建立輸卵管上皮細胞之初代培養以測定卵白蛋白啟動子活性

Abstract

由於家禽類生長周期較短以及穩定產蛋的特性，基因轉殖家禽的研究已經被廣泛的應用在產製外源蛋白質，尤其是以禽蛋為生產工具。而蛋白佔禽蛋內總蛋白質含量的一半以上，且卵白蛋白 (ovalbumin, OVA) 為蛋白中的主要蛋白質。因為蛋白相對簡單的組成以及卵白蛋白在輸卵管中的專一性表現，許多研究已利用卵白蛋白之啟動子引導下游目標基因表現，並成功於禽蛋中偵測到目標蛋白質。而家禽輸卵管中專一性生產蛋白的蛋白分泌部上皮細胞，其體外之培養系統尚未被完整的建立，因此本研究主要之目的為建立輸卵管上皮細胞(oviductal epithelial cells, OECs)之初代培養系統，並以此系統來測定卵白蛋白啟動子之活性。 性成熟之日本鵪鶉輸卵管的蛋白分泌部被解剖以收集OECs。在將上皮組織從輸卵管管腔刮取下來後，會以蛋白分解酶進行分離細胞作用。為了使細胞的存活率以及增殖效率提高，我們測試不同種類及濃度的蛋白分解酶以及細胞盤塗層材料，以便建立最佳的培養系統。結果發現使用含有第四型膠原蛋白酶 (275 Units/mL) 以及0.02%之胰蛋白酶的消化溶液時，細胞的分離效果為最好；而使用0.1%的Gelatin type B作為細胞盤的塗層物質時，細胞於細胞盤上的貼覆率比沒有處理或塗佈聚離胺酸為高。接著使用免疫染色法以及西方墨點法來標定細胞內的特定蛋白質 (OVA) 以及Estrogen receptor alpha (ESR 1)，確認本研究所建立的方法可以取得正確的OECs。為了探討OVA promoter之活性調控，我們將OECs以不同濃度之Estrogen (E2)、Progesterone (P4)及 Dexamethasone處理，結果發現E2處理都能增加OECs內之OVA mRNA的表現量，而且表現量隨劑量增加而上升，而P4和Dexamethasone處理在有2.5×10-7 M E2都能增加OECs內之OVA mRNA的表現量，尤其在同時加入2.5×10-7 M E2, 1×10-7 M P4 and 1×10-7 M Dexamethasone時會達到最高表現量。當含2.8 kb OVA promoter且帶有冷光報導基因的pGL3-OVA載體轉染到OECs及293T細胞時，以相同濃度及種類之內泌素處裡兩種細胞，結果顯示，OECs內的冷光表現量會受到E2、P4和Dexamethasone處理的調控，結果與OECs內內源性OVA mRNA之表現量相似，而293T細胞內的冷光表現量則不受這些內泌素調控。 綜合上述，我們成功地建立並且改善了日本鵪鶉輸卵管上皮細胞的初代培養系統，並釐清OVA promoter受內泌素調控之機制，建立增進此基因表現的條件。因此這個培養系統之後將可以應用在評估及調控OVA promoter的活性，用以產製外源蛋白質。

Research models of transgenic avian have been widely applied for generating exogenous proteins in eggs because of their relatively short generation interval and stable protein productions. Albumin constitutes more than half of the protein in eggs, and ovalbumin (OVA) is the primary component. According to the composition of egg white and the tissue-specific expression of ovalbumin, most of the published exogenous proteins are driven by ovalbumin promoter and detected in egg white. However, in vitro functional studies on avian oviductal epithelial cells (OECs) in magnum segments, where egg white are synthesized and secreted, are needed. In this study, we established and the primary culture system for OECs in order to study the regulation of ovalbumin promoter activity. The magnum segments of oviducts from sexually-mature female Japanese quails were dissected for OECs collection. After scraping epithelial cells from the lumen surface, the obtained cells were then underwent enzymatic dissociation. For improving cell survival rate and proliferation, different concentrations of digestion enzymes and culture dish coating materials were examined. Results showed that digestion solution containing collagenase type IV (275 units/ mL) and 0.02% trypsin worked the best for OECs isolation. The 0.1% gelatin solution is required for coating culture plate to enhance OECs attachment. We examined protein expression of OVA and estrogen receptor 1 (ESR1) on cultured OECs by immunofluorescent staining and Western blotting. For studying OVA promoter activity regulation, the OECs were treated with several concentrations of estrogen (E2), progesterone (P4) and Dexamethasone to examine the effects of different hormones on the expression of OVA. We found that OVA mRNA expression was significantly increased when treated with 2.5×10-7 M E2, 1×10-7 M P4 and 1×10-7 M Dexamethasone. Meanwhile, we cloned a 2.8-kb OVA promoter from chicken genomic DNA and constructed with the luciferase reporter vector (pGL3). We then transfected the pGL3-OVA construction into both OECs and 293T cell line and treated with E2, P4 and Dexamethasone to measure promoter activity using a reporter gene, luciferase. Results showed that under hormone stimulations, the expressions of OECs promoter regulated by hormones were similar to the expression of OVA mRNA in OECs, but there was no hormonal effect in 293T cells. In conclusion, we successfully established a primary culture system for Japanese quail oviductal epithelial cells, and this system can be applied to study the evaluation and regulation of the OVA promoter for exogenous protein production.