Abstract：
OBJECTIVE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro.
METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay.
RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2.
CONCLUSIONS: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.
METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay.
RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2.
CONCLUSIONS: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.
摘要：
目的：铜绿假单胞菌诱导的角膜炎是角膜感染的最严重和最有挑战性的形式之一，由于其相关的强烈的炎症反应导致角膜坏死和致密的角膜瘢痕与视力丧失。由于间充质干细胞（MSCs）被报道具有抗菌和免疫调节特性，它们可以与抗生素一起作为辅助治疗进行测试，抗生素是目前的护理标准。本研究旨在研究人骨髓MSC来源的条件培养基（MSC-CM）在体外对铜绿假单胞菌感染的人角膜上皮细胞（HCECs）的抗菌和免疫调节作用。
方法：通过集落形成单位试验评价MSC-CM对铜绿假单胞菌临床分离株生长的影响。通过ELISA分析了脂多糖处理的MSCs和HCECs中炎性细胞因子（IL-6和TNF-α）和抗微生物肽（脂质运载蛋白2）的表达。通过划痕试验研究了铜绿假单胞菌感染后的角膜上皮修复。
结果：与对照组相比（P.铜绿假单胞菌（5*105）在DMEM（1ml）中在37°C下孵育16小时），MSC-CM显着：i）抑制铜绿假单胞菌的生长（159*109vs.104*109CFU/ml)，ii）加速铜绿假单胞菌感染后的角膜上皮修复（9％vs.感染12小时后受伤区域闭合24%)，和iii）下调HCECs中脂多糖诱导的IL-6，TNF-α和脂质运载蛋白2的表达。MSC-CM与抗生素的组合，环丙沙星适度调节IL-6、TNF-α、和Lipocalin2。
结论：MSC-CM有望作为铜绿假单胞菌诱导的角膜上皮损伤的辅助治疗方法。
方法：通过集落形成单位试验评价MSC-CM对铜绿假单胞菌临床分离株生长的影响。通过ELISA分析了脂多糖处理的MSCs和HCECs中炎性细胞因子（IL-6和TNF-α）和抗微生物肽（脂质运载蛋白2）的表达。通过划痕试验研究了铜绿假单胞菌感染后的角膜上皮修复。
结果：与对照组相比（P.铜绿假单胞菌（5*105）在DMEM（1ml）中在37°C下孵育16小时），MSC-CM显着：i）抑制铜绿假单胞菌的生长（159*109vs.104*109CFU/ml)，ii）加速铜绿假单胞菌感染后的角膜上皮修复（9％vs.感染12小时后受伤区域闭合24%)，和iii）下调HCECs中脂多糖诱导的IL-6，TNF-α和脂质运载蛋白2的表达。MSC-CM与抗生素的组合，环丙沙星适度调节IL-6、TNF-α、和Lipocalin2。
结论：MSC-CM有望作为铜绿假单胞菌诱导的角膜上皮损伤的辅助治疗方法。
