The Research On The Anti-apoptotic Effect Of Polycationic Nanoparticle Compound On Melanocytes In Vitiligo Models

Vililigo is an acquired depigmentation, which caused by melanin depigmentation ofthe skin and hair follicles because of melanocytes’ specific injury. It’s readily diagnosablebut recalcitrant to cure. The etiology and pathogenesis has not yet been clearly elucidated.Recent studies indicate that defective melanocytes have become a leading cause formelanocytes loss on vitiligo lesions. Therefore, suppressing the apoptosis of melanocytesis a critical potential target for vitiligo treatment. Studies have shown that Bcl-2inmelanocytes is one of the principal apoptosis suppressing protein, while Bax (Bcl-2associated X protein) is one of the principal apoptosis promoting protein. Individual whois susceptivility to vitiligo has reduced expression of Bcl-2or overexpression of Bax.Regulating the expression of Bcl-2and Bax on a proper proportion can control apoptosisof melanocytes effectively, and provide a new idea for the treatment of vitiligo.We have successfully constructed the drug-delivery system P123-PEI-MSH/Bax/iRNAwhich targeted melanocytes. It relies on the specific binding role ofα-melanocyte-stimulating hormone (α-MSH) and the melanocortin-1receptor (MC-1R),taking polyethyleneimine (PEI) as gene carrier, using bidirectional gene regulationtechnology of RNA interference/DNA expression, wrapping the small interfering RNAfragment/plasmid up the gene carrier, in an effort to suppress the expression of Bax andboost the expression of Bcl-2. In vitro, it was confirmed that the targeted drug-deliverysystem P123-PEI-MSH/Bax/iRNA has the excellent characteristics of high targeting, lowtoxicity and so on. What’s more, it also significantly promotes the proliferation ofmelanocytes and supresses the apoptosis of melanocyte.The goal of this study is to establish the animal models of vitiligo by chemicaldecolorization and assess them, research the efficiency of the drug-delivery system whichestablished by bidrectional gene regulation technology of RNA interference and DNAexpression in vitiligo mice, and study the optimal proportion of small interfering RNAand plasmid DNA in an effort to provide an ideal treatment for vitiligo furtherly.Part one, Establish Animal Models of Vitiligo and Assess Them[Objective] To establish successful animal models of vitiligo and provide areliable basis for further study of efficacy.[Methods] Chemical decolorization. We selected C57black mice as experimentalmodels, and take a2cm2cm area hair removal on its back. The blank group wereapplied distilled water0.7ml twice a day for50consecutive days, while the model group applied5%solution of hydrogen peroxide0.7ml twice a day for50consecutive days.Take pictures and record changes in skin every five days. After modeling, randomlyselect two mice both in blank group and model group respectively and kill them bycervical dislocation. Then, take the test area of skin to do dopa-staining of melanocytesand observe the number of melanocytes.[Results] By visual observing, the skins of the mice models are obviously whiteand becoming white patches of some, even growing white hair of some. While, the blankgroup did not change.The number of melanocytes is significantly reduced compared with the controlgroup under the microscope of dopa-staining.[Conclusion] The mice models established by5%hydrogen peroxide chemicaldecolorization can simulate vitiligo in clinical.Part two, Study on the Anti-apoptotic Role of Melanocytes[Objective] To research the role of anti-apoptosis of melanocyte ofP123-PEI-MSH/Bax/iRNA, and study the optimal proportions of small interfering RNAfragment and plasmid DNA in vitiligo mice models.[Methods]10mice were randomly selected in each group, grouping andmedication are as follows:Group I-normal control group;Group II-the negative control group;Group III-the positive control group,0.1%topical tacrolimus ointment once a day for20consecutive days;Group IV-treated with0.5ml drug-containing solution (siRNA1ug, DNA0.5ug), once aday for20consecutive days;Group V-treated with0.5ml drug-containing solution (siRNA1ug, DNA1ug), once aday for20consecutive days;Group VI-treated with0.5ml drug-containing solution (siRNA1ug, DNA1.5ug), oncea day for20consecutive days;Randomly select5mice from each group, record the brightness differences of skinbetween back and abdominal by measuring skin brightness through Lab colorimeter.Take dopa-staining of skins to detect the number of melanocytes, modified Lillie’sstains to detect the number of melanin granules. And then, compare the number abovewith the negative control group and the positive control group. [Results] By measuring skin brightness, compared with normal control group(group II), the differences of brightness of III-VI groups decreased significantly (P＜0.05), and the differences of group V and VI decreased significantly compared withgroup III (P <0.05); the brightness of skin of the normal control group did notdecrease.Histologically, compared with group II, the number of melanocytes and melaningranules of medication group(group III-VI) significantly increased(P＜0.05), and thenumber of melanocytes and melanin granules of group V and group VI are significantlymore than that of group III(P <0.05).[Conclusion] The cationic polymerization drug-delivery systemP123-PEI-MSH/(DNA+RNA), which target melanocyte, can promote the number ofactive melanocytes and the formation of melanin. When the concentration ratio of siRNAand DNA is1:1, the therapeutic effect achieves the best. This study demonstrated thattargeted regulating the expression of Bcl-2and Bax of melanocytes can effectivelycontrol the apoptosis of melanocytes, which provides a new idea for the treatment ofvitiligo.