C57BL/6J小鼠Ⅱ型糖尿病模型的优化和评价
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摘要
研究背景与目的
糖尿病对人类造成的健康危害和经济负担,仅次于肿瘤和心脑血管疾病而高居第三位,且发病率仍呈上升趋势。非胰岛素依赖型糖尿病(NIDDM,Ⅱ型糖尿病)发病率占所有糖尿病的90%-95%,其中60%-90%的Ⅱ型糖病患者体重超重,能够模拟和体现各型糖尿病病理生理变化的动物模型将有助于人们更好地理解疾病的本质并提供更好的治疗方法。
高脂饮食+STZ(Streptozotocin,链脲佐菌素)诱导Ⅱ型糖尿病模型是一种常见的造模方法,通常是应用脂肪含量在30%左右的高脂饲料喂养后再辅以小剂量的STZ(小鼠的STZ剂量在200mg／kg左右),能够比较好地评价治疗Ⅱ型糖尿病的药物以及进行Ⅱ型糖尿病病理机理和并发症相关方面的研究,具备方法简单、经济、节省时间等优点。但是查阅早些的文献以及我们的一些实验情况来看,模型还存在着不少的缺点：比如有很多因素影响模型的成模效果,模型对于有些药物评价效果不好(例如格列美脲类的促胰岛素分泌药),把对胰岛素释放有促进效果的药物判为无效,因此在应用该模型对新型化合物进行体内药效评价和筛选时很有可能会漏筛一些极具开发价值的药物,所以很有必要对模型进行进一步的优化和改良。
通过查阅文献和对以往实验的总结我们发现主要有三大因素影响着模型,分别是STZ单次注射剂量、首次注射的时间以及注射的次数,为了找出这几个因素间的最佳组合,我们进行了三因素三水平的正交实验并为这些因素设计了如下的水平：三个注射剂量为75mg/kg、85mg/kg、100mg/kg,首次注射时间为第2周、第3周、第4周,注射次为1次、2次、3次,根据正交表来安排这些组合,通过正交实验筛选出其中的最佳组合,然后对该组合进行模型的稳定性和重复性的考察,最后进行药效学评价,明确模型的适用范围。
本课题的目的是进一步缩短造模时间,优化模型,使模型更加接近于Ⅱ型糖尿病并明确模型的适用范围,为抗糖尿病药物的筛选和评价提供一个更好的平台。
方法和内容
1.进行三因素三水平的正交实验,考察不同因素间的组合对模型的影响,筛选出最佳的组合。我们采用脂肪含量为30%的高脂饮食,各因素和水平分别为：首次STZ注射的时间a1=第2周,a2=第3周,a3=第4周；STZ单次注射剂量的三个水平为b1=75mg/kg, b2=85mg/kg, b3=100mg/kg; STZ注射的次数c1=1次,c2=2次,c3=3次。查正交表安排正交试验,组合安排如下：a1b1c1,a1b2C2,a1b3C(c2), a2b1c2,a2b2c3(c2),a2b3c1,a3b1c3,a3b2c1,a3b3c2；根据以往的文献剔出不符合的组合换成括号里面的组合。3周龄的断乳C57BL/6J小鼠分为9组,每组8只,每周观察体重、血糖指标的变化,注射STZ前检测部分动物空腹血糖或非空腹血糖,以观察血糖升高情况。空腹血糖检测方法是：晚上8点换垫料禁食但自由饮水,到第2天早上8点,禁食12h。剪尾取血,用美国强生公司稳步倍加型血糖仪及试纸条检测血糖。注射完STZ后继续高脂饲料喂养。每周观察体重、进食量、血糖的变化,5周后处死,取内脏做病理切片,处死前两天进行OGTT实验。
2.口服糖耐量/OGTT实验实验前小鼠禁食15个小时,给葡萄糖前先测小鼠的空腹血糖,根据空腹血糖的情况确定给予葡萄糖的浓度：当空腹血糖平均<150mg/dl时,用20%葡萄糖；当空腹血糖平均>150mg/dl时,用10%葡萄糖。经过30分钟、60分钟、120分钟测一次血糖,做成血糖曲线图,进行统计学的处理,比较各组间的差异,采用重复测量资料的方差分析,检验水准：a=0.05。
3.血脂以及胰岛素水平的测定采用血浆甘油三酯和游离脂肪酸检测试剂盒检测血脂水平；胰岛素的检测原理是采用ELISA方法,在96孔板上进行。检测方法和步骤：按照试剂盒的说明操作
4.组织病理学检测小鼠处死后,用组织剪和手术刀取合适大小的胰腺组织。经4%多聚甲醛固定24h以上,酒精梯度脱水、二甲苯梯度透明,浸蜡包埋,4度下过夜,切成4um切片贴于防脱硅化玻片上,烘干后备用。HE染色：经脱蜡、一系列的酒精梯度脱水、水洗、HE染色、脱水和封片固定,在光学显微镜下进行组织学病理变化的观察,对胰腺组织的坏死、凋亡以及变性情况进行评价。
5.对最佳组合进行模型稳定性和重复性考察：对最佳的组合进行8周以上的观察,以考察其稳定性。24只C57BL6J小鼠,分为模型组、对照组和高脂饮食组,每组8只,按照正交实验的结果进行造模；用7个不同批次小鼠进行造模以考察模型的重复性。
6.对优化后的模型进行药效学评价100只C57BL／6J小鼠中,80只喂饲高脂饲料,20只喂饲低脂饲料。高脂鼠其中的70只和低脂鼠其中的10只注射STZ(按优化后的方法)。70只高脂+STZ造模的小鼠中,选一部分用于药物试验,分为对照组和给药组,对照组为阴性对照和正常对照,每类药物分为高剂量和低剂量：二甲双胍组(剂量分别为200mg/kg/d,500mg/kg/d),格列美脲组(剂量为0.1mg/kg/d, 0.3mg/kg/d),匹格列酮组(剂量为10mg/kg/d,30mg/kg/d)。小鼠在给药前一天根据血糖和体重由计算机随机分组,各给药组体重和血糖平均值要接近(误差<5%)。药物配好后按剂量每天下午4：00灌胃给药,每天1次,给药体积均为10mL/kg,连续21天。
结果
1.正交实验筛选出的最佳组合是第三周开始注射streptozotocin(STZ),注射剂量为85mg/kg(注射两次,每次间隔时间为3天)。
2.模型具有很好的重复性和稳定性,造模的成功率在90%以上,血糖水平在注射STZ后稳定上升,注射1周后(第5周)维持稳定并且可以持续到8周以上,建议应用该模型的最佳时间是注射完STZ后至少1周以上。
3.模型对药物的评价效果：优化后的模型对二甲双胍和匹格列酮具有很好的评价效果；同时也很好地改善了对促胰岛素分泌类药物如格列美脲的评价功效。
4.血甘油三酯和游离脂肪酸检测结果表明所用的三种药物能够很好地改善模型的高甘油三酯和高脂肪酸,特别是二甲双胍和匹格列酮高剂量组,与阴性组比较,格列美脲低剂量组低16%左右。
结论：
1.成功地优化了以C57BL／6J小鼠糖尿病动物模型,使造模时间缩短,三个因素间最佳的组合是第3周开始注射STZ,单次剂量为85mg/kg连续注射两次,每次间隔3天模型更加接近于Ⅱ型糖尿病病理生理状态。
2.为评价和筛选治疗Ⅱ型糖尿病药物提供了一个廉价以及可靠的平台。
本研究的创新之处
利用正交设计的原理来优化动物模型,动物的使用量极大地减少,使动物模型的构建和优化的成本和时间尽量减少。
Background&Objective:
Diabetes, ranking just after cancer and cardiovascular and cerebrovascular diseases, is one of the most harmful diseases to human on health and economies.90-95% of the diabetic patients are belongs to NIDDM, and 60-90% of the NIDDM patients are overweight. Therefore, to establish an animal model whose pathophysiologic changes are similar to human diabetes, may provide better understanding and treatments on diabetes.
Combination of high-fat diet and STZ is commonly used in model establishment of typeⅡdiabetes. Generally, high-fat diet (30%of fat) feeding and a small amount of STZ (200mg/kg) injection in mice has been considered to be a simple, economical and time saving method to establish model of typeⅡdiabetes. Such model can provide good evaluation of potential medicine that may treat typeⅡdiabetes and make the pathomechanism and related complications of type II diabetes more clear so that further study can be continued. However, we found that there are still some disadvantages like unsteadiness, bad evaluation of some drug, invalid evaluation of insulin secretagoguesn in this model, which might cause omission when screening new drugs that have potential value in treating typeⅡdiabetes. Therefore, further optimization of this model is obviously necessary.
There are three main factors of this model including dose of single dose, time of the first injection and injected frequency according to many documents and previous study. In order to find out the best combination, three factors and three levels orthogonal test is adopted and three factors and three levels are:single does (75mg/kg、85mg/kg、100mg/kg), time of the first injection (the first、the second、the third week), injected frequency (1、2、3 times), respectively. Find out the best combination through orthogonal test according to the orthogonal layout. Finally, examine the stability and repeatability of the model and confirm the area of application. Aim:
To optimize a rodent model of type II diabetes that would replicate the metabolic characteristics of the human syndrome and the suitable for pharmaceutical research.
Methods:
1.The three factors and three levels orthogonal test:In order to find out the best combination, three factors and three levels orthogonal test is adopted and three factors and three levels are:time of the first STZ injection (a1=the first week、a2=the second week、a3=the third week), single does (b1=75mg/kg、b2=85mg/kg、b3=100mg/kg), injected frequency (c1=1 time、c2=2 times、c3=3times), respectively. According to the orthogonal layout, combinations are ranked as followed:a1b1c1, a1b2c2、a1b3c3(c2), a2b1c2, a2b2C3(c2), a2b3c1, a3b1c3,a3b2c1, a3b3c2, Inadequate combinations are rejected on the basis of the previous documents and replaced by the level in the brackets. There-week-old weaning C57BL/6J mice were fed with high-fat diet containing 30% of fat then divided into 9 groups (8 animals per group). Weight and blood glucose were recorded every week. Fasting blood glucose was measured before the STZ injection. The method of fasting blood glucose testing is as followed:mice were fasted but free access to water for 12 hours (08:00pm-08:00am). Blood samples were collected by cutting the tail tip and tested through blood glucose meters (type: steadily SureStep, supplied by Johnson & Johnson of the United States). Animals were fed with high-fat diet sequentially after STZ injection. The changes of body weight, food intake and blood glucose were measured every week. The mice were decapitated five weeks after STZ injection, and the pancreata were taken for pathological section.
2. Oral glucose tolerance test Overnight-fasted (15 hours) mice were given a glucose solution orally (20% glucose if fasting glucose<150mg/dl or 10% glucose) and blood samples were collected from tail vein at 30,60 and 120 minutes after the glucose administration. Blood glucose curve diagram was done. Deal with the data with repeated measurement and compare the diversity between groups, a=0.05)
3. Plasma chemistry:Plasma free fatty acids, insulin level were tested with corresponding testing kits. All detecting procedures were done according to the kit instructions.
4. Histopathological detection:Pancreata of the mice were collected with scissors and scalpel after decapitation. After fixed in 4% of paraformaldehyde for more than 24h, the pancreatic tissue was dehydrated by gradient alcohol, transparentized by dimethylbenzene, embedded by wax and kept overnight at 4℃. The samples were cut into slices of 4um then affixed to the slide on the antidepinning siliconization. The dried samples were stained with HE. Pathological changes were observed in the optical microscope. Evaluate the putrescence、apoptosis and denaturation of the pancreata.
5. Investigation of stability and reproducibility of the optimize model We used different batches of mice and observed for more than 8 weeks to investigate the stability and reproducibility of the best combination.24 C57BL/6J mice were divided into three groups (8 per group):model group, control group and high-fat diet group.
6. Pharmacodynamic evaluation of the optimized model:To determine Pharmacological effects of the optimized Fat-fed/STZ mice model, we tested three kinds of drugs widely used in type 2 diabetes.100 C57BL/6J mice were used in the test and 80 were fed with high-fat diet,20 low-fat diet.70 high-fat-diet mice and 10 low-fat-diet mice were injected with STZ according to the results of orthogonal experiment. Choose 64 animals from high-fat-diet/STZ mice and then divided them into 8 groups:normal control, negative control and medication administration group. Three kinds of drugs were choosen for testing:metformin(200mg/kg/d,500mg/kg/d), glimepiride(0.1mg/kg/d,0.3mg/kg/d) and pioglitazone(10mg/kg/d,30mg/kg/d). Mice were randomized by computer 1 day before administration according to blood glucose concentration and body weight. Intragastric administration were executed 1 time per day (10mL/kg) and continued for 21 days.
Results:
1. The result indicates that injection of streptozotocin(85mg/kg) twice every five days at the third week is the best for establishing rodent type II diabetes model. Biopsy also showed appropriate destruction of islet.
2. The optimized model is stable and reproducible. The success ratio of model establishment is 90%. The blood glucose concentration was stable and can sustain until eight weeks after STZ injection.
3. Fat-fed/STZ mice is sensitive to metformin and pioglitazone. Glimeperide also has hypoglycemic effect when administrating dose is up to 0.3 mg/kg.
4. Test results of blood triglyceride and free fatty acid shows that these three kinds of drugs used in the model could well ameliorate the high triglycerides and high-fatty acids, particularly metformin and pioglitazone high-dose group. Glimepiride low-dose group is about 16%lower than the negative control group.
Conclusions:
1. The rodent model of typeⅡdiabetes is successfully optimized (pathology is more similar to type 2 diabetes of human) and the time of inducing is shorter. The best combination is as followed:the first time of STZ injection (85mg/kg) is at the third week and twice totally.
2. Provide a cheap and reliable platform for the evaluation and selection of drugs have used or will use in typeⅡdiabetes.
糖尿病对人类造成的健康危害和经济负担,仅次于肿瘤和心脑血管疾病而高居第三位,且发病率仍呈上升趋势。非胰岛素依赖型糖尿病(NIDDM,Ⅱ型糖尿病)发病率占所有糖尿病的90%-95%,其中60%-90%的Ⅱ型糖病患者体重超重,能够模拟和体现各型糖尿病病理生理变化的动物模型将有助于人们更好地理解疾病的本质并提供更好的治疗方法。
高脂饮食+STZ(Streptozotocin,链脲佐菌素)诱导Ⅱ型糖尿病模型是一种常见的造模方法,通常是应用脂肪含量在30%左右的高脂饲料喂养后再辅以小剂量的STZ(小鼠的STZ剂量在200mg／kg左右),能够比较好地评价治疗Ⅱ型糖尿病的药物以及进行Ⅱ型糖尿病病理机理和并发症相关方面的研究,具备方法简单、经济、节省时间等优点。但是查阅早些的文献以及我们的一些实验情况来看,模型还存在着不少的缺点：比如有很多因素影响模型的成模效果,模型对于有些药物评价效果不好(例如格列美脲类的促胰岛素分泌药),把对胰岛素释放有促进效果的药物判为无效,因此在应用该模型对新型化合物进行体内药效评价和筛选时很有可能会漏筛一些极具开发价值的药物,所以很有必要对模型进行进一步的优化和改良。
通过查阅文献和对以往实验的总结我们发现主要有三大因素影响着模型,分别是STZ单次注射剂量、首次注射的时间以及注射的次数,为了找出这几个因素间的最佳组合,我们进行了三因素三水平的正交实验并为这些因素设计了如下的水平：三个注射剂量为75mg/kg、85mg/kg、100mg/kg,首次注射时间为第2周、第3周、第4周,注射次为1次、2次、3次,根据正交表来安排这些组合,通过正交实验筛选出其中的最佳组合,然后对该组合进行模型的稳定性和重复性的考察,最后进行药效学评价,明确模型的适用范围。
本课题的目的是进一步缩短造模时间,优化模型,使模型更加接近于Ⅱ型糖尿病并明确模型的适用范围,为抗糖尿病药物的筛选和评价提供一个更好的平台。
方法和内容
1.进行三因素三水平的正交实验,考察不同因素间的组合对模型的影响,筛选出最佳的组合。我们采用脂肪含量为30%的高脂饮食,各因素和水平分别为：首次STZ注射的时间a1=第2周,a2=第3周,a3=第4周；STZ单次注射剂量的三个水平为b1=75mg/kg, b2=85mg/kg, b3=100mg/kg; STZ注射的次数c1=1次,c2=2次,c3=3次。查正交表安排正交试验,组合安排如下：a1b1c1,a1b2C2,a1b3C(c2), a2b1c2,a2b2c3(c2),a2b3c1,a3b1c3,a3b2c1,a3b3c2；根据以往的文献剔出不符合的组合换成括号里面的组合。3周龄的断乳C57BL/6J小鼠分为9组,每组8只,每周观察体重、血糖指标的变化,注射STZ前检测部分动物空腹血糖或非空腹血糖,以观察血糖升高情况。空腹血糖检测方法是：晚上8点换垫料禁食但自由饮水,到第2天早上8点,禁食12h。剪尾取血,用美国强生公司稳步倍加型血糖仪及试纸条检测血糖。注射完STZ后继续高脂饲料喂养。每周观察体重、进食量、血糖的变化,5周后处死,取内脏做病理切片,处死前两天进行OGTT实验。
2.口服糖耐量/OGTT实验实验前小鼠禁食15个小时,给葡萄糖前先测小鼠的空腹血糖,根据空腹血糖的情况确定给予葡萄糖的浓度：当空腹血糖平均<150mg/dl时,用20%葡萄糖；当空腹血糖平均>150mg/dl时,用10%葡萄糖。经过30分钟、60分钟、120分钟测一次血糖,做成血糖曲线图,进行统计学的处理,比较各组间的差异,采用重复测量资料的方差分析,检验水准：a=0.05。
3.血脂以及胰岛素水平的测定采用血浆甘油三酯和游离脂肪酸检测试剂盒检测血脂水平；胰岛素的检测原理是采用ELISA方法,在96孔板上进行。检测方法和步骤：按照试剂盒的说明操作
4.组织病理学检测小鼠处死后,用组织剪和手术刀取合适大小的胰腺组织。经4%多聚甲醛固定24h以上,酒精梯度脱水、二甲苯梯度透明,浸蜡包埋,4度下过夜,切成4um切片贴于防脱硅化玻片上,烘干后备用。HE染色：经脱蜡、一系列的酒精梯度脱水、水洗、HE染色、脱水和封片固定,在光学显微镜下进行组织学病理变化的观察,对胰腺组织的坏死、凋亡以及变性情况进行评价。
5.对最佳组合进行模型稳定性和重复性考察：对最佳的组合进行8周以上的观察,以考察其稳定性。24只C57BL6J小鼠,分为模型组、对照组和高脂饮食组,每组8只,按照正交实验的结果进行造模；用7个不同批次小鼠进行造模以考察模型的重复性。
6.对优化后的模型进行药效学评价100只C57BL／6J小鼠中,80只喂饲高脂饲料,20只喂饲低脂饲料。高脂鼠其中的70只和低脂鼠其中的10只注射STZ(按优化后的方法)。70只高脂+STZ造模的小鼠中,选一部分用于药物试验,分为对照组和给药组,对照组为阴性对照和正常对照,每类药物分为高剂量和低剂量：二甲双胍组(剂量分别为200mg/kg/d,500mg/kg/d),格列美脲组(剂量为0.1mg/kg/d, 0.3mg/kg/d),匹格列酮组(剂量为10mg/kg/d,30mg/kg/d)。小鼠在给药前一天根据血糖和体重由计算机随机分组,各给药组体重和血糖平均值要接近(误差<5%)。药物配好后按剂量每天下午4：00灌胃给药,每天1次,给药体积均为10mL/kg,连续21天。
结果
1.正交实验筛选出的最佳组合是第三周开始注射streptozotocin(STZ),注射剂量为85mg/kg(注射两次,每次间隔时间为3天)。
2.模型具有很好的重复性和稳定性,造模的成功率在90%以上,血糖水平在注射STZ后稳定上升,注射1周后(第5周)维持稳定并且可以持续到8周以上,建议应用该模型的最佳时间是注射完STZ后至少1周以上。
3.模型对药物的评价效果：优化后的模型对二甲双胍和匹格列酮具有很好的评价效果；同时也很好地改善了对促胰岛素分泌类药物如格列美脲的评价功效。
4.血甘油三酯和游离脂肪酸检测结果表明所用的三种药物能够很好地改善模型的高甘油三酯和高脂肪酸,特别是二甲双胍和匹格列酮高剂量组,与阴性组比较,格列美脲低剂量组低16%左右。
结论：
1.成功地优化了以C57BL／6J小鼠糖尿病动物模型,使造模时间缩短,三个因素间最佳的组合是第3周开始注射STZ,单次剂量为85mg/kg连续注射两次,每次间隔3天模型更加接近于Ⅱ型糖尿病病理生理状态。
2.为评价和筛选治疗Ⅱ型糖尿病药物提供了一个廉价以及可靠的平台。
本研究的创新之处
利用正交设计的原理来优化动物模型,动物的使用量极大地减少,使动物模型的构建和优化的成本和时间尽量减少。
Background&Objective:
Diabetes, ranking just after cancer and cardiovascular and cerebrovascular diseases, is one of the most harmful diseases to human on health and economies.90-95% of the diabetic patients are belongs to NIDDM, and 60-90% of the NIDDM patients are overweight. Therefore, to establish an animal model whose pathophysiologic changes are similar to human diabetes, may provide better understanding and treatments on diabetes.
Combination of high-fat diet and STZ is commonly used in model establishment of typeⅡdiabetes. Generally, high-fat diet (30%of fat) feeding and a small amount of STZ (200mg/kg) injection in mice has been considered to be a simple, economical and time saving method to establish model of typeⅡdiabetes. Such model can provide good evaluation of potential medicine that may treat typeⅡdiabetes and make the pathomechanism and related complications of type II diabetes more clear so that further study can be continued. However, we found that there are still some disadvantages like unsteadiness, bad evaluation of some drug, invalid evaluation of insulin secretagoguesn in this model, which might cause omission when screening new drugs that have potential value in treating typeⅡdiabetes. Therefore, further optimization of this model is obviously necessary.
There are three main factors of this model including dose of single dose, time of the first injection and injected frequency according to many documents and previous study. In order to find out the best combination, three factors and three levels orthogonal test is adopted and three factors and three levels are:single does (75mg/kg、85mg/kg、100mg/kg), time of the first injection (the first、the second、the third week), injected frequency (1、2、3 times), respectively. Find out the best combination through orthogonal test according to the orthogonal layout. Finally, examine the stability and repeatability of the model and confirm the area of application. Aim:
To optimize a rodent model of type II diabetes that would replicate the metabolic characteristics of the human syndrome and the suitable for pharmaceutical research.
Methods:
1.The three factors and three levels orthogonal test:In order to find out the best combination, three factors and three levels orthogonal test is adopted and three factors and three levels are:time of the first STZ injection (a1=the first week、a2=the second week、a3=the third week), single does (b1=75mg/kg、b2=85mg/kg、b3=100mg/kg), injected frequency (c1=1 time、c2=2 times、c3=3times), respectively. According to the orthogonal layout, combinations are ranked as followed:a1b1c1, a1b2c2、a1b3c3(c2), a2b1c2, a2b2C3(c2), a2b3c1, a3b1c3,a3b2c1, a3b3c2, Inadequate combinations are rejected on the basis of the previous documents and replaced by the level in the brackets. There-week-old weaning C57BL/6J mice were fed with high-fat diet containing 30% of fat then divided into 9 groups (8 animals per group). Weight and blood glucose were recorded every week. Fasting blood glucose was measured before the STZ injection. The method of fasting blood glucose testing is as followed:mice were fasted but free access to water for 12 hours (08:00pm-08:00am). Blood samples were collected by cutting the tail tip and tested through blood glucose meters (type: steadily SureStep, supplied by Johnson & Johnson of the United States). Animals were fed with high-fat diet sequentially after STZ injection. The changes of body weight, food intake and blood glucose were measured every week. The mice were decapitated five weeks after STZ injection, and the pancreata were taken for pathological section.
2. Oral glucose tolerance test Overnight-fasted (15 hours) mice were given a glucose solution orally (20% glucose if fasting glucose<150mg/dl or 10% glucose) and blood samples were collected from tail vein at 30,60 and 120 minutes after the glucose administration. Blood glucose curve diagram was done. Deal with the data with repeated measurement and compare the diversity between groups, a=0.05)
3. Plasma chemistry:Plasma free fatty acids, insulin level were tested with corresponding testing kits. All detecting procedures were done according to the kit instructions.
4. Histopathological detection:Pancreata of the mice were collected with scissors and scalpel after decapitation. After fixed in 4% of paraformaldehyde for more than 24h, the pancreatic tissue was dehydrated by gradient alcohol, transparentized by dimethylbenzene, embedded by wax and kept overnight at 4℃. The samples were cut into slices of 4um then affixed to the slide on the antidepinning siliconization. The dried samples were stained with HE. Pathological changes were observed in the optical microscope. Evaluate the putrescence、apoptosis and denaturation of the pancreata.
5. Investigation of stability and reproducibility of the optimize model We used different batches of mice and observed for more than 8 weeks to investigate the stability and reproducibility of the best combination.24 C57BL/6J mice were divided into three groups (8 per group):model group, control group and high-fat diet group.
6. Pharmacodynamic evaluation of the optimized model:To determine Pharmacological effects of the optimized Fat-fed/STZ mice model, we tested three kinds of drugs widely used in type 2 diabetes.100 C57BL/6J mice were used in the test and 80 were fed with high-fat diet,20 low-fat diet.70 high-fat-diet mice and 10 low-fat-diet mice were injected with STZ according to the results of orthogonal experiment. Choose 64 animals from high-fat-diet/STZ mice and then divided them into 8 groups:normal control, negative control and medication administration group. Three kinds of drugs were choosen for testing:metformin(200mg/kg/d,500mg/kg/d), glimepiride(0.1mg/kg/d,0.3mg/kg/d) and pioglitazone(10mg/kg/d,30mg/kg/d). Mice were randomized by computer 1 day before administration according to blood glucose concentration and body weight. Intragastric administration were executed 1 time per day (10mL/kg) and continued for 21 days.
Results:
1. The result indicates that injection of streptozotocin(85mg/kg) twice every five days at the third week is the best for establishing rodent type II diabetes model. Biopsy also showed appropriate destruction of islet.
2. The optimized model is stable and reproducible. The success ratio of model establishment is 90%. The blood glucose concentration was stable and can sustain until eight weeks after STZ injection.
3. Fat-fed/STZ mice is sensitive to metformin and pioglitazone. Glimeperide also has hypoglycemic effect when administrating dose is up to 0.3 mg/kg.
4. Test results of blood triglyceride and free fatty acid shows that these three kinds of drugs used in the model could well ameliorate the high triglycerides and high-fatty acids, particularly metformin and pioglitazone high-dose group. Glimepiride low-dose group is about 16%lower than the negative control group.
Conclusions:
1. The rodent model of typeⅡdiabetes is successfully optimized (pathology is more similar to type 2 diabetes of human) and the time of inducing is shorter. The best combination is as followed:the first time of STZ injection (85mg/kg) is at the third week and twice totally.
2. Provide a cheap and reliable platform for the evaluation and selection of drugs have used or will use in typeⅡdiabetes.
引文
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[2]Malecki MT. Genetics of type 2 diabetes mellitus. Diabetes Res Clin Pract, 2005,68:S10-21.
[3]ParilloM, Riccardi G. Diet composition and the risk of type 2 diabetes: epidemiological and clinical evidence. Br J Nutr,2004,92:7-19.
[4]Kahn SE. The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of type 2 diabetes. Diabetologia,2003,46, 3-19.
[5]Fritsche L, Weigert C, Haring HU, et al. How insulin receptor substrate proteins regulate the metabolic capacity of the liver-implications for health and disease. Current Medicinal Chemistry,2008,15:1316-1329.
[6]Harding HP & Ron D. Endoplasmic reticulum stress and the development of diabetes:a review. Diabetes,2002,51:S455-S461.
[7]Kaneto H, Nakatani Y, Kawamori D, et al. Role of oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-cell dysfunction and insulin resistance, International Journal of Biochemistry and Cell Biology,2006,38:782-793.
[8]Ramarao P, Kaul CL. Insulin resistance:Current therapeutic approaches. Drugs Today,1999,35:895-911.
[9]Butler AE, Janson J, Bonner-WeirS, et al. Cell deficit and increased cell apoptosis in humans with type 2 diabetes. Diabetes,2003,52,102-110.
[10]Sakuraba H, Mizukami H, Yagihashi N, et al. Reduced beta-cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese type Ⅱ diabetic patients. Diabetologia,2002,45:85-96.
[11]Burke JP, Williams K, Gaskill SP, et al. Rapid rise in the incidence of type 2 diabetes from 1987 to 1996:Results from the San Antonio Heart Study. Arch. Int.Med,1999,159:1450-1456.
[12]Mokdad AH, Bowman BA, Ford ES, et al. The continuing epidemics of obesity and diabetes in the United States. JAMA,2001,286:1195-1200.
[13]Lingohr MK, Buettner R & Rhodes CJ. Pancreatic be cell growth and survival: A role in obesity-linked type 2 diabete Trends Mol. Med,2002,8,375-384.
[14]Boujendar S, Reusens B, Merezak S, et al. Taurine supplementation to a low protein die during foetal and early postnatal life restores a normal proliferation and apoptosis of rat pancreatic islets. Diabetologia,2002,45,856-866.
[15]Finegood DT, McArthur MD, Kojwangt D, et al. β-Cell mass dynamics in Zucker diabetic fatty rats:Rosiglitazone prevents the rise in net cell death. Diabetes,2001,50:1021-1029.
[16]Goto Y, Suzuki KI, Sasaki M, et al. GK rat as amodel of non-obese, non-insulin dependent diabetes:Selective breeding over 35 generations. In E. Shafrir & A. E. Renold (Eds.), Lessons from animal studies Ⅱ,1998, (pp.301-303). London: Libbey.
[17]Miralles, F & Portha, B. Early development of beta-cells is impaired in the GK rat model of type 2 diabetes. Diabetes,2001,50(Suppl.1), S84-S88.
[18]Briaud I, Kelpe CL, Johnson LM, et al. Differential effects of hyperlipidemia on insulin secretion in islet of Langerhans from hyperglycemic versus normoglycemic rats. Diabetes,2002,51:662-668.
[19]Staffers DA, Ferrer J, Clarke WL, et al. Early-onset type-Ⅱ diabetes mellitus (MODY4) linked to IPF1. Nat. Genet,1997,17:138-139.
[20]Edlund, H. Developmental biology of the pancreas. Diabetes,2001,50(Suppl. 1), S5-S9.
[21]Johnson JD, Ahmed NT, Luciani DS, et al. Increased islet apoptosis in Pdx1+/-mice. J. Clin.Invest,2003,111:1147-1160.
[22]Withers DJ, Gutierres JS, Towery H, et al. Disruption of IRS-2 causes type-2 diabetes in mice. Nature,1998,391:900-904.
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