I. Trip6 蛋白質在小鼠腦中之表現 II. 建立人類惡性腫瘤之斑馬魚異體移植模式

Abstract

壹、 Trip6 蛋白質在小鼠腦中之表現 甲狀腺素受體作用蛋白質 6 (Thyroid receptor-interacting protein 6, Trip6)是一種焦點連接(focal adhesion)分子，它調控一些細胞機制 如：細胞之間的連接(cell adhesion)、細胞的遷移(cell migration)以及 基因轉錄的活化 (gene transactivation)。過去研究指出 Trip6 屬於幹 細胞性(stemness)的基因，在不同的幹細胞中具有較高的表現量。為了探究Trip6 在神經幹細胞所扮演的角色，我們分別檢測了 Trip6 蛋白質在胚胎與成年小鼠大腦中的表現量。發現 Trip6 的 mRNA 主要在胚胎小鼠的腦中有表現，但在成年小鼠腦中則比較低或偵測不到。其蛋白質也只可以在胚胎小鼠的大腦中被偵測到，成年小鼠則否。另外我們在胚胎與成年小鼠的大腦組織切片中，以不同的細胞標誌與 Trip6 進行組織免疫螢光染色。包括幹細胞的標誌 Sox2、增殖中細胞的標誌 Ki67、室管膜細胞的標誌 S100β、神經母細胞的標誌 DCX、神經元的標誌 MAP2、星狀細胞的標誌 GFAP 以及小膠質細胞的標誌(Iba1)。我們發現 Trip6 主要表達在胚胎小鼠的腦室區(ventricular zone, VZ)以及出生後小鼠的腦室下區(subventricular zone, SVZ)內的神經幹細胞(neural stem cells, NSC)中。這樣的結果支持Trip6 可能在調控幹細胞的特性中是一個重要的關鍵。

貳、建立人類惡性腫瘤之斑馬魚異體移植模式 神經膠質母細胞瘤是成人最常見且高侵略性的原發惡性腦腫瘤。它的侵襲力和耐傳統療法使其成為極易復發的惡性腫瘤。Rac蛋白質屬於Rho GTP酶亞家族，其主要功能包括調節細胞運動，增殖和存活。為了探究Rac蛋白質是否可以作為膠質母細胞瘤的新治療標靶，特別是對於神經膠質母細胞瘤幹細胞，我們利用其類癌幹細胞株建立了斑馬魚的異體移植模式來研究抑制Rac蛋白質對於神經膠質母細胞瘤的致癌性影響。 我們將表達控制組的shRNA或者是針對Rac蛋白質做抑制的shRNA序列和綠螢光蛋白的神經膠質母細胞瘤細胞株U251-MG和U373-MG培養於低分化培養液中，以形成腫瘤細胞球(tumorspheroids)。這些體外培養的球體細胞有著幹細胞的特性。我們將這些細胞以顯微注射的方式注射進入受精後兩天大的血管紅螢光轉基因斑馬魚Tg(kdr: mCherry)的卵黃囊。觀察發現注入的癌細胞誘導了血管新生作用的發生，而表達shRacs細胞萎縮且並未引發血管新生作用。另外，注射shRacs細胞的魚隻生存率也較高。 從我們的研究結果，Rac蛋白質會誘導膠質瘤幹細胞引發血管新生作用，並且可做為一個生物標誌。因此，Rac蛋白質可能可以進一步應用在神經膠質母細胞瘤的標靶治療上。 另一方面，我們也利用注射肝癌細胞株Hep3B進入受精後兩天大的斑馬魚卵黃囊中，來觀察Hep3B細胞的遷移現象，此模式約有20%的魚隻可觀察到細胞遷移。I. The Expression Pattern ofTrip6 in Mouse Brain Trip6 (Thyroid receptor-interacting protein 6) is a focal adhesion molecule and its function is related to cell adhesion, cell migration and gene transactivation. Trip6 has been identified as a “stemness” gene which expresses higher in different lineage of stem cells. To investigate the role of Trip6 in neural stem cells, we examined the expression level of Trip6 in the brains of embryonic and adult mice, and found that the mRNA of Trip6 is detected in the embryonic mouse brain but low or undetectable in the adult mice brain. Similarly, the protein level of Trip6 is only detected in the embryonic mouse brains by western blot. We also performed double immunofluorecence with Trip6 and different cellular markers, including, stem cells marker- Sox2, proliferating cells marker- Ki67, ependymal cells marker- S100β, neuroblast marker- DCX, neuron maker- MAP2, astrocytes marker- GFAP and microglia marker- Iba1.We found that Trip6 was expressed by neural stem cells in the ventricular zone and subventricular zone of embryonic and postnatal mouse brains, respectively. Taken together, Trip6 may play an important role in regulation of neural stem cell (NSC) properties.

II. Establishing Zebrafish Xenotransplantation Model of Human Malignant Tumors Glioblastoma is the most common and aggressive malignant primary brain tumor in adults. Its invasiveness and resistance to traditional therapies make it a highly recurrent malignant disease with poor prognosis. Rac proteins belong to the Rho small GTPase subfamily, which major functions include regulation of cell movement, proliferation, and survival. To investigate whether Rac proteins can serve as new therapeutic targets for glioblastoma, especially for glioblastoma stem cells, we established a zebrafish xenotransplantation model to study the effects of Rac proteins in glioblastoma. Glioblastoma cell lines U373 and U251 expressing short hairpin RNA targeting Rac proteins or control RNA sequence and green fluorescence protein were generated and cultured in low differentiating medium to form tumorspheroids. These spheroid cells harbor stem cell properties and mimic stem cells in vitro. We injected these cells to egg yolk of zebrafish Tg(kdr: mCherry) on 2 post-fertilization-day stage(pfd). The behaviors of the injected cells were then monitored every day by the confocal microscope. We observed that the cells expressing control scramble RNA sequence induced angiogenesis in the egg yolk of the fish, while the cells expressing shRacs shrank inside the fish and did not induce angiogenesis. The overall survival rates of fishes were also higher in the fishes injected shRac cells instead of fishes with control scramble RNA cells. From our results, we conclude that Rac proteins induce angiogenesis of glioblastoma stem cells and serve as a poor prognosis marker. Therefore, Rac proteins could further be the potential therapeutic targets of glioblastoma and its stem cells. On the other hand, we also inject the hepatocellular carcinoma cell line- Hep3B into egg yolk of zebrafish on 2 pfd stage to observe the migration of Hep3B cells. We have successfully established the in vivo migration/metastasis model of Hep3B cells in zebrafish of which the metastasis rate is about 20%.