Objective To construct an artificial skin model of melanoma by mixed culture of human keratinocytes and MV3 melanoma cells on de-epidermized dermis (DED) in order to study the effect of keratinocytes on melanoma invasion. Methods Epidermal cell suspension was obtained by a two-step digestion method from the circumcised foreskin of a child, keratinocyte serum-free medium was applied to the culture and passage of keratinocytes. MV3 melanoma cells were cultured and passaged in RPMI 1640 medium. Log-phase keratinocytes and MV3 cells were mixed with a ratio of 3:1 and seeded onto the surface of DED followed by a liquid culture and air-liquid culture for a total of 2 weeks. Thereafter, the artificial tissue model was assessed by HE staining and immunohistochemical staining for S-100 protein, HM64S and keratin. Results HE staining showed that MV3 cells formed band-like tumor masses or foci on the surface of DED, with keratinocytes intermingling among the tumor cells, but no typical epidermis-like structure was observed. Some tumor cells infiltrated into the surface of DED and showed a cluster distribution; some tumor cells invaded the lumen of the DED, and attached to the luminal wall in a ring shape; some tumor cells penetrated through the wall into the surrounding dermal tissue. On the bottom and lateral side of DED, tumor cells were infiltrating dispersedly.The tumor loci stained positive for S-100 protein and keratin, and weakly positive for HMB45. Conclusion Keratinocytes enhance the invasion of MV3 melanoma cells into the skin tissue model of melanoma.

Objective To establish an in vitro model for malignant melanoma with a malignant melanoma cell line MV3 on de-epidermized dermis (DED) and to study the invasion mode of melanoma cells. Methods A human de-epidermized dermis was prepared with some elements of basal membrane (BM). Then, the reconstructed BM was identified by periodic acid schiff (PAS) staining and immunochemical staining for collagen Ⅳ. MV3 cells were seeded onto the prepared acellular dermis and maintained at the air-liquid interface for 13-15 days after 3-day submerged culture. Subsequently, the reconstructed malignant melanoma tissue was examined with hematoxylin and eosin (HE) staining and immunohistochemical staining with antibodies to S-100 protein and HMB45. Results No obvious changes were observed by naked eye in DED after the inoculation with MV3 cells. PAS staining and immunochemical staining for collagen Ⅳ confirmed the presence of BM component on the surface of DED and in the cavity of skin appendages in DED. Histological examination and immunochemical staining revealed that on the BM zone, MV3 cells grew into irregularly sized clusters; in the .cavity of skin appendages, they attached onto the BM and aggregated into circular or bandlike shape; and at the lateral side of DED, they invasively and diffusely grew, broke through the BM and intruded into the surrounding tissues of DED. The reconstructed tissue was positive for S-100 protein and weakly positive for HMB45. Conclusions The in vitro model of malignant melanoma could be reconstructed by skin organ culture system. And, the experiment suggests that BM could affect the invasive growth pattern of malignant melanoma cells.

BACKGROUND:We have built the three-dimensional human skin melanoma model with human epidermal keratinocytes and MV3 melanoma cel s co-cultured on the de-epidermized dermis in vitro.
OBJECTIVE:To establish a skin model of melanoma by mixed culture of MV3 melanoma cel s and HaCaT cel s on the de-epidermized dermis in vitro.
METHODS:MV3 melanoma cel s and HaCaT cel s were mixed with different percentages and inoculated on the surface of de-epidermized dermis fol owed by a liquid culture and air-liquid culture, and then the tissue-engineered skin model was established in vitro. Routine biopsy immunohistochemical observation was performed on the constructed skin melanoma model.
RESULTS AND CONCLUSION:Hematoxylin-eosin staining showed that MV3 melanoma cel s were distributed on the surface layer of de-epidermized dermis and formed tumor masses, while the HaCaT cel s were mixed growth with tumor cel s and formed a typical epidermoid structure. Some tumor cel s infiltrated into the surface or deep of de-epidermized dermis and showed a tumor foci distribution. The CK10, CK-pan and S-100 proteins were positive for immunohistochemical staining. With the increasing of MV3:HaCaT cel percentage, CK10 and CK-pan gradual y down-moved from the surface, and changed from layer distribution to lumpy distribution, while the staining of S-100 protein was gradual y distributed layer-by-layer, and some area showed tumor-like distribution. The results show that the skin model of melanoma can be in vitro constructed successful y by mixed culture of MV3 melanoma cel s and HaCaT cel s on de-epidermized dermis.

Objective To observe the changes of melanin granules in superficial corneocytes in vitiligo lesions after irradiation with narrow-band ultraviolet B (NB-UVB) by using an adhesive tape stripping technique.Methods Vitiligo lesions were selected from 6 patients and irradiated with NB-UVB every other day for 31 sessions.Superficial corneocytes were obtained by an adhesive tape stripping technique from the vitiligo lesions and perilesional normal skin before every treatment.The morphology,distribution and color of melanin granules were observed after Masson-Fontana silver staining.The percentage of area occupied by melanin granules in superficial corneocytes were calculated by using the Image-Pro Plus 6.0 software.Statistical analysis was conducted by the SPSS11.5 software.Results There were still a few superficial corneocytes containing melanin granules remaining in the vitiligo lesions before treatment.The percentage of area occupied by melanin granules was (5.31 ± 4.12)％ before treatment,(6.24 ± 2.65)％ after 10 treatment sessions,(10.14 ± 5.73)％ after 20 sessions,and (13.05 ± 6.17)％ after 30 sessions,with significant differences between these time points (F =4.334,P ＜ 0.05).Multiple comparisons revealed a significant increase in the percentage of area occupied by melanin granules after 30 treatment sessions compared with those before treatment and after 10 treatment sessions (both P ＜ 0.01 ).The morphology and color of melanin granules in repigmented lesions after treatment differed from those in perilesional normal skin before treatment.Conclusion The adhesive tape stripping technique may serve as a useful tool for the evaluation of repigmentation in vitiligo lesions after phototherapy.

Objective To construct tissue-engineered skin containing melanin with mixed culture of human keratinocytes (KCs) and melanocytes (MCs) on de-epidermized dermis (DED) in vitro. Methods Single-cell suspension was obtained by digestion of isolated preputial epidermis with pancreatin. Keratinocyte serum-free medium (K-SFM) and modified M254 culture medium were used to culture KCs and MCs respectively. Third-passage KCs were seeded into cell culture flasks and cultured for 48 hours; then, third-passage MCs were seeded into the same cell culture flasks with the MC/KC ratio being 1: 10 followed by a 5-day coculture. The suspension of third-passage KCs and MCs with the MC/KC ratio of 10:1 were seeded onto the surface of a prepared DED and maintained at the air-liquid interface for 11 days following a 4-day submerged culture.Subsequently, the constructed tissue-engineered skin was examined with HE staining, immunohistochemical staining for keratin and Masson-Fontana staining. Results After coculture in flasks for 5 days, KCs exhibited a typical paving-stone appearance, MCs with projected dendrites were scattered in the extracellular space between KCs. HE staining revealed 3 to 6 layers of cells with the formation of stratum corneum after mixed culture on DED for 15 days. Keratin protein was positive throughout the artificial epidermis, and melanin pigments were located in the basal layer of the epidermis as Masson-Fontana staining showed. Conclusions The co-culture of MCs and KCs can form single-cell layers with the contact between MCs and KCs in flasks, and construct tissue-engineered skin with melanin component on DED in vitro.

Objective:To reconstruct middle ear structure for open mastoid antrum with external auditorycanal after radical mastoidectomy in one-stage. Method: 71 ears of post-mastoidectomy (discharging 53 ears anddried up 18 ears) were undergone with reconstruction of middle ear. The posterior wall of external auditorycanal, mastoid cavity and chain of ossicles were reconstructed with homologous costal cartilage. Result: 69 ears of71 cases were near normal structure followed up 6 months to 5 years after operations. The result showed hearingimprovement over 15 dB were 55 ears (77.5％) and under 15 dB were 11 cases (15.5％). Five cases (7.0％)were failed. Conclusion: Reconstruction of middle ear with homologous costal cartilage is a ideal surgery toreconstruct hearing structure and avoid infection of middle cavity after radical mastoidectomy.

Objective To explore the impact of patients with esophageal cancer knowing diagnosis on the mental health of their children and investigate the factors.Methods 107 family carriers whose parent diagnosed esophageal cancer in General Hospital of Jinan Command were interviewed.Respondents were categorized into two groups:one group was those whose parents knew their diagnosis(Group A,n=45)and the other group was those whose parents did not know(Group B,n=62).The Chinese version of Self-reporting Inventory(SCL-90) was used.Results The scores of 107 children of esophageal cancer patients in depression(1.62±0.30),anxiety(1.78±0.34) and hostility (1.93±0.47) were higher than national norms (P＜0.01).Compared with group A,group B showed a significant higher degree of anxiety((1.89±0.33) vs (1.62±0.33),P＜0.01).In multiple regression analysis,age and the knowledge of esophageal cancer diagnosis had statistically significant difference (P＜ 0.05).Conclusion Patients' knowledge of the esophageal cancer diagnosis contribute to relieving family carers' symptoms of anxiety.

Objective To evaluate the role of nuclear factor erythroid 2?related factor 2 ( Nrf2)∕antioxidant response element( ARE) signaling pathway in inhibition of lipopolysaccharide ( LPS)?induced inflammatory factor release from macrophages by hydrogen. Methods RAW264. 7 macrophages of mice were cultured in 6?well plates (2×106 cells∕well) and were divided into 4 groups (n=24 each) using a random number table: control group ( group C); LPS group; hydrogen?rich saline+LPS group ( group LPS+H2); Nrf2 small interference RNA (siRNA)+LPS+hydrogen?rich saline group (siRNA+LPS+H2 group) . LPS 1 μg∕ml was added in group LPS. In group LPS+H2 , LPS 1μg∕ml was added, and the cul?ture medium was then replaced with the culture medium containing 0. 6 mmol∕L hydrogen?rich saline. In group siRNA+LPS+H2 , after Nrf2?siRN was successfully transfected into the cells, the cells were continu?ously incubated for 24 h, and the culture medium was then replaced with the culture medium containing 0.6 mmol∕L hydrogen?rich saline after LPS 1 μg∕ml was added. At 24 h of incubation, the supernatant was sep?arated for determination of the lactic dehydrogenase (LDH) activity (using colorimetric method) and for detection of the concentrations of tumor necrosis factor?alpha ( TNF?α) , interleukin?1 beta ( IL?1β) , high mobility group box?1 (HMGB1) and IL?6 (by ELISA). The cells were collected for measurement of the proliferation of cells ( by methyl thiazolyl tetrazolium assay) and for determination of the expression of Nrf2 and heme oxygenase?1 ( HO?1) in cells ( by Western blot) . Results Compared with group C, the LDH activity and concentrations of TNF?α, IL?1β, IL?6 and HMGB1 in the supernatant were significantly in?creased, the proliferation of cells was significantly decreased, and the expression of HO?1 in cells was sig?nificantly up?regulated in LPS and siRNA+LPS+H2 groups, and the expression of Nrf2 in cells was signifi?cantly up?regulated in LPS and LPS+H2 groups (P<0.05). Compared with group LPS, the LDH activity and concentrations of TNF?α, IL?1β, IL?6 and HMGB1 in the supernatant were significantly decreased, the proliferation of cells was significantly increased, and the expression of Nrf2 and HO?1 in cells was sig?nificantly up?regulated in group LPS+H2 , and the expression of Nrf2 and HO?1 in cells was significantly down?regulated in group siRNA+LPS+H2 ( P<0.05) . Compared with group LPS+H2 , the LDH activity and concentrations of TNF?α, IL?1β, IL?6 and HMGB1 in the supernatant were significantly increased, the proliferation of cells was significantly decreased, and the expression of Nrf2 and HO?1 in cells was signifi?cantly down?regulated in group LPS+H2+siRNA ( P<0.05) . Conclusion The mechanism by which hydro?gen inhibits LPS?induced inflammatory factor release from macrophages is related to the activation of Nrf2∕ARE signaling pathway in mice.

Objective To study the in vitro culture condition for melanoblasts from human foreskin tissue. Methods The skin tissue taken from foreskin of children was treated with 0.5% dispase Ⅱ to separate epidermis from dermis, then with trypsin to obtain single cell suspension, which was cultured in modified medium for melanoblasts, i.e., MCDB254 medium supplied with several cell growth factors. Finally, melanoblasts were obtained based on the difference of adhesion speed. The morphology and proliferation of cultured melanoblasts were observed under a light microscope. DOPA staining, immunostaining with anti- S-100 and -tyrosinase related protein 2 (TRP2) antibodies, and transmission electron microscopy were per- formed to identify the cultured melanoblasts. Results The cultured human melanocytes displayed a match-like shape, scattered arrangement, syrmnetric double poles, slim cell body, highly refractive nuclei; meanwhile, the melanoblasts exhibited plentiful cytoplasm, large volume, bipolar or irregular shape and clonal growth. Additionally, the melanocytes were positive for TRP2, S-100 and Dopa staining, while the melanoblasts were positive only for TRP2. Electron microscopy revealed the presence of mature melanin granules (stage Ⅲ-Ⅳ ) in melanocytes but immature melanin granules (stage Ⅰ ) in melanoblasts. Conclu- sion Stable pure culture of melanoblasts has been realized with the reformed medium, which may lay a foundation for the investigation into the mechanism of epidermal pigmentation.

Objective To investigate the application value of fusion indocyanine green fluorescence imaging (FIGFI) in the laparoscopic anatomical liver resection (ALR).Methods The retrospective crosssectional study was conducted.The clinical data of 21 patients who underwent laparoscopic ALR using FIGFI in the Chinese People's Liberation Army General Hospital between December 2015 and February 2017 were collected.Indocyanine green (ICG) staining included positive staining and negative staining.Observation indicators:(1) intraoperative situations:surgical procedures,extent of liver resection,methods and results of ICG staining,operation time,volume of intraoperative blood loss,cases with blood transfusion;(2) postoperative situations:postoperative complications,duration of postoperative hospital stay,postoperative pathological examination;(3) follow-up situations.Follow-up using outpatient examination and telephone interview was performed to detect the patients' survival and tumor recurrence or metastasis up to March 2017.Measurement data with normal distribution were represented as average (range).Results (1) Intraoperative situations:of 21 patients,20 underwent successful laparoscopic ALR and 1 had conversion to open surgery.The positive and negative stainings of ICG were respectively applied to 5 and 16 patients.Seventeen patients had successful staining and 4 had failed staining.Average operation time,average volume of intraoperative blood loss and cases with blood transfusion were respectively 268 minutes (range,120-360 minutes),388 mL (range,100-800 mL) and 3.(2) Postoperative situations:5 patients had postoperative complications,including 3 with Clavien-Dindo classification Ⅰ and 2 with Clavien-Dindo classification Ⅱ.Average duration of postoperative hospital stay of 21 patients was 9.3 days (range,6.0-14.0 days).Sixteen patients with malignant tumor had negative surgical margins.(3) Follow-up situations:all the 21 patients were followed up for 1.0-14.0 months,with a median time of 3.3 months.During follow-up,all the patients survived,and 1 patient had tumor recurrence.Conclusion The FIGFI is safe and feasible in the laparoscopic ALR,with a good short-term outcome.