可溶性重组脂联素融合蛋白（GST-gAD）在大肠杆菌中的优化表达及其生物活性鉴定
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摘要
目的:探讨可溶性重组脂联素球状结构域融合蛋白(GST-gAD)在大肠杆菌中的表达及其抑制人乳腺癌细胞MDA-MB-231增殖的生物学活性。
方法:①用CaCl2法制备感受态细菌JM109,质粒pGEX-KG-gAD转化大肠杆菌JM109,空载质粒pGEX-KG作为内对照同时转化JM109,筛选建株;②以正交设计软件正交设计助手Ⅱ选择L9(34)正交设计表在三个水平,四个因素(IPTG浓度,诱导时间,诱导温度,细菌密度OD600)下确定优化表达的最佳组合条件;③亲和层析法纯化GST-gAD;④用MTT法检测融合蛋白GST-gAD对人乳腺癌细胞MDA-MB-231增殖的影响。
结果:①重组质粒pGEX-KG-gAD成功转化大肠杆菌JM109;②对GST-gAD诱导表达影响最大的因素为温度,最佳诱导表达组合条件为温度32℃,IPTG的浓度1.0 mmol/L,诱导前细菌的生长密度OD600 1.0,诱导表达时间1.5h,此条件下其表达量占菌体总蛋白的6 %左右;③获得纯化的分子量为42KD的GST-gAD;④GST-gAD浓度高于0.5μmol/L时对人乳腺癌细胞MDA-MB-231的增殖具有明显的抑制作用
结论:①通过正交设计确定了可溶性GST-gAD融合蛋白在大肠杆菌中表达的优化条件;②GST-gAD融合蛋白在原核表达系统中可以有效表达,并且具有较好的生物学活性。
Objective: To explore the best expressing condition of the recombinant GST-gAD( globular domain of human adiponection,gAD) in E.coli and study its bioactivity in inhibiting the proliferation of human breast cancer cell MDA-MB-231.
Methods:①Competent bacterium JM109 was prepared with CaCl2. The plasmid pGEX-KG-gAD and the empty plasmid pGEX-KG, as control, were transformed into E.coli host JM109;②The best expressing condition of GST-gAD was explored through orthogonal design which took L 9(34),including: growth temperature, IPTG concentration,culture density,incubation time as indices by orthogonal design assistantⅡ;③GST- gAD and GST were purified by GSH-agarose;④The effect of GST—gAD on proliferation of MDA-MB-231 cells were measured by MTT essay.
Results:①The pGEX-KG-gAD recombinant vector was transformed successfully into E.coli host JM109;②The expression level was highest in the conditions of OD600 value 1.0,1.0mmol/L IPTG, 32℃and 1.5 hours of induction time. The amount of the fusion protein was about 6% of the total bacterial protein;③The molecular weight of GST-gAD was 42KD;④The GST-gAD can obviously inhibit the proliferation of MDA-MB-231 when its concentration was more than 0.5μmol/L. Conclusion:①The best expressing condition of the recombinant GST-gAD in Ecoli was determined through orthogonal design;②The GST-gAD fusion protein was efficiently expressed in E.coli and showed natural biological activities.
方法:①用CaCl2法制备感受态细菌JM109,质粒pGEX-KG-gAD转化大肠杆菌JM109,空载质粒pGEX-KG作为内对照同时转化JM109,筛选建株;②以正交设计软件正交设计助手Ⅱ选择L9(34)正交设计表在三个水平,四个因素(IPTG浓度,诱导时间,诱导温度,细菌密度OD600)下确定优化表达的最佳组合条件;③亲和层析法纯化GST-gAD;④用MTT法检测融合蛋白GST-gAD对人乳腺癌细胞MDA-MB-231增殖的影响。
结果:①重组质粒pGEX-KG-gAD成功转化大肠杆菌JM109;②对GST-gAD诱导表达影响最大的因素为温度,最佳诱导表达组合条件为温度32℃,IPTG的浓度1.0 mmol/L,诱导前细菌的生长密度OD600 1.0,诱导表达时间1.5h,此条件下其表达量占菌体总蛋白的6 %左右;③获得纯化的分子量为42KD的GST-gAD;④GST-gAD浓度高于0.5μmol/L时对人乳腺癌细胞MDA-MB-231的增殖具有明显的抑制作用
结论:①通过正交设计确定了可溶性GST-gAD融合蛋白在大肠杆菌中表达的优化条件;②GST-gAD融合蛋白在原核表达系统中可以有效表达,并且具有较好的生物学活性。
Objective: To explore the best expressing condition of the recombinant GST-gAD( globular domain of human adiponection,gAD) in E.coli and study its bioactivity in inhibiting the proliferation of human breast cancer cell MDA-MB-231.
Methods:①Competent bacterium JM109 was prepared with CaCl2. The plasmid pGEX-KG-gAD and the empty plasmid pGEX-KG, as control, were transformed into E.coli host JM109;②The best expressing condition of GST-gAD was explored through orthogonal design which took L 9(34),including: growth temperature, IPTG concentration,culture density,incubation time as indices by orthogonal design assistantⅡ;③GST- gAD and GST were purified by GSH-agarose;④The effect of GST—gAD on proliferation of MDA-MB-231 cells were measured by MTT essay.
Results:①The pGEX-KG-gAD recombinant vector was transformed successfully into E.coli host JM109;②The expression level was highest in the conditions of OD600 value 1.0,1.0mmol/L IPTG, 32℃and 1.5 hours of induction time. The amount of the fusion protein was about 6% of the total bacterial protein;③The molecular weight of GST-gAD was 42KD;④The GST-gAD can obviously inhibit the proliferation of MDA-MB-231 when its concentration was more than 0.5μmol/L. Conclusion:①The best expressing condition of the recombinant GST-gAD in Ecoli was determined through orthogonal design;②The GST-gAD fusion protein was efficiently expressed in E.coli and showed natural biological activities.
引文
[1]Chandran M, Phillips SA, Ciaraldi T, et al. Adiponectin: more than just another fat cell hormone? Diabetes Care. 2003,26(8):2442-2450.
[2]Housa D, Housova J, Vernerova Z, et al. Adipocytokines and cancer. Physiol Res, 2006,55(3): 233-244.
[3]Petridou E, Mantzoros C, Dessypris N, et al. Plasma adiponectin concentrations in relation to endometrial cancer: a case-control study in Greece. J Clin Endocrinol Metab, 2003,88(3): 993-997.
[4]Mantzoros C, Petridou E, Dessypris N, et al. Adiponectin and breast cancer risk. J Clin Endocrinol Metab,2004, 89(3): 1102– 1107.
[5]Petridou E, Mantzoros CS, Dessypris N,et al. Adiponectin in relation to childhood myeloblastic leukaemia. Br J Cancer. 2006 ,94(1):156-160.
[6]Wei EK, Giovannucci E, Fuchs CS, et al. Plasma adiponectin levels and the risk of colorectal cancer in men. J Natl Cancer Inst,2005, 97(22):1688-1694.
[7]Ishikawa M, Kitayama J, Kazama S,et al. Plasma adiponectin and gastric cancer. Clin Cancer Res, 2005 ,11(2 Pt 1):466-472.
[8]Goktas S, Yilmaz MI, Caglar K,et al. Prostate cancer and adiponectin. Urology,2005 ,65(6):1168-1172.
[9]Kadowaki T, Yamauchi T. Adiponectin and adiponectin receptors. Endocr Rev ,2005,26(3): 439–451.
[10]Tsao TS, Murrey HE, Hug C,et al. Oligomerization state-dependent activation of NF-kappa B signaling pathway by adipocyte complement-related protein of 30 kDa (Acrp30). J Biol Chem, 2002, 277(33):29359-29362.
[11]Goldstein BJ, Scalia R. Adiponectin: A novel adipokine linking adipocytes and vascular function. J Clin Endocrinol Metab ,2004,89(6): 2563– 2568.
[12]Tomas E,Tsao TS,Saha AK,et al. Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain:Acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation.Proc Natl Acad Sci USA,2002,99(25): 16309-16313.
[13]Motoshima H,Wu XD,Mah adev K,et al. Adiponectin suppresses proliferation and superoxide generation and enhances eNOS activity in endothelial cells treated with oxidized LDL.Biochem Biophys Res Commun,2004,315(2):264-271.
[14]贺石林 陈修编著. 医学科研方法导论〔M〕北京:人民卫生出版社 1998,239-273.
[15]Wolk A, Gridley G, Svensson M,et al. A prospective study of obesity and cancer risk (Sweden). Cancer Causes Control ,2001,12(1): 13–21.
[16]Chen DC, Chung YF, Yeh YT, et al. Serum adiponectin and leptin levels in Taiwanese breast cancer patients. Cancer Lett, 2005,237(1):109-114.
[17]Brakenhielm E, Veitonmaki N, Cao R,et al. Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis.Proc Natl Acad Sci U S A.,2004 ,101(8):2476-2481.
[18]Wang Y,Lam J B,Lam K S,et a1.Adiponectin modulates the glycogen synthase kinase-3beta / beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice.Cancer Res,2006,66(23):11462—11470.
[19]Philp AJ, Campbell IG, Leet C,et al. The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors. Cancer Res.,2001,61(20):7426-7429.
[20]Yokota T, Oritani K, Takahashi I,et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood ,2000,96(5) :1723–1732.
[21]Arditi JD, Venihaki M, Karalis KP,et al. Antiproliferative effect of adiponectin on MCF7 breast cancer cells: a potential hormonal link between obesity and cancer. Horm Metab Res., 2007 ,39(1):9-13.
[22]Kang J H,Lee Y Y,Yu B Y,et a1.Adiponectin induces growth arrest and apoptosis of MDA—MB-231 breast cancer cell. Arch Pharm Res,2005,28(11):1263- 1269.
[23]王胜芬,韩佩芬. 脂联素在原核系统中的表达及其抑瘤活性的研究.中国知网优秀硕博士论文库,2005
[24]Smith D B, Johnson K S. Single-step purification of polypeptides expressed in Escherichia coli as fusion with glutathione S-transferase. Gene,1988,67(1):31-40.
[25]孙汶生,曹英林,马春红. 基因工程学[M].北京: 科学出版社.2002,95-105.
[26]萨姆布鲁克,D.W.拉塞尔,著. 黄培堂.译.分子克隆实验指南(M) .第 3 版.北京科学出版社.2002,1217-1270.
[1]Schere PE,William S,Foglinano M,et al. A novel serum protein similar to C1q,produced exclucively in adipocytes [J]. J Biol chem,1995,270(45):26746-26749.
[2]Arita Y,Kihara S,Ouchi N.Paradoxiael decrease of an adipose—specific protein,adiponectin,in obesity [J]. Biochem Biophys Res Cornmun,1999,257(1):79-83.
[3] Yamauchi T,Kamon J,Minokoshi Y,et al..Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[J]. Nat Med,2002,8(11):1288-1295.
[4] Brakenhielm E,Veitonmaki N,Cao R,et al. Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis [J]. Proc Natl Acad Sci USA,2004,101(8):2476-2481.
[5] Kobayashi H,Ouchi N,Kihara S,et al . Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin [J]. Circ Res,2004,94(4):27-31.
[6]Trujillo ME, Scherer PE. Adiponectin—journey from an adipocyte secretor protein to biomarker of the metabolic syndrome [J]. Intern Med ,2005,257(2): 167–175.
[7] Qi L, Rimm E, Liu S, et al. Dietary glycemic index, glycemic load, cereal fiber, and plasma adiponectin concentration in diabetic men [J]. Diab Care,2005, 28(5): 1022– 1028.
[8] Hug C,Wang J,Ahmad NS ,et al. T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin [J]. Proc Natl Acad Sci USA,2004,101(28):10308-10313.
[9]Petridou E,Mantzoros C,Dessypris N,et al. Plasma adiponectin concentrations in relation to endometrial cancer:a case–control study in Greece [J]. J Clin Endocrino Meta,2003,88(3):993-997.
[10] Mantzoros C,Petridou E,Dessypris N,et al. Adiponectin and breast cancer risk [J]. J Clin Endocrinol Metab,2004,89(3):1102-1107.
[11] Wei EK,Giovannucci E,Fuchs CS,et al . Plasma adiponectin levels and the risk of colorectal cancer in men [J]. J Natl Cancer Inst,2005,97(22):1688-1694.
[12] Ishikawa M,Kitayama J,Kazama S,et al. Plasma adiponectin and gastric cancer [J]. Clin Cancer Res,2005,11(2):466-472.
[13] Goktas S,Yilmaz MI,Caglar K,et al. Rostate cancer and adiponectin [J]. Urology,2005,65(6):1168-1172.
[14] Yokota T,Oritani K,Takahashi I,et al. Adiponectin,a new member of the family of soluble defense collagens,negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages [J]. Blood,2000,96(5):1723-1732.
[15] Wolk A,Gridley G,Svensson M,et al. A prospective study of obesity and cancer risk [J]. Cancer Causes Control,2001,12(1):13-21.
[16] Dieudonne MN,Bussiere M,Dos Santos E,et al. adiponectin mediates antiproliferative and apoptotic responses in humanMCF7 breast cancer cells [J]. Biochem Biophys Res Commun, 2006,345(1):271-279.
[17] Arditi JD,Venihaki M,Karalis KP,et al. Antiproliferative effect of adiponectin on MCF7 breast cancer cells:A potential hormonal link between obesity and cancer [J]. Horm Metab Res, 2007,39(1):9-13.
[18] Chen DC,Chung YF,Yeh YT,et al . Serum adiponectin and leptin levels in Taiwanese breast cancer patients [J]. Cancer Lett,2006,237(1):109-114.
[19]Petridou E,Mantzoros C,Dessypris N,et al. Plasma adiponectin concentrations in relation to endometrial cancer:a case–control study in Greece [J]. J Clin Endocrinol Metab,2003,88(3):993-997.
[20] Mistry T,Digby JE,Chen J,et al. The regulation of adiponectin receptors in human prostate cancer cell lines[J]. Biochem Biophys Res Commun,2006,348(3):832-838.
[21] Ouchi N,Kihara S,Arita Y,et al.Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin [J]. Circulation,1999,100(25):2473- 2476.
[22] Wang Y,Lam KS,Xu JY,et al . Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner [J]. J Biol Chem,2005,280(18):18341-18347.
[23] Miyazaki T,Bub JD,Uzuki M,et al. Adiponectin activates c-Jun NH2-terminal kinase and inhibits signal transducer and activator of transcription 3 [J]. Biochem Biophys Res Commun, 2005,333(1):79-87
[24] Luo Z,Saha AK,Xiang X,et al. AMPK,the metabolic syndrome and cancer [J]. Trends Pharmacol Sci,2005,26(2):69- 76.
[2]Housa D, Housova J, Vernerova Z, et al. Adipocytokines and cancer. Physiol Res, 2006,55(3): 233-244.
[3]Petridou E, Mantzoros C, Dessypris N, et al. Plasma adiponectin concentrations in relation to endometrial cancer: a case-control study in Greece. J Clin Endocrinol Metab, 2003,88(3): 993-997.
[4]Mantzoros C, Petridou E, Dessypris N, et al. Adiponectin and breast cancer risk. J Clin Endocrinol Metab,2004, 89(3): 1102– 1107.
[5]Petridou E, Mantzoros CS, Dessypris N,et al. Adiponectin in relation to childhood myeloblastic leukaemia. Br J Cancer. 2006 ,94(1):156-160.
[6]Wei EK, Giovannucci E, Fuchs CS, et al. Plasma adiponectin levels and the risk of colorectal cancer in men. J Natl Cancer Inst,2005, 97(22):1688-1694.
[7]Ishikawa M, Kitayama J, Kazama S,et al. Plasma adiponectin and gastric cancer. Clin Cancer Res, 2005 ,11(2 Pt 1):466-472.
[8]Goktas S, Yilmaz MI, Caglar K,et al. Prostate cancer and adiponectin. Urology,2005 ,65(6):1168-1172.
[9]Kadowaki T, Yamauchi T. Adiponectin and adiponectin receptors. Endocr Rev ,2005,26(3): 439–451.
[10]Tsao TS, Murrey HE, Hug C,et al. Oligomerization state-dependent activation of NF-kappa B signaling pathway by adipocyte complement-related protein of 30 kDa (Acrp30). J Biol Chem, 2002, 277(33):29359-29362.
[11]Goldstein BJ, Scalia R. Adiponectin: A novel adipokine linking adipocytes and vascular function. J Clin Endocrinol Metab ,2004,89(6): 2563– 2568.
[12]Tomas E,Tsao TS,Saha AK,et al. Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain:Acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation.Proc Natl Acad Sci USA,2002,99(25): 16309-16313.
[13]Motoshima H,Wu XD,Mah adev K,et al. Adiponectin suppresses proliferation and superoxide generation and enhances eNOS activity in endothelial cells treated with oxidized LDL.Biochem Biophys Res Commun,2004,315(2):264-271.
[14]贺石林 陈修编著. 医学科研方法导论〔M〕北京:人民卫生出版社 1998,239-273.
[15]Wolk A, Gridley G, Svensson M,et al. A prospective study of obesity and cancer risk (Sweden). Cancer Causes Control ,2001,12(1): 13–21.
[16]Chen DC, Chung YF, Yeh YT, et al. Serum adiponectin and leptin levels in Taiwanese breast cancer patients. Cancer Lett, 2005,237(1):109-114.
[17]Brakenhielm E, Veitonmaki N, Cao R,et al. Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis.Proc Natl Acad Sci U S A.,2004 ,101(8):2476-2481.
[18]Wang Y,Lam J B,Lam K S,et a1.Adiponectin modulates the glycogen synthase kinase-3beta / beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice.Cancer Res,2006,66(23):11462—11470.
[19]Philp AJ, Campbell IG, Leet C,et al. The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors. Cancer Res.,2001,61(20):7426-7429.
[20]Yokota T, Oritani K, Takahashi I,et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood ,2000,96(5) :1723–1732.
[21]Arditi JD, Venihaki M, Karalis KP,et al. Antiproliferative effect of adiponectin on MCF7 breast cancer cells: a potential hormonal link between obesity and cancer. Horm Metab Res., 2007 ,39(1):9-13.
[22]Kang J H,Lee Y Y,Yu B Y,et a1.Adiponectin induces growth arrest and apoptosis of MDA—MB-231 breast cancer cell. Arch Pharm Res,2005,28(11):1263- 1269.
[23]王胜芬,韩佩芬. 脂联素在原核系统中的表达及其抑瘤活性的研究.中国知网优秀硕博士论文库,2005
[24]Smith D B, Johnson K S. Single-step purification of polypeptides expressed in Escherichia coli as fusion with glutathione S-transferase. Gene,1988,67(1):31-40.
[25]孙汶生,曹英林,马春红. 基因工程学[M].北京: 科学出版社.2002,95-105.
[26]萨姆布鲁克,D.W.拉塞尔,著. 黄培堂.译.分子克隆实验指南(M) .第 3 版.北京科学出版社.2002,1217-1270.
[1]Schere PE,William S,Foglinano M,et al. A novel serum protein similar to C1q,produced exclucively in adipocytes [J]. J Biol chem,1995,270(45):26746-26749.
[2]Arita Y,Kihara S,Ouchi N.Paradoxiael decrease of an adipose—specific protein,adiponectin,in obesity [J]. Biochem Biophys Res Cornmun,1999,257(1):79-83.
[3] Yamauchi T,Kamon J,Minokoshi Y,et al..Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[J]. Nat Med,2002,8(11):1288-1295.
[4] Brakenhielm E,Veitonmaki N,Cao R,et al. Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis [J]. Proc Natl Acad Sci USA,2004,101(8):2476-2481.
[5] Kobayashi H,Ouchi N,Kihara S,et al . Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin [J]. Circ Res,2004,94(4):27-31.
[6]Trujillo ME, Scherer PE. Adiponectin—journey from an adipocyte secretor protein to biomarker of the metabolic syndrome [J]. Intern Med ,2005,257(2): 167–175.
[7] Qi L, Rimm E, Liu S, et al. Dietary glycemic index, glycemic load, cereal fiber, and plasma adiponectin concentration in diabetic men [J]. Diab Care,2005, 28(5): 1022– 1028.
[8] Hug C,Wang J,Ahmad NS ,et al. T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin [J]. Proc Natl Acad Sci USA,2004,101(28):10308-10313.
[9]Petridou E,Mantzoros C,Dessypris N,et al. Plasma adiponectin concentrations in relation to endometrial cancer:a case–control study in Greece [J]. J Clin Endocrino Meta,2003,88(3):993-997.
[10] Mantzoros C,Petridou E,Dessypris N,et al. Adiponectin and breast cancer risk [J]. J Clin Endocrinol Metab,2004,89(3):1102-1107.
[11] Wei EK,Giovannucci E,Fuchs CS,et al . Plasma adiponectin levels and the risk of colorectal cancer in men [J]. J Natl Cancer Inst,2005,97(22):1688-1694.
[12] Ishikawa M,Kitayama J,Kazama S,et al. Plasma adiponectin and gastric cancer [J]. Clin Cancer Res,2005,11(2):466-472.
[13] Goktas S,Yilmaz MI,Caglar K,et al. Rostate cancer and adiponectin [J]. Urology,2005,65(6):1168-1172.
[14] Yokota T,Oritani K,Takahashi I,et al. Adiponectin,a new member of the family of soluble defense collagens,negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages [J]. Blood,2000,96(5):1723-1732.
[15] Wolk A,Gridley G,Svensson M,et al. A prospective study of obesity and cancer risk [J]. Cancer Causes Control,2001,12(1):13-21.
[16] Dieudonne MN,Bussiere M,Dos Santos E,et al. adiponectin mediates antiproliferative and apoptotic responses in humanMCF7 breast cancer cells [J]. Biochem Biophys Res Commun, 2006,345(1):271-279.
[17] Arditi JD,Venihaki M,Karalis KP,et al. Antiproliferative effect of adiponectin on MCF7 breast cancer cells:A potential hormonal link between obesity and cancer [J]. Horm Metab Res, 2007,39(1):9-13.
[18] Chen DC,Chung YF,Yeh YT,et al . Serum adiponectin and leptin levels in Taiwanese breast cancer patients [J]. Cancer Lett,2006,237(1):109-114.
[19]Petridou E,Mantzoros C,Dessypris N,et al. Plasma adiponectin concentrations in relation to endometrial cancer:a case–control study in Greece [J]. J Clin Endocrinol Metab,2003,88(3):993-997.
[20] Mistry T,Digby JE,Chen J,et al. The regulation of adiponectin receptors in human prostate cancer cell lines[J]. Biochem Biophys Res Commun,2006,348(3):832-838.
[21] Ouchi N,Kihara S,Arita Y,et al.Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin [J]. Circulation,1999,100(25):2473- 2476.
[22] Wang Y,Lam KS,Xu JY,et al . Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner [J]. J Biol Chem,2005,280(18):18341-18347.
[23] Miyazaki T,Bub JD,Uzuki M,et al. Adiponectin activates c-Jun NH2-terminal kinase and inhibits signal transducer and activator of transcription 3 [J]. Biochem Biophys Res Commun, 2005,333(1):79-87
[24] Luo Z,Saha AK,Xiang X,et al. AMPK,the metabolic syndrome and cancer [J]. Trends Pharmacol Sci,2005,26(2):69- 76.