臺灣博碩士論文加值系統

雄性哺乳動物之精原幹細胞（spermatogonial stem cells, SSCs）具有再生與分化之能力，且目前已發展出不同體外培養SSCs系統，其中包括含血清（serum）培養液或是飼養層細胞（feeder layer）之培養系統，然而，此兩種系統之成分複雜且變異頗大，如不同批號血清與相異批次之飼養層細胞，可能含有不同成分或濃度之各式因子，因此雖然一般而言均可使 SSCs 生長，但是卻難以界定維持幹細胞特性之關鍵物質。研究指出，似胜肽類激素（peptide-like hormones）之生長因子（growth factors）可藉由結合其細胞表面之特異受體，調節細胞各類活動與功能，以促進細胞分化與成熟，其中，神經膠質衍生神經營養因子（glial cell line-derived neurotrophic factor, GDNF）具有調控SSCs分化與再生之能力，而白血病抑制因子（leukemia inhibitory factor, LIF）則可調節 SSCs 形成聚落（colony）之能力。故本試驗之目的為建立可維持小鼠精原幹細胞（mouse spermatogonial stem cells, mSSCs）增生與分化能力之無血清與無飼養層之體外培養系統，並檢測LIF與GDNF生長因子，對其生長之影響。5日齡公鼠之睪丸去除被膜（testicular capsules）後，經胰蛋白酶與去氧核糖核酸酶消化清洗後，將分離之細胞分別置於無添加生長因子（Group 1），或添加不同生長因子（Group 2, 103/mL LIF; Group 3, 20 ng/mL GDNF; Group 4, 103/mL LIF + 20 ng/mL GDNF）之mSSCs培養液，觀察並分析各組細胞生長、聚落形成與幹細胞特性（stemness）之差異。試驗一分組培養之結果顯示，雖然各組培養至第五天，mSSCs 之細胞數不具顯著性差異，但Groups 2與4所形成之mSSCs聚落數，則顯著高於其他兩組；然而mSSCs相關指標基因（thy-1, -6 integrin 與 -1 integrin）以及特異性標誌蛋白質（Oct-4與Thy-1）之表現，於各組間均無顯著差異。在後續繼代培養時， 發現Groups 2與4之mSSCs聚落再形成時間較其他組別快約7-14天，且繼代至第二代時，其mSSCs聚落之特異性基因與蛋白質均仍持續表現。試驗二細胞冷凍解凍後之培養結果顯示，Groups 2與4之mSSCs冷凍解凍培養至第五代後，其大部分之特異性基因與蛋白質仍均持續表現，唯thy-1基因於兩組培養條件下皆無法偵測，且不論是否有無冷凍解凍處理， Group 4 之細胞其特異性基因表現量均顯著高於Group 2者。試驗三將體外培養之Groups 2與4 之綠螢光轉基因mSSCs （GFP-mSSCs）移植回不孕模式小鼠體內，檢視 GFP-mSSCs 於體內分化之情形。結果發現，移植 60 天後以免疫組織化學染色法可於睪丸生精小管內檢測到表現綠螢光蛋白質細胞，同時亦可偵測到表現精子分化標誌蛋白質（Stra8）與細胞增生指標蛋白質（PCNA）之細胞，顯示經白消安處理之生殖細胞於75天後可恢復增生與分化能力。綜上所述，為達成建立無血清與無飼養層之mSSCs培養系統，培養液中添加LIF+GDNF為必需之條件，而本試驗建立之系統可維持部分 mSSCs移回體內後之發育能力。

The mammalian spermatogonial stem cells (SSCs) possess self-renewal and differentiation ability. Several in vitro culture systems have been developed for maintenance of SSCs, including serum-containing media, or feeder-layer system. Although it has been reported that serum- or feeder layer-containing culture system could maintain SSCs, the components and their percentages in the culture system might be various and inconsistent due to the different batches of serum or feeder cells. Furthermore, it may hinder the study of key molecules involved in the maintenance of SSCs. Research results indicate that the growth factors of peptide-like hormones could regulate the activity and function of cells to enhance their differentiation and maturation through the specific receptors located on the cell surface. It has been shown that glial cell line-derived neurotrophic factor (GDNF) is able to regulate the differentiation and self-renewal of SSCs, and leukemia inhibitory factor (LIF) is recognized to promote colony formation in SSCs. Therefore, the objective of this study was to establish a serum- and feeder-free culture system for maintenance of proliferation and differentiation in mouse spermatogonial stem cells (mSSCs) in vitro. Additionally, the effect of LIF and GDNF on the growth of mSSCs was evaluated. Testes from 5-day old mice were treated with trypsin and DNase after removal of testicular capsules. The washed cells were allocated into 4 groups, which are Group 1, medium without growth factor; Group 2, medium supplemented with 103/mL LIF; Group 3, medium supplemented with 20 ng/mL GDNF; Group 4, medium supplemented with 103/mL LIF + 20 ng/mL GDNF. During cultivation, the growth, colonization and stemness of mSSCs were analyzed among groups. Experiment I results showed that no significant differences were identified on the cell numbers, the expressions of mSSC marker genes (thy-1, -6 integrin and -1 integrin) and proteins (Oct-4 and Thy-1) among groups 5 days after culture, but the number of colonies was significantly higher in the Groups of 2 and 4. Also, it was noticed that the formation of colonies in Groups of 2 and 4 was 7-14 days faster than the cells in the other groups after passages. The colonies formed in Groups 2 and 4 continuously expressed mSSC marker genes and proteins after 2 passages. Experiment II results indicated that the frozen-thawed mSSCs from Groups 2 and 4 could be maintained to passage 5. The colonies formed in these two groups continuously expressed mSSC marker genes and proteins, but thy-1 was no longer to detect. Additionally, it was found that the expression of SSC marker genes in Group 4 was significantly higher than those in Group 2, no matter in fresh or frozen-thawed mSSCs. Experiment III, the green fluorescent protein-transferred mSSCs (GFP-mSSCs) from Groups 2 and 4 culture conditions were injected into the infertility model mouse testes to further analyze the developmental ability of the cultured GFP-mSSCs. The results showed that some GFP positive cells were identified by immunohistochemistry in the host testes 60 days after transfer. Additionally, meiosis marker protein, Stra8, and cell proliferation marker protein, PCNA, could be detected, indicating that the germ cells restored their developmental ability 75 days after busulfan treatment. In conclusion, it is found that it is necessary to supplement both of LIF and GDNF in the current mSSC culture medium in order to establish serum- and feeder-free culture system. Also the current system partially supports the developmental ability of mSSCs after transfer into host testes.

目次
摘要 i
Abstract iii
目次 v
圖目次 viii
文獻探討 1
一、 精原幹細胞 1
二、 生精細管之微環境 1
三、 神經膠質細胞株衍生神經滋養因子 (GDNF) 2
(一)、 GDNF在細胞中訊號傳遞路徑 3
(二)、 GDNF對於生殖細胞之重要性與影響 4
四、 白血病抑制因子 (LIF) 4
（一） LIF於細胞中之訊號傳遞路徑 5
（二） LIF對於生殖細胞之重要性與影響 6
五、 精原幹細胞主要訊息傳遞路徑 6
（一） JAK/STAT訊息路徑 6
（二） Src 訊息路徑 7
（三） PI3K/Akt 訊息路徑 7
（四） Ras/Erk1/2 訊息路徑 8
（五） Smad 訊息路徑 8
六、 精原幹細胞相關細胞標誌 10
（一） 未分化精原幹細胞之細胞標誌 10
1. Pou5f1 (Octamer-binding transcription factor 4, Oct-4) 10
2. Thy-1 (CD90) 10
3. α6 - integrin (CD49f) 11
4. β1- integrin (CD29) 11
5. Zbtb16 （Zinc finger and BTB domain containing 16, PLZF） 11
（二） 分化之精原幹細胞細胞標誌 12
1. Dazl (Deleted in azoospermia like) 12
2. Stra8 (Stimulated by retinoic acid gene 8) 12
七、 精原幹細胞體外培養與應用 12
（一） 精原幹細胞之含血清 (serum) 與飼養層 (feeder cell layer) 之培養系統 …………………………………………………………………12
（二） 精原幹細胞無血清與飼養層培養系統 13
（三） 精原幹細胞體外培養之應用 13
八、 結論 14
試驗一、精原幹細胞體外培養 15
前言 15
試驗目的 15
材料與方法 16
一、 實驗小鼠來源與飼養管理 16
二、 分離小鼠精原幹細胞 16
三、 精原幹細胞培養液製備 16
四、 精原幹細胞培養與繼代 17
五、 細胞免疫化學染色法 （Immunocytochemistry, ICC） 17
六、 Total RNA 萃取 18
七、 核酸濃度之檢測 19
八、 反轉錄聚合酶鏈鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR) 19
九、 精原幹細胞多能性以及細胞特異性基因表現之分析 20
十、 統計分析 21
結果 22
討論 24
一、 分離精原幹細胞之方法 24
二、 分選精原幹細胞之方法 24
三、 無血清與無飼養層之培養系統比較 25
四、 精原幹細胞挑選後細胞生長遲緩 26
五、 精原幹細胞 mRNA表現量之差異 26
試驗一小結 27
試驗二、體外培養之精原幹細胞冷凍解凍試驗 38
前言 38
試驗目的 38
材料與方法 39
一、 精原幹細胞培養與繼代 39
二、 精原幹細胞冷凍解凍 39
（一） 精原幹細胞冷凍 39
（二） 精原幹細胞解凍 39
三、 細胞免疫化學染色法 （Immunocytochemistry, ICC） 39
四、 Total RNA萃取 39
五、 核酸濃度之檢測 39
六、 反轉錄聚合酶鏈鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR) 40
七、 精原幹細胞多能性以及細胞特異性基因表現之分析 40
八、 統計分析 40
結果 41
討論 43
一、 精原幹細胞不同抗凍劑使用之比較 43
二、 精原幹細胞冷凍解凍後colony形成能力之改變 43
三、 精原幹細胞冷凍解凍後相關基因表現之改變 44
試驗二小結 44
試驗三、精原幹細胞移植試驗 51
前言 51
試驗目的 51
材料與方法 52
一、 製備無內源性生殖幹細胞之模式公鼠 52
二、 綠螢光精原幹細胞之製備 52
三、 小鼠睪丸輸出小管 (Ductuli efferentes) 細胞移植 52
（一） 製作毛細玻璃管細胞移植之注射針 52
（二） 綠螢光精原幹細胞之輸出小管注射 53
四、 蘇木紫＆伊紅染色法 54
五、 免疫組織化學染色法 （Immunostaining, IHC） 54
（一） 抗原修復與染色 55
結果 56
討論 57
一、 精原幹細胞移植後形成精子之時間 57
二、 影響精原幹細胞移植至睪丸中成敗之可能因素 57
試驗三小結 58
總結 68
參考書目 69

 

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