摘要
第一部分胰岛β细胞的脂性凋亡及其机制
目的：证实脂毒性诱导胰岛β细胞凋亡；探讨脂毒性诱导β细胞凋亡的途径。
方法：体外培养胰岛β细胞，随机化分为：
①正常对照组（BSA）：含0.5%BSA的DMEM培养基培养24小时。②棕榈酸组(PA)：加入0.4mmol/l的棕榈酸(含0.5%BSA)培养24小时。③油酸脂组（OA）：加入0.4mmol/l的油酸脂(含0.5%BSA)培养24小时。④Z-LEHD-FMK组（LEHD）：40uM Z-LEHD-FMK预孵2小时后，加入0.4mmol/l的棕榈酸(含0.5%BSA)培养24小时。⑤Z-IETD-FMK组（IETD）：40uM Z-IETD-FMK预孵2小时后，加入0.4mmol/l的棕榈酸(含0.5%BSA)培养24小时。⑥Z-LEHD-FMK联合Z-IETD-FMK （LEHD/IETD）组：40uM Z-LEHD-FMK及40uMZ-IETD-FMK联合预孵2小时后，加入0.4mmol/l的棕榈酸(含0.5%BSA)培养24小时。
采用Annexin V-Cy3凋亡试剂盒检测细胞凋亡，Western blotting检测Cleaved caspase-3、8、9蛋白的表达。
结果：与对照组及油酸脂组比较，棕榈酸组Cleaved caspase3的表达增加（p<0.05），凋亡细胞数目明显增加（p<0.01），且内源性凋亡途
径的Cleaved caspase8（p<0.05）及外源性凋亡途径的Cleaved caspase9表达也增加（p<0.01），而Cleaved caspase9的升高更显著。使用Caspase抑制剂后发现，Z-LEHD-FMK（Caspase9抑制剂）显著减少Cleavedcaspase3的表达（p<0.05），而Z-IETD-FMK（Caspase8抑制剂）对Cleaved caspase3的表达有减少趋势，但没有统计学意义。同时使用Z-LEHD-FMK（Caspase9抑制剂）及Z-IETD-FMK（Caspase8）不能完全抑制棕榈酸诱导的Cleaved caspase3的表达。
结论：脂毒性通过同时激活内源性及外源性凋亡途径导致胰岛β细胞凋亡，其中内源性凋亡途径可能起着主要作用。脂毒性诱导的Cleaved caspase3激活中可能还存在非Caspase8及Caspase9的诱导因素。
第二部分脂联素抗脂性凋亡及其机制
目的：证实脂联素的抗凋亡特性；初步探讨脂联素抗脂性凋亡的机制。
方法：体外培养胰岛β细胞，随机分为①正常对照组（BSA）：含0.5%BSA的DMEM培养基培养24小时。②棕榈酸组(PA)：加入0.4mmol/l的棕榈酸(含0.5%BSA)培养24小时。③脂联素组OA）：加入脂联素预孵2小时后，加入0.4mmol/l的棕榈酸(含0.5%BSA)培养24小时。
采用Annexin V-Cy3凋亡检测细胞凋亡情况， Western blotting检测Cleaved caspase-3、8、9及Bax、Bcl-2蛋白的表达。
结果：与单纯棕榈酸组比较，脂联素预孵后Cleaved caspase3的表达减少、胰岛β细胞凋亡数目减少（p<0.01）；Cleaved caspase9表达减少（p<0.05）；而Cleaved caspase8的表达有减少趋势，但没有统计学意义。脂联素预孵后，Bax表达减少， Bcl-2表达增加（p<0.05）。
结论：脂联素具有抗脂性凋亡的作用，脂联素对内源性及外源性凋亡途径均有抑制作用，但对内源性凋亡途径的抑制作用可能起着主要作用。脂联素通过调节Bax、Bcl-2的表达抑制内源性凋亡途径。
第三部分表达小鼠脂联素蛋白的重组慢病毒载体干预糖尿病
目的：构建表达小鼠脂联素的重组慢病毒载体；通过慢病毒载体提高血循环脂联素浓度；探讨提高血循环脂联素浓度对糖尿病的干预作用。
方法：构建表达小鼠全长脂联素蛋白的重组慢病毒载体（携带绿色荧光蛋白）。小剂量链脲菌素多次注射联合高脂饮食喂养建立2型糖尿病小鼠模型。随机分组:(1)正常对照组（Control）：普通饮食喂养6周。(2)2型糖尿病组：小鼠禁食16小时后腹腔注射链尿菌素（55mg/kg体重，每天一次，连续5天），继续高脂饮食喂养4周。以随机血糖浓度>16.7mmol/L为糖尿病造模成功标准。4周后成膜的糖尿病小鼠随机分为①生理盐水注射组(Saline):尾静脉注射生理盐水100ul，继续高脂饮食喂养2周。②脂联素干预组(Plenti-Acdc-EGFP):尾静脉注射表达小鼠脂联素的重组慢病毒载体100ul，病毒滴度为1x108ifu/ml，继续高脂饮食喂养2周。③空病毒载体注射组（Plenti-EGFP）：尾静脉注射空病毒载体100ul，继续高脂饮食喂养2周。
采用荧光显微镜观察肝脏绿色荧光表达情况；Western blotting检测肝脏脂联素蛋白的表达；Elisa法检测血循环中脂联素的水平；TUNEL法检测胰岛凋亡；免疫组化检测Cleaved caspase3、8、9的表达；化学法检测血脂水平。指血糖仪检测血糖水平。
结果：小剂量多次链脲菌素注射联合高脂饮食喂养法成功建立了2型糖尿病小鼠模型，其血脂、血糖水平明显高于正常对照组（p<0.01）。
慢病毒尾静脉注射14天后，肝组织冰冻切片，荧光显微镜下见大量绿色荧光表达于脂联素干预组及空病毒载体注射组，而正常对照组及生理盐水注射组未见绿色荧光表达。Western blotting显示仅脂联素干预组见脂联素蛋白表达。Elisa检测结果显示脂联素干预组血脂联素水平明显高于其他三组（p<0.01）,为48.03±3.79ug/ml，空病毒载体组与生理盐水组水平相似，但显著低于正常对照组（p<0.05）。
与正常对照组比较，生理盐水注射组及空病毒注射组体重下降、血糖、血脂水平升高（p<0.01）。生理盐水注射组及空病毒注射组两组间比较，没有统计学差异意义。
与生理盐水注射组及空病毒注射组比较，脂联素干预组血糖降低（p<0.05），甘油三脂水平也降低（p<0.01）。脂联素干预组胰岛β凋亡细胞数目明显减少（p<0.05），胰岛Cleaved caspase3、8、9的表达显著降低（p<0.01）。
结论：小剂量STZ联合高脂饮食喂养法能成功构建2型糖尿病小鼠模型；慢病毒载体可用于基因治疗提高脂联素水平；提高血循环脂联素水平可抗胰岛β细胞凋亡、改善糖脂代谢，干预糖尿病。
Part1Pancreatic β-cell lipoapoptosis and it’s mechanism
Objectives: To confirm that lipotoxicity induce pancreatic β-cellapoptosis and investigate it’s possible apoptotic pathways.
Methods: Pancreatic β cells were cultured in vitro, and divided intosix groups randomly:①Control group (BSA): Min6cells were cultured inDMEM medium containing0.5%BSA for24hours.②Palmitate group(PA): Min6cells were cultured in DMEM medium containing0.5%BSAand0.4mmol/l palmitate for24hours.③Oleate group(OA): Min6cellswere cultured in DMEM medium containing0.5%BSA and0.4mmol/loleate for24hours.④Z-LEHD-FMK(LEHD): Min6cells werepreincubated with40uM Z-LEHD-FMK for2hour, then cultured inDMEM medium containing0.5%BSA and0.4mmol/l palmitate for24hours.⑤Z-IETD-FMK(IETD): Min6cells were preincubated with40uMZ-IETD-FMK for2hour, then cultured in DMEM medium containing0.5%BSA and0.4mmol/l palmitate for24hours.⑥Z-LEHD-FMK andZ-IETD-FMK group(LEHD/IETD): Min6cells were preincubated withboth Z-IETD-FMK(40uM) and Z-LEHD-FMK (40uM) for2hour, then cultured in DMEM medium containing0.5%BSA and0.4mmol/lpalmitate for24hours.
Annexin V-of Cy3apoptosis detection kit was used for cell apoptosis,and western blotting for caspase-3,8,9expression.
Results: Compared with control group and oleate group, Cleavedcaspase3expression increased in palmitate group (p <0.05). The number ofapoptotic cells increased significantly in palmitate group (p <0.01).Cleaved caspase8(p <0.05) and cleaved caspase9(p <0.01) both increasedin palmitate group, with high level for cleaved caspase9. Z-LEHD-FMK(Caspase9inhibitor) significantly reduced cleaved caspase3expression (p<0.05), there was a trend for Z-IETD-FMK (Caspase8inhibitor) with nostatistical differences. Z-LEHD-FMK (Caspase9inhibitor) combiningZ-IETD-FMK (Caspase8) couldn’t completely inhibited cleaved caspase3expression.
Conclusion: Lipotoxicity induced pancreatic β cell apoptosis byactivating both intrinsic and extrinsic apoptotic pathways, while theintrinsic pathway may play a major rolein it. Cleaved Caspase3activationinduced by lipotoxicity may also be partly independent of Caspase8andCaspase9expression.
Part2Inhibition of lipoapoptosis by adiponectin and it’smechanism
Objectives: To confirm that adiponectin has anti-apoptotic propertiesand explore it’ mechanism.
Methods: Pancreatic β cell lines were cultured in vitro, and dividedinto three groups.①Control group (BSA): Min6cells were cultured inDMEM medium containing0.5%BSA for24hours.②Palmitate group(PA): Min6cells were cultured in DMEM medium containing0.5%BSAand0.4mmol/l palmetate for24hours.③Adiponectin group(AD): Min6cells were preincubated with5ug/ml adiponectin for2hours, then culturedin DMEM medium containing0.5%BSA and0.4mmol/l palmitate for24hours.
Annexin V-of Cy3apoptosis detection kit was used for cell apoptosis,and western blotting for caspase-3,8,9and BAX、BCL-2expression.
Results: Compared with palmitate group, the number of apoptosiccells was significantly lower in adiponectin group (p <0.01). Cleavedcaspase3,9expression significantly decreased in adiponectin group (p<0.05). Caspase8expression also decreased in adiponectin group, but withno statistical differences. BAX expression decreased in adiponectin group,with BCL-2expression increasement (p <0.05).
Conclusion: Adiponectin has anti-lipotoxicity properties byinhibiting both intrinsic and extrinsic apoptotic pathways, however inhibition of intrinsic apoptosic pathways may play a major role in it.
Part3Diabetic intervention effect of recombinant lentiviral vectorexpressing mouse adiponectin protein
Objectives: To construct recombinant lentiviral vectors expressingmouse adiponectin. To increase blood adiponectin level by recombinantlentiviral vectors in diabetes. To investigate diabetic intervention effect ofadiponectin.
Methods: To construct recombinant lentiviral vectors expressingmouse full-length adiponectin protein (with green fluorescent protein).Multiple injections of small doses of streptozotocin plus high-fatdiet induced type2diabetes in mice. Diabetic mice were divided into twogroups randomly.(1) Control group (Control): Mice were fed on normaldiet for6weeks (2) Diabetic mice group: After fasting for16hours, micewere intraperitoneally injected with STZ (55mg/kg body weight, once dailyfor5days). If random blood glucose level exceeded16.7mmol/L, diabeticmodels were considered to be successful. Mice were fed on high-fat diet.4weeks later, diabetic mice were divided into three groups randomly:(1)Saline injection group (Saline,): Mice received100ul saline injection by tailvein,then continued high-fat diet for2weeks.(2) Adiponectin interventiongroup (Plenti-Acdc-EGFP): Mice received injection of100ul recombinant lentiviral vectors expressing mouse adiponectin by tail vein, thencontinued high-fat diet for2weeks. The virus titer was1x108ifu.(3)Vector injection group (Plenti-EGFP): Mice received injection of100ulempty viral vectors by tail vein, continued high-fat diet for2weeks.
Green fluorescent expression was observed in liver underfluorescence microscopy. Western blotting was used to detect adiponectinprotein expression in liver. Elisa was used for detecting blood adiponectinlevels. Islet apoptosis was detected by TUNEL assay. Expression ofCleaved caspase3,8,9was detected by immunohistochemistry. Blood lipidlevels were detected by chemical methods. Blood glucose level wasmeasured by blood glucose meter.
Results: Type2diabetic mice were induced successfully by multipleinjections of small doses of streptozotocin plus high-fat diet, with higherplasma blood lipids and glucose level than control group mice(p <0.01).
14days after injection, obvious green fluorescent proteins wereobserved in liver frozen sections under a fluorescence microscope inadiponectin intervention group and vector injection group. No expressionwere observed in control group and saline injection group. Western blottingshowed that adiponectin protein expressed only in adiponectin interventiongroup. Elisa also showed that plasma level of adiponectin in adiponectinintervention group was significantly higher than in other three groups (p<0.01). The concentration was48.03±3.79ug/ml in adiponectin intervention group. Adiponectin levels did not differ between vectorinjection group and saline injection group, but were significantly lowerthan in control group (p <0.05).
Compared with control group, low body weight and high bloodglucose/lipid level were found in saline injection group and vector injectiongroup. No difference was found between saline injection group and vectorinjection group,
Compared with saline injection group and vector injection group,adiponectin intervention group have lower blood glucose level and TGlevel（p<0.05,p<0.01）. Apoptotic β cell’s number significantly reduced andthe expressions of Cleaved caspase3,8,9in islet were significantly lowerin adiponectin intervention group.
Conclusion:Mutiply injection of small doses of STZ plus high fat dietcan successfully induce type2diabetes in mice. Lentiviral vectors for genetherapy can increase plasma adiponectin level. Improving bloodadiponectin level can be a way to inhibit pancreatic β cell apoptosis，toimprove glucose and lipid metabolisms，to intervene diabetes.
引文
[1] Yang SH, Dou KF, Song WJ. Prevalence of diabetes among men and women inChina.The New England journal of medicine[J].2010Jun24;362(25):2425-6.
[2] Helen E Thomas,Mark D McKenzie, Eveline Angstetra, et al. Beta cell apoptosisin diabetes[J]. Apoptosis2009,14:1389–1404.
[3] Engin Ulukaya1, Ceyda Acilan, Yusuf Yilmaz. Apoptosis: why and how does itoccur in biology?[J]. Cell Biochem Funct2011,29:468–480.
[4] Johnson JD, Luciani DS. Mechanisms of pancreatic beta-cell apoptosis in diabetesand its therapies[J]. Adv Exp Med Biol2010,654:447-62..
[5] Christine M, Kusminski, Shoba Shetty, et al. Diabetes and apoptosis: lipotoxicity[J].Apoptosis2009,14:1484–1495.
[6] Cui J, Panse S, Falkner B. The role of adiponectin in metabolic and vasculardisease: a review[J]. Clinical Nephrology2011,Jan;75(1):26-33.
[7] Peter Huypens, Karen Moens, Harry Heimberg. Adiponectin-mediated stimulationof AMP-activated protein kinase (AMPK) in pancreatic beta cells[J]. Life Sciences2005,77:1273–1282.
[8] Nadeeja Wijesekara, Mansa Krishnamurthy, Alpana Bhattacharjee, et al.Adiponectin-induced ERK and Akt phosphorylation protects against pancreatic betacell apoptosis and increases insulin gene expression and secretion[J]. The Journal ofBiological Chemistry2010,285:33623-31.
[9] Wijesekara N, Krishnamurthy M, Bhattacharjee A, et al. Receptor-mediatedactivation of ceramidase activity initiates the pleiotropic actions of adiponectin[J].Nature Medicine2011,17(1):55-63.
[10] Armoni M, Harel C, Karnieli E, et al. Transcriptional regulation of the GLUT4gene: from PPAR-gamma and FOXO1to FFA and inflammation[J]. TrendsEndocrinol Metab2007,18(3):100-7.
[11]Kehlenbrink S, Tonelli J, Koppaka S, et al. Inhibiting gluconeogenesis preventsfatty acid-induced increases in endogenous glucose production. Am J PhysiolEndocrinol Metab2009,297(1):E165-73.
[12]Nolan CJ, Madiraju MS, Delghingaro-Augusto V, et al. Fatty acid signaling in thebeta-cell and insulin secretion[J]. Diabetes2006,55Suppl2:S16-23.
[13]Ohneda M, Inman LR, Unger RH. Caloric restriction in obese pre-diabetic ratsprevents beta-cell depletion, loss of beta cell GLUT2and glucose incompetence[J].Diabetologia1995,38:173–179.
[14]Lang F, Ullrich S, Gulbins E. Ceramide formation as a target in beta-cell survivaland function. Expert opinion on therapeutic targets[J]2011,15(9):1061-71.
[15]Gehrmann W, Elsner M, Lenzen S. Role of metabolically generated reactiveoxygen species for lipotoxicity in pancreatic β-cells[J]. Diabetes Obesity Metablism2010,12Suppl2:149-58.
[16]Udayakumar Karunakaran, Han-Jong Kim. Guards and Culprits in the EndoplasmicReticulum: Glucolipotoxicity and β-Cell Failure in Type II Diabetes[J].Experimental Diabetes Research2012,
[17] Bachar E, Ariav Y, Cerasi E, et al. Neuronal nitric oxide synthase protects thepancreatic beta cell from glucolipotoxicity-induced endoplasmic reticulum stressand apoptosis[J].Diabetologia2010,53(10):2177-87.
[18]De Pablo MA, Susin SA, Jacotot E, et al.Palmitate induces apoptosis via a directeffect on mitochondria[J]. Apoptosis1999,4(2):81-7.
[19]Yaron Fuchs, Hermann Steller. Programmed Cell Death in Animal Developmentand Disease[J]. Cell2011,11;147(4):742-58.
[20]Sayers TJ. Targeting the extrinsic apoptosis signaling pathway for cancer therapy[J].Cancer immunology immunotherapy2011,60(8):1173-80.
[21]Brenner D, Mak TW. Mitochondrial cell death effectors[J]. Current Opinion in cellbiology2009,21(6):871-7.
[22]Hikita H, Takehara T, Kodama T, et al. Delayed-onset caspase-dependent massivehepatocyte apoptosis upon Fas activation in Bak/Bax-deficient mice[J]. Hepatology2011,54(1):240-51.
[23]Yinyuan Wu1, Dianjun Wang, Xiaodong Wang, et al. Caspase3is Activatedthrough Caspase8instead of Caspase9during H2O2-induced Apoptosis in HeLaCells[J]. Cell Physiology Biochemistry2011,27:539-546.
[24] Maedler K, Spinas GA, Lehmann R, et al. Glucose induces beta-cell apoptosis viaupregulation of the Fas receptor in human islets[J]. Diabetes2001,50(8):1683-90.
[25] McKenzie MD, Jamieson E, Jansen ES, et al. Glucose induces pancreatic islet cellapoptosis that requires the BH3-only proteins Bim and Puma and multi-BH domainprotein Bax[J].Diabetes2010,59(3):644-52.
[26]Rangnath Mishra, Michael S Simonson. Saturated free fatty acids and apoptosis inmicrovascular mesangial cells: palmitate activates pro-apoptotic signalinginvolving caspase9and mitochondrial release of endonuclease G[J].Cardiovascular Diabetology2005,4:2.
[27]Shehzad A, Iqbal W, Shehzad O,et al. Adiponectin: Regulation of its productionand its role in human diseases[J]. Hormones2012,11(1):8-20.
[28]Tishinsky JM, Robinson LE, Dyck DJ. Insulin-sensitizing properties ofadiponectin[J]. Biochimie2012, Jan31.
[29]Gastaldelli A, Miyazaki Y, Mahankali A, et al. The effect of pioglitazone on theliver: role of adiponectin[J]. Diabetes Care2006,29(10):2275-81.
[30]Sowers JR. Endocrine functions of adipose tissue: focus on adiponectin[J]. ClinCornerstone2008,9(1):32-8.
[31]Cui J, Panse S, Falkner B. The role of adiponectin in metabolic and vascular disease:a review[J]. Clin Nephrol2011,75(1):26-33.
[32]Buechler C, Wanninger J, Neumeier M.Adiponectin, a key adipokine in obesityrelated liver diseases[J].World J Gastroenterol2011,17(23):2801-11.
[33] Park M, Youn B, Zheng XL,et al.Globular adiponectin, acting viaAdipoR1/APPL1, protects H9c2cells from hypoxia/reoxygenation-inducedapoptosis. PLoS One2011,28;6(4):e19143.
[34]Lin LY, Lin CY, Su TC,et al. Angiotensin II-induced apoptosis in human endothelialcells is inhibited by adiponectin through restoration of the association betweenendothelial nitric oxide synthase and heat shock protein90[J]. FEBS Lett2004,10;574(1-3):106-10.
[35]Qiu G, Wan R, Hu J,et al. Adiponectin protects rat hippocampal neurons againstexcitotoxicity[J].Age2011,33(2):155-65.
[36]Niu K, Asada M, Okazaki T, et al.Adiponectin pathway attenuates malignantmesothelioma cell growth[J]. Am J Respir Cell Mol Biol2012,46(4):515-23.
[37]Akifusa S, Kamio N, Shimazaki Y, et al.Globular adiponectin-induced RAW264apoptosis is regulated by a reactive oxygen species-dependent pathway involvingBcl-2[J]. Free Radic Biol Med2009,1;46(9):1308-16.
[38]Li G, Cong L, Gasser J, Zhao J, et al. Mechanisms underlying the anti-proliferativeactions of adiponectin in human breast cancer cells, MCF7-dependency on thecAMP/protein kinase-A pathway[J].Nutr Cancer2011,63(1):80-8.
[39]Konturek PC, Burnat G, Rau T,et al. Effect of adiponectin and ghrelin on apoptosisof Barrett adenocarcinoma cell line[J]. Dig Dis Sci2008,53(3):597-605.
[40]Cong L, Gasser J, Zhao J, et al. Human adiponectin inhibits cell growth and inducesapoptosis in human endometrial carcinoma cells, HEC-1-A and RL952[J]. EndocrRelat Cancer2007,14(3):713-20.
[41]Rakatzi I, Mueller H, Ritzeler O, et al. Adiponectin counteracts cytokine-and fattyacid-induced apoptosis in the pancreatic beta-cell line INS-1[J]. Diabetologia2004,47(2):249-58.
[42]Lin P, Chen L, Li D, Liu J, et al. Adiponectin reduces glucotoxicity-inducedapoptosis of INS-1rat insulin-secreting cells on a microfluidic chip[J]. Tohoku JExp Med2009,217(1):59-65.
[43]Holland WL, Miller RA, Wang ZV,et al. Receptor-mediated activation ofceramidase activity initiates the pleiotropic actions ofadiponectin[J]. Nat Med2011,17(1):55-63.
[44]Wijesekara N, Krishnamurthy M, Bhattacharjee A, et al. Adiponectin-induced ERKand Akt phosphorylation protects against pancreatic beta cell apoptosis andincreases insulin gene expression and secretion[J]. J Biol Chem2010,29;285(44):33623-31.
[45]Chetboun M, Abitbol G, Rozenberg K, et al.Maintenance of redox state andpancreatic beta-cell function: Role of leptin and adiponectin[J]. J Cell Biochem2012,1:17.
[46]Staiger K, Stefan N, Staiger H, et al.Adiponectin is functionally active in humanislets but does not affect insulin secretory function or beta-cell lipoapoptosis[J]. JClin Endocrinol Metab2005,90(12):6707-13.
[47]Martinou JC, Youle RJ. Mitochondria in apoptosis: Bcl-2family members andmitochondrial dynamics[J].Dev Cell2011,19;21(1):92-101.
[48]Iwabu M, Yamauchi T, Okada-Iwabu M, et al. Adiponectin and AdipoR1regulatePGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1[J]. Nature2010,464(7293):1313-9.
[49]Ola MS, Nawaz M, Ahsan H. Role of Bcl-2family proteins and caspases in theregulation of apoptosis[J].Mol Cell Biochem2011,351(1-2):41-58.
[50]Akifusa S, Kamio N, Shimazaki Y, et al.Globular adiponectin-induced RAW264apoptosis is regulated by a reactive oxygen species-dependent pathway involvingBcl-2[J]. Free Radic Biol Med2009,46(9):1308-16.
[51]Yang ZX, Wang D, Wang G, et al. Clinical study of recombinant adenovirus-p53combined with fractionated stereotactic radiotherapy for hepatocellularcarcinoma[J].J Cancer Res Clin Oncol2010,136(4):625-30.
[52]Xie YS, Zhang YH, Liu SP, et al.Synergistic gastric cancer inhibition bychemogenetherapy with recombinant human adenovirus p53and epirubicin: an invitro and in vivo study[J].Oncol Rep2010,24(6):1613-20.
[53]Huang PI, Chang JF, Kirn DH, et al. Targeted genetic and viral therapy foradvanced head and neck cancers[J].Drug Discov Today2009,14(11-12):570-8.
[54]PL Sinn1, SL Sauter, PB McCray Jr, et al. Gene Therapy Progress and Prospects:Development of improved lentiviral and retroviral vectors–design, biosafety andproduction[J]. Gene Therapy2005,12:1089–1098.
[55]Islam MS, Loots du T.Experimental rodent models of type2diabetes: a review[J].Methods Find Exp Clin Pharmacol2009,31(4):249-61.
[56]Joost HG. The genetic basis of obesity and type2diabetes: lessons from the newzealand obese mouse, a polygenic model of the metabolic syndrome[J].ResultsProbl Cell Differ2010,52:1-11.
[57]Deeds MC, Anderson JM, Armstrong AS, et al. Single dosestreptozotocin-induced diabetes: considerations for study design in islettransplantation models[J].Lab Anim2011,45(3):131-40.
[58]Tani H.[Development of viral vectors and the application for viral entrymechanisms[J].Uirusu2011,61(1):99-107.
[59]Nowrouzi A, Glimm H, von Kalle C, et al. Retroviral vectors: post entry events andgenomic alterations[J]. Viruses2011,3(5):429-55.
[60]Ryan M, O’Connell, Alejandro B, et al. Lentiviral Vector Delivery of HumanInterleukin-7(hIL-7) to Human Immune System (HIS) Mice Expands TLymphocyte Populations[J].PLoS One2010,6;5(8):e12009.
[61] Satoh H, Nguyen MT, Trujillo M, et al. Adenovirus-mediated adiponectinexpression augments skeletal muscleinsulin sensitivity in male Wistar rats[J].Diabetes2005,54(5):1304-13.
[62]Okamoto Y, Kihara S, Ouchi N, et al. Adiponectin reduces atherosclerosis inapolipoprotein E-deficient mice. Circulation2002,26;106(22):2767-70.
[63]Thierry Sulpice, Be ne dicte, Prunet Marcassus, et al. An Adiponectin-LikeMolecule with Antidiabetic Properties[J]. Endocrinology2009,150(10):4493–4501.
[1] Fantuzzi G. Adipose tissue, adipokines, and inflammation[J].J Allergy ClinImmunol2005,115(5):911-9.
[2] Shehzad A, Iqbal W, Shehzad O,et al. Adiponectin: Regulation of its productionand its role in human diseases[J]. Hormones2012,11(1):8-20.
[3] Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific genedysregulated in obesity[J].J Biol Chem1996,3;271(18):10697-703.
[4] Maeda K, Okubo K, Shimomura I,et al.cDNA cloning and expression of a noveladipose specific collagen-like factor, apM1(AdiPose Most abundant Genetranscript1)[J].Biochem Biophys Res Commun1996,16;221(2):286-9.
[5] Nakamura Y, Shimada K, Fukuda D, et al. Implications of plasma concentrations ofadiponectin in patients with coronary artery disease[J].Heart2004,90(5):528-33.
[6] Waki H, Yamauchi T, Kamon J, et al.Impaired multimerization ofhuman adiponectin mutants associated with diabetes. Molecular structure andmultimer formation of adiponectin[J].J Biol Chem2003,10;278(41):40352-63.
[7] Kharroubi I, Rasschaert J, Eizirik DL, et al. Expression of adiponectin receptors inpancreatic beta cells[J]. Biochem Biophys Res Commun2003,26;312(4):1118-22.
[8] Okamoto M, Ohara-Imaizumi M, Kubota N,et al.Adiponectin induces insulinsecretion in vitro and in vivo at a low glucose concentration[J]. Diabetologia2008,51(5):827-35.
[9] Chetboun M, Abitbol G, Rozenberg K, et al. Maintenance of redox state andpancreatic beta-cell function: Role of leptin and adiponectin[J]. J CellBiochem.2012Jan17.
[10]Winzell MS, Nogueiras R, Dieguez C, et al. Dual action of adiponectin on insulinsecretion in insulin-resistant mice[J].Biochem Biophys Res Commun.2004Aug13;321(1):154-60.
[11]Rakatzi I, Mueller H, Ritzeler O, et al.Adiponectin counteracts cytokine-and fattyacid-induced apoptosis in the pancreatic beta-cell line INS-1[J].Diabetologia2004,47(2):249-58.
[12]Lin P, Chen L, Li D, Liu J, et al. Adiponectin reduces glucotoxicity-inducedapoptosis of INS-1rat insulin-secreting cells on a microfluidic chip[J]. Tohoku JExp Med2009,217(1):59-65
[13]Wijesekara N, Krishnamurthy M, Bhattacharjee A, et al.Adiponectin-induced ERKand Akt phosphorylation protects against pancreatic beta cell apoptosis andincreases insulin gene expression and secretion[J]. J Biol Chem2010,29;285(44):33623-31.
[14]Brown JE, Conner AC, Digby JE, et al. Regulation of beta-cell viability and geneexpression by distinct agonist fragments of adiponectin[J].Peptides2010,31(5):944-9.
[15]Holland WL, Miller RA, Wang ZV, et al. Receptor-mediated activation ofceramidase activity initiates the pleiotropic actions of adiponectin[J].Nat Med2011,17(1):55-63.
[16]K Staiger,N Stefan, H Staiger,et al. Adiponectin Is Functionally Active in HumanIslets but Does Not Affect Insulin Secretory Function or Cell Lipoapoptosis[J]. TheJournal of Clinical Endocrinology&Metabolism2005,90(12):6707–6713.
[17]French SS, Dearing MD, Demas GE. Leptin as a physiological mediator ofenergetic trade-offs in ecoimmunology: implications for disease[J]. Integr CompBiol2011,51(4):505-13.
[18]Gema FR UHBECK. Intracellular signalling pathways activated by leptin[J].Biochem. J2006,393,7–20.
[19]Tsiotra PC, Tsigos C, Raptis SA.TNFalpha and leptin inhibit basal andglucose-stimulated insulin secretion and gene transcription in the HIT-T15pancreatic cells[J].Int J Obes Relat Metab Disord2001,25(7):1018-26.]
[20]Kulkarni RN, Wang ZL, Wang RM, et al. Leptin rapidly suppresses insulin releasefrom insulinoma cells, rat and human islets and, in vivo, in mice[J]. J Clin Invest1997,1;100(11):2729-36.
[21]Laubner K, Kieffer TJ, Lam NT,et al. Inhibition of preproinsulin gene expressionby leptin induction of suppressor of cytokine signaling3in pancreaticbeta-cells[J].Diabetes2005,54(12):3410-7.
[22]Tanabe K, Okuya S, Tanizawa Y, et al. Leptin induces proliferation of pancreaticbeta cell line MIN6through activation of mitogen-activated proteinkinase[J].Biochem Biophys Res Commun1997,29;241(3):765-8.
[23]Islam MS, Morton NM, Hansson A, et al. Rat insulinoma-derived pancreaticbeta-cells express a functional leptinreceptor that mediates a proliferativeresponse[J]. Biochem Biophys Res Commun1997,29;238(3):851-5.
[24]Chetboun M, Abitbol G, Rozenberg K, et al. Maintenance of redox state andpancreatic beta-cell function: Role ofleptin and adiponectin[j].J Cell Biochem2012,17.
[25]Lam QL, Wang S, Ko OK, et al. Leptin signaling maintains B-cell homeostasis viainduction of Bcl-2and Cyclin D1[J]. Proc Natl Acad Sci U S A2010,3;107(31):13812-7.
[26]Maedler K, Schulthess FT, Bielman C, Glucose and leptin induce apoptosis inhuman beta-cells and impair glucose-stimulated insulin secretion through activationof c-Jun N-terminal kinases[j]. FASEB J2008,22(6):1905-13.
[27]Sonoli SS, Shivprasad S, Prasad CV, et al.Visfatin--a review[J]. Eur Rev MedPharmacol Sci2011,15(1):9-14.
[28]Stofkova A. Resistin and visfatin: regulators of insulin sensitivity, inflammationand immunity[J].Endocr Regul2010,44(1):25-36.
[29]Zhu J, Schott M, Liu R, et al. Intensive glycemic control lowers plasma visfatinlevels in patients with type2diabetes[J].Horm Metab Res2008,40(11):801-5.
[30]Brown JE, Onyango DJ, Ramanjaneya M, et al. Visfatin regulates insulin secretion,insulin receptor signalling and mRNA expression of diabetes-related genes inmouse pancreatic beta-cells[J].J Mol Endocrinol2010,44(3):171-8.
[31]Cheng Q, Dong W, Qian L, et al.Visfatin inhibits apoptosis of pancreatic β-cell line,MIN6, via the mitogen-activated protein kinase/phosphoinositide3-kinasepathway[J]. J Mol Endocrinol2011,4;47(1):13-21.
