發展腎小管細胞應用於角膜內皮功能缺陷疾病之細胞療法

Abstract

角膜內皮細胞(Corneal Endothelial Cells, CECs)具有主動排水(pump)功能，還有角膜房水屏障能力，此功能對於角膜維持透明極其重要。若角膜內皮細胞失去排水功能，可能造成角膜水腫和大泡性角膜病變。從文獻中可得知，腎小管細胞水分運輸的機制與角膜內皮細胞有許多相似之處，水可以經由鈉鉀幫浦主動運輸鈉離子所造成的滲透壓梯度被動地運輸。因此，本實驗希望能發展腎小管細胞應用於角膜內皮功能病變病人的治療可能性。

本研究使用人類腎小管細胞 (human Renal Proximal Tubular Cells, hRPTCs) 並培養在牛角膜的後彈力層上，在體外模擬腎小管細胞生長於角膜的環境，評估細胞型態與功能。接下來，將人類腎小管細胞與人類角膜內皮細胞株 (HCEC-B4G12) 共同培養在角膜後彈力層上，觀察兩者細胞之間的相互作用以及相容性。此外會用共軛焦螢光顯微鏡(confocal microscope)建立3D影像，確定兩者細胞功能蛋白Na+-K+ ATPase和ZO1的表現位置。最後，建立動物模型，進行體內測試並評估治療效果。

體外實驗結果顯示，當人類腎小管細胞培養在角膜環境，可保持正常型態、表現重要的功能蛋白Na+-K+ ATPase和ZO1。在細胞共培養的情況，兩種細胞可以共同生存，相同的細胞會聚集在同一區域，形成明顯的細胞分界。並且，我們發現兩種細胞的Na+-K+ ATPase蛋白皆表現在基底外側 (basolateral side)。因此我們推測，如果腎小管細胞移植到角膜內皮環境中，有可能會受內部環境調控與角膜內皮細胞產生相同的水分運輸方向。從動物實驗初步的結果可知，腎小管細胞可以貼附在兔子的角膜後彈力層上，但還需要更進一步的研究，確認是否能在活體實驗中達到有效的治療結果。

Corneal endothelial cells have an active pumping function and corneal-aqueous humor-barrier capability, which is extremely important for maintaining normal thickness and transparency of the cornea. If the corneal endothelial cells lose pumping function, it may cause corneal edema and bullous keratopathy. From other literatures, we know that the water transport mechanism of renal tubular cells was similar with corneal endothelial cells. The water is passively transported through the osmotic pressure gradient caused by the sodium potassium ATPase (Na+-K+ ATPase) active pumping sodium ion. Therefore, the aim of this study is to develop an alternative therapy of renal tubular cells for the corneal endothelial dysfunction.

In this study, human renal proximal tubular cells (hRPTCs) were cultured on the Descemet’s membrane of the bovine cornea, and evaluated cell morphology and function in vitro. Next, hRPTCs were co-cultured with corneal endothelial cell line (HCEC-B4G12) on the Descemet’s membrane of the bovine cornea to evaluate cell-to-cell interaction and compatibility. In addition, 3D images will be created using a confocal microscope to determine the functional proteins (Na+-K+ ATPase and ZO1) expression sites of the two cells. Finally, an animal model is established to evaluate the therapeutic effect in vivo.

In vitro, the results showed that when hRPTCs were cultured in the corneal environment, the normal morphology and important functional proteins Na+-K+ ATPase and ZO1 were expressed. In co-culture system, the two cells can co-exist. And the same cells will accumulate together, forming a distinct cell boundary. Moreover, we found that both Na+-K+ ATPase proteins of the two cells are expressed on the basolateral side. Therefore, we speculate that if the hRPTCs are transplanted into the corneal endothelium environment, it may be regulated by the internal environment to produce the same water transport direction as corneal endothelial cells. From the preliminary results of animal experiments, it can be seen that hRPTCs can be attached to the Descemet’s membrane of the rabbit cornea. However, further study is needed to confirm whether the hRPTCs can achieve effective therapeutic results in vivo.