分析豬輸卵管幹細胞之細胞外囊泡及探討miRNA對於卵母細胞影響

The analysis of extracellular vesicles from porcine fallopian tube stem cells and the potential effects of selected miRNAs on porcine oocytes

王恕旋(Wang Shu-Hsuan)

指導教授 ： 彭劭于 ； 沈朋志 ； 林郁涵

國立屏東科技大學/農學院/動物科學與畜產系所/碩士(2020年)

https://doi.org/10.6346/NPUST202100209

摘要

隨著年齡增長以及生活環境型態等因素，使婦女發生不孕症比例年年增加。輸卵管在生殖系統中扮演重要的角色，不只是精子卵子結合的地方更是能幫助早期胚發育，而輸卵管細胞所分泌細胞外囊泡（extracellular vesicles, EVs）包含脂質、蛋白質、核醣核酸和短片段核醣核酸（microRNAs, miRNAs）等生物調控因子，推測可以幫助細胞與細胞之間的交互作用促進卵母細胞的受精及發育。目前豬輸卵管細胞對於卵母細胞之研究甚少，並且無深入探討輸卵管分泌EVs之成分。故擬探討豬輸卵管幹細胞（Porcine fallopian tube stem cells, PFTSCs）所分泌之細胞外囊泡對於卵母細胞之影響，並鑑定PFTSCs中的EVs並分析高表現之miRNAs，期望找出促進豬胚發育之因子。因此將PFTSCs培養至第五代，待細胞密度達80~90%時，換成豬體外成熟培養液（M199）培養3小時，再將其條件培養液（condition medium, CM）培養卵母細胞，發現卵母細胞成熟率顯著提升。為定義CM中EVs，以沉降法的方式收集EVs。先檢測肌動蛋白（β-actin）、熱休克蛋白（heat shock proteins 70, HSP70）及細胞色素c（cytochrome c, CYT C）等蛋白質的存在狀態以確認EVs無細胞參雜。同時使用超高解析度熱場發射掃描式電子顯微鏡（Field Emission Scanning Electron Microscope, FE-SEM）和奈米粒子追蹤分析儀來觀察及定義EVs型態。再利用NGS分析EVs並檢測出267種miRNAs，其中選出表現量最高的14種分析其GO（Gene Ontology）及KEGG（Kyoto Encyclopedia of Genes and Genomes）路徑富集圖。這14種miRNAs的可能功能和MAPK路徑與減數分裂有高度相關性，故我們選擇了miR-152-3p、miR-148a-3p、miR-320a-3p、let-7f-5p及miR-22-3p等五種miRNAs，皆有可能影響標的CEPB基因，而再影響MAPK路徑。五種miRNAs分別於體外成熟（IVM）添加培養後，發現添加miR-320a-3p之組別成熟率顯著最高，則後續孤雌激活豬胚之囊胚率以IVM添加50 nM的miR-320a-3p顯著最高。其餘miRNAs處理下，卵母細胞成熟率、後續孤雌激活豬胚之卵裂率、囊胚率及細胞數不受影響。於體外培養（IVC）添加miR-320a-3p組別之囊胚率顯著高於對照組，但不影響其卵裂率及細胞數。綜合之結果說明，豬輸卵管幹細胞之條件培養液能助於卵母細胞的發育，收集條件培養液中細胞外囊泡與分析，也鑑定出最有可能影響卵母細胞的miRNA為miR-320a-3p，除了不只可以幫助卵母細胞的成熟，更能有效提升囊胚率。

關鍵字

細胞外囊泡 ； 輸卵管 ； 豬卵母細胞 ； microRNA ； miR-320a-3p

並列摘要

With the increase of age and the types of living environments and other factors, the infertility of Taiwanese women increases yearly. The fallopian tube is essential in the reproductive system, for combining sperms and eggs and helping early embryonic development. Extracellular vesicles (EVs) secreted by fallopian tube contain biological regulatory factors, such as lipids, proteins and microRNAs (miRNAs). It’s suggested that EVs can help the fertilization and development of oocytes. At present, the researches on oocytes from porcine oviduct cells are rare, and the study of the components from oviduct secreting EVs remains vague. Therefore, to explore the effect of EVs secreted by porcine fallopian tube stem cells (PFTSCs) on oocytes, to identify EVs functions in PFTSCs and to uncover potential miRNAs, are the goals to promote the porcine embryonic developments. First of all, when the fifth-generation PFTSCs were reached 80-90% confluency, cultural medium was replaced with pig in vitro maturation medium (M199) and cultured for 3 hours, and collected the condition medium (CM) for oocyte incubations. The maturation rate was significantly increased after CM incubation. To understand functions of EVs in CM, EVs were collected by sedimentation approach. Proteins as ACTIN, HSP70 (heat shock proteins 70) and CYT C (cytochrome c) were used to determine whether extracted EVs were cell-free. Field Emission Scanning Electron Microscope (FE-SEM) and Nanoparticle Tracking Analyzer were used to observe EVs morphology, with a defined particle size range from 100-200 nm. The EVs were further analyzed by NGS and identified 267 miRNAs, and the 14 with the highest expression levels were selected to analyze the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment maps, and the results suggested that the function and MAPK pathway are highly related to meiosis. The selected miR-152-3p, miR-148a-3p, miR-320a-3p, let-7f-5p and miR-22-3p, were predicted to target Cepb1 gene, Cepb1 was also reported to affect MAPK pathway. After separately adding five miRNAs and cultured in vitro maturation, the maturation rate of the group with miR-320a-3p was significantly increased. The subsequent parthenogenetic activation of the blastocyst rate of pig embryos was also significantly enhanced by adding 50 nM miR-320a-3p. The other miRNAs shared no effects on oocyte maturations, on cleavage rates, blastocyst rates, and neither on cell numbers of subsequent parthenogenetic porcine embryos. In vitro culture with miR-320a-3p, the blastocyst rate was significantly higher than control group, and the cleavages rate and cell numbers were not affected by miR-320a-3p. The results showed that the CM of PFTSCs can effectively improve porcine oocyte development, and the miRNAs in EVs are sequenced and identified. It was found that miR-320a-3p can not only help the maturation, but also can effectively increase the blastocyst rates.

並列關鍵字

Extracellular vesicles ； Fallopian tube ； microRNA ； miR-320a-3p ； Oocytes

參考文獻

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