| Keratin is a main component of intermediate filaments and have a molecules weights of 40 000~70 000 Da. As a major structure element of epithelial cells in cytoskeletons, keratins are recognized as a marker protein of epithelial cells. The function of these proteins has long been thought to be only structural. However, Evidence is now emerging that IF composed of keratins also act as an important framework for the modulation and control of essential cell processes, in particular, cell differentiation, signal transduction and gene regulations.Keratin 17(K17) is highly expressed in psoriatic lesions. In normal epidermis, K17 expression is only detected in hair follicle of growth phase and sweat gland. Further experimental findings have also suggested the participation of K17 in pathogenesis of psoriasis. It has been found that K17 shares a similar antigenic epitope with M protein in streptococcus. Both K17 and M protein have the capacity of activating T cells and promoting the release of Interferon Y (IFNy). The IFNysecreted by T cells is able to increase the expression of K17 in epithelial cells inversely. Accordingly, a positive feedback loop is established and leads to persistent inflammatory response and abnormal proliferation within psoriatic lesions. Our previous studies have showed that monoclonal antibody (Ab) against keratins, especially Ab against K17, are capable of inhibiting proliferation of keratinocytes and displayed a curative effect on psoriatic lesions. Based on previous studies, we proposed anovel therapeutics targets firstly, i.e. interruption or block of K17, to treat psoriasis. In present studies, antisense oligonuleotides targeting K17 mRNA were designed to modulate the gene expression of K17. The effects of antisense oligonucleotides against K17 on proliferation, differentiation and apoptosis of keratinocytes were observed in vitro primarily. Subsequently, their efficacy on psoriasiform lesions in animal models was also evaluated. Our studies were attempted to validate the novel pharmacological target against K17 and lay a foundation for biologic therapy of psoriasis.The main experimental procedures and results are described as follows:1 Computer assisted design and screen of antisense drug against K17The secondary structures of K17 mRNA were predicted according to the lowest free energy method by the computer program mRNA structure. Four antisense drugs directly against the secondary structural elements bulge loops, internal loops, hairpins and knots, which had a positive free energy were designed and designated as AK37, AK102, AK279 and AK1200 (anti-keratin was abbreviated as AK, the number followed by AK indicated the 5' start position of antisense oligonucleotide in mRNA).The four antisense oligonucleotides were introduced into human HaCaT cells by liposome. To evaluate their efficacy of antisense oligonucleotide primarily, the growth of HaCaT cells was tracked by MTT colorimetric determination and the cell cycles were detected by flow cytometry. It was showed that AK279 inhibited the proliferation of HaCaT cells significantly. Regarding the cycles of HaCaT cells treated by AK279, Gl phase was significantly increased accompanied by remarkable decrease of S phase. The distinct sub-Gl peak indicated that AK279 might induce apoptosis of keratinocytes. And then AK279 was choosed to perform the following experiments.2 Effects of K17 antisense oligonucleotides on proliferation and apoptosis of cultured KCIt has been proved that IFNy increases the expression of K17 in HaCaTcells. To some extent, this effect may model the pathogenic mechanisms of psoriasis and was used widely as an in vitro model of psoriasis researches. Using this method, We introduced AK279 into HaCaT cells pre-incubated with IFNy. The proliferation, differentiation and apoptosis of cultured KC were observed after intervention with K17 antisense oligonucleotides.The results of RT-PCR showed that AK279 specifically blocked the targeted genes and decreased the expression of K17 mRNA. The results of Western blot revealed that AK279 suppressed the translation of K17 genes and decreased the expression of K17 protein. In view of absent effects in control group, mis-match group and sense oligonucleotides group, it indicated the specialty of K17 antisense oligonucleotides on the expression of K17 at mRNA and protein level. After transfection of AK279, MTT colometric dermination displayed growth arrest of HaCaT cells with an extension of logarithmic growth and platform phases. Annexin V/PI double staining showed that apoptotic or necrotic ratio of HaCaT cells in the presence of AK279 were higher than control group, mismatch group and sense oligonucleotides group. Moreover, the effects on apoptosis of KCs were associated closely with the concentrations of AK279.The morphological changes of KCs were also observed under light microscopy and transmission electron microscopy (TEM). It was found that some HaCaT cells treated with AK279 were detached and floated with cell debris formed, presenting a striking contrast to controlled HaCaT cells. Significant changes of HaCaT cells were also found under TEM, which displayed a significantly reduced IF in cytoplasm. Typical apoptotic cells were also visualized with high condensed and marginalized chromatins in nuclei. Under TEM, apoptotic bodies were also found in HaCaT cells treated with AK279.To assure the entry of AK279 into HaCaT cells, the K17 antisense oligonucleotides were labeled with FITC and transfected into cultured KCs by liposome. Fluorescent-stained nuclei were apparent under laser confocal scanning microscopy, which occurred rapidly (within 6 h) after incubation with AK279. The fluorescent signal in nuclei peaked after 24h with appearance of fluorescent staining in cytoplasm. Using immunocyto-fluorescence stainings and laser scanning confocal microscopy, it also showed the decrease of K17 expression after introduction of AK279 into KCs.
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