探討血管生成素對於鼠胚著床前發育之影響與體外著床模式之建立

Abstract

不同種類生物個體其體內存在多種核醣核酸水解酵素（ribonucleases, RNases），經多年之研究，許多RNases之DNA序列、酵素活性與分子結構，均已有一定程度之瞭解，但對此普遍存在於生物體內之蛋白質，如屬於RNase A家族之血管生成素（angiogenin, Ang），其於胚發育過程中所扮演之角色並未有深入之研究。應用基因轉殖（transgenesis）、基因剔除（knockout）以及基因突變（mutagenesis）等技術可探討基因之生理功能，但其除了耗時、費力外，亦有基因表達專一性不高或不易選殖某特定基因之缺失。因此，本研究之目的為利用核醣核酸干擾（RNA interference, RNAi）作用降解鼠胚Ang mRNA，以產生’似基因剔除’（knockout-like）或’似基因減弱’（knockdown-like）之現象。此外，本研究亦嘗試並建立模擬子宮環境之體外培養系統，期未來能應用於經RNAi處理且Ang表現量降低之鼠胚，在置於此體外培養系統後探討Ang對鼠胚著床之影響。試驗一，設計小鼠Ang家族基因群，包含Ang-1, -2, -4等四型之專一性引子，利用反轉錄聚合酶連鎖反應分析小鼠囊胚各型Ang之表現。結果顯示，僅Ang-1表現於小鼠囊胚。試驗二，針對Ang-1與Ang-2不同序列構築多組不同之RNAi表現質體，以小鼠U6為啟動子並藉由轉染（transfection）技術，將質體送至小鼠B16-F10細胞株內測試各RNAi表現片段（RNAi expression cassette）之效率，試驗結果顯示，所構築之質體均有至少40%之抑制效果，最大之抑制效果甚至可達70%。試驗三，利用顯微注射將Ang-1 RNAi表現片段（U6-sh-1）導入小鼠原核期胚內，觀察其對胚後續發育能力之影響。經體外培養72 h，注射U6-sh-1或U6-shR-1（RNase-1 RNAi表現片段）片段之胚分裂率顯著低於未經任何處理之對照組者（77%與81% v.s. 95%，P<0.05），而注射U6-sh-1或U6-shR-1與對照組之胚發育到桑椹期與囊胚期之百分比率分別具有統計上之顯著差異（49%與48% v.s. 90%, P<0.05），顯示注射外源性基因構築對早期鼠胚之發育有不良影響。以免疫化學染色法分析Ang-1之表現量，可見Ang-1於U6-sh-1注射後鼠胚之表現較未注射組低，RT-PCR之分析結果經數量化後，可見經注射U6-sh-1後Ang-1表現量僅為未注射組之41%。試驗四嘗試以小鼠子宮內膜上皮細胞（endometrial epithelial cells）、基質細胞（stromal cells）與細胞外基質（extracellular matrix, ECM）建立小鼠胚體外著床培養系統，使鼠胚於此培養系統下表現正常侵入（invasion）子宮內膜之行為，以彌補體內著床適期（implantation window）觀察不易之缺點。利用石臘切片與掃描式電子顯微鏡觀察子宮內膜上皮細胞於此培養系統之形態，發現上皮細胞呈現柱狀、極化之現象，可形成類似胞飲突（pinopodes）之結構物。綜合上述結果，本研究構築之RNAi質體具有抑制血管生成素之效果，且所建立之培養系統，雖能提供子宮內膜上皮細胞-表現如體內般之正常形態，然如何可使其形成胞飲突之培養條件仍需更進一步探討，使此系統可應用於未來探討Ang對鼠胚著床前之影響。

A group of homologous ribonucleases (RNases) belonged to the RNase A superfamily has been isolated and characterized from various species. During the past decades, a lot of efforts have been devoted to the study of RNases, including DNA cloning, analyses of their catalytic activities and structures. However, little is known about the biological functions of the ubiquitous RNases in vivo, especially RNase 5, the angiogenin. Recently, it was demonstrated that the gene expression could be blocked after injection of double-stranded RNA (dsRNA) into organisms and the phenomenon is called RNA interference (RNAi). The RNAi is mediated by 19-23 nucleotide dsRNAs homologous in sequence to the target genes to silence cognate genes post-transcriptionally, which involves mRNA degradation. Consequently, it shows the gene knockdown or knockdown-like effects. Hence, the aims of this study were to investigate the effects of angiogenin on the embryonic implantation by knocking down the expression of angiogenin (Ang) in mouse blastocysts, in addition to establishment of an in vitro culture system for early implantation study. In Experiment 1, the specific primers for Ang-1, Ang-2 and Ang-4 were designed to analyze the expression of pattern in mouse blastocysts by reverse transcription polymerase chain reaction (RT-PCR). The results showed that only Ang-1 was expressed at the blastocyst stage in mice. In Experiment 2, the different regions in the DNA sequences of Ang-1 and Ang-2 were selected and subcloned into RNAi expression vector, in which short hairpin RNAs were derived by U6 promoter. After transfected into B16-F10 mouse cell line, the expression of Ang-1 and Ang-2 were analyzed by RT-PCR. The RT-PCR results demonstrated that the expression of angiogenin was decreased to at least 40% by various RNAi expression vectors. In Experiment 3, the U6-sh-1 (RNAi expression cassette for knocking down Ang-1) or U6-shR-1 (RNAi expression cassette for knocking down RNase 1) fragments were injected into the pronuclei of mouse zygotes. The cleavage rates in the U6-sh-1-injected and U6-shR-1 injected groups were significantly lower than those in the uninjected control group after 72 h culture in vitro (77 % and 81% v.s. 95%, P < 0.05). No significant differences of morula/blastocyst formation were found between U6-sh-1-injected and U6-shR-1-injected groups (77% v.s. 81%, P > 0.05), but the development of morula/blastocyst in the U6-sh-1-injected or U6-shR-1 group was significantly decreased, compared to the control group (49% and 48% v.s. 90%, P < 0.05). The defective effects of U6-sh-1 and U6-shR-1 on the development of preimplantation embryos were demonstrated. Immunofluorescent staining showed that U6-sh-1 injection reduced Ang-1 protein levels in the blastocysts compared to those in the control group. In Experiment 4, the endometrial epithelial cells and stromal cells were isolated from mouse uteruses. For construction of the 3-dimensional culture system, epithelial cells were seeded on an artificial basal membrane (ECMatrix™) with underlying stromal cells embedded in the type I collagen matrix. The whole system was settled in a Millicell&reg; (Millipore) hanging in a 24-well culture plate. The morphology of epithelial cells on the matrix became cuboidal after culture. Additionally, the columnar appearance with a basal nucleus was observed on the paraffin wax sections of epithelial cells. The mouse blastocysts were recovered and cultured in this model system. Normal hatching and attachment of the blastocyst were observed. The endometrial cells grown in the established in vitro culture system appeared similar morphology as those in vivo, suggesting this model system might facilitate further understanding of the cellular and molecular mechanisms involving in the implantation of mammalian embryos. In conclusion, the DNA-based shRNA constructs produced in this study could effectively decrease the expression of Ang-1. Further studies would be required to elicit the optimal culture conditions for the in vitro implantation model before applied to the study of the effect of angiogenin on the implantation.