SPARC通过磷酸化p38-MAPK信号通路调控角膜缘干细胞生物学特性的机制研究

Limbal stem cells (LSCs) are the basis of cornea physiological function. Severe ocular surface disease could cause limbal stem cell deficiency (LSCD), and lead to irreversible visual damage. LSCs transplantation is the best way to cure LSCD. The success rate of LSCs transplantation is severely influenced by the number of LSCs in graft—when LSCs number was less than 3% in graft, the transplantation success rate was no more than 10%. Our previous research showed that secreted protein acidic and rich in cysteine (SPARC) only existed in the basement membrane of limbus, and interestingly LSCs were also only found in the region where SPARC expressed. Hereby, we infer that SPARC might promote LSCs physiological function. The phosphorylation of p38-mitogen-activated protein kinase (p38-MAPK) signal pathway could enhance the proliferation of stem cell, mediate asymmetric division of stem cell, and replenish the stem pool; and the expression of phosphorylated p38-MAPK was observed in LSCs. Recent limited research proved that SPARC could enhance the proliferation and migration of tumor stem cell by the activation of p38-MAPK phosphorylation. However, how SPARC and the phosphorylation of p38-MAPK signal pathway mediate LSCs physiological function was still unclear. Accordingly, we hypothesize that 1) SPARC is indispensable to the maintenance of LSCs physiological function; 2) SPARC mediate the physiological function of LSCs mainly through the phosphorylation of p38-MAPK signal pathway. Our research could deepen the comprehension of the molecular mechanism in LSCs maintenance, promote the transplantation success rate, and provide a new method for LSCD therapy.

角膜缘干细胞（LSCs）是维持角膜生理功能的关键, LSCs失代偿（LSCD）会导致不可逆视力损害。LSCD治疗主要依赖LSCs移植。植片中LSCs数量显著影响移植成功率：植片内LSCs＜3%时移植成功率<10% 。我们前期研究表明，富含半胱氨酸的酸性分泌蛋白（SPARC）在眼表仅表达于角膜缘基底膜，LSCs仅见于SPARC表达区。故我们推断SPARC与LSCs特性维持密切相关。SPARC可激活p38-MAPK信号通路磷酸化，增强细胞的增殖及迁移能力，维持干细胞池稳定；p38-MAPK信号通路在LSCs中亦有表达。而SPARC与p38-MAPK通路在LSCs特性维持中的机制尚不清楚。据此，本研究提出①SPARC有助于LSCs特性维持②SPARC通过p38-MAPK信号通路磷酸化调控LSCs特性维持。本研究将深化对LSCs特性维持机制的认识，为提高LSCs移植成功率，治疗LSCD开拓新思路。

角膜缘干细胞（LSCs）是维持角膜正常生理功能的基础，LSCs失代偿（LSCD）会导致不可逆视力损害。目前LSCD治疗主要依赖LSCs移植，而植片中LSCs数量显著影响移植成功率：当植片内 LSCs＞3%时移植成功率约为 78%；当 LSCs＜3%时，移植成功率骤降为不足 10%。我们的前期研究证实富含半胱氨酸的酸性分泌蛋白（SPARC）在眼表仅表达于角膜缘基底膜，与LSCs存在空间高度相关性。在2016－2018年度本课题主要研究了①SPARC在LSCs 生物学特性维持中的作用，证实外源性SPARC能够促进LSCs体外扩增，增强传代能力及克隆形成能力，维持干细胞特异性蛋白及基因表达，抑制自发分化，并进一步筛选了SPARC最适培养浓度；②阐明了SPARC通过激活JNK及p38-MAPK 信号通路磷酸化调控LSCs 特性的维持；③在角膜上皮损伤动物试验中，证实了外源性SPARC能够促进LSCs增殖，加速角膜上皮创伤愈合；同时阐明了内源性SPARC在角膜上皮修复中的作用。本研究为改善LSCs 存活率、扩充LSCs 移植种子来源、有效治疗角膜缘干细胞失代偿、改善不可逆视力损害开拓了新的思路和提供理论依据。