Terc(−/−)模式小鼠的退化病徵研究

Abstract

端粒長度與細胞衰老已被證實與多種衰老疾病有高度相關。端粒酶複合物(Telomerase complex)能在特定細胞類型中維持端粒長度並保護染色體完整性,而其中端粒酶的 RNA 成分 Terc(也稱為 TR)含有端粒序列合成的模板。先前的研究顯示,在小鼠中,Terc 缺失引起會多種衰老表型,其中包括生殖缺陷。在端粒酶基因剔除小鼠後代中,隨著端粒持續的短化,Terc 剔除小鼠繁殖至第六代(G6)便無法生育。 目前,在端粒相關的老化動物模型中,需花費大量的時間繁殖端粒酶功能缺失的後代。因此,本研究的目的是建立在較年輕以及前期世代便能進行老化實驗的端粒缺失動物模型。在本研究中,我們使用先前研究所使用的同一品系 Terc 缺失小鼠(JAX stock #004132)與本地野生型 C57BL/6 品系進行交配,以獲得異質性 Terc小鼠,並進一步繁殖 Terc 缺失子代(tTerc),發現由此繁殖的 tTerc 品系僅能繁殖到第三代(G3)。我們推測是因為本地野生型 C57BL/6J 小鼠的端粒長度較短,因而繁殖出端粒較短且無法維持的 tTerc 小鼠。我們進一步針對 tTerc 小鼠進行老化程度表徵和基礎生理參數檢測,包括外觀、組織學以及血液學分析。在結果中,我們觀察到 6 個月和 9 個月的 G2 tTerc 小鼠中,各種器官(如肺、腸道和生殖器官)的退化表現。在外觀變化中,體重積累率在 tTerc 提前下降。另外透過組織切片觀察到 G2tTerc 小鼠體內高度增殖器官(如生殖器官和腸道隱窩)受損,進而造成生殖能力下降,無法產生 G3 後代。而器官的受損引發體內發炎現象與相關症狀在周邊血液分析得到證實。除此之外,我們使用 G2 tTerc 小鼠與異質性 Terc 小鼠交配以獲得G3 tTerc(iG3)和異質性 Terc 小鼠,並分析 iG3 脾臟內血球組成,結果亦顯示端粒酶缺失會造成血球比例變化。 在本研究中,我們使用組織學、血液學和生理學研究對端粒酶缺失小鼠的 tTerc品系進行了表徵分析,並證明了 tTerc 模式動物中 Terc 缺失會引起不正常衰老與發炎,其中包含: (1)產生 G3 的生殖能力顯著下降 (p-value = 0.0047); (2)提前出現體重增長停滯; (3)生殖細胞生長障礙; (4)小腸隱窩數量減少; (5)周邊血液組成變化;以及(6) iG3 中脾臟白血球變化。這些結果顯示 tTerc 小鼠與先前的端粒酶缺失品系中,提前展現了退化表現。而在未來相關的端粒研究中,tTerc 模型能加速生成端粒缺失的小鼠模型, 將此模式動物應用於多種與衰老相關的疾病。包含促進藥物開發、臨床應用,甚至是癌症治療的研究。

Telomere length is highly correlated with cellular senescence and several aging-associated diseases. The telomerase complex maintains telomere length and protects chromosome integrity in specific cell types. The RNA component of telomerase Terc(also known as TR) contains the template for telomeric sequence synthesis. Previous studies have shown that Terc deficiency in mice induces multiple aging phenotypes, including reproductive defects. Along with continuous telomere shortening displayed by the offspring of telomerase knockout mice, Terc-deficient mice are infertile by generation six (G6). Currently, significant time and cost have been expended on generating offspring and studying the effects of telomerase deficiency in aging animal models. Therefore, the aim of this study is to generate a more suitable animal model in both younger age and earlier generations for telomerase research. When the same strain of Terc-deletion mice used in above mentioned studies (JAX stock #004132) were crossed it with the local wild type C57BL/6 strain to obtain heterozygous Terc mice, which were then crossed to generate Terc-deletion progeny (tTerc), it was found that the generated tTerc strain could only be bred to generation 3 (G3), presumably because of the shorter telomere length of local wild type C57BL/6J mice. Upon characterizing aging progression in wild type and the generated tTerc line at G2, as well as basal physiological parameters, including appearance observation, histology and hematology, degenerative phenotypes in various organs, such as lungs, intestine, and sexual organs of 6- and 9-month-old G2 tTerc mice were observed. In the appearance record, we noted that the body weight accumulation rate lost was at an earlier age in G2 tTerc mice. Through histology, we observed that G2 tTerc mice showed organ damage in some highly proliferative structures. Additionally, physiological inflammation symptoms were confirmed by peripheral complete blood count. Furthermore, when we crossed G2 tTerc with heterozygous Terc mice to acquire inbred G3 tTerc (iG3) and heterozygous Terc, the analysis of iG3 splenic blood showed changes in blood cell composition in telomerase deficiency mice. In our study, we characterized the local background strain of telomerase knockout mice using histology, hematology, and physiological measures. We demonstrated that tTerc mice exhibit degenerative phenotypes at earlier generations than previous model strains. These include: (1) a significant reduction in reproductive capability to produce G3 (p-value = 0.0047); (2) an earlier arrest of body weight gain; (3) an increase in sexual organ atrophy (6 months of WT vs. G2: 99% vs. 10%, p-value <0.0001); (4) intestinal crypts distortion (9 months of WT vs. G2: 20 vs 11 crypts number/mm, p-value <0.0001); (5) changes in peripheral blood composition; and (6) an increase in granulocytes (p-value = 0.0159) and inflammatory monocytes (p-value = 0.0317), and a decrease in CD8+ T cells (p-value = 0.0159) in the analysis of iG3 splenic blood. Our thesis presents a novel strategy that accelerates the generation of a valuable mouse model for telomere research. This model can provide fundamental insights into biology and be used to study multiple aging-related diseases. By facilitating the study of drug development, clinical applications, and even cancer therapies, this model has the potential to make significant contributions to the field of telomere research.