CN110476877A - 一种β细胞中特异性Raptor敲除同时GFP示踪的小鼠的制备方法 - Google Patents
一种β细胞中特异性Raptor敲除同时GFP示踪的小鼠的制备方法 Download PDFInfo
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Abstract
本发明涉及一种β细胞中特异性Raptor敲除同时GFP示踪的小鼠的制备方法,包括:将ROSA26‑EGFPf与RIP‑Cre小鼠交配,获得带有RIP‑Cre及EGFP序列的纯合型小鼠;再将上述纯合型小鼠与Raptorlox/lox小鼠交配后获得带有RIP‑Cre及EGFP纯合子序列的Raptorlox/w小鼠;再将这种Raptor杂合子小鼠的进行自交,获得带有RIP‑Cre及EGFP纯合子序列的Raptorlox/lox。本发明旨在说明糖尿病发病之前mTORC1对于β细胞身份和维持所起的直接作用,为mTORC1作为药物作用靶点提供了进一步的依据。
Description
技术领域
本发明属于2型糖尿病领域,特别涉及一种β细胞中特异性Raptor敲除同时GFP示踪的小鼠的制备方法。
背景技术
糖尿病是一种危害人类健康的最常见的代谢紊乱性疾病,根据国际糖尿病联盟(IDF)资料,全球有超过3亿人遭受糖尿病的困扰,到2030年将增加到5亿,而同期中国的糖尿病患病率高达11.6%,其中仅有10%的患者通过治疗得到较好的血糖控制。糖尿病已成为继肿瘤和心脑血管疾病之后的第三大非传染性疾病,对国民健康和经济发展造成了严重的影响,因此加强对糖尿病发病机制和治疗靶点的研究是十分必要和迫切的。2型糖尿病以胰岛β细胞功能障碍伴随外周胰岛素抵抗为主要特点,其发病的中心环节是功能性β细胞质量的逐渐丢失,最终导致胰岛素分泌不能满足代谢需求,功能性β细胞质量不仅取决于单个β细胞的功能,还受β细胞绝对数量的控制,而后者又受增殖、新生、凋亡以及内分泌细胞之间的相互转化等多种因素平衡。最近一系列的研究提示胰岛β细胞存在身份脆弱性和内在可塑性,分化成熟的β细胞在某些条件下可不同程度地丧失其身份属性并且退化到分化程度较低的内分泌前体细胞状态,这个概念被称为β细胞的去分化,去分化细胞具有以下几个特征:1)包含关键转录因子、葡萄糖代谢和胰岛素加工分泌相关基因在内的β细胞重要基因下调;2)正常β细胞中不表达或者表达量很低的不允许基因上调;3)内分泌祖细胞相关基因的上调。
雷帕霉素理论性靶点蛋白(mTOR)是一个高度保守的丝氨酸/苏氨酸激酶,mTOR信号通路依赖于它的两个复合体mTORC1和mTORC2而起作用。mTORC1由RAPTOR,mLST8,PRAS40,DEPTOR和mTOR组成,对于雷帕霉素的抑制作用比较敏感,感应生长因子、激素、能量状态、供氧及氨基酸等上游的信号,从而对细胞的蛋白质和脂质合成、自噬等生理过程起到关键调控作用。mTORC1活性在2型糖尿病病人和糖尿病小鼠模型中上调表明其在糖尿病进展期间的适应和失代偿中起到关键作用。广泛的研究表明,生理状态下mTORC1的激活对于β细胞的发育、生长、功能和存活至关重要,而其过度激活可能导致β细胞衰竭。在体外通过过表达脑Ras同系物蛋白(Rheb)或者敲除结节性硬化综合征复合物1/2(TSC1/TSC2)从而持续性激活mTORC1可导致β细胞功能性质量和胰岛素分泌的增加,相反,过表达mTOR突变体的小鼠由于功能相关性因子Pdx1的缺陷,导致其在高脂饮食下出现葡萄糖不耐受。
目前尚不清楚mTORC1是否可以调节β细胞的身份属性以及Raptor敲除小鼠功能障碍是否不依赖于糖代谢水平。
发明内容
本发明所要解决的技术问题是提供一种β细胞中特异性Raptor敲除同时GFP示踪的小鼠的制备方法,该方法旨在说明糖尿病发病之前mTORC1对于β细胞身份和维持所起的直接作用,为mTORC1作为药物作用靶点提供了进一步的依据。
本发明提供了一种β细胞中特异性Raptor敲除同时GFP示踪的小鼠的制备方法,包括:
将ROSA26-EGFPf与RIP-Cre小鼠交配,获得带有RIP-Cre及EGFP序列的纯合型小鼠;再将带有RIP-Cre及EGFP序列的纯合型小鼠与Raptorlox/lox小鼠交配后获得带有RIP-Cre及EGFP纯合子序列的Raptorlox/w小鼠;再将这种Raptor杂合子小鼠的进行自交,获得带有RIP-Cre及EGFP纯合子序列的Raptorlox/lox和Raptorw/w小鼠,前者即为β细胞中特异性Raptor敲除同时GFP示踪的小鼠βRapKOGFP。
通过流式分选技术验证小鼠GFP阳性细胞分选效率。
通过全式金DNA抽提试剂盒进行小鼠DNA抽提及基因分型鉴定。
有益效果
本发明构建了特异性的β细胞Raptor敲除绿色荧光蛋白GFP示踪小鼠,通过对野生型小鼠、糖尿病敲除小鼠和正常血糖敲除小鼠进行比较,探究在无高血糖影响下,特异性Raptor敲除后的β细胞是否存在身份改变和功能障碍,并且结合野生型小鼠、糖尿病敲除小鼠以及正常血糖敲除小鼠三组β细胞的RNA测序结果,旨在说明糖尿病发病之前mTORC1对于β细胞身份和维持所起的直接作用,为mTORC1作为药物作用靶点提供了进一步的依据。
附图说明
图1为本发明小鼠的构建;其中,A为GFP谱系追踪的βRapKOGFP小鼠胰岛中GFP(红色)与胰岛素(绿色)阳性细胞免疫荧光;B为Raptor在8周龄小鼠胰岛β细胞、心脏、肾脏、肝脏和下丘脑的相对表达量;C为westernblot显示Raptor以及mTORC1下游靶分子PS6和4EBP-1的蛋白表达水平;D为8周龄对照及敲除小鼠打散的胰岛进行PS6(红色)和胰岛素(绿色)的免疫荧光染色。
图2为βRapKOGFP小鼠β细胞中α细胞特征性分子的表达;其中,A为8周龄WT和βRapKOGFP小鼠胰腺切片中进行GFP(红色)与胰岛素(白色)和胰高血糖素(绿色)免疫共染色;B为8周龄WT和βRapKOGFP小鼠中GFP与胰高血糖素共染的细胞占所有GFP阳性细胞的比例;C和D为8周龄WT和βRapKOGFP小鼠胰腺切片中对胰岛素(白色),胰高血糖素(绿色)和Arx/MafB(红色)进行免疫荧光共染。
图3为βRapKOGFP小鼠治疗模型构建;其中,A为胰岛素缓释泵植入示意图;B为从4周龄开始隔天监测βRapKOGFP小鼠和胰岛素治疗βRapKOGFP小鼠的随机血糖直至8周龄。
图4为高血糖纠正后βRapKOGFP小鼠胰岛结构及MafA的表达;其中,A为在8周龄WT、糖尿病βRapKOGFP和正常血糖βRapKOGFP小鼠胰腺切片中对胰岛素(绿色)和胰高血糖素(红色)进行免疫荧光共染;B为在8周龄WT、βRapKOGFP和正常血糖βRapKOGFP小鼠胰腺切片中进行胰岛素(白色)、胰高血糖素(绿色)和MafA(红色)的免疫荧光共染。
图5为糖尿病βRapKOGFP和正常血糖βRapKOGFP小鼠β细胞RNA测序结果分析;其中,A为三组小鼠β细胞RNA测序结果差异基因数目;B为受Raptor调控的所有基因在热图中的层级聚类。
图6为qRT-PCR验证β细胞功能相关基因在WT,糖尿病βRapKOGFP以及正常血糖βRapKOGFP三组β细胞中的相对表达水平。
图7为三组小鼠胰岛素分泌相关蛋白表达水平和胰岛内ATP含量;其中,A为在WT,βRapKOGFP以及正常血糖βRapKOGFP胰腺切片中对Glut2(红色)和胰岛素(绿色)进行免疫荧光共染;B为在WT,βRapKOGFP以及正常血糖βRapKOGFP胰腺切片中对Ucn3(红色)和胰岛素(绿色)进行免疫荧光共染;C为测定WT,糖尿病βRapKOGFP以及正常血糖βRapKOGFP三组小鼠的胰岛内ATP含量。
图8为三组小鼠葡萄糖刺激胰岛素合成及分泌测定;其中,A为三组小鼠胰腺内C肽含量测定;B为三组小鼠胰腺内胰岛素原含量测定;C为体外低糖(2.8mM)和高糖(16.7mM)刺激WT,糖尿病βRapKOGFP以及正常血糖βRapKOGFP三组小鼠胰岛后C肽的分泌;D为高糖(16.7mM)相对于低糖(2.8mM)C肽分泌量的相对指数。
图9为三组小鼠胰岛素剪切相关指标变化;其中,A为胰腺内胰岛素原与C肽含量比率;B为胰岛内胰岛素原与C肽含量比率;C为显示胰岛素原在三组小鼠胰岛中的分布情况。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
1.βRapKOGFP小鼠的构建
SPF(Specific pathogen Free,无特定病原体)级Raptorlox/lox的C57BL/6小鼠和ROSA26-EGFPf混合背景小鼠购自Jackson Laboratory。
将8周龄性成熟以后的雄性小鼠与雌性小鼠合笼,在所生小鼠满3周龄后进行分笼,同时剪取少量指甲组织编号并用于后续基因型鉴定试验。将ROSA26-EGFPf与RIP-Cre小鼠交配,获得带有RIP-Cre及EGFP序列的纯合型小鼠。通过流式分选技术验证小鼠GFP阳性细胞分选效率。按同样的方式带有RIP-Cre及EGFP序列的纯合型小鼠与Raptorlox/lox小鼠交配后获得带有RIP-Cre及EGFP纯合子序列的Raptorlox/w小鼠。再将这种Raptor杂合子小鼠的进行自交,获得带有RIP-Cre及EGFP纯合子序列的Raptorlox/lox和Raptorw/w小鼠,前者即为β细胞中特异性Raptor敲除同时GFP示踪的小鼠(βRapKOGFP),后者为带有GFP荧光蛋白的野生型小鼠(WT),两组均将雄性小鼠用作后续实验。
2.胰岛素缓释泵灌注方法及体内植入手术
在灌注和植入过程中应全程佩戴手套,皮肤表面的大量油脂可能会影响胶囊性能,胶囊一旦污染后,应使用70%的异丙醇擦拭,切忌使用异丙醇浸泡,注意灌注药物要在常温下进行。
1)取10ml注射器及1ml注射器若干,准备好生理盐水以及短效胰岛素笔芯诺和灵R,称量空胶囊以及它的流量调节器(总重);
2)先将胰岛素及生理盐水按比例稀释混匀后置于0.6ml的EP管内(胶囊可容纳100μl液体,但配200μl);
3)拔掉流量调节器,使胶囊直立,首先用1ml注射器吸取生理盐水冲洗灌流器,然后将灌流管插入胶囊顶部的小开口,直到底为止,此时灌流管的顶端已接近胶囊的底部;
4)利用1ml的注射器抽取稀释好的胰岛素缓慢灌装胶囊,若使用较大注射器,由于灌装速度过快,可能会在胶囊中产生气泡;
5)保持胶囊直立,缓慢推注射器的活塞,当药物溶液到达胶囊顶部即将溢出时停止灌注,移去灌注管;
6)擦掉溢出的胰岛素溶液,将流量调节器插入直到白色帽子到达胶囊顶部为止,插入的流量调节器将会取代部分已经灌注到胶囊中的药物溶液,溢出的溶液擦拭干净,确保该装置正常运行,需要将流量调节器全部插进胶囊中。胶囊植入小鼠体内前将其浸泡在生理盐水中;
7)将小鼠用戊巴比妥麻醉后,在其背部肩胛骨间的皮肤处剪开一小口,然后用止血钳将皮肤和皮下结缔组织分开,形成一个小袋结构,将胶囊置于此小袋中,流量调节器方向朝里,皮肤开口缝合后使用抗生素溶液擦拭;
8)小鼠麻醉苏醒后立即监测其血糖水平,从手术第二天起隔天监测胰岛素泵植入小鼠的随机血糖水平。
3.测试结果
1)β细胞特异性GFP示踪的Raptor敲除小鼠(βRapKOGFP)构建
①为了探究mTORC1在β细胞分化命运中的调控作用,构建了β细胞特异性Raptor敲除GFP示踪的小鼠,Raptor作为mTORC1复合物中的重要组成部分,其蛋白表达的下降使mTORC1复合物无法形成。验证了βRapKOGFP小鼠的构建效能,首先GFP信号在βRapKOGFP小鼠中仅表达于胰岛素阳性细胞中,证明GFP阳性细胞全部来源于β细胞(图1A)。Raptor的mRNA水平在β细胞中几乎检测不到,但在其他组织如心脏、肾脏、肌肉、肝脏和下丘脑中大量表达(图1B)。western blot结果显示胰岛β细胞内Raptor蛋白表达水平显著下调,mTORC1两个重要的下游分子PS6和4EBP-1的磷酸化水平也明显降低(图1C)。此外,敲除小鼠胰岛打散后在胰岛素阳性细胞中检到mTORC1活性的丧失(图1D)。以上结果证明βRapKOGFP小鼠模型已构建成功。
②Raptor敲除的β细胞获得α细胞身份特征
βRapKOGFP小鼠的代谢表型与之前的研究结果一致:1)βRapKOGFP小鼠4周起出现随机血糖和6小时空腹血糖的升高;2)βRapKOGFP小鼠8周出现糖耐量异常;3)8周βRapKOGFP小鼠血浆胰岛素水平下降。已对8周龄βRapKOGFP小鼠和野生型小鼠(WT)的胰腺切片进行GFP,胰岛素和胰高血糖素的免疫荧光共染色,发现在敲除小鼠中的胰岛中,由GFP阳性信号示踪的β细胞来源细胞中出现明显的胰岛素和胰高血糖素的共染(图2A),计数后发现相较于对照小鼠,这样的细胞在所有GFP阳性细胞中的比例明显增高(6.65±0.27%vs 0.10±0.01%,P<0.001)(图2B)。
由β细胞来源的GFP阳性细胞中出现了重要的α细胞重要蛋白胰高血糖素的表达,首先对两组小鼠中胰高血糖素和Ki67共表达细胞进行计数后发现并无差异,这提示增多的α细胞并非来源于新的增殖,由此提出假设,这些β细胞可能发生了向α细胞的转化,接下来验证了调控α细胞命运的关键转录因子在这些激素共表达的细胞中的水平,发现α细胞转录因子无芒相关同源框(Arx)和肌腱膜纤维肉瘤癌基因同系物B(MafB)均在多激素细胞中出现表达(图2C)。以上结果提示Raptor敲除的β细胞获得了α细胞身份特征。
2)βRapKOGFP小鼠胰岛素治疗模型构建
①βRapKOGFP小鼠胰岛素治疗方案及血糖监测
为了探究Raptor敲除和高血糖两者分别在胰岛β细胞向α细胞的转化中所起的独立作用,将含有胰岛素的缓释胶囊植入在4周龄的βRapKOGFP小鼠体内(随机血糖以及空腹6小时血糖开始升高的时间),使其血糖在整个观察周期中维持在与WT同等的水平(图3A)。正如预期的那样,胰岛素缓释泵(每天释放约0.2-0.3U)植入当天,βRapKOGFP小鼠的平均血糖由12.86±0.37mM迅速下降至5.43±0.96mM,植入后小鼠平均血糖维持在8.78±0.62mM(图2B)。每隔一天对治疗小鼠的血糖进行监测持续共4周时间,而未经治疗的βRapKOGFP小鼠从4周龄起表现出逐渐严重的高血糖(22.45±0.73)(图3B),因此在后文中将未经治疗的小鼠称为糖尿病βRapKOGFP小鼠,而胰岛素治疗的小鼠称为血糖正常βRapKOGFP小鼠。
②通过胰岛素治疗维持βRapKOGFP小鼠血糖在正常水平
利用免疫荧光技术对胰岛素治疗以后的血糖正常βRapKOGFP小鼠的胰岛形态和MafA的表达进行了评估,发现胰岛形态在正常血糖小鼠中胰岛结构(图4A)与MafA的蛋白水平也与野生型小鼠无异常(图4B),以上结构均证实我们的胰岛素治疗模型是行之有效的。
③糖尿病βRapKOGFP小鼠和正常血糖βRapKOGFP小鼠的β细胞基因表达谱具有高度的相似性
进一步,在胰岛素治疗4周以后利用流式技术分选出8周龄WT、糖尿病βRapKOGFP和正常血糖βRapKOGFP三组小鼠胰岛中的β细胞来源的GFP阳性细胞,抽提mRNA逆转录成cDNA后建库进行RNA测序分析。测序结果显示WT与糖尿病βRapKOGFP两组间有334个差异基因(FoldChange≥1.5,Pvalue<0.05),WT与正常血糖βRapKOGFP两组间有113个差异基因,而糖尿病βRapKOGFP和正常血糖βRapKOGFP两组间仅有61个差异基因(图5A)。接着对Raptor调控的334个差异基因在三组间进行分层聚类分析,结果显示糖尿病βRapKOGFP和正常血糖βRapKOGFP两组聚集最为紧密,而糖尿病βRapKOGFP和WT两组之间则最为遥远(图5B),以上结果提示糖尿病βRapKOGFP小鼠和正常血糖βRapKOGFP小鼠的β细胞基因表达谱具有高度的相似性,说明Raptor敲除后引起的β细胞身份和代谢表型的改变可能主要来源于mTOR本身。
3)高血糖和Raptor对β细胞功能的不同影响
①正常血糖βRapKOGFP小鼠存在代谢异常
利用RT-PCR确认了一些重要基因mRNA水平的改变。观测到β细胞葡萄糖转运体Glut2,细胞色素c氧化酶复合物Cox6a2以及NAD(P)H脱氢酶(醌)家族成员Nqo1的显著下降(图6);此外,胰岛素结晶/分泌所需的Slc30a8和参与胰岛素颗粒运输和对接的Sytl4,Rab37的表达水平在糖尿病βRapKOGFP和正常血糖βRapKOGFP两组中均是显著降低的,RT-PCR的结果也显示出β细胞“不允许基因”的异常上调。
Glut2是胰岛素分泌途径中的重要分子。免疫荧光实验证实糖尿病βRapKOGFP胰岛中Glut2蛋白的表达水平明显降低,并且这种显著变化在高血糖纠正以后并没有逆转(图7A)。3型尿皮质激素(Ucn3)是由Melton实验室通过不同年龄小鼠的基因表达谱筛选出来的β细胞功能性成熟的标记物,Ucn3仅表达于β细胞中,并且是小鼠葡萄糖刺激胰岛素分泌过程中所必须的一个重要蛋白,对Ucn3进行免疫荧光的染色发现,其蛋白表达在糖尿病βRapKOGFP胰岛中严重受损,并且在正常血糖βRapKOGFP的胰岛中没有得到改善(图7B)。
线粒体代谢异常是β细胞衰竭过程中的一个重要特征,其可能是β细胞去分化的一个重要致病因素,线粒体复合物功能异常可直接导致ATP生成减少。糖尿病βRapKOGFP和正常血糖βRapKOGFP两组胰岛中葡萄糖刺激胰岛素分泌所需要的ATP水平严重降低(图7C)。
②正常血糖βRapKOGFP小鼠胰岛素合成与分泌功能障碍
在外源性胰岛素治疗后正常血糖βRapKOGFP小鼠胰腺中C肽的含量完全恢复至野生型小鼠的水平(图8A),但与WT小鼠相比,胰腺内胰岛素原的含量仍然显著降低(图8B),提示Raptor直接导致胰岛素合成功能障碍。为了进一步探究正常血糖βRapKOGFP在面对高糖负荷时的胰岛素分泌能力,分离了三组小鼠的胰岛,在体外分别用低浓度葡萄糖(2.8mM)和高浓度葡萄糖(16.7mM)对其进行刺激,为排除外源性胰岛素治疗的干扰,用C肽的分泌量来衡量三组小鼠胰岛的分泌能力。结果显示,与WT组相比,糖尿病βRapKOGFP和正常血糖βRapKOGFP两组的胰岛在低糖情况下均表现出基础C肽分泌量的增加,提示胰岛素分泌的葡萄糖阈值增加,但是在高糖情况下,葡萄糖刺激的C肽分泌量显著减少(图8C),正常血糖βRapKOGFP组的胰岛其高糖相较于低糖分泌指数的变化(图8D)提示了Raptor敲除本身是胰岛素分泌反应受损的原因。
③糖尿病βRapKOGFP小鼠胰岛素加工剪切过程,活性氧生成和抗炎相关基因的表达受高血糖影响
胰腺(图9A)和胰岛(图9B)中胰岛素原/C肽的比例在糖尿病βRapKOGFP小鼠中均有所增加,但在WT和正常血糖βRapKOGFP小鼠中这种异常被完全纠正,Manuel Blandino-Rosano等人报道了特异性Raptor敲除的β细胞存在存在胰岛素剪切加工障碍,而结果提示在糖尿病βRapKOGFP小鼠中观察的胰岛素加工剪切过程的缺陷可能是由持续的高血糖代谢压力所造成的。
通过对三组小鼠进行胰岛素原的免疫荧光染色现,在糖尿病βRapKOGFP胰岛中,胰岛素原的染色则显示出较为弥散的细胞定位,表明胰岛素原加工成成熟胰岛素的减少,当高血糖得以纠正之后,胰岛素原则可被正常加工并保持其在细胞内的正常定位(图9C)。以上结果进一步提示糖尿病βRapKOGFP小鼠中观察的胰岛素加工剪切过程的缺陷可能是由持续的高血糖代谢压力所造成的。
4.讨论
在本实施例中,通过给4周龄βRapKOGFP小鼠植入胰岛素缓释泵的方法以消除高血糖带来的代谢压力以研究mTORC1信号通路对于β细胞身份特性和可塑性的直接影响。
在血糖正常的βRapKOGFP小鼠中观察到胰岛结构和MafA的表达已经被完全恢复,然而在去除代谢应激后β细胞去分化和重编程现象依旧存在。即使在正常血糖的βRapKOGFP小鼠中β细胞的身份也极为不稳定并且获得了α细胞的身份特征,包括:1)正常血糖的βRapKOGFP小鼠中存在部分β细胞重要身份标记蛋白Ucn3、Nkx6.1和Glut2的表达缺失,β细胞去分化标记物Aldh1a3的表达增加;2)GFP谱系追踪发现8周龄正常血糖的βRapKOGFP小鼠中所有GFP阳性细胞中GFP和胰高血糖素共阳性细胞的百分比仍然是WT小鼠中的13倍之多;3)RNA测序结果发现8周龄正常血糖的βRapKOGFP小鼠胰岛β细胞中有一组α细胞富集基因(Pyy,Etv1,Ace2,Slc38a5等)的上调;4)RNA测序结果发现8周龄正常血糖的βRapKOGFP小鼠胰岛β细胞中线粒体代谢相关基因(Idh2,Atp4a,Cox6a2,mt-Co3,mt-Co1,mt-Nd2,Nqo1,)发生明显下调,ATP合成受损;5)2周龄βRapKOGFP小鼠胰岛中出现α细胞重要转录因子MafB的表达。
根据以上实验结果,可以认为mTORC1信号通路的缺失主要介导βRapKOGFP小鼠胰岛β细胞的功能障碍,而高血糖参与了部分代偿性过程。
Claims (3)
1.一种β细胞中特异性Raptor敲除同时GFP示踪的小鼠的制备方法,包括:
将ROSA26-EGFPf与RIP-Cre小鼠交配,获得带有RIP-Cre及EGFP序列的纯合型小鼠;再将带有RIP-Cre及EGFP序列的纯合型小鼠与Raptorlox/lox小鼠交配后获得带有RIP-Cre及EGFP纯合子序列的Raptorlox/w小鼠;再将这种Raptor杂合子小鼠的进行自交,获得带有RIP-Cre及EGFP纯合子序列的Raptorlox/lox和Raptorw/w小鼠,前者即为β细胞中特异性Raptor敲除同时GFP示踪的小鼠βRapKOGFP。
2.根据权利要求1所述的制备方法,其特征在于:通过流式分选技术验证小鼠GFP阳性细胞分选效率。
3.根据权利要求1所述的制备方法,其特征在于:通过全式金DNA抽提试剂盒进行小鼠DNA抽提及基因分型鉴定。
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CN113025707A (zh) * | 2021-05-19 | 2021-06-25 | 北京中医药大学 | 生物标志物在制备诊断湿热困脾型2型糖尿病的产品中的应用、试剂盒 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2755027A1 (en) * | 2013-01-11 | 2014-07-16 | Albert-Ludwigs-Universität Freiburg | Modulators of the interaction of astrin and raptor, and uses thereof in cancer therapy |
CN106172183A (zh) * | 2016-07-06 | 2016-12-07 | 上海市内分泌代谢病研究所 | 一种用于mTORC1通路的小鼠模型及其建立方法 |
CN107684622A (zh) * | 2017-09-05 | 2018-02-13 | 上海市内分泌代谢病研究所 | mTORC1在调控胰岛β细胞功能和转化药物中的用途 |
CN107858373A (zh) * | 2017-11-16 | 2018-03-30 | 山东省千佛山医院 | 内皮细胞条件性敲除ccr5基因小鼠模型的构建方法 |
-
2019
- 2019-08-15 CN CN201910753780.XA patent/CN110476877A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2755027A1 (en) * | 2013-01-11 | 2014-07-16 | Albert-Ludwigs-Universität Freiburg | Modulators of the interaction of astrin and raptor, and uses thereof in cancer therapy |
CN106172183A (zh) * | 2016-07-06 | 2016-12-07 | 上海市内分泌代谢病研究所 | 一种用于mTORC1通路的小鼠模型及其建立方法 |
CN107684622A (zh) * | 2017-09-05 | 2018-02-13 | 上海市内分泌代谢病研究所 | mTORC1在调控胰岛β细胞功能和转化药物中的用途 |
CN107858373A (zh) * | 2017-11-16 | 2018-03-30 | 山东省千佛山医院 | 内皮细胞条件性敲除ccr5基因小鼠模型的构建方法 |
Non-Patent Citations (1)
Title |
---|
QINGLEI YIN等: "Raptor determines β-cell identity and plasticity independent of hyperglycemia in mice", 《NATURE COMMUNICATIONS》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113025707A (zh) * | 2021-05-19 | 2021-06-25 | 北京中医药大学 | 生物标志物在制备诊断湿热困脾型2型糖尿病的产品中的应用、试剂盒 |
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