AIM: To explore the expression of Dickkopf-1 (DKK1) in human gastric carcinoma cells, and the influences of DKK1 gene silencing on cell invasion.METHODS: The levels of DKK1 in the human gastric mucosa cell line GES-1 and gastric carcinoma cell lines MKN-45 and SGC-7901 were detected by real-time PCR and Western blot.DKK1 gene was silenced by RNA interference, which was verified by real-time PCR, Western blot and ELISA.The cell invasion ability was determined by Transwell assay, and the cell proliferation was inhibited by mitomycin C.The levels of E-cadherin, N-cadherin, vimentin and β-catenin were determined by real-time PCR and Western blot.RESULTS: The expression of DKK1 was significantly higher in MKN-45 cells and SGC-7901 cells than that in GES-1 cells, indicating that DKK1 expression was obviously increased in gastric carcinoma cells.After successful silencing of DKK1 gene in the MKN-45 cells and SGC-7901 cells, the cell invasion ability was markedly decreased in a time-dependent pattern with increased expression of E-cadherin and decreased expression of N-cadherin and vimentin, indicating that DKK1 silencing dramatically inhibited gastric carcinoma cell invasion and epithelial-mesenchymal transition (EMT).The introduction of exogenous recombinant DKK1 (rDKK1) demonstrated the promoting effect of DKK1 on gastric carcinoma cell invasion and EMT.In addition, the inhibitory effects of DKK1 silencing on gastric carcinoma cell invasion and EMT were fulfilled by down-regulating β-catenin.CONCLUSION: The expression of DKK1 is significantly increased in human gastric carcinoma cells.Silencing of DKK1 markedly inhibits gastric carcinoma cell invasion and EMT by down-regulating β-catenin.

Objective To evaluate the protective effects of tea polyphenols against the destruction of melanocytes by CD8+ T cells from vitiligo patients.Methods Skin tissue was resected from the margin of vitiligo lesions followed by the isolation and culture of CD8+ T lymphocytes,and from the normal skin of vitiligo patients followed by the isolation and culture of melanocytes.Flow cytometry was carried out to evaluate the purity of CD8+ T cells.The melanocytes were cocultured with the CD8 + T cells at different ratios followed by the evaluation of killing effect of CD8+ T cells.Various concentrations (200 and 400 μg/ml) of tea polyphenols were added into the co-culture system of CD8+ T cells and melanocytes at a ratio of 5 ∶ 1 followed by an additional culture of 48 hours.Then,flow cytometry was performed to detect the apoptosis in melanocytes in the coculture system.Results CD8+ T lymphocytes were successfully obtained from the marginal area of vitiligo lesions with a purity of more than 90％,which highly expressed the antigens CD137 and CD69.The coculture with CD8+ T cells markedly accelerated the apoptosis in melanocytes,while the accelerative effect was inhibited by tea polyphenols of 200 and 400 μg/ml.Conclusions The CD8+ T cells infiltrating the edge of vitiligo lesions display a potential destructive effect on autologous melanocytes from vitiligo patients,and tea polyphenols have a protective effect against the destruction of melanocytes by CD8+ T cells.

Objective To study the feasibility of using chitosan membrane to carry and transport melanocytes, in order to refine the technique for melanocyte transplantation with chitosan membrane. Methods Melanocytes were inoculated onto chitosan membrane and cultured for a period of time, then, electron microscopy,MTT assay and NaOH assay were carried out to estimate the adherence, growth and melanogenesis of the melanocytes. Skin wound surface was prepared in 12 nude mice, which were equally divided into 3 groups, test group inoculated with melanocytes on chitosan membrane, negative control group I treated with chitosan membrane without melanocytes, and negative control group II directly dressed immediately after the preparation of wound surface. On day 10 and 20 after the transplantation, confocal laser microscopy and immunohistochemistry were performed to observe the migration of melanocytes into the skin wound surface. Results Scanning electron microscopy and inverted microscopy showed that melanocytes were evenly distributed on and adhered well to the underlying chitosan membrane. As the growth curve of melanocytes demonstrated, chitosan membrane could support the normal growth of melanocytes, and no significant difference was observed in the synthesized melanin content between melanocytes cultured on the chitosan membrane and those in culture disks (0.087 ± 0.027 vs. 0.101 ± 0.036, t = 0.79, P > 0.05). Melanocytes were seen at the transplantation sites by confocal laser microscopy, and biopsy specimens from the transplantation sites stained positive for antimelan-A monoclonal antibody. Conclusions Melanocytes can adhere to and grow on the chitosan membrane,which can facilitate the migration of melanocytes to the transplantation sites in animals with the maintenance of biological activity of melanocytes.

Objective To evaluate the relationship between cell seeding density and clinical efficacy of autologous cultured melanocyte transplantation in the treatment of vitiligo.Methods A total of 632 patients with vitiligo were enrolled in this study,and randomly classified into 4 groups to be treated with transplantation of autologous cultured melanocytes at 4 different seeding densities respectively,i.e.,(3.0-4.9)× 104/cm2 (n =201),(5.0-7.9) × 104/cm2 (n =303),(8.0-9.9) × 104/m2 (n =82),(10.0-12.0) × 104/cm2 (n =46).Epidermal sheets were obtained by suction blister biopsy from the normal skin of the vitiligo patients,and subjected to the isolation and culture of melanocytes.After 2 to 5 passages,the cultured autologous melanocytes were transplanted at different seeding densities to vitiligous lesions,which were abraded previously by ultra-pulsed CO2 laser,of these patients.All the patients were followed for 6-12 months.Results At 6 months after the transplantation,52.85％of these patients achieved more than 90％ repigmentation,and 82.28％ more than 50％ repigmentation,with no differences in the cure rate and response rate between the 4 groups (both P ＜ 0.05).The percentage of patients obtaining excellent color matching was significantly higher in the group treated with transplantation of melanocytes at a seeding density of (5.0-7.9) × 104/cm2 than in the other 3 groups at 6,12 and 24 months after treatment (all P ＜ 0.05),and higher in all the 4 groups at 12-and 24-month points compared with the 6-month point (all P ＜ 0.05),but no statistical difference was observed between the 12-and 24-month point in any of these groups (all P ＞ 0.05).Conclusions The transplantation of autologous cultured pure melanocytes is effective for the treatment of stable vitiligo with the optimal cell seeding density of melanocytes being (5.0-7.9) × 104/cm2,and the color matching appears to improve with time.

ObjectiveTo establish an individualized culture system for melanocytes, and to estimate its efficacy for the treatment of large-area vitiligo. MethodsHu 16 medium was used for in vitro primary culture of melanocytes isolated from patients with stable segmental vitiligo.Doubling time(DOT), melanin content (M), melanin production(MP) and number of dendrites were examined to evaluate the biological activity of melanocytes. To obtain melanocytes with better biological activity, the components of Hu16 culture medium were adjusted. Ultra pulse CO2 laser was utilized to shave the vitiligous lesions and remove the epidermis followed by autologous transplantation. Follow-up was carried out. ResultsMelanocytes were obtained from 10 patients with stable segmental vitiligo and cultured. The melanocytes from 6 patients showed relatively short DOT, stable M and MP during the first and seventh passage, and were considered to be at initial or growth stage and applicable to transplantation. The remaining melanocytes from the other 4 patients had displayed long DOT, instable M, MP and dendrite quantity since the third passage; by adjusting the components of culture medium, these cells were induced into growth stage and finally applied to transplantation. A 12-month follow-up revealed that the repigmentation rate was higher than 90％ in 7 patients, ranged between 70％ and 80％ in the remaining 3 patients, with the transplantation area being 116.8 + 75.6 cm2. ConclusionsThe individualized culture system with adjusted components in culture medium yields melanocytes with satisfying biological activity, which are proved to be effective for the treatment of large-area, segmental and stable vitiligo.

Objective To evaluate the therapeutic effect of transplantation of autologous melanocytes cultured with individualized medium in vitiligo. Methods Donor skin was obtained by suction blisters from a normally pigmented area of the abdomen of 155 patients with vitiligo. The roof of the blisters was clipped and digested with trypsin, then the suspension of epidermal cells and melanocytes were cultured in Hu16 medium.The cell division time (DOT) and melanin content of cultured melanocytes were measured followed by the adjustment of concentration of fetal calf serum, cytokines and cAMP elevating agents based on the DOT,melanin content and morphology of melanocytes for the individualized culture of melanocytes. After 2 - 5 passages, melanocytes were harvested and inoculated into ultrapluse CO2 laser-denuded lesions. All patients were followed up for at least 6 months. Results One hundred and fifty-five vitiligo patients with 204 lesions were treated with transplantation of autologous melanocytes. Of the 155 patients, 119 received 1 session of transplantation, 36 received 2 to 4 session of transplantation. Cells were expanded by 50 - 80 times in vitro after individualized culture. Repigmentation was more than 50% in 84.8% of these lesions, more than 90% in 52.94% of the lesions. A homogeneous skin color was obtained in repigmented skin, and no scarring or other side effects were observed. No influence was noted on the outcome of transplantation for sex, age, course of disease or lesion size of patients. Segmental vitiligo showed better response than vitiligo vulgaris: the effective rate and cure rate were 93.62% and 65.96% respectively for segmental vitiligo, 82.16% and 49.04% respectively for vitiligo vulgaris. Lesions located on the arms and legs (not including elbows and knees) showed the best response, with a cure rate of 73.08%, whereas acral sites were the most difficult area to repigment, with a cure rate of just 25.93%. Conclusions Individualized culture can significantly increase the success rate of melanocyte culture and expanding times of melanocytes. Transplantation of cultured autologous melanocytes is an effective modality deserving clinical application in the treatment of stable vitiligo, with the advantage of treating large depigmented area with melanocytes from a small donor site.

Objective To evaluate the therapeutic efficacy of autologous melanocyte transplantation for the treatment of vitiligo in patients with abnormal thyroid function.Methods A total of 60 patients with vitiligo were enrolled in this study,including 30 with abnormal thyroid function and 30 without.Epidermal sheets were obtained by suction blister biopsy from the normal skin of all the patients followed by melanocyte isolation and culture.After 2-5 passages of subculture,the melanocytes were transplanted onto vitiliginous lesions,which were abraded previously by ultra-pulsed CO2 laser,in the corresponding patients.All the patients were followed for 6-12 months.Results Of the 30 patients with abnormal thyroid function,7 patients achieved more than 90％ repigmentation,9 patients 50％-89％ repigmentation,53.3％ more than 50％ repigmentation,with the average repigmentation rate being 47％ within 6 months after the transplantation.Meanwhile,13 out of the 30 patients without abnormal thyroid function showed more than 90％ repigmentation,11 showed 50％-89％ repigmentation,with the average repigmentation rate being 75％.Both the cure rate and response rate were significantly higher in the patients without abnormal thyroid function than in those with (cure rate,43.3％ vs.23.3％,P＜ 0.05; response rate,80％ vs.53.3％,P＜ 0.05).Significant differences were also found in the response rate for lesions on the face or neck and for those sized more than 20 cm2 between the two groups of patients (both P ＜ 0.05).The lesions transplanted with epidermal melanocytes from the waist exhibited the lowest cure rate and response rate.Conclusion Clinical or subclinical thyroid dysfunction may have a negative impact on the efficacy of autologous melanocyte transplantation in vitiligo.

Dihydroartemisinin (DHA),the major active metabolite of artemisinin,participates in tumor progression through the following ways:forming free radicals to induce cancer cells death dependent on iron,inducing apoptosis,inhibiting angiogenesis,tumor cells invasion and metastasis,modulating muhidrug resistance,controlling intracellular Ca2+ concentration,regulating cell cycles,cell autophagy and the immune system and so on.Generally,it is considered to be a potential anti-tumor drug.

Objective To explore CT diagnosis of atypical meningioma .Methods 18 cases of atypical meningioma were undergone MR plan scans, among them,17 cases were examined by CT contrast scans. All cases were proved by operation and pathology.Results The tumors appeared as mixed density in 11 cases,cystic in 4 cases,complete enhancement in 3 cases.Conclusion The tumors to be comfirmed at external cerebra is the key in diagnosing atypical meningioma exactly by CT.Atypical manifestations can be seen in a few meningiomas,therefore, it is significant in differential diagnosis of meningioma.

Objective: To study the role of auto-antibody in the diagnosis of patients with PBC. Methods: ANA, SMA and AMA in serum of 58 PBC patients were tested by indirect immune fluorescence and Western blot. Such auto-antibodies as Anti-type AMAM2,anti-SLA/LP, anti-LKM-1 and anti-LC-1 were also identified. Results: Auto-antibodies existing in patients with PBC were mainly AMA(96.5%) and AMAM2(93.1%) and the titer was beyond 1∶100. 8 cases of those patients were positive with ANA and SMA simultaneously. One case had positive AMA and SLA/LP in serum and the clinical appearances were the same as those of type Ⅰ and type Ⅲ autoimmune hepatitis. 19 patients with positive AMAM2 in serum had liver-puncture and the results suggested the diagnosis of PBC in 63.7%(12/9). Conclusion: Test of auto-antibodies is clinically significant for the diagnosis of autoimmune hepatitis.