shRNA沉默人肝癌细胞株SK-Hep1 Kir6.2基因的研究
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摘要
离子通道是生命活动的基础,离子通道与肿瘤的关系逐渐引起人们关注,深入了解离子通道在肿瘤形成、发展和转移过程中的作用,不仅有利于更加全面地阐明肿瘤发病机制,也将可能为肿瘤预防和治疗提供新靶标和新的生物学干预手段。钾离子通道是种类最多、最为复杂的一类离子通道,在可兴奋细胞和非可兴奋细胞的信号转导过程中都起着非常重要的作用,也参与肿瘤细胞的增殖和分化的调节。钾离子通道与肿瘤细胞增殖的关系已成为肿瘤基础研究的新热点。ATP敏感性钾通道(ATP-sensitive potassium channels,KATP)将细胞的能量代谢和生物膜电活动耦联起来。细胞生物学和药理学的许多实验证实,钾离子通道在肿瘤细胞增殖和生存的调节机制中起作用。RNAi技术不仅可以发现肿瘤发展过程中基因的变化,而且可能作为肿瘤治疗的一种方法。shRNA(short hairpin RNA,shRNA)可以稳定抑制目的基因。原发性肝癌是一种常见的恶性肿瘤,发病率有逐年上升的趋势。虽然诊断和治疗取得了很大进步,但是原发性肝癌患者的生存率低,预后差,仍然需要加强基础研究。
目的:构建shRNA质粒表达载体。脂质体法转染肝癌SK-Hep1细胞株,G418筛选,挑选荧光单克隆、扩增。RT-PCR、Western Blot检测基因表达情况。膜片钳检测KATP通道电流。研究细胞生物学特性改变情况。
方法:
1.构建shRNA质粒表达载体根据shRNA设计原则,设计两条干扰序列1、2和一条非特异性阴性对照序列。shRNA的表达插入的片段由互补的寡核苷酸链退火生成,和经BamH I和Hind III双酶切的线性化空载体pGenesil-3连接,得到三种质粒K1(携带干扰序列1)、K2(携带干扰序列2)、HK(携带非特异性阴性对照序列),转化感受态细菌DH5a,培养后提取质粒。酶切、测序。
2.SK-Hep1细胞的培养、传代、转染、筛选SK-Hep1细胞复苏后,在含有10% FBS的DMEM培养液中连续培养。细胞生长到80-90%运用0.25%胰蛋白酶溶液消化、传代。利用脂质体法转染构建的质粒,经G418进行筛选后,挑选荧光单克隆进行扩增。实验分4组:SK (空白对照)组、SK-K1(转染质粒K1)组、SK-K2(转染质粒K2)组、SK-HK(转染质粒HK)组。提取各组细胞总RNA进行RT-PCR检测Kir6.2基因的表达。WestrenBlot检测Kir6.2蛋白的相对量。
3.KATP通道电流的变化采用膜片钳全细胞记录方法记录各组细胞KATP通道电流。高阻封接后,首先用细胞外液进行灌流,再用10mmol/l 2-脱氧-D-葡萄糖(2-deoxy-D-glucose,DOG)灌流液灌流10分钟,测量各组的电流,最后换成100uM格列本脲(glybenclimade,Gly)灌流液灌流10分钟,再次测量电流,两次电流相减即可得格列本脲敏感的KATP电流。采用斜坡电压刺激,得到各组的I-V曲线。。
4.shRNA沉默SK-Hep1细胞Kir6.2基因后对于细胞生物学特性的影响流式细胞仪检测各组细胞周期。荧光探针Fluo4-AM孵育各组细胞,流式细胞仪检测荧光强度,根据公式计算细胞内钙离子浓度。MTT法检测各组细胞增殖情况,Transwell小室法检测各组细胞体外侵袭能力。
结果:
1.酶切和测序鉴定显示shRNA构建成功。
2.转染、筛选、单克隆扩增RT-PCR结果显示SK-K1组、SK-K2组Kir6.2 mRNA的表达与SK组、SK-HK组比较均明显降低。SK组、SK-HK组Kir6.2蛋白的量与SK-K1组、SK-K2组比较均明显增多。
3.KATP通道电流的记录SK组、SK-HK组、SK-K1组、SK-K2组的KATP电流的分别为(n=7)(nA)1.80±0.673,1.87±0.541,5.16±1.011,3.89±1.287。SK-K1组、SK-K2组与SK组、SK-HK组比较电流明显增强(p<0.01),SK-K1组与SK-K2组比较电流明显增强(p<0.05)。
4.细胞生物学特性的变化SK组、SK-HK组与SK-K1、SK-K2组细胞周期相比,G0-G1期细胞明显增加(p<0.05),S期细胞减少显著(p<0.05)。SK-K1和SK-K2组荧光强度明显弱于SK组(p<0.05),SK-K1和SK-K2组钙离子浓度远远低于SK组(p<0.05)。Transwell小室法显示SK组、SK-HK组、SK-K1组、SK-K2组细胞的增殖分数分别为0.7523±0.0681,0.7351±0.0589,0.2494±0.0485,0.2261±0.0298。SK-K1组、SK-K2组与SK组比较增殖分数明显降低(p<0.05)。Transwell小室法结果显示SK-K1组、SK-K2组、SK-HK组细胞的穿膜数分别为47.0±7.1,40.7±8.0,14.3±5.1,15.0±3.7。SK-K1组、SK-K2组与SK组比较穿膜细胞明显减少(p<0.05),SK-HK组穿膜细胞明显多于K1组和K2组(p<0.05)。
结论:
RT-PCR、Western Blot证实shRNA沉默了SK-Hep1 Kir6.2基因。膜片钳实验证明了干扰后KATP通道电流明显增强,通道开放时K+外流增加。SK-K1、SK-K2组细胞周期G0-G1期细胞明显减少,S期细胞增加显著。SK-K1、SK-K2组荧光强度、细胞内钙离子浓度远远低于对照组。SK-K1、SK-K2组细胞增殖受到抑制,穿膜细胞数明显减少。shRNA沉默SK-Hep1细胞Kir6.2基因影响了细胞生物学特性。
Ion channels are fundamental to the life.The relationship between ion channels and cancer is now gradually attracting the interest,To deeply understanf the effect of ion chanels on the tumor development and metastasis will be helpful not only to completely elucidate the cancer mechanism but also to provide new targets and biological intervention methods for the cancer prevention and ttreatment.Potassium channels have many kinds and are the more complicated ion channel,they play the key role on the signal transduction of the excitable cells and non-excitable cells,meanwhile take part in the regulation of tumour cell proliferation and differentiation.The relationship between the potassium channels and tumour cell proliferation has been the new hot spot of the tumour fundamental research.ATP-sensitive potassium channels couple membrane excitability to energy metabolism.Many studies in the cell biology and pharmacology have proved that potassium channels have effect on the tumour cell proliferation and survival. RNAi technology can not only discover the gene change bot also be a method to treat cancer.ShRNA (short hairpin RNA) can stably silence target gene.Human primary hepatocellular carcinoma (HCC) is one of the most common maligent tumour in our country,the tendency of HCC incidence is gradually increasing.Though great progress in diagnosis and treatment of HCC has been made over the past decades,the lower survival rate and poorer prognosis remains in patients with HCC.Basic research should be strengthened
Objective: To construct the plasmid vector expresses shRNA.The recombinant plasmid was transfectd to SK-Hep1 cell strains, KATP currents were detected by patch clamp. The characteristic of cell biology was investigated.
Methods
1.To construct the plasmid-mediated shRNA Fragment inserting the plasmid was obtained from the complementary oligonucleotide annealing was linked to the linearization empty pGenesil-3 zymased by ,The recombinant plasmid was transformed into the competence germ DH5a,The germ was cultured by using LB medium to extract the plasmid which was incisived and sequenced.
2.Culture,passage,transfection,screen of the SK-Hep1 cell strains SK-Hep1 was continuously cultured in use of DMEM with 10% FBS(fetal bovine serum),passage was done when the cell density reached 80-90%.Transfection was done with the help of liposome 2000.
3. I-V curves was drawed.4.Effect of shRNA silencing Kir6.2 gene on the characteristic of cell biology Cell cycle was detected with flow cytometry. probe incubated cells in each group .Fluorescent density of intracellular Ca2+ concentration was achieved. Cell proliferation was detectd with MTT assay. The cell invasive ability in vivo was obtained by using Transwell cabin.
Results
1.Identification by incision enzyme and sequencing Report showed the recombinant plasmid expressing shRNA was successful.
2.Transfection,screening,amplification RT-PCR showed that expression of Kir6.2 gene in SK-K1 and SK-K2 groups remarkably decreased compared with that in SK and SK-HK groups,Kir6.2 protein in SK and SK-HK groups was more than that in SK-K1 and SK-K2 groups.
3.Current of KATP channels in each group The KATP current in SK, SK-HK, SK-K1, SK-K2 groups (nA) was 1.80±0.673,1.87±0.541,5.16±1.011,3.89±1.287, respectively. Compared the curren in K1and K2 groups with that in SK group, there was significantly statistical. the current in HK group was much lower than that in K1 and K2 groups. I-V curves in K1 and K2 groups were close to the vertical axis.
4.Change of the characteristic of cell biology Cells in G0-G1 of SK, SK-HK groups were more than that in SK-K1, SK-K2 groups.Cells in S phase of SK-K1, SK-K2 groups were more than that in SK, SK-HK groups. Calcium concentration SK-K1, SK-K2 groupswas much lower than that in SK group. Calcium concentration in HK groups was remarkably higher than that in SK-K1, SK-K2 groups Underexpression of kir6.2 reduced significantly cell proliferation and invasion..
Conclusion
RT-PCR and Western Blot identified that shRNA successfully silenced Kir6.2 gene.Current of KATP channels in SK-K1, SK-K2 groups enchanced more. The characteristic of cell biology in SK-K1, SK-K2 groups was affected.
目的:构建shRNA质粒表达载体。脂质体法转染肝癌SK-Hep1细胞株,G418筛选,挑选荧光单克隆、扩增。RT-PCR、Western Blot检测基因表达情况。膜片钳检测KATP通道电流。研究细胞生物学特性改变情况。
方法:
1.构建shRNA质粒表达载体根据shRNA设计原则,设计两条干扰序列1、2和一条非特异性阴性对照序列。shRNA的表达插入的片段由互补的寡核苷酸链退火生成,和经BamH I和Hind III双酶切的线性化空载体pGenesil-3连接,得到三种质粒K1(携带干扰序列1)、K2(携带干扰序列2)、HK(携带非特异性阴性对照序列),转化感受态细菌DH5a,培养后提取质粒。酶切、测序。
2.SK-Hep1细胞的培养、传代、转染、筛选SK-Hep1细胞复苏后,在含有10% FBS的DMEM培养液中连续培养。细胞生长到80-90%运用0.25%胰蛋白酶溶液消化、传代。利用脂质体法转染构建的质粒,经G418进行筛选后,挑选荧光单克隆进行扩增。实验分4组:SK (空白对照)组、SK-K1(转染质粒K1)组、SK-K2(转染质粒K2)组、SK-HK(转染质粒HK)组。提取各组细胞总RNA进行RT-PCR检测Kir6.2基因的表达。WestrenBlot检测Kir6.2蛋白的相对量。
3.KATP通道电流的变化采用膜片钳全细胞记录方法记录各组细胞KATP通道电流。高阻封接后,首先用细胞外液进行灌流,再用10mmol/l 2-脱氧-D-葡萄糖(2-deoxy-D-glucose,DOG)灌流液灌流10分钟,测量各组的电流,最后换成100uM格列本脲(glybenclimade,Gly)灌流液灌流10分钟,再次测量电流,两次电流相减即可得格列本脲敏感的KATP电流。采用斜坡电压刺激,得到各组的I-V曲线。。
4.shRNA沉默SK-Hep1细胞Kir6.2基因后对于细胞生物学特性的影响流式细胞仪检测各组细胞周期。荧光探针Fluo4-AM孵育各组细胞,流式细胞仪检测荧光强度,根据公式计算细胞内钙离子浓度。MTT法检测各组细胞增殖情况,Transwell小室法检测各组细胞体外侵袭能力。
结果:
1.酶切和测序鉴定显示shRNA构建成功。
2.转染、筛选、单克隆扩增RT-PCR结果显示SK-K1组、SK-K2组Kir6.2 mRNA的表达与SK组、SK-HK组比较均明显降低。SK组、SK-HK组Kir6.2蛋白的量与SK-K1组、SK-K2组比较均明显增多。
3.KATP通道电流的记录SK组、SK-HK组、SK-K1组、SK-K2组的KATP电流的分别为(n=7)(nA)1.80±0.673,1.87±0.541,5.16±1.011,3.89±1.287。SK-K1组、SK-K2组与SK组、SK-HK组比较电流明显增强(p<0.01),SK-K1组与SK-K2组比较电流明显增强(p<0.05)。
4.细胞生物学特性的变化SK组、SK-HK组与SK-K1、SK-K2组细胞周期相比,G0-G1期细胞明显增加(p<0.05),S期细胞减少显著(p<0.05)。SK-K1和SK-K2组荧光强度明显弱于SK组(p<0.05),SK-K1和SK-K2组钙离子浓度远远低于SK组(p<0.05)。Transwell小室法显示SK组、SK-HK组、SK-K1组、SK-K2组细胞的增殖分数分别为0.7523±0.0681,0.7351±0.0589,0.2494±0.0485,0.2261±0.0298。SK-K1组、SK-K2组与SK组比较增殖分数明显降低(p<0.05)。Transwell小室法结果显示SK-K1组、SK-K2组、SK-HK组细胞的穿膜数分别为47.0±7.1,40.7±8.0,14.3±5.1,15.0±3.7。SK-K1组、SK-K2组与SK组比较穿膜细胞明显减少(p<0.05),SK-HK组穿膜细胞明显多于K1组和K2组(p<0.05)。
结论:
RT-PCR、Western Blot证实shRNA沉默了SK-Hep1 Kir6.2基因。膜片钳实验证明了干扰后KATP通道电流明显增强,通道开放时K+外流增加。SK-K1、SK-K2组细胞周期G0-G1期细胞明显减少,S期细胞增加显著。SK-K1、SK-K2组荧光强度、细胞内钙离子浓度远远低于对照组。SK-K1、SK-K2组细胞增殖受到抑制,穿膜细胞数明显减少。shRNA沉默SK-Hep1细胞Kir6.2基因影响了细胞生物学特性。
Ion channels are fundamental to the life.The relationship between ion channels and cancer is now gradually attracting the interest,To deeply understanf the effect of ion chanels on the tumor development and metastasis will be helpful not only to completely elucidate the cancer mechanism but also to provide new targets and biological intervention methods for the cancer prevention and ttreatment.Potassium channels have many kinds and are the more complicated ion channel,they play the key role on the signal transduction of the excitable cells and non-excitable cells,meanwhile take part in the regulation of tumour cell proliferation and differentiation.The relationship between the potassium channels and tumour cell proliferation has been the new hot spot of the tumour fundamental research.ATP-sensitive potassium channels couple membrane excitability to energy metabolism.Many studies in the cell biology and pharmacology have proved that potassium channels have effect on the tumour cell proliferation and survival. RNAi technology can not only discover the gene change bot also be a method to treat cancer.ShRNA (short hairpin RNA) can stably silence target gene.Human primary hepatocellular carcinoma (HCC) is one of the most common maligent tumour in our country,the tendency of HCC incidence is gradually increasing.Though great progress in diagnosis and treatment of HCC has been made over the past decades,the lower survival rate and poorer prognosis remains in patients with HCC.Basic research should be strengthened
Objective: To construct the plasmid vector expresses shRNA.The recombinant plasmid was transfectd to SK-Hep1 cell strains, KATP currents were detected by patch clamp. The characteristic of cell biology was investigated.
Methods
1.To construct the plasmid-mediated shRNA Fragment inserting the plasmid was obtained from the complementary oligonucleotide annealing was linked to the linearization empty pGenesil-3 zymased by ,The recombinant plasmid was transformed into the competence germ DH5a,The germ was cultured by using LB medium to extract the plasmid which was incisived and sequenced.
2.Culture,passage,transfection,screen of the SK-Hep1 cell strains SK-Hep1 was continuously cultured in use of DMEM with 10% FBS(fetal bovine serum),passage was done when the cell density reached 80-90%.Transfection was done with the help of liposome 2000.
3. I-V curves was drawed.4.Effect of shRNA silencing Kir6.2 gene on the characteristic of cell biology Cell cycle was detected with flow cytometry. probe incubated cells in each group .Fluorescent density of intracellular Ca2+ concentration was achieved. Cell proliferation was detectd with MTT assay. The cell invasive ability in vivo was obtained by using Transwell cabin.
Results
1.Identification by incision enzyme and sequencing Report showed the recombinant plasmid expressing shRNA was successful.
2.Transfection,screening,amplification RT-PCR showed that expression of Kir6.2 gene in SK-K1 and SK-K2 groups remarkably decreased compared with that in SK and SK-HK groups,Kir6.2 protein in SK and SK-HK groups was more than that in SK-K1 and SK-K2 groups.
3.Current of KATP channels in each group The KATP current in SK, SK-HK, SK-K1, SK-K2 groups (nA) was 1.80±0.673,1.87±0.541,5.16±1.011,3.89±1.287, respectively. Compared the curren in K1and K2 groups with that in SK group, there was significantly statistical. the current in HK group was much lower than that in K1 and K2 groups. I-V curves in K1 and K2 groups were close to the vertical axis.
4.Change of the characteristic of cell biology Cells in G0-G1 of SK, SK-HK groups were more than that in SK-K1, SK-K2 groups.Cells in S phase of SK-K1, SK-K2 groups were more than that in SK, SK-HK groups. Calcium concentration SK-K1, SK-K2 groupswas much lower than that in SK group. Calcium concentration in HK groups was remarkably higher than that in SK-K1, SK-K2 groups Underexpression of kir6.2 reduced significantly cell proliferation and invasion..
Conclusion
RT-PCR and Western Blot identified that shRNA successfully silenced Kir6.2 gene.Current of KATP channels in SK-K1, SK-K2 groups enchanced more. The characteristic of cell biology in SK-K1, SK-K2 groups was affected.
引文
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6. C.P. Paul, P.D. Good, I. Winer, D.R. Engelke, Effective expression of small interfering RNA in human cells, Nat. Biotechnol. 2002,20(5): 505–508.
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23. Claudia Weber, Fernanda Mello de Queiroz, Bryan R. Downie, et al. Silencing the activity and proliferative properties of the human EagI Potassium Channel by RNA Interference. 2006,281(19):13030-13037
24. Jie Zhao , Xiao-Li Wei , Yong-Sheng Jia,et al. Silencing of herg gene by shRNA inhibits SH-SY5Y cell growth in vitro and in vivo. European Journal of Pharmacology , 2008,579 : 50–57
1. Mittal V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet 2004;5:355–365.
2. Sioud M. On the delivery of small interfering RNAs into mammalian cells. Expert Opin Drug Deliv 2005;2:639–651.
3. Ryther RC, Flynt AS, Phillips 3rd JA, Patton JG. siRNA therapeutics: big potential from small RNAs. Gene Ther 2005;12:5–11.
4. Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference. Nature 2004;431:371–378.
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6. C.P. Paul, P.D. Good, I. Winer, D.R. Engelke, Effective expression of small interfering RNA in human cells, Nat. Biotechnol. 2002,20(5): 505–508.
7. N.S. Lee, T. Dohjima, G. Bauer, H. Li, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells, Nat. Biotechnol. 2002,20(5):500–505.
8. Cornelis S,Vandenbranden M,Ruysschaert J M.Role of intracellular cationic liposome-DNA complex dissoiation in transfection mediated cationic lipids.DNA Cell Biol.2002,21(2):91-97.
9. Dalby B,Cates S,Harris A,et al.Advanced transfection with Lipofectamine2000 regent:primary neurons,siRNA and high-throughput applications.Methods, 2004, 33(2):95-103.
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12. Ichiki Y, Kitamura K, Kangawa K,et al. Distribution and charac-terization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Letters, 1994,328(1):6-10
13. Wichterman KA, Baue AE, Chaudry IH. Sepsis and septic shock—A review of laboratory models and a proposal. J Surg Res,1980,29:189-201.
14. Abbort RE,Schachter D. Regional differentiation in rat aorta: L-arginine metabolism and cGMP content in vitro. Am J Physiol,1994,266:H2287-2295.
15. Noma, A. ATP-regulated K channels in cardic muscle. Nature. 1983 (305):147-155.
16. eino,S. ATP-sensitive potassium channels: A model of Heteromultimeric potassium channel/receptor assemblies. Annual Review of Physiology. Annu Rev Physiol. 1999;61:337-62.
17. YokoshikiH, SunagawaM, SekiT, eta.l ATP-sensitive K channels in pancreatic, cardiac and vascular smooth muscle cells[J]. Am J Physio,l 1998, 274(43): C25-C37.
18. CuongDV, Kim N, JooH, et a.l Subunit composition of ATP-sensitive potassium channels in mitochondria of rat hearts[J]. Mitochondrion, 2005, 5(2): 121-133.
19. Harmeet Malhi, Adil N. Irani, Pankaj Rajvanshi . et al. KATP Channels Regulate Mitogenically Induced Proliferation in Primary Rat Hepatocytes and Human Liver Cell Lines. Joural of Biologyical Chemistry..2000, 275(34):26050-26057.
20. Sebolt-Leopold, J. S., Dudley, D. T., Herrera, Leopold,. et al. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat Med. 1999 ,5(7):810-816.
21. (Paolo Tammaro and Frances Ashcroft) The Kir6.2-F333I mutation differentially modulates KATP channels composed of SUR1 or SUR2 subunits. J Physiol .2007,581(3) pp 1259–1269
22.万军,吴钢,黄从新等。ATP敏感性钾通道P266T突变对离子通道特性的影响。中国病理生理杂志。2007,23(1):18-21。
23. Claudia Weber, Fernanda Mello de Queiroz, Bryan R. Downie, et al. Silencing the activity and proliferative properties of the human EagI Potassium Channel by RNA Interference. 2006,281(19):13030-13037
24. Jie Zhao , Xiao-Li Wei , Yong-Sheng Jia,et al. Silencing of herg gene by shRNA inhibits SH-SY5Y cell growth in vitro and in vivo. European Journal of Pharmacology , 2008,579 : 50–57
1. Mittal V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet 2004;5:355–365.
2. Sioud M. On the delivery of small interfering RNAs into mammalian cells. Expert Opin Drug Deliv 2005;2:639–651.
3. Ryther RC, Flynt AS, Phillips 3rd JA, Patton JG. siRNA therapeutics: big potential from small RNAs. Gene Ther 2005;12:5–11.
4. Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference. Nature 2004;431:371–378.
5. Dorsett Y, Tuschl T. siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 2004;3:318–329.
6. Caplen NJ, Mousses S. Short interfering RNA (siRNA)-mediated RNA interference (RNAi) in human cells. Ann NY Acad Sci 2003;1002:56–62.
7. Fiona McLaughlin, Paul Finn and Nicholas B. La Thangue. The cell cycle, chromatin and cancer: mechanism-based therapeutics come of age. DDT, 2003,8( 17):793-802.
8. WondergemR,CreganM,Strickler L,Miller R.Membrane potassium channel and human bladder trmer cells:ⅡGrowth properties [J].Membr Biol,1998,161(3):257-262
9. KlimatcgevaE,wonderlin W F.An ATP-sensitive K+current that regulates progression through early G1phase of the cell cycle in MCF- 7 human breast cells[J]. J MemberBiol,1999,171(10):35-46
10. Dubois,J.M,and B.Riuzaire-Dubois.Role of potassium xhannels in mitogenesis[J].Prog Biophys Mol Biol,1993,59:1-21
11. Lewis,R.S,and M.D.Cahalan.Ion channels and signal transduction in lymphocytes[J].Annu Rev Physiol,1990,52:415-430
12. Wonderlin WF,strobl Js.Patassium,proliferation and G1 progression[J].J member Biol,1996,154(2):91-107
13. Chittajallu R,chen Y,Wang H,Yuan X.Regulation of Kvl subunit expression in oligodendrocyte progenitor cells and their role in G/S phase progression of the cellcycle[J].Proc Natl Acad Sci SA,2002,19:99(4):2350-235
14. Day ML, Pickering SJ, Johnson MH, et,al .Cell-cycle control of a large-conductance K+ channel in mouse early embryos.Nature. 1993,365(6446):560-562
15. ]Wonderlin WF, Strobl JS. Potassium channels, proliferation and G1 progression.J Membr Biol. 1996,154(2):91-107.
16.戴天阳,周清华.人非小细胞肺癌细胞膜钾离子通道特性的研究[J].Chin J lung cancer2001,4(4):281-286
17.王立平,邵国光.4氨基吡啶对小细胞肺癌中电压激活性钾离子电流和细胞增殖的影响[J].中华肿瘤杂志,2002,24(3):230-233.
18. Santella L, De Riso L, Gragnaniello G, et al.Separate activation of the cytoplasmic and nuclear calcium pools in maturing starfish oocytes.Biochem Biophys Res Commun. 1998 Nov 9;252(1):1-4.
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