TTR基因Gly83Arg突变致家族遗传性玻璃体淀粉样变性小鼠模型基因表达的鉴定

发布时间：2018-05-30 06:12

本文选题：甲状腺激素结合蛋白(TTR) + 玻璃体淀粉样变性　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:鉴定采用基因打靶技术的TTR基因Gly83Arg突变小鼠模型是否是研究Gly83Arg家族遗传性玻璃体淀粉样变性的稳定模型,并确证该突变点是否为该病遗传学分子学特征。方法:饲养SPF级F3代小鼠15只(NEO-;FLP-;TTR+/-),6只雄小鼠,9只雌小鼠。对照组小鼠(C57BL/6)10只。每周腹腔注射一次2.5%水合氯醛(0.1ml/25g)麻醉,采用复方托吡卡胺滴眼液散瞳,裂隙灯观察小鼠屈光介质,发现玻璃体浑浊即处死小鼠,送肝脏组织行基因测序;随机数字表选实验组4只与对照组小鼠4只取心脏、脑组织、肝脏、肾脏、眼球做石蜡切片,采用刚果红染色及偏振光检测淀粉样物质,免疫组化定位检测TTR蛋白;研磨肝脏提取RNA与蛋白质,荧光定量PCR检测TTR基因mRNA表达、Western Blot检测TTR基因蛋白质表达。结果:送检测序的C57BL/6模型小鼠8只均发生基因突变,第3外显子的107位碱基处出现杂合突变,第83氨基酸的密码子由GGC突变成CGC,即Gly83Arg;刚果红染色及偏振光检测TTR突变小鼠玻璃体呈阳性,肝脏、肾脏、心脏、脑组织均呈阴性;免疫组化检测结果显示TTR对照组肝脏呈阳性,玻璃体、肾脏、心脏、脑组织为阴性,模型小鼠玻璃体呈阳性,肝脏、肾脏、心脏、脑组织均为阴性;荧光定量PCR结果显示模型小鼠mRNA表达低于对照组,差异有统计学意义(t=3.030,P=0.023);Western Blot检测模型小鼠肝脏TTR基因蛋白质表达低于对照组,差异有统计学意义(t=3.224,P=0.018)。结论:1.TTR Gly83Arg确为家族遗传性玻璃体淀粉样变性的分子学特征且仅表现为眼部发病;2.TTR Gly83Arg突变的C57BL/6小鼠模型建立成功,该品系小鼠可用于家族遗传性玻璃体淀粉样变性的研究。
[Abstract]:Objective: to identify whether the mouse model of TTR gene Gly83Arg mutation using gene targeting technique is a stable model for studying Gly83Arg family hereditary vitreous amyloidosis and to confirm whether the mutation site is a genetic molecular characteristic of the disease. Methods: 15 SPF grade F3 mice were fed with 6 male mice and 9 female mice. There were 10 C57BL / 6 mice in the control group. Intraperitoneal injection of 2.5% chloral hydrate 0.1 ml / 25 g was performed once a week. Compound topiramine eye drops were used to dilate the pupil, slit lamp was used to observe the diopter of the mice, and the mice were killed when the vitreous body was opacified. The mice were sent to the liver for gene sequencing. The heart, brain, liver, kidney and eyeball of the experimental group and the control group were selected as paraffin sections. The amyloid was detected by Congo red staining and polarizing light, and TTR protein was detected by immunohistochemistry. RNA and protein were extracted from ground liver, TTR gene mRNA expression was detected by fluorescence quantitative PCR and TTR gene protein expression was detected by Western Blot. Results: all the 8 C57BL/6 model mice were mutated and heterozygous mutations were found at the 107th base of exon 3. The 83rd amino acid codon was mutated from GGC to CGC, that is Gly83 Arg; Congo red staining and polarizing light showed that the vitreous bodies of TTR mutant mice were positive, but the liver, kidney, heart and brain were all negative. The results of immunohistochemistry showed that the liver of TTR control group was positive. Vitreous body, kidney, heart and brain tissue were negative, model mice were positive in vitreous body, liver, kidney, heart and brain tissue were all negative. Fluorescence quantitative PCR showed that the expression of mRNA in model mice was lower than that in control group. The expression of TTR gene protein in the liver of the model mice was significantly lower than that of the control group (P < 0.05), and the difference was statistically significant (P < 0.05). Conclusion: 1. TTR Gly83Arg is a molecular characteristic of familial vitreous amyloidosis and only shows eye disease. 2. The model of C57BL/6 mice with TTR Gly83Arg mutation is established successfully, and this strain can be used for the study of familial inherited vitreous amyloidosis.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R776.4

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