细胞早期凋亡率AnnexinV明显高于对照K562细胞(P<0.01),而且随着时间延长和剂量的增大早期凋亡率也增加(P<0.05)。20和15 μmol/L SV组早期凋亡率均高于10 μmol/L SV组,而前两者之间差异无统计学意义(P>0.05)。晚期凋亡细胞率(PI)各组中差异均无统计学意义(P>0.05)。结论 SV 体外抑制K562细胞增殖及诱导细胞凋亡,SV 与Ara-C具有协同作用,增加了K562细胞对化疗药物的敏感性。15 μmol/L 可能为SV体外最佳作用浓度。  相似文献   

7.
目的 :观察逆转录病毒介导的双自杀基因对K5 6 2细胞的杀伤作用 ,探讨慢性粒细胞白血病的基因治疗方法。方法 :通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,经G4 18筛选后大量培养产病毒的阳性克隆PA317 CD +tk细胞株 ,收集病毒上清 ,浓缩后转染K5 6 2细胞 ,再次经G4 18筛选 ,获得稳定表达双自杀基因的K5 6 2 CD +tk细胞株。用RT PCR检测双自杀基因的表达。给予前体药物 5 氟胞嘧啶 (5 flourocytosine ,5 FC)和 或无环鸟苷 (Ganciciovir,GCV)后MTT法测定转基因组及未转基因组K5 6 2细胞的存活率。结果 :双自杀基因在K5 6 2细胞中可稳定表达 ,联合使用 5 FC和GCV对细胞增殖的杀伤作用及旁杀伤效应高于单独使用 5 FC或GCV。结论 :逆转录病毒介导自杀基因可有效杀死K5 6 2细胞 ,双自杀基因较单一自杀基因具有更强的抗肿瘤作用  相似文献   

8.
目的:研究CCAAT/增强子结合蛋白α(CCAAT/enhancer-binding protein-alpha,C/EBPα)对K562细胞株分化和凋亡的影响及对相关基因的调控,为慢性粒细胞白血病的治疗提供新的治疗靶点.方法:将C/EBPα表达质粒pEGFP-C/EBPα及空载体对照质粒pEGFP分别经阳离子脂质体2000介导转染K562细胞,用G418筛选出C/EBPα稳定表达细胞株.Wright-Giemsa染色观察细胞形态学变化,FCM分析细胞表面分化抗原CD11b的表达、细胞周期及细胞凋亡,电子显微镜观察细胞凋亡,RT-PCR和Western印迹法检测细胞中相关基因Per 2、cyclin B1和C-myc的表达.结果:筛选得到稳定表达C/EBPα的细胞株pEGFP-C/EBPα-K562.与空载体转染组及对照组细胞相比,转染组K562细胞分化明显,同时粒系细胞表面分化抗原CD11b表达增加;细胞周期分析发现,转染组细胞中G2期细胞增多,与空载体组和对照组相比,差异有统计学意义(P<0.05),同时出现细胞凋亡峰.细胞凋亡检测结果显示,转染组细胞凋亡明显增加(21.1%),与空载体组(6.0%)和对照组(4.2%)比较,差异有统计学意义(P<0.05);电子显微镜观察发现,转染组细胞中出现染色质浓集、断裂和核固缩等现象,并见凋亡小体;RT-PCR和Western印迹法检测发现,C/EBPα明显上调Per 2 mRNA和蛋白表达,抑制cyclinB1、C-myc mRNA和蛋白的表达.结论:C/EBPα能促进K562细胞分化,并诱导细胞凋亡,其机制可能是通过对细胞周期相关基因的调控来实现的.  相似文献   

9.
10.
微波和足叶乙甙诱导K562细胞的凋亡   总被引:4,自引:0,他引:4  
目的:探讨微波和足叶乙甙体外诱导K562细胞凋亡和可能性及其机制。方法:将微波和足叶乙甙分别单位以及联合作用于正常骨髓细胞和K562细胞株,通过细胞凋亡率和bcl-2和p21^ras蛋白阳性率的测定来评价细胞凋亡的程度并研究其机制。结果:微波和足叶乙甙各自都能诱导正常骨髓细胞和K562细胞的少量凋亡,两者联合作用所诱导的细胞凋亡率较之于足叶乙甙有显著性增加(P〈0.05),其中K562细胞凋亡率的  相似文献   

11.

Background

Clear cell renal cell carcinoma (ccRCC) is the most frequently encountered tumor in the adult kidney. Many factors are known to take part in the development and progression of this tumor. Nuclear factor kappa B (NF-κB) is a family of the genes that includes five members acting in events such as inflammation and apoptosis. In this study, the role of NF-κB (p50 subunit) in ccRCC and its relation to angiogenesis and apoptosis were investigated.

Methods

Formalin-fixed and paraffin embedded tissue blocks from 40 patients with ccRCC were studied. Expressions of NF-κB (p50), VEGF, EGFR, bc1-2 and p53 were detected immunohistochemically. The relationship of NF-κB with these markers and clinicopathological findings were evaluated.

Results

The expression of NF-κB was detected in 35 (85%), VEGF in 37 (92.5%), EGFR in 38 (95%), bc1-2 in 33 (82.5%) and p53 in 13 (32.5%) of 40 ccRCC patients. Statistical analyses revealed a significant relation between NF-κB expression and VEGF (p = 0.001), EGFR (p = 0.004), bc1-2 (p = 0.010) and p53 (p = 0.037). There was no significant correlation between NF-κB and such parameters as tumor grade, stage, age and sex.

Conclusion

The results of this study indicated that in ccRCC cases NF-κB was associated with markers of angiogenesis and apoptosis such as VEGF, EGFR, bc1-2 and p53. In addition, the results did not only suggest a close relationship between NF-κB and VEGF, EGFR, bc1-2 and p53 in ccRCC, but also indicate that NF-κB was a potential therapeutic target in the treatment of ccRCC resistant to chemotherapy.  相似文献   

12.
目的:探讨BCR/ABL融合基因对抑癌基因PTEN介导的信号传导通路在K562细胞增殖和凋亡中的影响。方法:用不同浓度的格列卫(0.5、1、2、5、10μg/ml)干预K562细胞不同时间,观察其对细胞增殖的影响。用1μg/ml浓度的格列卫干预K562细胞不同时间(12、24、36、48、72h)后,通过荧光定量PCR检测细胞BCR/ABL、PTEN、mTOR mRNA水平的变化,并分析它们之间的相互关系。Western blotting检测细胞Akt和p-Akt水平,分析BCR/ABL融合基因对HEN mRNA表达的影响。结果:1μg/ml格列卫作用K562细胞后随着BCR/ABL融合基因表达减低,PTEN mRNA表达上调,mTOR mRNA表达下调,p-Akt表达下调。48h后随着BCR/ABL融合基因的抑制减弱,PTEN mRNA表达进而减低,而mTOR mRNA表达升高,p-Akt持续降低。BCR/ABL mRNA表达与PTEN mRNA表达呈负相关(r=-0.881,P〈0.05),与mTOR mRNA及p-Akt表达呈正相关(依次为r=0.961,r=0.879,P均〈0.05)。结论:BCR/ABL融合基因可以通过抑制PTEN基因表达,调控P13K/Akt、mTOR信号传导通路,参与细胞增殖、凋亡及细胞周期调控等生物学特性。  相似文献   

13.
14.
目的:观察雷帕霉素(rapamycin,rapa)对食管鳞癌细胞系EC9706的mTOR/p70S6K信号通路的影响。方法:采用免疫细胞化学证实mTOR/p70S6K信号通路的存在,然后通过DNALadder、RT—PCR、Westernblot及流式细胞术分别从DNA、RNA、蛋白及细胞水平研究rapa对细胞凋亡和信号通路的影响。结果:免疫细胞化学结果显示,在细胞核及细胞质中mTOR均呈阳性;rapa处理后有明显DNALadder产生,且mTOR的mRNA水平及蛋白水平下调。但是,mTOR下游的直接靶点p70S6K的mRNA水平及蛋白水平则升高,二者的变化程度均与rapa剂量的相关;流式细胞术检测结果表明,rapa可使细胞停滞于G1期。结论:食管鳞癌细胞系EC9706中存在mTOR/p70S6K信号通路并且处于激活状态,rapa能明显促进细胞凋亡并抑制该通路激活,从而间接抑制翻译的进行。  相似文献   

15.
目的 探讨异土木香内酯对慢性粒细胞白血病耐药细胞K562/A02增殖的抑制作用及其机制.方法6.25、12.5、25、50、100μmol/L的异土木香内酯作用于K562/A02细胞24、48 h,四甲基偶氮唑盐(MTT)法检测其对K562/A02细胞的增殖抑制效果;10、15、20μmol/L异土木香内酯作用于K562/A02细胞24 h,流式细胞术检测细胞周期及凋亡;Western blot法检测增殖相关蛋白表达水平.多组间比较采用单因素方差分析.结果异土木香内酯能够显著抑制K562/A02细胞增殖,并呈浓度依赖性(P<0.05),作用24 h的半数抑制浓度(IC50)值为(15.00±1.03)μmol/L;阴性对照组及10、15、20μmol/L异土木香内酯作用后K562/A02细胞凋亡率分别为(2.71±0.52)%、(19.10±1.55)%、(27.61±2.32)%和(32.01±3.01)%,呈浓度依赖性(F=33.901,P<0.05);S期细胞比例分别为(57.80±2.11)%、(68.62±2.89)%、(78.44±3.51)%和(80.61±2.90)%,呈浓度依赖性(F=51.328,P<0.05).异土木香内酯明显降低bcl-2、p-bcr-abl、p-STAT5、细胞周期蛋白依赖性激酶2(CDK2)和细胞周期蛋白A(cyclin A)的表达(P<0.05),上调细胞色素C、Bax和p21的表达(P<0.05).结论异土木香内酯能够通过bcr-abl-STAT5信号通路抑制K562/A02细胞增殖.  相似文献   

16.
Characterization of a K562 multidrug-resistant cell line   总被引:2,自引:0,他引:2  
A daunorubicin-resistant variant of the K562 human leukemia cell line (K562-R), which demonstrates cross-resistance to other anthracycline antibiotics and Vinca alkaloids, has been developed in vitro by continuous exposure to daunorubicin. Cross-resistance to anthracyclines and Vinca alkaloids is reversed when cells are exposed to drugs in the presence of verapamil, a calcium channel blocker. The K562-R cell line overexpresses a 4.5-kilobase mRNA, which is thought to code for the Mr 170,000 membrane glycoprotein associated with multidrug resistance. Transport studies indicate reduced intracellular accumulation and retention of daunorubicin in the K562-R cells as compared to the parent cell line. These studies further suggest the presence of distinct cellular pools composed of both rapidly and slowly exchanging drug, with the rapidly exchanging pool being more pronounced in the resistant line. The development of multidrug resistance in the K562-R cell line is also associated with the overexpression of five different cell surface membrane proteins ranging in molecular weight between 50,000 and 210,000, whose function remains to be defined.  相似文献   

17.
Yin DD  Fan FY  Hu XB  Hou LH  Zhang XP  Liu L  Liang YM  Han H 《Leukemia research》2009,33(1):109-114
Notch signaling functions in the development of some types of leukemia and lymphoma, but the relationship between Notch signaling and chronic myeloid leukemia (CML) remains to be elucidated. In this study, we examined the expression of Notch receptors and ligands in the human CML cell line K562. When the active form of Notch1, the Notch intra-cellular domain (NIC), was over-expressed in K562, the proliferation of K562 was mildly but significantly inhibited, accompanied by increased Hes1 mRNA level. On the other hand, when Notch signaling was attenuated by over-expression of a dominant-negative RBP-J, RBP-J(R218H), in K562 cells, the proliferation of K562 was increased. Moreover, we found that activation of Notch signaling inhibited while repression of Notch signaling promoted the colony-forming activity of K562 cells. We examined cell cycle-related molecules in K562 transfected with NIC or RBP-J(R218H), and found that the protein level of the retinoblastoma gene product (the Rb protein) was induced in K562 expressing NIC, and down-regulated in K562 expressing RBP-J(R218H). These data suggest that the Notch signaling may function as a tumor inhibitor in human CML cells.  相似文献   

18.
K562--a human erythroleukemic cell line.   总被引:36,自引:0,他引:36  
We have studied the surface membrane properties of the human leukemic cell line K562 which previously has been reported to represent an early stage of granulocyte maturation. The surface glycoprotein pattern of the K562 cells obtained after galactose oxidase-NaB[3H]4 labelling and slab gel electrophoresis shows striking similarities with that of normal erythrocytes but is completely different from the patterns of normal and malignant cells of various stages of the myeloblast to granulocyte differentiation. Moreover, the K562 cell expressed the major red cell sialoglycoprotein, glycophorin, on its surface as shown by immunofluorescence and by immunoprecipitation from labelled membrane preparations. As glycophorin is exclusively found on erythroid cells in human bone marrow we conclude that the K562 is a human erythroleukemic line.  相似文献   

19.
目的为肿瘤细胞自律性生长的细胞内自激因子假设提供实验证据。方法将新发现的造血相关核蛋白EDAG基因转染人白血病细胞系K562,比较过表达EDAG对K562细胞的增生能力,以及对细胞的造血调控、凋亡及细胞周期相关蛋白表达的影响。结果在无血清培养条件下,过表达EDAG的K562细胞的增生能力明显高于两组对照细胞,并且有c—myb和bcl-2mRNA表达的显著上调。结论过表达的EDAG可以起细胞内自激因子作用。为核蛋白异常表达可以起细胞内自激因子作用提供了实验证据。  相似文献   

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