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標題: | NRIP的表現會隨著老化下降且AAV-NRIP治療可以增加老化小鼠的肌肉纖維面積 The NRIP expression declines with aging and AAV-NRIP can increase muscle fiber size in aging mice |
作者: | 謝欣芸 Hsin-Yun Hsieh |
指導教授: | 陳小梨 Show-Li Chen |
關鍵字: | 核受體結合蛋白,基因治療,轉基因小鼠,人類肌肉組織,免疫組織化學染色, NRIP,gene therapy,aging,transgenic mice,human muscle tissue,IHC, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 核受體相互作用蛋白(NRIP),又稱為DCAF6(DDB1和CUL4相關因子6)或IQWD1,是一種核蛋白,由860個氨基酸組成,具有七個WD-40結構域和一個IQ結構。在我們之前的研究中,我們發現NRIP在老化的老鼠骨骼肌和脊髓中的表達下降。此外,16週大的特定於肌肉的NRIP基因敲除(cKO)小鼠表現出異常肌肉和運動缺陷,與老化小鼠相似。這表明NRIP可能參與與年齡相關的肌肉功能障礙。
因此,我們希望通過進行AAV-NRIP基因治療來研究NRIP是否能改善老化小鼠的運動功能。結果顯示,AAV-NRIP能夠恢復老化小鼠的肌肉力量和神經肌肉接合點(NMJ)大小。為進一步研究AAV-NRIP基因治療在老化小鼠中的效果,本研究評估了肌纖維大小、氧化纖維表達水平、肌纖維類型分佈以及myogenin的表達。我們發現AAV-NRIP基因治療能夠恢復肌纖維大小,並提高老化小鼠中AAV-NRIP感染細胞中的myogenin表達。 接下來,我們對老化小鼠全身肌肉中NRIP過度表達的影響很感興趣。因此,在我們的實驗室中生成了特定表達肌肉中NRIP的NRIP轉基因小鼠(Rosa-NRIP-cre小鼠)。我們測量了NRIP轉基因小鼠的運動能力,包括運動活動能力、旋轉桿測試和握力測試。這些行為測試的結果到小鼠60週為止都沒有顯示出顯著差異,這表明NRIP轉基因小鼠到60週大時都沒有改善運動能力的情況。因此後續還需繼續進行運動能力測試以觀察NRIP是否有助改善小鼠老化時的運動功能。 最後,為了驗證NRIP在人類骨骼肌中的表達水平隨年齡的變化,我們對人類骨骼肌組織的微陣列進行了免疫組織化學染色。在對正常骨骼肌組織和疾病骨骼肌組織(尤其是橫紋肌肉瘤)進行NRIP表達評分後,我們通過統計分析分析了不同病例的評分。結果表明,正常人類骨骼肌中的NRIP表達隨著年齡的增長而下降,這與先前的研究結果相一致,顯示NRIP在老鼠老化模式中也會隨著年齡的增長而下降。此外,橫紋肌肉瘤(一種人類骨骼肌疾病)中的NRIP表達在老化過程中略微增加。此外,比較正常和疾病骨骼肌中的NRIP評分,我們發現橫紋肌肉瘤中的NRIP表達水平高於正常骨骼肌。 綜上所述,NRIP可能在老化過程中在骨骼肌中發揮重要作用。在這項研究中,我們發現AAV-NRIP基因治療可以恢復老化小鼠的肌纖維大小並提高myogenin的表達。此外,NRIP在老化的人類正常骨骼肌中表達的趨勢與老化小鼠相似。以上所有結果表明,NRIP在老化過程中可能成為治療肌肉異常的潛在方法,需要進一步的研究。 Nuclear receptor interaction protein (NRIP), also known as DCAF6 (DDB1 and CUL4 associated factor 6) or IQWD1, is a nuclear protein consisting of 860 amino acids with seven WD-40 domains and one IQ motif. In our previous study, NRIP expression declined in the skeletal muscle and spinal cord of aging mice. Furthermore, 16-week-old muscle-specific NRIP knockout (cKO) mice displayed abnormal muscles and motor deficits, similar to those observed in aging mice. This indicates that NRIP may participate in age-related muscle dysfunction. Therefore, we wanted to investigate whether NRIP could improve the motor function of aging mice by conducting AAV-NRIP gene therapy. The results showed that AAV-NRIP rescued muscular strength and NMJ size in aging mice. To further examine the effects of AAV-NRIP gene therapy in aging mice, this study assessed muscle fiber size, oxidative fiber expression levels, myofiber type distribution, and myogenin expression. We discovered that AAV-NRIP gene therapy rescued myofiber size and elevated myogenin expression in AAV-NRIP infected cells of aging mice. Next, were highly interested in investigating the effects of overexpressing NRIP in the muscles of aging mice. Therefore, we generated NRIP transgenic mice with NRIP specifically expressed in muscle (Rosa-NRIP-cre mice) in our laboratory. We measured the motor functions of the NRIP transgenic mice, including locomotor activity, rotarod test, and grip force test. The results of these behavioral tests did not show significant differences in mice up to 60 weeks of age, indicating that the NRIP transgenic mice did not exhibit improved motor functions by the age of 60 weeks. Therefore, further ongoing motor function tests is required to observe whether NRIP contributes to improving motor function in aging mice. Finally, to verify the expression level of NRIP in human skeletal muscle with age, we performed IHC staining on a microarray of human skeletal muscle tissue. After scoring the NRIP expression in normal skeletal muscle tissues and diseased skeletal muscle tissues, specifically rhabdomyosarcoma, we analyzed the scores of different cases using statistical analysis. The results indicate that NRIP expression declines with aging in normal human skeletal muscle, which corresponds to the previous study showing NRIP decline with age in a mouse aging model. Moreover, NRIP expression slightly increases with aging in rhabdomyosarcoma, a human skeletal muscle disease. Additionally, when comparing the NRIP score between normal and diseased human skeletal muscle, we found that NRIP expression level is higher in rhabdomyosarcoma tissues than in normal skeletal muscle tissues. Collectively, NRIP may play an important role in skeletal muscle during aging. In this study, we discovered that AAV-NRIP gene therapy can rescue muscle fiber size and elevate myogenin expression in aging mice. Additionally, NRIP expression follows a similar declining trend in aging human normal skeletal muscle as in aging mice. All of the above findings suggest that NRIP has the potential to be a possible treatment for muscle abnormalities in aging, which warrants further research. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90453 |
DOI: | 10.6342/NTU202303610 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 微生物學科所 |
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