Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns · 2011
[Expression of integrin-linked kinase in human hypertrophic scar and its relationship with angiogenesis].
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Li Ye-yang, Mi Lan, Li Gang, Lin Wei-hua, Sun Jing-en, Wang Ren-kun, Liang Zhen-wen
Аннотация
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 observation 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 1 group (transfected with empty plasmid), and transfection 2 group (transfected with ILK cDNA plasmid) according to the random number table. After 24 hours, the expressions of ILK mRNA, Flt-1 mRNA, and KDR mRNA were determined with real time fluorescence quantitative RT-PCR. Data were processed 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, F(KDR) = 11.241, F(Flt) = 18.199, with P 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.
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 observation 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 1 group (transfected with empty plasmid), and transfection 2 group (transfected with ILK cDNA plasmid) according to the random number table. After 24 hours, the expressions of ILK mRNA, Flt-1 mRNA, and KDR mRNA were determined with real time fluorescence quantitative RT-PCR. Data were processed 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, F(KDR) = 11.241, F(Flt) = 18.199, with P 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.