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.
揹景 角膜化學燒傷是緻盲眼病之一,先前的各種療法在臨床上的應用均受到一定的限製,而脂肪間充質榦細胞(ADSCs)移植療法受到密切關註. 目的 觀察誘導分化的ADSCs羊膜片移植治療兔角膜堿燒傷的療效及其機製.方法 無菌條件下剪取兔角膜,用懸浮基質片法分離併培養兔角膜基質細胞(CSCs).取兔腹股溝脂肪組織,以酶消化法(質量分數0.25％胰蛋白酶)分離及培養兔ADSCs,併用流式細胞術鑒定ADSCs錶麵抗原的錶達.將CSCs與ADSCs共培養,採用免疫熒光技術和逆轉錄PCR(RT-PCR)鑒定CSCs誘導後ADSCs嚮角膜基質樣細胞分化的錶達,分彆將誘導或未誘導的ADSCs種植于羊膜上製備成細胞羊膜片.選擇60隻新西蘭白兔,右眼用浸有質量分數1％ NaOH的濾紙貼敷于角膜中央50 s製備兔角膜堿燒傷模型,按隨機數字錶法將模型眼隨機分為誘導ADSCs+羊膜移植組、未誘導ADSCs±羊膜移植組、單純羊膜移植組及模型組.分彆于術後1週、2週和1箇月在裂隙燈顯微鏡下觀察各組兔模型眼角膜的混濁程度和新生血管形成情況,併對角膜炎癥進行評分,採用ELISA法檢測術後1箇月角膜組織勻漿中CD45、γ榦擾素(IFN-γ)、白細胞介素-10(IL-10)蛋白及房水中血管內皮生長因子(VEGF)蛋白的質量濃度.結果 從脂肪組織分離培養的ADSCs錶麵抗原CD105、CD29、CD44的暘性錶達率分彆為90.23％、88.56％、98.88％;分離培養的CSCs波形蛋白呈暘性錶達;CSCs與ADSCs共培養後多數細胞雙標記染色暘性.囌木精-伊紅染色顯示,ADSCs種植于羊膜後生長良好.術後1箇月,模型組角膜混濁呈瓷白色,新生血管麵積最大,而誘導ADSCs+羊膜移植組角膜透明,新生血管最少.術後1箇月,誘導ADSCs+羊膜移植組、未誘導ADSCs+羊膜移植組、單純羊膜移植組和模型組兔角膜混濁程度評分分彆為1.65±0.18、2.05±0.17、2.68±0.25和2.90±0.18,新生血管麵積分彆為(10.59±1.78)、(22.58±1.63)、(37.98±1.90)和(45.37±1.65) mm2,術後1週、2週及1箇月4箇組間角膜混濁程度評分和角膜新生血管(CNV)麵積的差異均有統計學意義(F=280.826、330.172、465.707,均P=0.000;F=60.020、670.811、1 510.231,均P=0.000),其中誘導ADSCs+羊膜移植組角膜炎癥評分明顯低于其他3箇組,CNV麵積小于其他3箇組,差異均有統計學意義(均P＜0.01).術後1箇月,4箇組間角膜組織勻漿中CD45、IL-10、IFN-γ質量濃度的差異均有統計學意義(F=916.545、1 739.358、462.134,均P=0.000),其中與未誘導ADSCs+羊膜移植組、單純羊膜移植組和模型組比較,誘導ADSCs+羊膜移植組CD45、IFN-γ質量濃度明顯降低,而IL-10質量濃度明顯升高,差異均有統計學意義(均P＜0.01).4箇組兔房水中VEGF質量濃度的總體比較差異有統計學意義(F=129.126,P=0.000),其中誘導ADSCs+羊膜移植組兔房水中VEGF質量濃度明顯低于未誘導ADSCs+羊膜移植組、單純羊膜移植組和模型組,差異均有統計學意義(均P＜0.01).結論 CSCs誘導的ADSCs羊膜片移植法治療兔角膜堿燒傷有較好的療效,其作用機製可能是ADSCs在CECs誘導下分化為具有角膜上皮細胞特徵的細胞,同時羊膜可減輕免疫炎癥反應,抑製新生血管形成.
배경 각막화학소상시치맹안병지일,선전적각충요법재림상상적응용균수도일정적한제,이지방간충질간세포(ADSCs)이식요법수도밀절관주. 목적 관찰유도분화적ADSCs양막편이식치료토각막감소상적료효급기궤제.방법 무균조건하전취토각막,용현부기질편법분리병배양토각막기질세포(CSCs).취토복고구지방조직,이매소화법(질량분수0.25％이단백매)분리급배양토ADSCs,병용류식세포술감정ADSCs표면항원적표체.장CSCs여ADSCs공배양,채용면역형광기술화역전록PCR(RT-PCR)감정CSCs유도후ADSCs향각막기질양세포분화적표체,분별장유도혹미유도적ADSCs충식우양막상제비성세포양막편.선택60지신서란백토,우안용침유질량분수1％ NaOH적려지첩부우각막중앙50 s제비토각막감소상모형,안수궤수자표법장모형안수궤분위유도ADSCs+양막이식조、미유도ADSCs±양막이식조、단순양막이식조급모형조.분별우술후1주、2주화1개월재렬극등현미경하관찰각조토모형안각막적혼탁정도화신생혈관형성정황,병대각막염증진행평분,채용ELISA법검측술후1개월각막조직균장중CD45、γ간우소(IFN-γ)、백세포개소-10(IL-10)단백급방수중혈관내피생장인자(VEGF)단백적질량농도.결과 종지방조직분리배양적ADSCs표면항원CD105、CD29、CD44적양성표체솔분별위90.23％、88.56％、98.88％;분리배양적CSCs파형단백정양성표체;CSCs여ADSCs공배양후다수세포쌍표기염색양성.소목정-이홍염색현시,ADSCs충식우양막후생장량호.술후1개월,모형조각막혼탁정자백색,신생혈관면적최대,이유도ADSCs+양막이식조각막투명,신생혈관최소.술후1개월,유도ADSCs+양막이식조、미유도ADSCs+양막이식조、단순양막이식조화모형조토각막혼탁정도평분분별위1.65±0.18、2.05±0.17、2.68±0.25화2.90±0.18,신생혈관면적분별위(10.59±1.78)、(22.58±1.63)、(37.98±1.90)화(45.37±1.65) mm2,술후1주、2주급1개월4개조간각막혼탁정도평분화각막신생혈관(CNV)면적적차이균유통계학의의(F=280.826、330.172、465.707,균P=0.000;F=60.020、670.811、1 510.231,균P=0.000),기중유도ADSCs+양막이식조각막염증평분명현저우기타3개조,CNV면적소우기타3개조,차이균유통계학의의(균P＜0.01).술후1개월,4개조간각막조직균장중CD45、IL-10、IFN-γ질량농도적차이균유통계학의의(F=916.545、1 739.358、462.134,균P=0.000),기중여미유도ADSCs+양막이식조、단순양막이식조화모형조비교,유도ADSCs+양막이식조CD45、IFN-γ질량농도명현강저,이IL-10질량농도명현승고,차이균유통계학의의(균P＜0.01).4개조토방수중VEGF질량농도적총체비교차이유통계학의의(F=129.126,P=0.000),기중유도ADSCs+양막이식조토방수중VEGF질량농도명현저우미유도ADSCs+양막이식조、단순양막이식조화모형조,차이균유통계학의의(균P＜0.01).결론 CSCs유도적ADSCs양막편이식법치료토각막감소상유교호적료효,기작용궤제가능시ADSCs재CECs유도하분화위구유각막상피세포특정적세포,동시양막가감경면역염증반응,억제신생혈관형성.
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.
