| BackgroudHypertrophic scar is known to result from excessive tissue repair after woundhealing. The scar formation involves multiple kinds of cells and extracellular matrix(ECM), as well as many signal paths. It is characterized by excessively proliferationof fibroblasts and collagen fibers. These processes rely on the nutrition offered by scarmicrovasculature. Since the importance of angiogenesis in hypertrophic scar has beenrecognized gradually in recent years, exploring the mechanism of angiogenesis hasbecome very urgent issue.Scar angiogensis needs the interaction between endothelial cells and ECM, whileintegrin is responsible for linking up their connection. Integrin-linked kinase is thekey enzyme in integrin pathway. As a signal path, it mediates the signal transmissionbetween cells and ECM and regulates the cellular biological characteristics such asproliferation, apoptosis, migration and angiogenesis. ILK is of promotingangiogenesis by restructuring endothelial cells’ cytoskeleton, accelerating cellmigration, preserving cell survival and proliferation in the researches about tumorangiogenesis, diabetic retinopathy (DR) and embryonic development. But there havebeen not any study about the effects and mechanisms of ILK in scar formation.The human scar microvascular endothelial cells (HSMECs) isolated and purifiedfrom hypertrophic scar were transfected with small molecules interference RNA tosilence the expression of ILK. At the same time, the PI3K-AKT pathway was blockedwith the specific inhibitor of PI3K (LY294002) to observe the ability of migration ofHSMECs and investigate the effects of ILK in scar angiogenesis in cytomorphology.PART Ⅰ Isolation, culture, purification, identification and transfection ofhuman scar microvascular endothelial cellsObjective To explore the method to isolate and purify HSMECs from hypertrophicscar and investigate the effects on ILK expression after interference with siRNA andinhibiting from specific signal path with LY294002. Methods Primary HSMECs were isolated from12patients’ hypertrophic scar withmechanical method combining type Ⅰ collagenase digestion. Then they were purifiedby immunomagnetic beads, identified by immunofluorescence and flow cytometry.The HSMECs with good condition in2thto4thgeneration were selected asexperimental objectives. They were divided into4groups:(1) blank control group,(2)negative control group,(3) LY294002of50nM group,(4) ILK siRNA group. RT-PCRand Western Blot were used to detect the expression of ILK mRNA and its protein.Result Immunofluorescence of Factor Ⅷ and flow cytometry of CD31showed thatthe isolated HSMECs’ purity were more than90%. The results of RT-PCR andWestern Blot showed that the expression of ILK mRNA and protein were bothsuppressed obviously in ILK siRNA group (t=13.151, P=0.006). And, the expressionof ILK in LY294002group was slightly lower than that of black control group, butthere ws no statistical difference.Conclusion High purified HSMECs can be obtained from hypertrophic scar with typeⅠ collagenase digestion combining immunomagnetic beads. The expression of ILKmRNA and protein in HSMECs is decreased effectively by transfecting ILK siRNA.PART Ⅱ Study on effect of integrin-linked kinase (ILK) to HSMECs’ migrationabilityObjective To explore the effect of ILK and LY294002on migration ability of humanscar microvascular endothelial cells.Methods The digested HSMECs were resuspended with DMEM without serum andthen seeded onto the upper compartment of transwell insert. The cell migrationexperiment was stopped in2h,4h,6h,8h,10h and12h and then erased the cells whichwere still rest on upper compartment. The transwell membrane were fixed, stainedand covered by a slide. Hereafter, pictures harvesting, migrated cells calculation andtime plot drawing were completed respectively. As the same with Part I, theexperiment was divided into4groups to be carried out. The effects of differentinterventions on the migration ability of HSMECs were evaluated at the best time which was derived from time plot.Result The time plot showed that10thhour，in which a large amount of cells hadmigrated and distributed evenly，was the best time to observe cell migration. At thistime, the amounts of migration cells in ILK siRNA group (t=71.938, P=0.000) andLY294002group (t=84.111, P=0.000) were both decreased significantly.Conclusion The migration ability of HSMECs is proportion to ILK’s expression.When ILK is silented by siRNA, the migration ability of HSMECs could besuppressed obviously.PART Ⅲ The regulation of integrin-linked kianse (ILK) to human scarmicrovascular endothelial cells in angiogenesis in vitroObjective To investigate the effects of ILK and LY294002on angiogenesis mediatedby HSMECs in vitro.Methods The thawed ECMatrixTMwas put into each well of pre-colled48-well tissueculture plate, and then the plate was put into the incubator at37℃to make it tobecome gel. The HSMECs treated with different factors were seeded onto the surfaceof the ECMatrix gel and were put into incubator. The experiments were stopped in2h,4h,6h,8h,10h and12h respectively. The ECMatrix gel contained HSMECs should betaken out from the incubator to fix and stain while the angiogenesis would be stoppedto analyze the results. Eight random view-fields per well should be examined at100×magnification and valued by the sheet of pattern recognition about angiogenesis. Fourgrouping experiments like Part I were carried out and stopped at the best time fromthe time plot to analyze the effect of ILK and LY294002on the anigogenesis in vitroafter completing time plot.Result The time plot showed that the complex mesh-like vascular structures haddeveloped and the HSMECs were well distributed at8thhour. The ability ofangiogenesis in vitro decreased significantly in ILK siRNA group, in which, thevascular network structures were not formed perfectly in8thhour (t=12.965, P=0.006).The same results were observed in LY294002group (t=6.653, P=0.003).Conclusion The angiogenesis mediated by HSMECs can be postponed significantly when the expression of ILK is down-regulated. It revealed that ILK may regulate scarangiogenesis. |
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