Background Corneal chemical burn is one of blinding eye diseases.Previous therapies for corneal chemical burn is limited to certain extent.However,transplantation of adipose-derived mesenchymal stem cells (ADSCs) for corneal diseases is drawing more and more attention.Objective This study was to observe the effect of rabbit ADSCs transplantation for ocular alkali burns and explore its mechanism.Methods Rabbit corneal stromal cells (CSCs) were isolated and cultured by suspended matrix method,and rabbit ADSCs were obtained and digested from inguinal fat tissue with enzyme digestion method (0.25％ trypsin) and identified by flow cytometry.CSCs cocultured with ADSCs,and CSCs-induced ADSCs were identified by double-label of with immunofluorescence and reverse transcription PCR (RT-PCR).Then induced or uninduced ADSCs were inoculated on amniotic membrane to prepared ADSCs-amnion patch.Corneal alkali burn models were established in the right eyes of 60 New Zealand rabbits by placing a filter paper with 1％ NaOH solution at the central cornea for 50 seconds.The models were randomized into the induced ADSCs+ amnion implanted group,the uninduced ADSCs + amnion implanting group,amnion implanted group and model group.Corneal opacification and neovascular area were examined and corneal inflammation was graded by slit lamp microscope 1 week,2 weeks and 1 month after surgery.The contents of CD45,interferon-γ (IFN-γ) and interleukin-10 (IL-10) in corneal homogenate as well as vascular endothelial growth factor (VEGF) in aqueous humor were detected by ELISA assay.The use and care of experiment animals followed the Statement of ARVO.Results ADSCs showed the positive responses for CD105,CD29,CD44 with the positive rate 90.23 ％,88.56％ and 98.88％,respectively.CSCs was positively reactive for vimentin.The double-label staining was positive after coculture of CSCs with ADSCs.Hematoxylin-eosin stain exhibited that ADSCs grew well on the amnion.Corneal porcelain opacity and a lot of new blood vessels were seen in the model group,and corneal was clear in the induced ADSCs+ amnion implanted group 1 month after surgery.The inflammatory scores were 1.65 ±0.18,2.05 ± 0.17,2.68±0.25,2.90 ±0.18,and the areas of neovasculization were (10.59 ± 1.78),(22.58 ± 1.63),(37.98 ± 1.90),(45.37±1.65)mm2 respectively in the induced ADSCs+ amnion implanted group,uninduced ADSCs+ amnion implanted group,amnion implanted group and the model group.The inflammatory scores of 1 week,2 weeks,1 month after operation among the four groups had statistically significant differences (F =280.826,330.172,465.707,all at P =0.000),and the areas of neovasculization of 1 week,2 weeks,1 month after operation among the four groups had statistically significant differences (F=60.020,670.811,1 510.231,all at P =0.000),the inflammatory scores in the induced ADSCs + amnion implanted group were remarkably lower than those of the other groups,the areas of neovasculization in the induced ADSCs+ amnion inplanted group were smaller than those of the other groups (all at P＜0.01).In 1 month after surgery,the contents of CD45,IL-10,IFN-γ in cornea and VEGF in aqueous humor were statistically different among the groups(F =916.545,1 739.358,462.134,129.126,all at P =0.000).Compared with the uninduced ADSCs+ amnion implanted group,amnion implanted group and the model group,CD45 and IFN-γ contents were declined,and IL-10 content was elavated in the induced ADSCs+ amnion implanted group (all at P＜ 0.01).In addition,VEGF contents in aqueous humor were significantly lower than those in the other groups (all at P＜0.01).Conclusions Rabbit CSCs-induced ADSCs amnion patch transplantation is effective for the reconstruction of ocular surface after alkali damage probably by differentiation of ADSCs into epithelial-like cell after CSCs induced.Moreover,amnion can alleviate immuno-inflammatory response and suppress neovascularization.%背景 角膜化学烧伤是致盲眼病之一,先前的各种疗法在临床上的应用均受到一定的限制,而脂肪间充质干细胞(ADSCs)移植疗法受到密切关注. 目的 观察诱导分化的ADSCs羊膜片移植治疗兔角膜碱烧伤的疗效及其机制.方法 无菌条件下剪取兔角膜,用悬浮基质片法分离并培养兔角膜基质细胞(CSCs).取兔腹股沟脂肪组织,以酶消化法(质量分数0.25％胰蛋白酶)分离及培养兔ADSCs,并用流式细胞术鉴定ADSCs表面抗原的表达.将CSCs与ADSCs共培养,采用免疫荧光技术和逆转录PCR(RT-PCR)鉴定CSCs诱导后ADSCs向角膜基质样细胞分化的表达,分别将诱导或未诱导的ADSCs种植于羊膜上制备成细胞羊膜片.选择60只新西兰白兔,右眼用浸有质量分数1％ NaOH的滤纸贴敷于角膜中央50 s制备兔角膜碱烧伤模型,按随机数字表法将模型眼随机分为诱导ADSCs+羊膜移植组、未诱导ADSCs±羊膜移植组、单纯羊膜移植组及模型组.分别于术后1周、2周和1个月在裂隙灯显微镜下观察各组兔模型眼角膜的混浊程度和新生血管形成情况,并对角膜炎症进行评分,采用ELISA法检测术后1个月角膜组织匀浆中CD45、γ干扰素(IFN-γ)、白细胞介素-10(IL-10)蛋白及房水中血管内皮生长因子(VEGF)蛋白的质量浓度.结果 从脂肪组织分离培养的ADSCs表面抗原CD105、CD29、CD44的阳性表达率分别为90.23％、88.56％、98.88％;分离培养的CSCs波形蛋白呈阳性表达;CSCs与ADSCs共培养后多数细胞双标记染色阳性.苏木精-伊红染色显示,ADSCs种植于羊膜后生长良好.术后1个月,模型组角膜混浊呈瓷白色,新生血管面积最大,而诱导ADSCs+羊膜移植组角膜透明,新生血管最少.术后1个月,诱导ADSCs+羊膜移植组、未诱导ADSCs+羊膜移植组、单纯羊膜移植组和模型组兔角膜混浊程度评分分别为1.65±0.18、2.05±0.17、2.68±0.25和2.90±0.18,新生血管面积分别为(10.59±1.78)、(22.58±1.63)、(37.98±1.90)和(45.37±1.65) mm2,术后1周、2周及1个月4个组间角膜混浊程度评分和角膜新生血管(CNV)面积的差异均有统计学意义(F=280.826、330.172、465.707,均P=0.000;F=60.020、670.811、1 510.231,均P=0.000),其中诱导ADSCs+羊膜移植组角膜炎症评分明显低于其他3个组,CNV面积小于其他3个组,差异均有统计学意义(均P＜0.01).术后1个月,4个组间角膜组织匀浆中CD45、IL-10、IFN-γ质量浓度的差异均有统计学意义(F=916.545、1 739.358、462.134,均P=0.000),其中与未诱导ADSCs+羊膜移植组、单纯羊膜移植组和模型组比较,诱导ADSCs+羊膜移植组CD45、IFN-γ质量浓度明显降低,而IL-10质量浓度明显升高,差异均有统计学意义(均P＜0.01).4个组兔房水中VEGF质量浓度的总体比较差异有统计学意义(F=129.126,P=0.000),其中诱导ADSCs+羊膜移植组兔房水中VEGF质量浓度明显低于未诱导ADSCs+羊膜移植组、单纯羊膜移植组和模型组,差异均有统计学意义(均P＜0.01).结论 CSCs诱导的ADSCs羊膜片移植法治疗兔角膜碱烧伤有较好的疗效,其作用机制可能是ADSCs在CECs诱导下分化为具有角膜上皮细胞特征的细胞,同时羊膜可减轻免疫炎症反应,抑制新生血管形成.
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