Objective:To develop an efficient and rapid method for the isolation and cultivation of human scalp dermal papilla cells from small specimens.Methods:Hair-bearing skin specimens measuring 0.5 cm × 0.5 cm -0.5 cm × 1 cm in size were obtained from the scalp of 3 patients with pigmented nevus and 6 with sebaceous nevus during surgery in Department of Dermatology, the First Hospital Affiliated to Army Medical University from September 2018 to January 2019. The subcutaneous fat layer containing hair follicles was cut out of the specimens, and hair follicles were sorted with ophthalmic forceps, which were subsequently digested with 0.6% dispase Ⅱ for 30 minutes, then with 0.2% collagenase Ⅳ at 37 ℃ for 30 - 60 minutes, and were centrifuged to obtain hair papillae. Morphological observation was performed on the isolated hair papillae, and dermal papilla cells were cultured, passaged and identified.Results:Under the microscope, the hair papillae isolated by two-step enzyme digestion of small scalp specimens were intact, and showed an inverted pear-like shape, and residual dermal sheaths could be observed around some hair papillae. However, no hair papilla was isolated by one-step enzyme digestion. With the two-step enzyme digestion method, the hair papilla separation rate was 60.8% ± 2.1%, the adherence rate of the dermal papilla cells at 72 hours was 86.6% ± 3.9%, the time for cells to emigrate out of hair papillae was 0.5 - 3.0 days, the total operation duration was 2.0 - 3.0 hours, and the actual operation duration after subtraction of digestion duration was 1.0 - 1.5 hours. The dermal papilla cells isolated by the two-step enzyme digestion method could grow in an aggregative pattern in early stage, but grew in a non-aggregative pattern after 8 passages.Conclusion:The two-step enzyme digestion of small specimens is a simple and efficient method for isolating human scalp dermal papilla cells.

Objective To investigate an efficient rapid method for the isolation and cultivation of human axillary dermal papilla cells.Methods Skin specimens with hair follicles were obtained from the axillary area of patients who received bromhidrosis surgery in the Department of Dermatology of the First Affiliated Hospital to Army Medical University from October 2015 to May 2016.The axillary dermal papilla cells were isolated by two-step enzyme digestion method,one-step digestion method and micro-dissection method separately.Then,axillary dermal papilla cells were cultured and identified.Differences in the operative procedure,separation efficiency and adhesion efficiency of dermal papilla cells,cell emigration duration,total operation duration and actual operation duration were compared among the above 3 methods.Results Compared with the one-step digestion method and micro-dissection method,the two-step enzyme digestion method showed simpler operative procedure,more than 30％ separation rate and 96％ adhesion rate of dermal papilla cells after 1 week.Moreover,the cell emigration duration was shortened by 3-4 days by the two-step enzyme digestion method.The two-step enzyme digestion method also showed longer total operation duration,but shorter actual operation duration compared with the one-step digestion method and micro-dissection method,as well as lower contamination rate compared with the micro-dissection method.Cultured axillary dermal papilla cells grew in an aggregative pattern in the early stage,but grew in a nonaggregative pattern after 6 passages.Immunofluorescence assay showed positive staining for laminin and collagen Ⅳ in axillary dermal papilla cells.Conclusion The modified two-step enzyme digestion method is a kind of simple,efficient and rapid method for the isolation of human axillary dermal papilla cells,and axillary dermal papilla cells can be harvested through this method by using a few specimens.

Enhancing the heat-sensitivity of tumor cells provides an alternative solution to maintaining the therapeutic outcome of photothermal therapy (PTT). In this study, we constructed a therapeutic system, which was composed of methoxy-polyethylene-glycol-coated-gold-nanorods (MPEG-AuNR) and VER-155008-micelles, to evaluate the effect of VER-155008 on the sensitivity of tumor cells to heat, and further investigate the therapeutic outcome of MPEG-AuNR mediated PTT combined with VER-155008- micelles. VER-155008- micelles down-regulate the expression of heat shock proteins and attenuate the heat-resistance of tumor cell. The survival of HCT116 cells treated with VER-155008- micelles under 45 °C is equal to that treated with high temperature hyperthermia (55 °C) . Furthermore, we proved either the MPEG-AuNR or VER-155008- micelles can be accumulate in the tumor site by photoacoustic imaging and fluorescent imaging. anti-cancer evaluation showed that tumor size remarkably decreased (smaller than 100 mm or vanished) when treated with combing 45 °C mild PTT system, which contrasted to the tumor size when treated with individual 45 °C mild PTT (around 500 nm) or normal saline as control (larger than 2000 nm). These results proved that the VER-155008- micelles can attenuate the heat-resistance of tumor cells and enhance the therapeutic outcome of mild-temperature photothermal therapy.

Objective To evaluate the efficacy of combined therapies of ablative fractional Er ∶YAG laser (2,940 nm) and ALA-PDT on refractory verruca plana.Methods 120 cases of refractory verruca plana patients were randomly divided into two groups:60 cases in the control group and 60 cases in the experimental group.The control group used 10％ ALA-PDT with LED irradiation of a power density 70 of mW/cm2 at a distance of 20 cm,which lasted for 20 min each time.The experimental group was treated with ablative fractional Er ∶ YAG laser (2,940 nm) first with the fluence of 500P/cm2 and short pulse duration,and then treated 10％ ALA-PDT as mentioned before.Every patient was treated twice at two-week intervals.Three independent investigators evaluated subject outcomes at 3 months post-treatment including efficacy and side-effects.Results The effective rate of the experimental group was 86.44 ％ at 3 months post-treatment after one to two times.Meanwhile,the control group was 59.65 ％.The clinical outcome of experimental group was better than the control group.There was significant difference between the two groups (P＜0.05).The recurrent rate of experimental group was 3.39％ and 12.30％ in control group.There was significant difference between the two groups (P＜0.05).There were no obvious side-effects in both groups.Conclusions Ablative fractional laser with low fluence promotes the transdermal absorption of ALA and enhances the efficacy of PDT.

OBJECTIVETo study the clinicopathologic features, immunophenotype and gene rearrangement of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL).

METHODSSeven cases of PCLBCL were enrolled into the study. Clinicopathologic analysis, immunohistochemical staining and gene rearrangement for IgH and Igκ were undertaken in the study.

RESULTSAll the seven cases were male, and the median age was 72 years. Patients usually presented with multiple purple tumors, nodules, papules and infiltrative plaques. Two patients had a history of leg injury before onset, and one had mosquito bites. Histologically, the tumor involved the dermis and subcutis with dense and diffuse infiltrative pattern composing of centroblasts and/or immunoblasts. Immunohistochemical staining showed that seven cases (7/7) expressed CD20, six (6/6) expressed bcl-2, four (4/4) expressed MUM-1, four (4/5) expressed CD79a, four (4/5) expressed PAX-5 and four (4/6) expressed bcl-6, respectively. All cases did not express CD3ε, CD45RO, CD10 and CD30. IgH gene rearranged bands were detected in three (3/6) cases and Igκ was detected in one (1/5) case. Six of the seven cases died and the remaining patient, who was 44-year-old, was alive after 22 months of follow-up.

CONCLUSIONSPCLBCL is rare, predominantly affects elderly male patients. PCLBCL has poor prognosis and high mortality, but younger patients seem to have better prognosis. Some cases had a history of trauma or mosquito bites. The relationship between the history and the onset of PCLBCL needs further evaluation.

Aged ; Aged, 80 and over ; Animals ; Antigens, CD ; analysis ; Culicidae ; Gene Rearrangement ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Immunophenotyping ; Insect Bites and Stings ; complications ; Leg ; Leg Injuries ; complications ; Lymphoma, Large B-Cell, Diffuse ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Prognosis ; Proto-Oncogene Proteins c-bcl-6 ; metabolism ; Skin Neoplasms ; genetics ; pathology

In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; Chromatography ; Dexamethasone ; chemistry ; Molecular Weight ; Pseudomonas alcaligenes ; enzymology

BACKGROUND:Currently, antisense oligonucleotides (AS-ODN) have a good prospect in gene therapy, but AS-ODN with smal molecular weight cannot easily enter into the cels, which is susceptible to nuclease degradation. Therefore, there is stil a lack of fundamental understanding about how to improve their transfection efficiency, and target-based transferring. OBJECTIVE:To investigate whether a weak cationic and phosphorylcholine-containing diblock copolymer (MPC30-DEA70) can act as a carrier system to deliver a chemicaly synthesized transforming growth factor-β1 (TGF-β1) AS-ODN into myocardial cels. METHODS: MPC30-DEA70 was compounded with TGF-β1 AS-ODN at various N/P ratios and the MPC30-DEA70/TGF-β1 AS-ODN complexes were characterized by DNA electrophoresis. MTT assay was used to observe the biocompatibility. Confocal laser scanning microscope was used to observe the distribution and location of MPC30- DEA70/TGF-β1 AS-ODN in cells. Flow cytometry was used to detect the transfection efficiency and fluorescence intensity of MPC30-DEA70/TGF-β1 AS-ODN in cells. Western blot and RT-PCR methods were employed to measure the expression of TGF-β1 in cells. RESULTS AND CONCLUSION: Cell growth inhibition showed that the MPC30-DEA70 had low cytotoxicity to myocardial cells within the effective transfection dosage range (< 20 mg/L). Data from the flow cytometry test indicated a clear trend of increasing transfection efficiency with the increasing of N/P ratios. At high N/P ratios, the expression levels of TGF-β1 mRNA and protein in myocardial cells were significantly lower. This study shows that MPC30-DEA70 can work as an effective transgenic vector in myocardial cells. TGF-β1 AS-ODN can silence the expression of TGF-β1 gene efficiently and specially, and may antagonize TGF-β1-mediated biological function.

To enable students to better grasp the basic skills of pathogenic biology and im-munology experimental teaching , and make full use of the characteristics of experimental teaching to train students' scientific quality and innovative consciousness , the reform of pathogenic biology and immunology experiment teaching was explored. Microbiology, Parasitology and Immunology experiment were integrated into an experimental course , and corresponding laboratory was set up to take an in-dependent experimental teaching. Through renewing experiment teaching idea, some measures were taken such as modularization and personalization of the teaching content, the establishment of a com-plete management system , writing a new experimental course to match the experiment , improving teaching methods and developing students' innovative experiments to improve their enthusiasm and in-terest for experimental class learning, thus enhancing their innovation ability.

Objective To evaluate the efficacy and safety of 308-nm excimer light in the treatment of stable vitiligo. Methods Thirty patients with stable vitiligo were enrolled in this clinical trial. All the subjects received the treatment with 308-nm excimer light on a twice-weekly schedule for 3 months. Results The repigmentation rate was 95.0%, 75.0% and 66.7% for lesions in the face and neck, trunk and limbs, with the treatment sessions averaging 10.22 ± 1.60, 19.10 ± 2.38, 37.74 ± 3.06, respectively, and accumulative irradiation dose averaging 7.50 ± 3.45, 10.60 ± 1.01, 18.56 ± 3.05 J/cm2 respectively. Significant differences were observed in the repigmentation rate and treatment sessions between the lesions in the face and neck, trunk and limbs (all P ＜ 0.05). No severe side effects were seen during the treatment. Conclusion 308-nm excimer light is effective and safe for the treatment of vitiligo.

Objective To study the survival and melanogenic potential of human melanocytes reversibly immortalized via SV40T antigen gene and Cre/loxP system in Guinea pigs. Methods The supernatants of retrovirus vector Cre-ERT2 were used to infect melanocytes which had been successfully transfected by SV40TAg gene (MCT), then the expression of Cre recombinase was induced with tamoxifen in infected cells; subsequently, the surviving cells, which were named as MCTC, were subjected to expansion culture. Guinea pigs were utilized to establish animal models of vitiligo, then MCTC and primary melanocytes were transplanted respectively into the animal models. The repigmentation at the transplanted area was observed with naked eyes successively until 3 months after the transplantation when tissue samples were obtained from implanted area and nonimplanted area of guinea pigs and subjected to Masson-Fontana silver stain and Hematoxylin-eosin stain for the analysis of melanocyte distribution and melanin deposition in epidermis. Results Repigmentation started 4 weeks after the transplantation, and dark or brown patches, which ranged in size from 0.5 to 1 cm, were observed in the implanted area 3 months after the transplantation. The repigmentation rate was of no significant difference between pigs transplanted with MCTC and those with primary melanocytes (82.5% vs 76.7%, P > 0.05). Pathological examination revealed melanin deposition in the basal layer of epidermis and some hair follicles in transplanted area. Conclusions SV40T antigen gene combined with Cre/loxP site-specific recombinase system can induce the reversible immortalization of human melanocytes, and the immortalized melanocytes have a favorable profile of biological safety and similarity in survival rate and melanogenic potential to primary melanocytes.