The Study On Polycation Targeted Nanoparticle Drug Delivery System For The Synergistic Regulation Of Gene Expression To The Apoptosis Of The Melanocyte

Vitiligo is an acquired depigmentation caused by melanocytes' specific injury,depigmentation of the skin and hair follicles to be more specific, characterized bydepigmented macules. It's readily diagnosable but recalcitrant to cure. Recent studiesindicate that defective melanocytes have become a leading cause for melanocytes loss onvitiligo legions. Therefore, suppressing the apoptosis of melanocyte is a critical potentialtarget for vitiligo treatment. Studies show that Bcl-2in melanocytes is the principalapoptosis suppressing gene, while Bax (Bcl-2associated X protein) is the principalapoptosis promoting gene. The apoptosis regulation of Bcl-2and Bax depends on the ratioof homodimer to heterodimer which they integrate into. Therefore, Bcl-2plasmid andsmall interfering RNA fragment of Bax on a proper proportion introduced into melanocytescould effectively contain melanocytes apoptosis.The goal of this study is to build a polycation gene nanocomposite drug deliverysystem with low molecular weight polyethyleneimine as gene carrier by using bidirectionalgene regulation technology of RNA interference and DNA expression and integratingspecifically α-melanocyte stimulating hormone and melanocortin-1receptor onmelanocytes surface. Intervention of melanocytes apoptosis by using bidirectional generegulation technology of RNA interference and DNA expression has two parts. The firstpart is to generate high molecular polymer by connecting lipohydrophilic prownic with lowmolecular weight polyethyleneimine. By discussing its molecular weight, structuralfeatures, modification and hydrolysis in different biological circumstances, a safer andmore effective polycation gene carriers can be created. At the same time, connect theligand of the melanocortin-1receptor with the carrier as the targeting group to realize thetargeting at the melanocyte. On the other hand, using the bidirectional gene regulationtechnology of RNA interference/DNA expression, the small RNA interference fragment/plasmid is to be encapsulated in the above carrier to inhibit the bax overexpression whilepromoting the bcl-2expression, thus preventing the melanocyte apoptosis.Part one, synthetization of low molecular weight polyethyleneimine and evaluation of the property, in vitro transfection and toxicity of polycation nanocomposite. Usetriphosgene plus N-hydroxy-succinamide (NHS) to activate hydroxy on double ends ofdifferent types of pluronic, which interacts with low molecular weight PEI. Determine themolecular weight of the products by gel permeation tomographic chromatography. UseMRI to asertain its structure, in an effort to determine the modificatory degree of PEI.Hydrolysis of the polymers in different biological circumstances will also be tested inorder to create polycation nanocomposite with different N/P ratio. Malvernmicromerigraph is to be used to determine the factors against diameter of polycation, Zetapotential and its stability. Use agarose gel electrophoresis to test the polycation'sconcentrating capacity on different N/P ratio condition for DNA/RNA, and DNA/RNApolycation anti-enzymatic ability will also be tested. We will choose mice melanoma cellline B16to do the transfection experiment. Two nucleic acids will be picked (pEGFP,green fluorescence protein reporter gene plasmid and small RNA fragment marked byFITC); use CCK-8to test toxicity of melanoma cells.Part two, in vitro evaluation of bidirectional regulation of Bax/Bcl-2apoptosis gene.Build Bcl-2expression plasmid to prove its in vitro expression degree; Generate and provesmall interference RNA fragment by reference literature in order to gain effective smallBax interference RNA; Optimize the feeding ratio of small interference RNA andexpression plasmid to figure out the optimum ratio of expression of Bax/Bcl-2affectedjointly by both. Melanoma cells will be in vitro transfected by mixed nucleic acids onoptimum ratio, wrapped up by polycation nanocomposite, to evaluate gene expression andapoptosis.Part three, modify the product polymer of the first part with α-melanocyte stimulatinghormone to gain carrier materials with targeting group; in vitro cultivate polycation andmelanoma wrapped by DNA and RNA to observe the integration of polycation andmelanoma, and evaluate primarily its targeting; generate frozen section by subcutaneousinduction of the DNA/RNA compound marked by quantum dot after corneum damaged bymicropins. use fluorescent confocal microscope to observe the layout of melanocytes marked by Melanoma Specific Fluorescent Antibody, and its perienchyma on dermis.Part four, apoptosis suppression of bidirectional gene regulated polycationnanocomposite. Establish vitiligo mouse model; apply drugs onto the localized vitiligo ofpathological model induced by adjuvant, and then test the melanocytes Bax/Bcl-2expression, and changes of melanocytes apoptosis on the pathological tissue by TUNELmethod.To sum up, in order to suppress melanocytes apoptosis, using RNA interference/DNAexpression bidirectional gene regulation technology, taking melanocortin-1receptormediated low molecular weight Polyethyleneimine targeting melanocytes as carrier,foreign gene is induced into biological cell to suppress Bax expression while boostingBcl-2expression, in an effort to prevent melanocytes apoptosis, and guaranteemelanocytes' biological role in vivo. So far, no report of study on suppressing melanocytesapoptosis by bidirectional gene regulation is seen abroad, and report of study onα-melanocyte stimulating hormone targeting melanocytes as ligand is also unseen.Therefore, this study is expected to provide a new perspective for treatment of vitiligo andother skin diseases.