Study On The Influence Of SCF/c-kit Signaling Pathway On Stable Vitiligo Vulgaris By NB-UVB Therapy

ObjectivesTo detect and compare the expression of SCF/c-kit and the downstream factors MITF, Bcl-2 in non-lesional skin (normal appearance skin far from lesions, normal appearance skin at the edge of lesions), lesional skin (marginal white patches, central white patches) of stable vitiligo vulgaris before and after NB-UVB irradiation therapy, to investigate: 1. The expression of SCF/c-kit in different regions of vitiligo and its clinical significance. 2. The effect of SCF/c-kit signal pathway on stable vitiligo vulgaris by NB-UVB phototherapy. Methods1. Seventeen patients diagnosed with stable vitiligo vulgaris were included. Skin biopsy specimens were taken from normal appearance skin far from lesions at least 1.5cm, normal appearance skin at the edge of lesions, marginal white patches and central white patches, respectively. Normal control: Ten specimens of normal control skin were obtained from cosmetic plastic operation and trauma surgery.2. Tissue sections were tested using immunohistochemistry and RT-PCR analysis to determine whether the expression of SCF in epidermal keratinocytes, and the expression of c-kit, MITF-M, Bcl-2 in epidermal melanocytes were different from normal controls, normal appearance skin far from lesions, normal appearance skin at the edge of lesions, marginal and central white patches.3. Using the BY-Ⅱ suction blister device, human epidermal sheets (blister roofs) were obtained from normal appearance skin far from lesions, normal appearance skin atthe edge of lesions, marginal and central white patches. The mRNA expression of SCF/c-kit, MITF-M, Bcl-2 in corresponding regions were also investigated by RT-PCR.4. All patients were treated twice weekly with 311nm UVB as a monotherapy using UVIOOIKLNB-UVB phototherapy system ( Waldmann company, Germany) for six months in all.5. After finishing the last treatment, the skin samples were taken immediately for the immunohistochemistry and RT-PCR detection again, and the expression of SCF/c-kit, MITF-M, Bcl-2 was compared in corresponding regions before and after NB-UVB radiation therapy.Results1. Location, intensity, positive rates and positive area of SCF immunohistochemistry stainingPositive staining was located at the cytoplasm and/or the cytomembrane. The SCF positive reactivity was found in all ten controls and seventeen vitiligo patients. SCF was expressed throughout the epidermis in all sections, but with less intense staining in control epidermis and non-lesional epidermis compared with lesional epidermis. There was no significant statistical difference in the positive staining area between non-lesional epidermis and normal control epidermis (P>0.05) , whereas the positive staining extension of lesional epidermis was significantly larger than those of normal control and lesional epidermis (P<0.05) .2. Location, intensity, positive rates and positive staining melanocyte number of c-kit immunohistochemistry stainingC-kit positive staining was located at the cytoplasm and/or the cytomembrane. The positive rates in normal control skin, non-lesional skin and marginal white patches were 100%, 100% and 88.24% respectively, and there was no c-kit positive staining melanocyte at the center of the white patches. Melanocytes positive expression with strong stain intensity-for c-kit were observed in the basal layer in normal appearance skin far from lesions and the mean number of (19.82±4.59)/(per 200 basal cells) compared with that of (22.40 + 3.86) from ten control subjects, the difference had no statistical significance (P>0.05);The mean number of c-kit positive melanocytes in normal appearance skin at the edge of white patches (17.88 + 3.77) was fewer than that of normal control skin (P<0.05 ), but when compared with normal appearance skin far from white patches, the difference had no statistical significance (P>0.05) , stainingintension coincided with normal appearance skin;The mean number of c-kit positive melanocytes at the edge of white patches (17.88 + 3.77) was obviously fewer than those of normal appearance skin at the edge of white patches, normal appearance skin far from white patches and normal skin (P<0.05) , and they were stained lightly.3. Location, intensity, positive rates and positive melanocyte number of MITF-M immunohistochemistry stainingMITF-M positive staining should be located at the nuclear of melanocytes in the basal layer, but no MITF-M positive staining melanocyte was found in all the samples from this study.4. Location, intensity, positive rates and positive melanocyte number of Bcl-2 immunohistochemistry stainingBcl-2 positive staining was located at the cytoplasm. The positive rates in normal control skin, non-lesional skin and marginal white patches were 100%, 100% and 82.35% respectively, and there was no Bcl-2 positive staining melanocyte at the center of the white patches. Both in control and non-lesional epidermis , melanocytes positive expression with strong staining intensity for Bcl-2 were found in the basal layer in non-lesional skin and control epidermis, and there was no significant statistic difference in the mean number of melanocytes staining positive with Bcl-2 between normal appearance skin far from lesions (17.59 + 2.18) /(per 200 basal cells) and normal controls (18.90±1.91)and also between normal appearance skin at the edge of white patches (17.29 + 2.05) and normal controls (Z^O.05 ) . Compared with normal appearance skin at the edge of white patches, normal appearance skin far from white patches and normal controls, there was a sharp reduction in the mean number and faint staining intensity of melanocytes positive for Bcl-2 at the edge of white patches(1.94 + 1.39) (PO.05) .5. RT-PCR examination results? The mRNA expression level of SCF in non-lesional epidermis corresponded with that of normal controls, whereas SCF expression level of lesional sites was higher than that of normal contols. (2) The mRNA expression level of c-kit in non-lesional epidermis corresponded with that of normal controls, and the c-kit expression level in marginal white patches was obviously lower than those of normal control and non-lesional epidermis, there was also a slight c-kit mRNA expression at the center ofwhite patches. ? MITF-M specific electrophoresis bands weren't seen in all samples. (4) The mRNA expression level of Bcl-2 in both non-lesional and lesional epidermis corresponded with that of normal controls.6. SCF expression in the corresponding vitiliginous regions before and after NB-UVB therapyAfter NB-UVB treatment, both the SCF antibody positive expression extension and mRNA transcription were not significantly altered than before in the corresponding sites of normal appearance skin far from lesional skin(l7.84+ 2.43)%, marginal white patches (30.45+ 5.99)%and central white patches(33.63 + 8.48)% (P>0.05) except for the marginal normal appearance skin, in which both the SCF positive expression extension(18.12 + 3.11)% and mRNA transcription decreased markedly than before(PO.05).7. C-kit expression in the corresponding vitiliginous regions before and after NB-UVB therapyAfter NB-UVB treatment, the mean number of c-kit positive melanocytes was not markedly altered in the corresponding sites of normal appearance skin far from lesinal skin (16.94+23.94) , marginal white patches (2.29 + 1.61) and central white patches than that of pretreatment (P>0.05) . But the mean number of c-kit positive melanocytes in normal appearance skin at the edge of lesions (14.82+3.30) was significantly fewer than that of pretherapy (P<0.05) . There was no significant difference in the mRNA expression of c-kit in normal appearance skin far from lesional skin posttreatment and pretreatment;There was a light reduction in the mRNA expression of c-kit in marginal normal appearance skin than before, and the expression level of c-kit mRNA in cental white patches was slightly higher than that of pretherapy, but still significantly lower than those of control.8. Bcl-2 expression in the corresponding vitiliginous regions before and after NB-UVB therapyThere was no significant statistical difference in the mean number of Bcl-2 positive melanocytes before and after treatment in corresponding regions of lesional sites and non-lesional sites (P>0.05 ) . After radiation therapy, the average level of Bcl-2 mRNA expression was not changed in non-lesional sites , whereas the Bcl-2 mRNA expression level of lesional area after radiation was lower than that of pre-therapy.Conclusion:1. There was a normal SCF/c-kit signal pathway in normal appearance skin far from white patches, the SCF/c-kit signal pathway function disordered in peri-lesional normal appearance skin and marginal white patches, and the SCF/c-kit signal pathway was blocked or obviously weakened at the central white patches. Disturbance of SCF/c-kit signal pathway may be related to pigment loss or hypopigmentation of vitiligo.2. NB-UVB may regulate the imbalance of epidermal cytokines expressed on keratinocytes and melanocytes of vitiligo at the level of genetic transcription at first, promoted melanocytes of lesional sites to transcribe and express c-kit, then c-kit combined with SCF of keratinocytes, recovered SCF/c-kit signal pathway, and controlled melanocytes' function.3. After NB-UVB treatment, The SCF/c-kit signal transduction may have not recovered completely in some repigmentation sites, further study is needed for this issue.