分泌性Lcn2蛋白在斑馬魚與小鼠腦部組織基因表現的研究

Abstract

Lcn2是疏水性結合蛋白家族的一員，初期研究被認為是一急性期蛋白，許多的研究指出其參與了多種生理功能，包括免疫功能、細胞凋亡、細胞分化與增生與各種疾病的發生等。近期的研究則多集中於該蛋白與癌症之間的關係。由於先前有研究指出Lcn2參與了胚胎期的腎臟發育，本實驗著重於探討Lcn2在斑馬魚與小鼠的胚胎發育時的表現，利用RT-PCR技術與即時定量PCR分析了Lcn2基因在斑馬魚及小鼠各組織及不同胚胎期Lcn2的表現，發現在斑馬魚的胚胎受精後10小時開始表現，並持續表現到72小時。此外以小鼠為動物模式將研究重點集中於胚胎期腦部的發育過程Lcn2基因的表現時，利用RT-PCR與qPCR的技術，觀察了在不同胚胎期的腦部Lcn2的表現量，結果發現E10.5即有Lcn2基因的表現，但E16.5的胚胎腦部Lcn2基因表現量下降，到E18.5後表現量又恢復，在成鼠亦能持續表現。利用免疫沉澱法與西方墨點術觀察Lcn2蛋白的表現也有類似的結果，但在不同時期的胚胎腦組織中，Lcn2蛋白表現量不多且不具顯著的差異。利用免疫染色法則無法觀察到小鼠胚胎期的腦部組織切片有Lcn2蛋白的表現，推測Lcn2基因在小鼠胚胎期的腦部表現量很低。Lcn2是否在發育過程中有重要功能則仍有待進一步的研究。

Lcn2 is a member of the lipocalin family and has been well known as an acute phase protein. Several evidences showed that Lcn2 is a multi-function protein, participating in such as immunity, apoptosis, ion transport, cell differenciation and proliferation. It may also be related to the occurrence of diseases. Subsequently, it has been found that the expression of Lcn2 gene triggered embryonic kidney development. The finding encourages us to characterize the Lcn2 expression in zebrafish and mice, and look for an animal model to elucidate the biological function of Lcn2. The mRNA level of Lcn2 in tissue distribution and stage profile of embryos in zebrafish and mice were analyzed by RT-PCR and Real-time PCR. Western blotting and immunohistochemical staining have performed for protein assay. The data showed that Lcn2 gene expression initiated in 10 hpf embryo and sustained till 72 hpf. Furthermore, with a mouse brain model, Lcn2 expressed in E10.5, decreased significantly in E16.5 but regained in E18.5, and expressed persistently in the mature mice. The results of immunoprecipitation and Western blotting technique coincided with the mRNA levels in these periods. Unfortunately, we did not find a significant protein expression in between the each stage of embryonic development, and neither in immunohistochemistry analysis. The data revealed that Lcn2 gene expressed in low level during the mouse brain development.