胰岛β细胞及脑组织特异性杀除的糖尿病模型构建及分析

发布时间：2018-09-10 06:11

【摘要】：糖尿病是由多种病因引起的以血糖紊乱为主要特征及多种并发症为主要损害的代谢紊乱性疾病,其发病机制尚未完全阐明。传统的观点认为,胰脏是血糖调节的唯一中心。最新的研究结果表明,体内血糖水平主要是受脑部神经系统和胰岛共同调节的。在本研究中我们尝试用遗传学手段,通过特异性β细胞和脑细胞杀除来直接研究胰岛β细胞和脑细胞在糖尿病中的调控机制。首先,本研究制备了β细胞特异性表达CreERT2的小鼠(Ins1CreERT2),与Cre/loxP条件性表达白喉毒素的Rosa26DTA176小鼠进行杂交,获得Ins1CreERT2;Rosa26DTA176小鼠。之后,我们通过他莫昔芬诱导白喉毒素表达,实现时空特异性的胰岛β细胞杀除。组织学鉴定结果显示,经过他莫昔芬诱导2周后,该小鼠模型的胰岛β细胞出现大面积的死亡现象,接近80%,成功实现了胰岛β细胞特异性杀除。然而,值得关注的是,血糖浓度测定和血糖耐受实验结果却显示,本模型中并未观察到血糖升高及血糖耐受等糖尿病表型。通过免疫荧光染色实验发现,在这些β细胞杀除小鼠的胰岛结构之外存在一些能够分泌胰岛素的Ngn3阳性细胞。然而,不同于传统的小鼠糖尿病模型,β细胞特异性杀除之后,我们的小鼠模型并没有检测到相应的脑部损伤。以上结果证实,利用β细胞特异性启动子Ins1结合Cre/loxP白喉毒素自杀系统,我们成功获得了小鼠胰岛β细胞特异性杀除模型,本模型表现出β细胞杀除与糖尿病表型不对应的特点,为研究非胰岛区域,特别是脑部神经系统对体内血糖水平的调控打下了良好的基础。为了进一步探索脑部神经系统与胰岛素分泌之间的关系,本研究构建了脑靶向性穿膜肽RVG介导的RVG-Cre重组酶递送系统。本研究首先在分子、细胞等不同水平对RVG-Cre重组酶递送系统的定点重组酶的活性进行了验证。之后我们利用mTmG和Rosa26lacZ转基因小鼠对RVG-Cre重组酶递送系统的酶活性及脑组织特异性进行了进一步地验证。结果表明,RVG-Cre重组酶递送系统可以高效率地穿过血脑屏障进入小鼠脑部细胞,并且能够在活体水平对脑部组织进行高效率、高特异性的基因编缉工作。进一步实验表明,用RVG-Cre重组酶递送系统注射Rosa26DTA176小鼠,可以在活体水平诱导其脑部特异性损伤;HE染色显示脑部与血糖调节相关的A5区域受到了损伤,甚至形成了脑组织坏死空洞。以上结果证实,RVG-Cre重组酶递送系统的建立,能够实现脑部精确的定位以及基因编辑能力,并特异性的在白喉毒素Rosaa26DTA176小鼠体内实现小鼠脑部精确杀伤。综上所述,本研究成功获得了胰岛β细胞特异性杀伤的Ins1CreERT2;Rosa26DTA176小鼠模型。同时,我们获得了能够导致Rosa26DTA176小鼠脑部细胞特异性杀伤的RVG-Cre重组酶递送系统。该小鼠模型和递送系统的建立,为研究脑部神经系统和体内血糖代谢之间的调控关系奠定了理论和实验的基础。
[Abstract]:Diabetes mellitus is a metabolic disorder disease caused by many kinds of etiology, whose main characteristics are blood sugar disorder and many complications. The pathogenesis of diabetes mellitus has not been fully elucidated. Traditionally, the pancreas is the only center of blood glucose regulation. The latest findings suggest that blood sugar levels are mainly regulated by the brain's nervous system and islets. In this study, we tried to study directly the regulation mechanism of islet 尾 cells and brain cells in diabetes mellitus by means of genetics and specific 尾 cell and brain cell killing. Firstly, the 尾 -cell-specific CreERT2 expressing mice (Ins1CreERT2) were prepared and hybridized with Cre/loxP conditioned diphtheria toxin Rosa26DTA176 mice to obtain Ins1CreERT2;Rosa26DTA176 mice. After that, diphtheria toxin expression was induced by tamoxifen to achieve space-time specific islet 尾 cell killing. The histological results showed that after 2 weeks of tamoxifen induction, the islet 尾 cells of the mouse model had a large area of death, which was close to 80%, and the specific killing of islet 尾 cells was achieved successfully. However, the results of blood glucose concentration determination and glucose tolerance test showed that no diabetes phenotypes such as hyperglycemia and glucose tolerance were observed in this model. By immunofluorescence staining, it was found that there were some Ngn3 positive cells which could secrete insulin besides the islet structure of mice killed by these 尾 cells. However, unlike the traditional mice model of diabetes mellitus, after the specific killing of 尾 cells, our mouse model did not detect the corresponding brain damage. The above results confirmed that the murine islet 尾 -cell specific killing model was successfully obtained by using 尾 -cell-specific promoter Ins1 and Cre/loxP diphtheria toxin suicide system. The model showed that 尾 -cell killing did not correspond to the phenotype of diabetes mellitus. It lays a good foundation for the study of the regulation of blood glucose levels in the non-islet area, especially in the nervous system of the brain. In order to further explore the relationship between the brain nervous system and insulin secretion, a brain targeted transmembrane peptide (RVG) mediated RVG-Cre recombinant enzyme delivery system was constructed. In this study, the activity of site-directed recombinant enzyme of RVG-Cre recombined enzyme delivery system was verified at different molecular and cellular levels. Then we used mTmG and Rosa26lacZ transgenic mice to further verify the enzyme activity and brain tissue specificity of RVG-Cre recombinant enzyme delivery system. The results showed that the RVG-Cre recombinant enzyme delivery system could efficiently penetrate the blood-brain barrier into the brain cells of mice and perform efficient and highly specific gene coding on the brain tissue at the in vivo level. Further experiments showed that injection of Rosa26DTA176 mice with RVG-Cre recombinant enzyme delivery system could induce specific brain injury in vivo. He staining showed that the A5 region related to blood glucose regulation was damaged, and even formed a necrotic cavity in brain tissue. The above results confirm that the RVG-Cre recombination enzyme delivery system can realize the precise localization and gene editing of the brain and the specific killing of mouse brain in diphtheria toxin Rosaa26DTA176 mice. In conclusion, the Ins1CreERT2;Rosa26DTA176 mouse model of islet 尾-cell specific killing was successfully obtained. At the same time, we have obtained the RVG-Cre recombinant enzyme delivery system which can induce specific killing of brain cells in Rosa26DTA176 mice. The establishment of the mouse model and delivery system provides a theoretical and experimental basis for the study of the regulation of blood glucose metabolism in the brain nervous system and in vivo.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.1;R-332

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