以人類臍帶間質幹細胞做為角膜內皮失償性水腫的細胞療法

Abstract

在人類、非人靈長類、以及犬貓的角膜內皮細胞再生能力有限，而角膜內皮細胞含有Na+/K+ ATPase 幫浦，對於維持角膜的透明度和控制角膜含水量有很大的重要性。因此若無法代償便會產生角膜水腫，唯有角膜移植手術能有效改善，然而全球都有角膜來源欠缺及移植後排斥反應的問題存在。人類臍帶間質幹細胞(human umbilical mesenchymal stem cells, HUMSCs)在近年來已被證明含有許多種分化潛能，而且在體外能以不同細胞培養環境來誘導，可應用在再生醫學方面。然而目前並沒有任何研究證明臍帶間質幹細胞可以分化為角膜內皮細胞。 本研究評估以HUMSC治療白兔角膜內皮細胞缺損之潛能，以兩種方式於紐西蘭白兔測試並建立眼角膜內皮細胞缺損的動物模式，包括眼前房注射mitomycin-C (MMC)及角膜彈力層刮除術，以模擬在人類和多數動物角膜內皮缺損的情形。結果顯示刮除術組能維持角膜混濁第4級，更可有效建立長久的重度角膜內皮缺損，然而MMC組方面只能達到中等程度的水腫，維持約少於一個月的角膜內皮缺損。HUMSC的移植採用細胞懸浮液(1x107 cell/μl)前房注射的方式，並在術後保持側臥姿6小時，角膜上皮朝下的方式以重力幫助移植細胞服貼於角膜內層。觀察其治療失償性水腫的效果，結果顯示臨床上角膜水腫情形並未改善；病理評估後發現，HUMSC移植後能存活但往多方面分化，如虹膜上皮、類似睫狀體的增生組織和肉芽腫。其中處於前房和虹膜的密集細胞團塊，因其細胞核質比高並出現有絲分裂，高度懷疑為HUMSC。此外CD31與VEGF染色證實有血管增生的情形。顯示雖然移植後的HUMSC能存活並保有分化能力，但有可能HUMSC分化成同樣是神經脊來源的間質組織。而以CFDA-SE測試體外標定HUMSC，結果顯示持續時期過短，螢光在約維持一周後便逐漸衰弱，而無法做為追蹤移植後HUMSC的細胞數量和位置。

The ability of human, nonhuman primate, canine and feline corneal endothelium to proliferate is severely limited, and corneal endothelial cells hold Na+/K+ ATPase and contribute to the maintenance of the corneal transparency by their pump and barrier functions. Therefore, decompensation of endothelium would produce corneal edema. Keratoplasty is the only effective treatment for endothelial dysfunction, but there is a worldwide shortage of human donor corneas. Human umbilical mesenchymal stem cells (HUMSCs) have the potential to develop into multicells in different ex vivo culture environment, improving regenerative medicine. However, there is no evidence to prove HUMSCs could differentiate into corneal endothelium. Hence, the objective of this study was to investigate the potential of HUMSCs transplantation to treat rabbit corneal endothelium deficiency edema. To create an animal model of corneal endothelium deficiency in New Zealand white rabbit, Mitomycin-C (MMC) anterior chamber injection and descemetohexis were used to enable the mimicking of human cornea. While descemetohexis group resulted in permanent defect of endothelium with corneal haze grade 4, the MMC group only maintained moderate cornea endothelium deficiency less than a month. HUMSCs were transplanted by the injection into anterior chamber in cell suspension (1x107 cell /μl). After transplantation, rabbits were kept in the prone position (epithelium down) for 6 hours, to improve HUMSC attachment to posterior cornea by gravity. As a result, post-transplantation did not decrease clinical corneal edema level, and histological examination revealed that dense cell clusters resided in the anterior chamber and iris. Given the fact that they possessed high N/C ratio and mitosis, it is highly suspected that the dense cell clusters were the transplanted HUMSCs. The HUMSCs survived and differentiated into various kinds of cells, and sequentially proliferated into iris epithelium and ciliary body-like tissue, in which CD31 and VEGF stain showed angiogenesis. The finding of this study also suggested that HUMSCs can survive after anterior chamber transplantation, and possess differentiation capacity. Thus, HUMSCs may have the potential to differentiate into various cells originating from the neural crest-derived mesenchymal tissue. Another discovery made by the study is the ex vivo labeled HUMSCs with CFDA-SE resulted in gradually weaker fluorescent after one week, a reason why CFDA-SE is probably not a suitable marker to trace the number and location of transplanted HUMSCs.