Objective To explore the expression of integrin-linked kinase(ILK) in scar in different growth stages,as well as its relationship with angiogenesis.Methods ( 1 ) Fifteen burn patients with scar formation time shorter than 6 months,ranging from 6 to 12 months,and longer than 12 months were hospitalized from December 2009 to December 2010.They were divided into A,B,and C groups according to the scar formation time,with 5 patients in each group.Scar specimens were harvested for obviation of ILK expression with immunohistochemistry method,and ILK mRNA expression with real time fluorescence quantitative RT-PCR.(2) Microvascular endothelial cells ( MEC ) were isolated from scar tissue in A group and cultured in vitro,and then they were purified by immunomagnetic beads and identified with coagulation factor Ⅷ marked by immunofluorescence (fibroblasts from human normal skin were used as control).The cultured cells in logarithmic growth phase were divided into control group ( cultured with M131 medium containing microvascular growth supplement ),transfection Ⅰ group (transfected with empty plasmid),and transfection 2 group ( transfected with ILK cDNA plasmid) according to the random number table.After 24 hour,the expressions of ILK mRNA,Flt-1 mRNA,and KDR mRNA were determined with real time fluorescence quantitative RT-PCR,Data were proceessed with one way analysis of variance.Results Immunohistochemical observation showed that ILK in A group mainly expressed in the basal layer cells of epidermis,cytoplasm of fibroblasts,and MEC in scar,while ILK in B group only distributed in the basal layer cells of epidermis,but ILK expression in C group was not obvious.The expression of ILK mRNA in A group (0.34 ±0.16) was significantly higher than those in B and C groups (0.17 ± 0.06,0.07 ± 0.13,F =37.007,P =0.000).MEC grew up showing cobble stone formation after purification.The expression of coagulation factor Ⅷ was positive in cytoplasm of purified MEC,while that was negative in fibroblast of human normal skin.The expressions of ILK mRNA (57.807 ± 5.556 ),KDR mRNA (0.836 ± 0.014),and Flt-1 mRNA (0.162 ± 0.005 ) in transfection 2 group were higher than those in control and transfection 1 groups (0.018 ±0.003,0.028 ±0.020,0.023 ±0.004 and 0.042 ±0.005,0.039 -±0.007,0.046 ±0.003;F ILK =87.110,FKDR =11.241,FFlt =18.199,withP values all below 0.01). Conclusions ILK mainly expressed in scar tissue with formation time shorter than 6 months,and it may affect vascularization of scar by regulating gene expressions of KDR and Flt-1 in MEC,which plays an important role in early scar formation.%目的 探讨不同生长期人类瘢痕中整合素连接激酶(ILK)表达及与血管生成的关系.方法 (1)将笔者单位2009年12月-2010年12月收治的15例烧伤瘢痕患者按瘢痕生长时间分为:小于6个月组、6～12个月组、大于12个月组,每组5例.采集各组瘢痕组织,用免疫组织化学法观察ILK的表达分布,实时荧光定量RT-PCR法检测ILK mRNA的表达水平.(2)取小于6个月组瘢痕组织,分离培养瘢痕微血管内皮细胞(MEC),免疫磁珠法纯化后用花青类荧光染料Cy3标记的凝血因子Ⅷ进行鉴定,以人皮肤Fb为对照.取对数生长期MEC,按随机数字表法分为3组:对照组,仅用含微血管生长添加剂的M131培养液培养；空质粒组,用空载质粒转染；ILK互补DNA转染组,用ILK互补DNA表达质粒转染.转染24h后,实时荧光定量RT-PCR法检测各组细胞中ILK、激酶功能区受体(KD R)、fms样酪氨酸激酶1(Flt1)的mRNA表达情况.对数据进行单因素方差分析. 结果 (1)小于6个月组瘢痕组织表皮基底细胞、MEC及Fb胞质中均有ILK阳性表达,6～12个月组瘢痕组织中仅表皮基底细胞可见ILK阳性表达,大于12个月组瘢痕组织中ILK阳性表达不明显.(2)小于6个月组瘢痕组织中ILK mRNA表达水平(0.34±0.16)明显高于6～12个月组(0.17±0.06)及大于12个月组(0.07±0.13),F=37.007,P - 0.000.(3)纯化后MEC呈铺路石样密集生长,胞质中凝血因子Ⅷ呈阳性表达；人皮肤Fb中未见凝血因子Ⅷ表达.(4)ILK互补DNA转染组瘢痕MEC中ILK、KDR及Flt-1的mRNA表达水平分别为57.807±5.556、0.836±0.014、0.162±0.005,均显著高于对照组的0.018±0.003、0.028±0.020、0.023±0.004和空质粒组的0.042±0.005、0.039±0.0070.046±0.003(F值分别为87.110、11.241、18.199,P值均小于0.01).结论 ILK主要表达于小于6个月的早期瘢痕组织中,并可以通过调节瘢痕MEC中的KDR及Flt-1 mRNA表达来影响早期瘢痕的血管生成,在早期瘢痕增生过程中具有重要作用.
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