本文選題：甲狀腺激素結(jié)合蛋白(TTR) + 玻璃體淀粉樣變性　； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:鑒定采用基因打靶技術(shù)的TTR基因Gly83Arg突變小鼠模型是否是研究Gly83Arg家族遺傳性玻璃體淀粉樣變性的穩(wěn)定模型,并確證該突變點(diǎn)是否為該病遺傳學(xué)分子學(xué)特征。方法:飼養(yǎng)SPF級(jí)F3代小鼠15只(NEO-;FLP-;TTR+/-),6只雄小鼠,9只雌小鼠。對(duì)照組小鼠(C57BL/6)10只。每周腹腔注射一次2.5%水合氯醛(0.1ml/25g)麻醉,采用復(fù)方托吡卡胺滴眼液散瞳,裂隙燈觀察小鼠屈光介質(zhì),發(fā)現(xiàn)玻璃體渾濁即處死小鼠,送肝臟組織行基因測(cè)序;隨機(jī)數(shù)字表選實(shí)驗(yàn)組4只與對(duì)照組小鼠4只取心臟、腦組織、肝臟、腎臟、眼球做石蠟切片,采用剛果紅染色及偏振光檢測(cè)淀粉樣物質(zhì),免疫組化定位檢測(cè)TTR蛋白;研磨肝臟提取RNA與蛋白質(zhì),熒光定量PCR檢測(cè)TTR基因mRNA表達(dá)、Western Blot檢測(cè)TTR基因蛋白質(zhì)表達(dá)。結(jié)果:送檢測(cè)序的C57BL/6模型小鼠8只均發(fā)生基因突變,第3外顯子的107位堿基處出現(xiàn)雜合突變,第83氨基酸的密碼子由GGC突變成CGC,即Gly83Arg;剛果紅染色及偏振光檢測(cè)TTR突變小鼠玻璃體呈陽(yáng)性,肝臟、腎臟、心臟、腦組織均呈陰性;免疫組化檢測(cè)結(jié)果顯示TTR對(duì)照組肝臟呈陽(yáng)性,玻璃體、腎臟、心臟、腦組織為陰性,模型小鼠玻璃體呈陽(yáng)性,肝臟、腎臟、心臟、腦組織均為陰性;熒光定量PCR結(jié)果顯示模型小鼠mRNA表達(dá)低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=3.030,P=0.023);Western Blot檢測(cè)模型小鼠肝臟TTR基因蛋白質(zhì)表達(dá)低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=3.224,P=0.018)。結(jié)論:1.TTR Gly83Arg確為家族遺傳性玻璃體淀粉樣變性的分子學(xué)特征且僅表現(xiàn)為眼部發(fā)病;2.TTR Gly83Arg突變的C57BL/6小鼠模型建立成功,該品系小鼠可用于家族遺傳性玻璃體淀粉樣變性的研究。
[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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R776.4

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本文編號(hào)：1954230