【作者】 崔明霞；

【导师】 郑荣梁；

【作者基本信息】 兰州大学 ， 细胞生物学， 2009， 博士

【摘要】 一、重组人睫状神经营养因子（rhCNTF）类似物抗代谢综合征作用中心性肥胖是代谢综合征（metabolic syndrom,MS）的核心环节,与胰岛素抵抗,2型糖尿病,非酒精性脂肪肝及心血管疾病的发生发展密切相关。因此对代谢综合征的治疗必须同时注重减重、降血糖、调脂和抗高血压等多方面的综合治疗措施。本实验分别用高脂、高营养饲料,小剂量链脲菌素静脉注射配合高营养饲料来诱导大鼠或小鼠形成饮食诱导性肥胖、糖尿病性肥胖、非酒精性脂肪肝或代谢综合征模型。探讨重组人睫状神经营养因子（rhCNTF）类似物（C-16和rhmCNTF）对肥胖相关指标,血脂水平,肝抗氧化能力,肝脏中细胞色素P450 2E1（CYP2E1）、硬脂酰CoA去饱和酶-1（SCD-1）和肉碱脂酰转移酶-1（CPT-1）表达的影响。研究结果显示,高脂高营养饲料,或以小剂量链脲菌素静脉注射配合高营养饲料长期喂养大鼠或小鼠均可诱导肥胖、腹腔内脏脂肪堆积、血脂异常、高血糖、高胰岛素血症、胰岛素抵抗和非酒精性脂肪肝。rhCNTF类似物均可显著、并呈剂量依赖性地减轻模型动物的体重及体脂含量,降低体重的作用在给药8-9天后有所减弱,表现为体重稳定在一定水平或轻微回升,且与摄食量的变化相关;在治疗停止后,降低的体重不会出现急速反弹,且在停药3周后仍然显著低于空白对照组;rhCNTF类似物还可不同程度的改善模型动物的血脂异常、高胰岛素血症和胰岛素抵抗;可抑制肝脏中脂肪沉积,明显减轻肝脏脂肪变性程度;可改善肝脏功能、增强肝脏抗氧化能力;促进肝脏CPT-1的表达,同时抑制SCD-1和CYP 2E1的表达;并且,rhCNTF类似物在减重的有效剂量下不会引起条件性厌食反应,不会使机体出现类似短期过度限食所致的应激反应。rhCNTF类似物在动物实验中表现出良好的抗代谢综合征作用,对代谢综合征中多种症状均有对抗,提示rhCNTF类似物在治疗以中心性肥胖为核心的代谢综合征方面具有重要的临床应用价值。二、重组人睫状神经营养因子（rhCNTF）类似物C-16的药代动力学为阐明注射用重组人睫状神经营养因子（rhCNTF）类似物（C-16）的体内过程特点,本实验用C-16对大鼠、小鼠和猴静脉注射（i.v.）和皮下注射（s.c.）给药后,用双抗体夹心ELISA法测定C-16在血清、胆汁、尿液及组织中的药物浓度,研究其在大鼠和猴体内i.v.和s.c.时的药-时曲线,用DAS药动学统计软件进行血药浓度-时间曲线拟合及药动学参数计算。结果显示:1.rhCNTF检测试剂盒特异性及检测灵敏度高（0.0625 ng/ml）,线性范围0.0625-16 ng/ml（r²＞0.9999）。在所有考察的生物介质中的最低检测限为0.5ng/ml,线性范围0.5-32 ng/ml（r²＞0.9997）。2.C-16在大鼠体内的药动学特性:C-16对大鼠i.v.300μg/kg的药动学行为符合二室模型,一级动力学消除,消除半衰期(t_1/2β)为1.39 h,清除率（CL）为0.036 L/h/kg,表观分布容积（Vd）为0.20 L/kg,曲线下面积（AUC）_0-6为8580μg/L·h,AUC_0-∞为9006μg/L·h。C-16对大鼠s.c.100、300、900μg/kg的药动学特征均符合一室模型,血药峰浓度(C_max)分别为10.8、202.0、508.7μg/L,AUC_0-t分别为42.2、876.5、1810.6μg/L·h,AUC_0-∞分别为46.4、939.8、2072.1μg/L·h,均随给药剂量的增加而增加,但低、中剂量组的AUC和C_max与给药剂量不成比例;达峰时间(T_max)分别为1.35、0.65和0.75 h;t_1/2分别为1.94、2.33、2.90 h;CL/F分别为2.23、0.33、0.54 L/h/kg,低剂量清除率明显较高（与中、高剂量组比,均P＜0.001）;平均滞留时间（MRT）_0-t分别为2.82、3.25、3.15h,MRT_0-∞分别为3.88、3.61、4.09 h。C-16对大鼠s.c.的生物利用度为10.4%。3.C-16在猴体内的药动学特性:C-16对猴i.v.150μg/kg的药动学行为符合二室模型,一级动力学消除,t_1/2β为2.39 h,CL为0.064 L/h/kg,Vd为0.302 L/kg,AUC_0-6为2438μg/L·h,AUC_0-∞为2553μg/L·h。C-16对猴s.c.50、150、450μg/kg的药动学特征均符合一室模型,C_max分别为9.9、29.5、73.5μg/L,AUC_0-t分别为58.8、157.8、491.9μg/L·h,AUC_0-∞分别为66.4、174.1、540.9μg/L·h,C_max和AUC值均随给药剂量的增加而成比例增加,与给药剂量之间有线性关系（r²均＞0.996）;T_max分别为3.50、3.00和2.50 h;t_1/2分别为2.08、2.09、2.93 h;CL/F分别为1.102、0.931、0.892 L/h/kg:MRT_0-t分别为4.07、4.04、5.26 h,MRT_0-∞分别为5.52、5.54、7.59 h。C-16对猴s.c.的生物利用度为6.82%。4.C-16在小鼠体内的分布特性:C-16对小鼠i.v.600μg/kg后15 min时卵巢组织中药物分布最高,脂肪、心肌组织次之,在肝、肾、胸腺和子宫组织中有中等量分布,在脾脏、肺、肌肉组织中含量较低（小于血清药物浓度的4%）,脑、胰腺和睾丸组织中未检测到药物。45 min时药物在脂肪和卵巢组织中分布最高,均高于血清中的药物浓度,肾组织中有中等量分布,在肝和子宫组织中的浓度约为血清中浓度的25%。在心肌、脾脏、肺、肌肉和胸腺组织中含量较低（小于血清药物浓度的20%）,脑组织中可检测出较低的药物浓度（为血清药物浓度的5.07%）,而胰腺和睾丸组织中仍未检测到药物。3 h时药物在脂肪组织中的分布依然很高,为血清药物浓度的2倍左右,在肾、卵巢和子宫组织中的分布程度相似,约为血清药物浓度的80%,心肌和肝组织中的药物浓度约为血清中的50%左右,在脾脏、肺、脑、肌肉、胸腺、胰腺和睾丸组织中均未检测到药物。5.C-16在小鼠尿中的排泄特性:C-16对小鼠i.v.600μg/kg后36 h尿累计排泄量为给药量的4.24/百万,在给药1 h后的尿样中均未检测到药物。6.C-16在大鼠胆汁中的排泄特性:C-16对大鼠i.v.300μg/kg后24 h在胆汁中的累计排泄量为给药量的0.31%。综上,C-16 i.v.后在大鼠和猴体内的药动学特征均符合二室一级消除,s.c.后均符合一室一级消除,主要药动学参数在同种属动物体内无明显差异。分布平衡后,C-16在脂肪组织中分布量最高,在卵巢、肝、肾中也有较高的分布,脑组织中可检到小量药物,而胰腺和睾丸中未检测到药物。该药在尿和胆汁中的原形排泄率均较低。更多还原

【Abstract】 ⅠTherapetic effects on metabolic syndrome of recombinant human ciliary neurotrophic factor （rhCNTF） analogsAbdominal obesity is the dominant underlying risk factor for metabolic syndrome and links with insulin resistance,type 2 diabetes,and coronary heart disease tightly.Each factor in metabolic syndrome is the risk factor for coronary heart disease,so therapy on metabolic syndrome is a whole concept of diagnosis and treatment of a group of high-related diseases.Loss of body weight,reduction of blood glucose and pressure,and blood lipid regulation are all same important.This experiment is to prepare models of diet-induced obesity,diabetes mellitus obesity,nonalcoholic fatty liver disease,and metabolic syndrome in rats or mice and to study the influence of rhCNTF analogs on activity of stearoyl-coenzyme A desaturase-1（SCD-1） and camitine palmitoyltransferase-1（CPT-1） and on free radical formation,blood level and expression of CYP2E1.The results will provide the theoretical basis for clinical therapy of obesity,nonalcoholic fatty liver disease,and metabolic syndrome with recombinant human ciliary neurotrophic factor（rhCNTF） analogs（C-16 and rhmCNTF）.The results showed that animal models had obesity,visceral adipose accumulation, blood lipid abnormality,hyperglycemia,hyperinsulinemia,insulin resistance,and hepatic steatosis.The two analogs of rhCNTF both significantly decreased body weight and fat content with dose-dependent manner.This effect was weakened after 8-9 days of treatment and correlated with changes of food intake.The body weight had no rapidly rebound after treatment cessation and obviously lower than blank group after 3 weeks of treatment cessation.The analogs of rhCNTF also ameliorated glucose and lipid metabolism abnormality,and enhanced insulin sensitivity.In addition,after treatment of rhCNTF analogs,hepatic steatosis and function of model animals were ameliorated and antioxidative activity of liver was enhanced.The expression of CPT-1 in liver was promoted,and SCD-1 and CYP 2E1 were inhibited. Furthermore,rhCNTF analogs had not cause conditioned taste aversion or stress response like short-term excessive food restriction when rats were administered a dose known to produce significant weight loss.The analogs of rhCNTF showed good theraputic effects on metabolic syndrome in animal model.It suggests that rhCNTF analogs have important clinical therapy value on metabolic syndrome. ⅡPharmacokinetics of recombinant human ciliary neurotrophic（rhCNTF） analogur C-16To study pharmacokinetic property of recombinant human ciliary neurotrophic factor（rhCNTF） analogue C-16 after intravenous（i.v.） and subcutaneous（s.c.） injection to mice,rats,or monkeys.Double antibody sandwich ELISA analysis was used for testing drug concentration in serum,bile,urine,and tissues.Pharmacokinetic parameters were calculated by DAS software.The results showed that:1.The specificity and sensitivity of rhCNTF assay kit were high.The detection limit was 0.0625 ng/ml and the linear range was within 0.0625-16 ng/ml（r²＞0.9999）. The detection limit of C-16 in biological media was 0.5 ng/ml and the linear range was within 0.5-32 ng/ml（r²＞0.9997）.2.Pharmacokinetic property of C-16 in rats after i.v.300μg/kg was consistent with two-compartment open model and first-order kinetic elimination.The main pharmacokinetic parameters were as follows:t_1/2β:1.39 h,CL:0.036 L/h/kg,Vd:0.20 L/kg,AUC_0-6:8580μg/L·h,AUC_0-∞:9006μg/L·h.Pharmacokinetic property of C-16 in rats after s.c.100,300,and 900μg/kg were consistent with one-compartment open model.C_max:10.8,202.0 and 508.7μg/L,AUC_0-t:42.2,876.5 and 1810.6μg/L·h, AUC_0-∞:46.4,939.8 and 2072.1μg/L·h,T_max:1.35,0.65 and 0.75 h,t_1/2:1.94,2.33 and 2.90 h,CL/F:2.23,0.33 and 0.54 L/h/kg,MRT_0-t:2.82,3.25 and 3.15 h,MRT_0-∞: 3.88,3.61 and 4.09 h,respectively.Bioavailability of C-16 s.c.in rats:10.4%.3.Pharmacokinetic property of C-16 in monkeys after i.v.300μg/kg was consistent with two-compartment open model and first-order kinetic elimination.The main Pharmacokinetic parameters were as follows:t_1/2α:2.39 h,CL:0.064 L/h/kg,Vd: 0.302 L/kg,AUC_0-6:2438μg/L·h,AUC_0-∞:2553μg/L·h.Pharmacokinetic property of C-16 in monkeys after s.c.50,150,and 450μg/kg were consistent with one-compartment open model.C_max:9.9,29.5 and 73.5μg/L,AUC_0-t:58.8,157.8 and 491.9μ/L·h,AUC_0-∞:66.4,174.1 and 540.9μg/L·h,T_max:3.50,3.00 and 2.50 h,t_1/2: 2.08,2.09 and 2.93 h,CL/F:1.10,0.93 and 0.89 L/h/kg,MRT_0-t:4.07,4.04 and 5.26 h, MRT_0-∞:5.52,5.54 and 7.59 h,respectively.Bioavailability of C-16 s.c.in monkeys: 6.82%.4.Distribution property of C-16 in mice:At 15 min after C-16 i.v.600μg/kg to mice,ovarian had the highest drug concentration.Fat and heart tissues took the second place.Live,kidney,thymus,and uterus tissues had middle level,while spleen、lung,muscle tissues had low level（less than 4%of that in serum）.Drug had not been detected in brain,pancreas,and testis tissues.At 45 min,ovarian and fat tissues had the highest drug concentration.Kidney took the second place.Drug concentration was 25%in liver and uterus and was less than 20%in heart,lung,muscle,spleen,and thymus of that in serum concentration.Drug was low（5.07%of that in serum） in brain and had not detected in pancreas and testis.At 3 h,Drug concentration was still high in fat tissues（about two times than that in serum）,was about 80%in kidney, ovarian,and uterus,and 50%in heart and liver of that in serum.Drug had not been detected in spleen,lung,brain,muscle,thymus,pancreas,and testis tissues.5.Elimination character of C-16 in urine:The total drug accumulative excretion ratio in urine was 4.24 per million during 36 h after i.v.600μg/kg and had not been detected after 1 h.6.Elimination character of C-16 in bile:The total drug accumulative excretion ratio in bile was 0.31%during 24 h after i.v.300μg/kg.In conclusion,pharmacokinetic models of C-16 are two-compartment open model and first-order kinetic elimination after i.v.and are one-compartment open model and first-order kinetic elimination after s.c.to rats and monkeys.The main pharmacokinetic parameters in the same genus animal are not significantly different. After balanced distribution,the concentration of C-16 in fat was the highest,which in ovarian,liver and kidney still maintained in high level,whilein in brain declined to low level,however which could not be detected in testis and pancreas.The excretion ratio of C-16 is very low in urine and bile.更多还原