摘要
目的：制备胰腺组织特异性Grb1O基因敲除小鼠并确定其基因敲除表型
方法用flox-loxp技术制备Grb10flox/-小鼠,并通过传代以及杂合子交配得到Grb10loxp+/+小鼠,与PDX-Cre+/-小鼠交配,得到胰腺组织特异性Grb10基因敲除小鼠(下称"pGrb10 KO小鼠”)以及Grb10flox/-小鼠(下称“野生型小鼠”)作为对照组。从各个主要组织中提取DNA并用Southern Blot以及PCR方法在DNA层面确定Grb10基因敲除情况。用Western Blot以及免疫组化的方法从蛋白层面了解Grb10基因敲除情况以及蛋白表达情况。
结果Southern Blot显示Grb10flox/-小鼠基因标记位置正确,PCR显示在各个组织中,基因敲除仅发生在胰腺,其他组织没有影响。Western Blot显示,野生型小鼠体内Grb10在胰腺中表达量最高,在基因敲除小鼠中Grb10蛋白被完全敲除；比较pGrb10 KO小鼠和野生型小鼠,其他各组织中的Grb10蛋白表达量没有改变。免疫组化显示,Grb10在胰腺各种细胞中都有表达,在胰岛中表达量相对较高,但在pGrb10 KO小鼠胰腺中Grb10蛋白完全无表达。
结论成功制备胰腺组织特异性Grb10基因敲除小鼠,对于pGrb10 KO小鼠,Grb10蛋白在胰腺中完全敲除,在身体其他组织中没有变化。
目的研究Grb10基因在beta细胞中敲除对胰岛beta细胞胰岛素释放、胰岛大小、以及血糖代谢、胰岛beta细胞增殖的影响。
方法小鼠出生4周后,根据PCR结果设置pGrb10 KO小鼠组及野生型小鼠组(n=8-13),以标准食物喂养18周,测量其体重、摄食(从第4周开始,每周一次,至22周)、以及身体脂肪比重(第22周)、身体各个器官重量。小鼠成年后实行葡萄糖耐量实验(GTT)(了解beta细胞对葡萄糖的敏感性)、胰岛素耐量实验(ITT)(了解周围组织对胰岛素的敏感性)、空腹胰岛素水平、总胰岛细胞重量。用BrdU标记法检测野生型小鼠与pGrb10 KO小鼠胰岛beta细胞增殖率的区别。
结果pGrb10 KO小鼠在体重、摄食、身体脂肪比重、各个器官重量(除胰腺外)、周围组织对胰岛素的敏感性没有明显改变。pGrb10KO小鼠有更大的胰腺,较低的血糖水平以及明显增高的beta细胞增殖率,但空腹胰岛素水平以及总胰岛细胞重量无明显区别。
结论与野生型小鼠比较,基因敲除小鼠血糖水平相对较低,但空腹胰岛素水平以及胰岛beta细胞团重量无明显差异。pGrb10 KO小鼠有明显升高的beta细胞增殖率。
目的在2型糖尿病小鼠模型中研究Grb10基因在beta细胞中敲除对beta细胞胰岛素释放、胰岛大小、以及血糖代谢的影响
方法小鼠出生4周后,根据PCR结果设置pGrb 10 KO小鼠组及野生型小鼠组(n≥10),以60%高脂饮食喂养18周测量其体重、摄食(从第4周开始,每周一次,至22周)、以及身体脂肪比重(第22周)。实行葡萄糖耐量实验(GTT)(了解beta细胞对葡萄糖的敏感性)、胰岛素耐量实验(ITT)(了解周围组织对胰岛素的敏感性)、空腹胰岛素水平、所有胰岛细胞重量。用胰酶消化胰腺后,分离得到胰岛细胞,用IGF-1和Insulin分别刺激后,用Western Blot测定不同组小鼠p-AKT及其下游通路p-Foxo以及MAK激酶磷酸化是否改变。用透射电镜检测野生型和pGrb10 KO小鼠胰岛beta细胞内胰岛素颗粒有无区别。
结果pGrb 10 KO小鼠在体重、摄食、身体脂肪比重、周围组织对胰岛素的敏感性没有明显改变。pGrb 10 KO小鼠有明显低血糖水平(与正常饮食小鼠近)以及高胰岛素水平,明显增多的胰岛beta细胞团以及胰岛beta细胞内明显增多的胰岛素颗粒。pGrb10 KO小鼠胰岛beta细胞内Akt磷酸化水平及其下游通路Foxo磷酸化水平明显上调,MAP激酶信号通路明显上调。电镜下显示pGrb10 KO小鼠胰岛beta细胞内胰岛素颗粒明显增多。
结论在2型糖尿病模型中,与野生型小鼠比较,pGrb 10 KO小鼠血糖水平相对正常,胰岛素水平增高,胰岛beta细胞明显增多以及beta细胞内胰岛素颗粒明显增多。pGrb10 KO小鼠胰岛beta细胞内胰岛素信号通路明显上调,胰岛beta细胞内胰岛素颗粒明显增多。
目的在糖尿病小鼠模型中研究Grb10基因在beta细胞中敲除对beta细胞胰岛素释放、胰岛大小、以及血糖代谢的影响
方法选取3个月大小鼠,用STZ 75mg/kg(雄性,n=8-14)80mg/kg(雌性,n=14)注射5天后每3日在同一时间点测量血糖,体重。21天后处死小鼠,测量胰腺重量,胰岛细胞重量以及胰岛素水平。并用In Situ Cell Death Detection Kit (Roche)测定不同小鼠beta细胞凋亡情况。
结果基因敲除小鼠相对对照组小鼠有明显低的血糖水平,体重维持较平稳,beta细胞明显较多,血胰岛素水平明显较高。TUNEL实验显示pGrb10 KO小鼠比野生型小鼠有明显减低的胰岛beta细胞凋亡率。
结论基因敲除小鼠对STZ诱导的beta细胞凋亡有更好的抵抗作用
Objectives:To generate pancreas-specific Grb10 knockout mice and test the phenotype.
Methods:Grb10flox/- mice have been generated and bred for 6 generation, and then heterozygote mice have been bred with heterozygote mice to get homozygote mice. Breed Grb10flox/flox mice with PDX-Cre+/-mice then get pancreas-specific Grb10 knockout mice (pGrb10 KO mice) and Grb10flox/- mice (WT control mice). DNA has been digested from tails for Sounthern blot, and DNA has been digested from different tissues for PCR to confirm in DNA level that Grb10 has been deleted only in pancreas. Western Blot and Immunohistochemistry have been used to detect whether Grb10 protein has been removed yet.
Results:Southern Blot shows that in pGrb10 KO mice the gene target sites locate right. PCR shows that deletion happens only in pancreas, no any effect on other tissues. According to Western Blot, Grb10 is highest expressed in pancreas, but completely deleted in pGrb10 KO mice. Immunohistochemistry of pancreas shows that Grb10 expresses in all of the endocrine cells and exocrine cells, conparablly higher expresses in islets.
Conclusion:pGrb10 KO mice have been successfully generated. Grb10 has been completely deleted in pancreas in pGrb10 KO mice, without effect on any other tissues.
Objectives:Study the effect on insulin releasing from the beta cell, islet mass size and glucose tolerance after deleting Grb10 in pancreas.
Methods:PGrb10 KO mice group and WT mice group(n=8-13) have been set to measure body weight, food intake(from 4 weeks old to 22 weeks, once per week), fat percentage(22 weeks old), organ weight. Feed with normal chow for 18 weeks, Glucose Tolerance Test(GTT) will be carried out to study the insulin secretion after glucose stimulation and periphery tissues'insulin sensitivity; Insulin Tolerance Test(ITT) will be carried out to study periphery tissues'insulin sensitivity. Fasting plasma insulin level and beta cell mass weight will also be tested. Pancreatic beta cell proliferation rate will be tested by BrdU labeling.
Results:pGrb10 KO mice have no difference with WT mice in Body weight, food intake, fat percentage, different organs (excluding pancreas), and periphery tisuue insulin sensitivity. pGrb10 KO mice have bigger pancreas, lower glucose level but pGrb10 KO mice have similar insulin level and beta cell mass weight. pGrb10 KO mice have higher proliferation rate than WT mice.
Conclusion:Compared with WT control mice, pGrb10 KO mice have bigger pancreas, lower Glucose level, and much higher pancreatic beta cell proliferation rate.
Objectives:Study the effect on insulin releasing from the beta cell, islet mass size and glucose tolerance in High Fat Diet induced type 2 diabetes mice model after deleting Grb10 in pancreas.
Methods:When mice are 4 weeks old, feed them with 60%HFD for 18 weeks. pGrb10 KO micegroup and WT mice group(n≥10) have been set up to measure body weight, food intake(from 4 weeks old to 22 weeks, once per week), fat percentage(22 weeks old). Glucose Tolerance Test(GTT) will be carried out to study the insulin secretion after glucose stimulation and periphery tissues'insulin sensitivity; Insulin Tolerance Test(ITT) will be carried out to study periphery tissues'insulin sensitivity. Fasting plasma insulin level and beta cell mass weight will also be tested. Islet isolation will be done to study the detail mechanism of Grb10 effect in beta cells. After digested with Collagenase P, the pancreas tissue will be dispersed into plate and then islets will be hand-picked and culture in 12-well plate. After stimulated with IGF-1 and insulin, cells will be collected to test whether p-AKT and downstream have been changed in pGrb10 KO mice. TEM will be used to test insulin granule in pancreatic beta cell in both WT and pGrb10 KO mice.
Results:Under high fat diet, PGrb10 KO micehave no difference with WT mice in Body weight, food intake, fat percentage, and periphery tissue insulin sensitivity. pGrb10 KO micehave significantly more beta cells, much lower glucose level, higher insulin level and much more insulin granule in pancreatic beta cells. In pGrb10 KO mice, p-Akt, p-Foxo and p-Erk have been upregulated.
Conclusion:In type 2 diabetes model, compared with WT control mice, pGrb10 KO mice have lower Glucose level, higher insulin lever, much more beta cell mass and insulin granules in pancreatic beta cells. Insulin signaling has been upregulated in pGrb10 KO mice.
Objectives:Study the effect on insulin releasing from the beta cell, islet mass size and glucose tolerance in STZ-induced type 1 diabetes mice model after deleting Grb10 in pancreas.
Methods:3-months-old normal chow mice have been chosen in pGrb10 KO mice and WT control mice(n=8-14). The male mice have been injected STZ 75mg/kgBW and females have been injected 80mg/kgBW for 5 days, and then test blood glucose every 3 days at the same time point.21 days later, the mice have been sacrificed, and the pancreas weight, islets weight and blood insulin will be tested. Similar age of mice will be chosen and inject with STZ for 2days,48 hours later, mice will be sacrificed and the pancreas will be taken, fixed in 10%formalin, bedded in wax, and cut sections. In Situ Cell Death Detection Kit (Roche) will be used to detect beta cell apoptosis.
Results:Compared with Wt control mice, pGrb10 KO mice have significant low glucose level much less weight losing, significant more beta cells and much higher plasma insulin level. TUNEL assay shows that pGrb10 KO mice have lower apoptosis rate than the WT mice.
Conclusion:In type 1 diabetes model, Compared with WT control mice, pGrb10 KO micehave higher ability to conquer apoptosis.
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