端粒酶基因缺失對全能幹細胞分化潛能之影響

Abstract

端粒酶（Telomerase）是細胞中負責合成延長端粒的酵素，避免細胞產生端粒缺乏的問題。端粒酶是由一反轉錄酵素telomerase reverse transcriptase（TERT）、一RNA模板telomerase RNA component（TERC）及其他穩定結構的蛋白所組成，當其中任一組成發生突變時便會使端粒酶功能喪失，造成許多老化相關疾病；當端粒酶缺失發生在人類時，多會引發端粒綜合症（telomere syndrome）等罕見疾病，此疾病多帶有短端粒以及中胚層發育缺陷的問題。藉由產製嵌合體小鼠以及四倍體囊胚互補試驗（tetroploid embryo complementation）等幹細胞全能性檢測試驗中也指出，端粒酶缺失（Terc-/-）的幹細胞如胚幹細胞（embryonic stem cells, ESCs）及誘導性全能幹細胞 （induced pluripotent stem cells, iPSCs）存在全能性分化上的缺陷；而另一研究也指出短端粒的iPSCs存在中胚層的分化缺陷，此結果可推測幹細胞的端粒酶缺失可能為造成其分化潛能缺陷的原因之一。此外補救端粒酶缺失所造成的短端粒問題也是重要的研究方向，其中SCR7和RS-1是具有促進同源性重組（homologous recombination, HR）發生的小分子藥物，可藉由非依靠端粒酶活性（telomerease-independent）的HR路徑延長端粒。故本研究藉由野生型、Terc+/-與Terc-/-的核移殖胚幹細胞（nuclear transfer embryonic stem cells, ntESCs）為基礎，針對端粒酶缺失的Terc+/-ntESCs與Terc-/-ntESCs是否存在中胚層分化缺陷，以及SCR7和RS-1能否透過HR補救端粒酶缺失之核移殖胚幹細胞的短端粒及分化潛能問題進行探討。試驗結果發現野生型、Terc+/-與Terc-/-的ntESCs在分化早期並無缺陷產生，但於後期30天的軟骨誘導分化中，證實Terc-/-ntESCs確實存在中胚層的分化缺陷問題，但此分化潛能缺陷卻無法藉由SCR7和RS-1進行補救，即使SCR7和RS-1可有效降低Terc+/-、Terc-/-ntESCs在軟骨分化過程中的細胞凋亡比率，但因端粒延長幅度過小，仍無法彌補端粒酶缺失所造成的短端粒缺陷。然而實際抑制中胚層分化的主因及機制至今仍未明瞭，仍需更多的實驗研究釐清，以利未來應用於治療端粒綜合症病患的臨床研究。

Telomerase is an enzyme for telomere elongation to protect cells from telomere insufficient, which is composed of telomerase reverse transcriptase (TERT), telomerase RNA component (TERC) and other stabilizing proteins. Mutations of genes that encode telomerase components like TERT or TERC would lead to premature aging and age-related diseases. In human, telomerase haplo-insufficient would cause the telomere syndromes with symptoms of short telomere and mesodermal defects. Several reports demonstrated that telomerase-deficient (Terc-/-) caused the developmental defects in pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), evidenced by production of mouse chimeras and tetraploid embryo complementation. The short telomeres also affect differentiation potential of PSCs, especially for mesodermal differentiation. On the other hand, SCR7 and RS-1 are small molecule drugs to stimulate homologous recombination (HR) happened. Telomere also can be elongated by HR in T-loop structure without telomerase participation. We hypothesis that SCR7 and RS-1 might an alternative way to elongate the telomere length and futher rescue the pluripotency of telomerase deficient ESCs. Therefore, by using telomerase- deficient (Terc+/- and Terc-/-) nuclear transfer embryonic stem cells (ntESCs), the aim of this project is to study: 1) whether telomerase-deficient affects mesodermal differentiation potential and 2) whether pluripotency of telomerase-deficient ntESCs can be rescued by SCR7 or RS-1. In our data, although there are not significant difference between Terc+/+, Terc+/- and Terc-/- ntESCs in 10 days of spontaneous differentiation, but the mesodermal defect of Terc-/- ntESCs during the late stage of differentiation process was confirmed by the chondrocyte differentiation. Besides, SCR7 and RS-1 can elongate the telomere length and decrease apoptotic rate of Terc-/- ntESCs during the chondrocyte differentiation. However, the defect of mesodermal differentiation in Terc-/- ntESCs wasn’t rescued by SCR7 or RS-1 treatment. Understanding the mechanism between mesodermal defect and short telomere in telomerase-deficient PSCs and the appropriate way for telomere elongation in telomerase-deficient PSCs would provide insights to how to optimally reset telomeres in patient-specific PSCs especially for telomere syndrome patients.