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角膜上皮细胞和培养的自体口腔黏膜上皮细胞(CAOMECS)移植物中的蛋白酶体用于眼表的再生

Bardag-Gorce, F., Hoft, R., Meepe, I., et al.

期刊名称:The Ocular Surface

卷期:2017年第15卷第4期

摘要

摘要:目的:本研究重点是描述用于再生眼表面的角膜上皮细胞(CEC)和培养的自体口腔黏膜上皮细胞(CAOMECS)中的蛋白酶体特征。方法:手术诱导兔子角膜出现角膜缘干细胞缺乏(LSCD)。 设计CAOMECS并移植到LSCD角膜以再生眼表面。结果:LSCD引起眼表面炎症细胞增加,免疫蛋白酶原(IPR)形成增加,以及组成型蛋白酶体(CPR)形成减少。具体来说,LSCD患病CECD-CEC)显示CPR糜蛋白酶样,胰蛋白酶样和胱天蛋白酶样活性降低,而健康CECHECC)和CAOMECS显示较高的活性。进行IPR诱导亚单位(B5iB2iB1i)定量分析,并与CPR亚单位(B5B2B1)水平进行比较。结果显示,D-CECB5i / B5B2i / B2B1i / B1比值较高,说明D-CECCAOMECSH-CEC相比,IPR量增加了近2倍。组织学分析表明,CAOMECS移植角膜具有再生上皮表面,CPR亚单位染色阳性,IPR亚基弱染色。此外,荧光强度数字定量测量显示,与未移植的LSCD角膜相比,CAOMECS移植角膜中CPR B5亚基表达显着增加。结论:CAOMECS移植成功的用高水平CPR替代了D-CEC口腔粘膜上皮细胞。组成型蛋白酶体表达的增加可能是CAOMECS移植角膜恢复和改善的原因。

PURPOSE:This study focuses on characterizing proteasomes in corneal epithelial cells (CEC) and in cultured autologous oral mucosal epithelial cell sheets (CAOMECS) used to regenerate the ocular surface.METHODS:Limbal stem cell deficiency (LSCD) was surgically induced in rabbit corneas. CAOMECS was engineered and grafted onto corneas with LSCD to regenerate the ocular surface.RESULTS:LSCD caused an increase in inflammatory cells in the ocular surface, an increase in the formation of immunoproteasomes (IPR), and a decrease in the formation of constitutive proteasome (CPR). Specifically, LSCD-diseased CEC (D-CEC) showed a decrease in the CPR chymotrypsin-like, trypsin-like and caspase-like activities, while healthy CEC (H-CEC) and CAOMECS showed higher activities. Quantitative analysis of IPR inducible subunit (B5i, B2i, and B1i) were performed and compared to CPR subunit (B5, B2, and B1) levels. Results showed that ratios B5i/B5, B2i/B2 and B1i/B1 were higher in D-CEC, indicating that D-CEC had approximately a two-fold increase in the amount of IPR compared to CAOMECS and H-CEC. Histological analysis demonstrated that CAOMECS-grafted corneas had a re-epithelialized surface, positive staining for CPR subunits, and weak staining for IPR subunits. In addition, digital quantitative measurement of fluorescent intensity showed that the CPR B5 subunit was significantly more expressed in CAOMECS-grafted corneas compared to non-grafted corneas with LSCD.CONCLUSION:CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.


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