臺灣博碩士論文加值系統

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® (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.

目錄 頁次
文獻檢討 ............................................................................................................. 1
一、哺乳動物胚著床之調控............................................................................... 1
（一）不同動物之著床模式與現象........................................................... 1
（二）著床前子宮內膜之變化................................................................... 2
1. 子宮內膜容受期.............................................................................. 2
2. 蛻膜化.............................................................................................. 3
3. 胞飲突.............................................................................................. 4
4. 腔狀上皮細胞連結之變化.............................................................. 4
5. 上皮細胞醣蛋白複合物之變化...................................................... 4
（三）囊胚之黏附....................................................................................... 5
1. 整合素.............................................................................................. 5
2. Basigin.............................................................................................. 6
3. Trophinin-tastin-bystin complex.................................................... 6
（四） 滋養葉細胞之侵入.......................................................................... 7
（五） 其他調控著床之因子...................................................................... 7
1. 卵巢荷爾蒙下流調控基因.............................................................. 7
2. 上皮細胞生長因子家族.................................................................. 8
3. 血管內皮生長因子.......................................................................... 8
4. 細胞素.............................................................................................. 9
二、小分子RNA對基因轉錄後之調控.......................................................... 10
（一）小分子干擾素RNA......................................................................... 10
（二）基因轉錄後之寂靜機制-RNAi作用............................................... 10
（三）導入dsRNA或siRNA表現型載體之策略................................... 12
1. 浸泡法與餵飼法.............................................................................. 12
2. 顯微注射法...................................................................................... 12
3. 電穿孔法.......................................................................................... 12
4. 陽離子脂質體轉染法...................................................................... 13
5. 病毒感染法...................................................................................... 13
三、血管生成素之發現..................................................................................... 13
（一）血管生成素家族............................................................................... 14
（二）血管生成素之功能........................................................................... 14
1. 促血管生成素之機制...................................................................... 14
（1）內皮細胞增生之調控................................................................. 15
（2）Ang於內皮細胞之訊息途徑..................................................... 16
2. 核糖核酸水解作用.......................................................................... 16
3. 病原菌毒殺與抗發炎作用.............................................................. 17
4. 協助卵巢形態改變與卵子成熟...................................................... 18
試驗一 Ang家族於小鼠E3.5胚之表現........................................................... 19
ㄧ、前言........................................................................................................... 19
二、材料與方法 ............................................................................................. 19
（一）小鼠Ang-1、Ang-2與Ang-4引子專一性之測試......................... 19
（二）試驗小鼠之取得及飼養管理........................................................... 26
（三）小鼠超級排卵與配種....................................................................... 26
（四）著床前E3.5胚之取得..................................................................... 26
（五）胚之免疫細胞化學染色................................................................... 26
（六）著床前小鼠胚total RNA之萃取.................................................... 27
（七）反轉錄酶連鎖反應分析小鼠Ang家族之表現.............................. 28
三、結果........................................................................................................... 31
四、討論........................................................................................................... 34
試驗二 不同序列片段Ang-1-RNAi與Ang-2-RNAi抑制載體之抑制效果 35
一、前言 .......................................................................................................... 35
二、材料與方法 .............................................................................................. 35
（一）細胞株之體外培養與繼代............................................................... 35
（二）Ang-1 shRNA與Ang-2 shRNA載體之製備................................. 36
（三）Ang-1-shRNA與Ang-2-shRNA表現載體之轉染........................ 43
（四）反轉錄聚合酶鏈鎖反應分析轉染後Ang-1與Ang-2之表現....... 43
（五）影像與統計分析 ............................................................................. 47
三、結果 ......................................................................................................... 47
（一）B16-F10細胞株之轉染效果 ......................................................... 47
（二）分析轉染不同 Ang 1-shRNA與Ang2-shRNA表現載體後Ang-1與Ang-2之表現量..........................................................................
48
四、討論 ......................................................................................................... 54
試驗三 顯微注射U6-sh1對於鼠胚體內與體外發育之影響 ......................... 55
一、前言 ......................................................................................................... 55
二、材料與方法 ............................................................................................. 55
（一）試驗小鼠之來源與飼養管理 ......................................................... 55
（二）小鼠超級排卵與配種 ..................................................................... 55
（三）胚操作液及培養液之配製............................................................... 55
（四）原核期鼠胚之取得........................................................................... 56
（五）顯微注射用基因片段之製備........................................................... 56
（六）顯微注射工具之備製 ..................................................................... 57
（七）原核期鼠胚之基因顯微注射操作 ................................................. 58
（八）注射後鼠胚之體外培養 ................................................................. 58
（九）胚之免疫螢光細胞染色 ................................................................. 61
（十）反轉錄聚合酶鏈鎖反應分析注射後之鼠胚 ................................. 61
（十一）統計分析....................................................................................... 63
三、結果 ......................................................................................................... 63
四、討論 ......................................................................................................... 67
試驗四 小鼠體外體外著床培養系統之建立..................................................... 68
ㄧ、前言........................................................................................................... 68
二、材料與方法 ............................................................................................. 68
（一）試驗小鼠之來源及飼養管理........................................................... 68
（二）小鼠超級排卵與配種....................................................................... 68
（三）細胞培養液之配置........................................................................... 68
（四）懷孕母鼠子宮內膜上皮細胞與基質細胞之分離........................... 69
（五）免疫化學染色................................................................................... 69
（六）子宮內膜上皮細胞與基質細胞共培養所構築之體外著床培養系統.......................................................................................................
70
（七）光學與電子顯微鏡之觀察............................................................... 70
（八）助孕素對於培養系統之影響........................................................... 72
（九）E3.5胚之取得與共培養.................................................................. 72
三、結果 ......................................................................................................... 72
（一）分離之子宮內膜上皮細胞與基質細胞之細胞特性分析............... 72
（二）體外著床培養系統之石蠟切片與掃描式電子顯微鏡之觀察....... 74
（三）囊胚於體外系統之共培養............................................................... 80
四、討論 ......................................................................................................... 81
結論 ..................................................................................................................... 84
參考文獻............................................................................................................... 85
附錄1.................................................................................................................... 97
附錄2 ................................................................................................................... 98

圖目錄
圖一、著床模式之分類……............................................................................... 1
圖二、Ang family 序列之比較......................................................................... 25
圖三、血管生成素家族引子專一性測試......................................................... 32
圖四、Ang家族於E3.5胚之表現..................................................................... 33
圖五、小鼠Ang-1序列與shRNA之標定區域................................................. 38
圖六、小鼠Ang-2序列與shRNA之標定區域................................................. 38
圖七、pUC-19表現載體、Ang-1-RNAi與Ang-2-RNAi表現片段之構築...
40
圖八、B16-F10細胞株轉染後GFP之表現.....................................................
48
圖九、B16-F10細胞株轉染U6-sh-1表現載體後Ang-1與actin之表現量. 49
圖十、B16-F10細胞株轉染Ang1-RNAi表現載體後Ang-1/actin之表現
量.............................................................................................................
50
圖十一、B16-F10細胞株轉染U6-sh-2-1表現載體後Ang-2與actin之表
現量.........................................................................................................
51
圖十二、B16-F10細胞株轉染U6-sh-2-2表現載體後Ang-2與actin之表
現量.........................................................................................................
51
圖十三、B16-F10細胞株轉染U6-sh-2-3表現載體後Ang-2與actin之表
現量.........................................................................................................
52
圖十四、B16-F10細胞株轉染Ang1-RNAi表現載體後Ang-2/actin之表現
量...,,........................................................................................................
53
圖十五、小鼠之原核顯微注射與體外培養....................................................... 59
圖十六、免疫螢光染色法分析注射U6-sh-1、U6-shR-1片段後之囊胚與
經處理之囊胚….....................................................................................
64
圖十七、小鼠受精卵經顯微注射U6-sh-1與U6-shR-1，以RT-PCR分析其
Ang-1與actin之表現量….....................................................................
65
圖十八、鼠胚經U6-sh-1或U6-shR-1注射至原核後後，Ang-1 / actin之表
現.............................................................................................................
66
圖十九、上皮細胞與基質細胞共培養所構築之體外著床培養系統…........... 71
圖二十、小鼠子宮內膜上皮細胞與基質細胞之免疫化學染色....................... 73
圖二十一、小鼠子宮內膜上皮細胞之分離、培養與蘇木紫－伊紅染色......... 75
圖二十二、掃描式電子顯微鏡觀察E3.5小鼠之子宮角.................................. 77
圖二十三、掃描式電子顯微鏡觀察小鼠體外著床培養系統........................... 78
圖二十四、小鼠囊胚黏附於體外著床系統.......................................................
80

表次
表一、小鼠受精卵經顯微注射U6-sh-1與U6-shR-1體外培養72 h之發
育……………………………………………………………………….
60

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