| Objective:1.Analysis of serum glutamic acid and cystine levels in patients with vitiligo and healthy person.2.To explore the effects of myosin X cell formation in human keratinocytes.Methods:1.Levels of Glu and Cys in fasting plasma were measured by high performance liquid chromatography(HPLC) in in 34 vitiligo patients and 31 healthy persons.2.With the aid of small interfering RNA mediated RNA interference technology, using Myo X siRNA gene transfection of HaCaT cells, and RT-PCR method to observe the most effective siRNA sequence.3.To synthesize Myo X-GFP-shRNA lentivirus recombinant based on the most effective siRNA sequence above and knock down Myo X gene expression,and synthesize Myo X-GFP-shRNA lentivirus recombinant targeting none of the genes of HaCaT cells. RT-PCR and Western blotting method to observe the changes of HaCaT cells in Myo X gene mRNA and protein levels of transfection before and after treatment.4.To observe the morphological changes of HaCaT cell filopodia with scanning electron microscope.5.To observe the phagocytic quantity changes of HaCaT cell with flow cytometry.6.To observe the phagocytic ability changes of HaCaT cell with laser scanning confocal microscope. Result:1.The patients with vitiligo had a lower serum glutamic acid level and a higher cystine level.The serum glutamic acid and cystine levels had statistical significance in patients with vitiligo and healthy person.(P<0.05)2.The serum glutamic acid and cystine levels in patients with vitiligo had no statistical significance in sex, age, course of disease.(P>0.05)3.The designed siRNA can effectively down-regulate Myo X gene mRNA and protein expression level.4.Scanning electron microscopic observation of shRNA transfection down-regulates the expression of Myo X gene in HaCaT cells and the number of filopodia reduced.5.Flow cytometry observation of shRNA transfection down-regulates the expression of Myo X gene in HaCaT cells and the phagocytic quantity reduced.6.Laser scanning confocal microscope observation of shRNA transfection down-regulates the expression of Myo X gene in HaCaT cells and the phagocytic ability of filopodia reduced. Conclusions:1.Vitiligo patients with abnormal metabolism of glutamic acid and cystine. In the treatment of vitiligo, patients should be paid attention to glutamic acid and cystine level change and its influence besides the conventional method, to further improve the curative effect.2.The formation process of Myo X involved in HaCaT cell filopodia, inhibit the expression of Myo X in HaCaT cells, HaCaT can reduce the formation of filopodia. |
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