低氧處理羊水幹細胞治療大鼠腎間質纖維化之療效

腎間質纖維化 (renal interstitial fibrosis, RIF) 為慢性腎臟病 (chronic kidney disease, CKD) 進展之主要途徑，而 CKD 為臺灣常見之慢性疾病，其盛行率於 2016 年達 9.8%，同年因腎炎、腎病症候群及腎病變造成之死亡率更列為十大死因之一。CKD 若嚴重會導致末期腎衰竭，治療方式以血液或腹膜透析或腎臟移植為主。先前之研究證實幹細胞具有組織再生之臨床應用潛能，期望能藉此找到更有效用之替代療法。羊水幹細胞 (amniotic fluid stem cells, AFSCs) 因應用上較無倫理道德爭議及形成畸胎瘤之風險，另具有低免疫原性、來源取得容易且低侵入性之優點，且胎兒發育期間之環境氧濃度僅約為 4 至 5%，故本試驗擬藉由分離自大鼠胎兒羊水之 AFSCs 進行低氧 (5% O2) 處理後再移植於單側輸尿管梗阻 (unilateral ureteral obstruction, UUO) 引發 RIF 之大鼠模式，探討低氧處理是否能維持體外培養 AFSCs 之幹性，進而增加移植後之 RIF 治療之可行性。試驗分二部分進行，試驗一探討低氧培養之大鼠羊水幹細胞 (rat amniotic fluid stem cells, rAFSCs)對其幹性、增殖及分化潛能之影響，自懷孕大鼠胎兒羊水分離之細胞培養於常氧 (normoxia, Nor) 19.9％ O2及低氧 (hypoxia, Hyp) 5％ O2環境。結果顯示，Nor-rAFSCs 與 Hyp-rAFSCs 隨著繼代培養，皆呈現類纖維母細胞之紡錘狀形態，生長方向為螺旋狀排列。將二種處理之 rAFSCs (P11) 以流式細胞儀進行分析結果顯示，Hyp-rAFSCs 之細胞形態顯著較 Nor-rAFSCs 小 (P < 0.05)，且於 CD29及 CD90之表現量皆有高於 Nor-rAFSCs 之趨勢，而其 octamer-binding transcription factor 4 (Oct-4) 之基因表現量倍率亦顯著較後者增加 (P < 0.05)；同時，Hyp 處理亦有顯著提高 rAFSCs 之增值倍率 (P < 0.05)。此外，rAFSCs 脂肪誘導分化試驗中，Hyp 處理無顯著提高其分化為脂肪細胞之能力；而 rAFSCs 之硬骨誘導分化試驗中，Hyp 之誘導處理組其硬骨分化能力顯著低於 Nor 處理組 (P < 0.05)，且於軟骨誘導分化之培養中無法形成細胞球，因此推測 Hyp 處理可能降低 rAFSCs 分化為軟骨及硬骨細胞之潛能。試驗二探討Hyp-rAFSCs及其條件培養液 (condition medium, CM) 對其移植於經UUO 大鼠尾靜脈後緩解其RIF之影響，其移植一週及二週後將大鼠犧牲，取其腎臟進行檢測分析，外觀型態上，無論 Nor 或 Hyp處理之 CM 或 rAFSCs 經移植一週與二週後之左腎皆因尿液積累而有較右腎膨大之現象；另將其組織切片以 H&E 與 Masson’s Trichome 染色進行組織分析，發現經移植一週與二週後之左腎皆有發炎細胞之浸潤，顯示腎臟組織受到損傷，經由Nor 或 Hyp處理之 CM 或 rAFSCs 移植後一週之 RIF 均較 vehicle 組有緩解之趨勢，移植二週後則除 Nor-CM 組外，其餘組別皆有緩解纖維化之趨勢，再者，RIF大鼠腎臟之纖維化相關因子 TGF-β1、collagen Ⅰ 及 α-SMA 之表現量倍率分析，除 Nor-rAFSCs 組，其他組別經移植一週後之纖維化因子之表現量倍率皆有低於 vehicle 組之趨勢；移植二週後，少數組別與 vehicle 組之基因表現量倍率趨近相同，然多數組別則有高於 vehicle 之趨勢，由此推測，rAFSCs 或其 CM 於體內無法改善 RIF 至二週，其原因可能與同種異體移植之幹細胞及其分泌因子於宿主體內存活及降解時間有關。綜上所述，Hyp 處理具有提高 rAFSCs 之增殖倍率，即有利於其體外增殖，同時亦能增加 rAFSCs 表現典型幹細胞表面抗原 CD90、MSCs 表面抗原 CD29 之比例，與幹性及多能性相關之分子標誌 Oct-4 之基因表現量倍率，並具降低其分化為硬骨之潛能，而 Hyp-rAFSCs 與其 CM 之移植對 RIF 大鼠之療效僅於移植一週與二週後有緩解纖維化之趨勢。

關鍵字

腎間質纖維化；羊水水幹細胞；低氧；常氧

並列摘要

Renal interstitial fibrosis (RIF) is a main progression pathway of chronic kidney disease (CKD). Besides, CKD is a common chronic disease in Taiwan and its prevalence rate was 9.8% in 2016. Moreover, mortality caused by nephritis, nephrotic syndrome and nephropathy were ranked as one of the top ten causes of death in the same year. If CKD is severe, it will lead to end-stage of renal failure, and therefore the treatment now is mainly based on hemodialysis or peritoneal dialysis or kidney transplantation. Previous researches confirmed that stem cells have the clinical potential of tissue regeneration and are expected to find more effective alternative therapies. Application of amniotic fluid stem cells (AFSCs) do not have ethical dispute and the risk of teratoma formation, besides, ASFCs have advantage such as low immunogenicity, easy and non-invasive accessibility. In addition, environmental oxygen concentration of fetal development process is only about 4 to 5%. For this reason, the aim of my research intended to isolate AFSCs from amniotic fluid of fetus during pregnant rats, treat it by hypoxia and then transplant it to RIF models induced by unilateral ureteral obstruction (UUO) in order to discuss whether hypoxia-treated AFSCs can maintain their stemness, proliferation and differentiation potentials, even increase feasibility of RIF treatment after AFSCs transplantation. There were two parts of experiment. In part one, I investigated the effect of hypoxia on stemness, proliferation and differentiation potentials of rAFSCs. After isolated rAFSCs from amniotic fluid of fetus during pregnant rats, cultured them under nomoxia (Nor) 19.9% O2 and hypoxia (Hyp) 5% O2 respectively. The result demonstrated that both Nor-rAFSCs and Hyp-rAFSCs showed spindle-like morphology of fibroblasts and their growth direction exhibit spiral order in subculture. Moreover, Hyp-rAFSCs presented significantly smaller morphology than Nor-rAFSCs (P < 0.05), had higher expression of surface markers such as CD29 and CD90 by flow cytometry, and even exhibited higher Oct-4 gene expression fold change than the latter. Simultaneously, Hyp-treatment significantly increased proliferation rate of Hyp-rAFSCs (P < 0.05). Furthermore, it showed that Hyp did not significantly increased adipogenesis but significantly decreased osteogenesis of Hyp-rAFSCs (P < 0.05), and even caused Hyp-rAFSCs did not form chondrogentic sphere. As a result, I speculated Hyp-treatment might lowered the potentials of Hyp-rAFSCs to differentiate into cartilage and osteoblasts. In part two of experiment, I further investigated whether the rAFSCs and rAFSCs-conditioned medium under hypoxia can alleviate the renal interstitial fibrosis of rat by tail vein injection. After 1 and 2 weeks of transplantation, rats were sacrificed and isolated kidneys for analysis. First, no matter Nor or Hyp-treated rAFSCs or CM groups, their left kidneys showed ectatic morphology due to urine accumulation after 1 and 2 weeks of transplantation. Next, stained renal tissues with H&E and Masson’s Trichrome stain for histological examination. The results demonstrated that infiltration of inflammatory cell in H&E stain, showing renal tissues were damaged. Besides, transplanted Nor or Hyp-treated rAFSCs or CM had alleviated trend on rat RIF after 1 week, and except Nor-CM group, other transplant groups had alleviated trend on fibrosis after 2 weeks. Moreover, analysis of fibrosis related gene such as TGF-β1, collagen I and α-SMA mentioned that except the Nor-rAFSCs group, others had lower trend of fibrosis related gene expression after 1 week-transplantation. However, fibrosis related gene expression fold change in minority of groups were similar to vehicle groups, majority of groups were higher than vehicle group instead after 2 weeks. Therefore, it was speculated that rAFSCs or their CM could not improve RIF in vivo for two weeks, which may be related to the survival time of allogeneic stem cells and decay of their secreted factors in the host. In summary, Hyp-treatment has the advantage of increasing the proliferation rate of rAFSCs, which means Hyp is beneficial to rAFSCs proliferation in vitro. Simultaneously, Hyp-treatment could also increase the ratio of typical stem cell surface antigens such as CD90, MSCs surface antigens such as CD29, the gene expression fold change of stemness and pluripotency related molecular markers such as Oct-4, and reduce the osteogenesis potential of Hyp-rAFSCs. Besides, the therapeutic effect of Hyp-rAFSCs and Hyp-CM on RIF rat only had alleviated trend after one and two-week-transplantation.

並列關鍵字

renal interstitial fibrosis ； amniotic fluid stem cells ； hypoxia ； normoxia

參考文獻

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低氧處理羊水幹細胞治療大鼠腎間質纖維化之療效

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