Objective To establish a primary culture of human melanocytes from tiny skin sheets harvested by using a suction blister method, to carry out a serial subcultivation of the melanocytes with human amniotic membrane (AM) as a scaffold, and to observe the influence of AM on the adhesion, proliferation and dendrite development of melanocytes. Methods Tiny skin sheets were collected from the flexual forearm or lower abdomen of a healthy male volunteer by a suction blister method and melanocytes in the skin sheet were counted following Dopa staining under a microscope. The trypsinized skin sheets were scraped with a scalpel to harvest melanocytes which were subjected to a primary culture. Then, the melanocytes were inoculated onto fresh or cryopreserved AM followed by a culture for various durations (4, 8 and 12 days). The morphology and dendrite development of melanocytes were visualized under an inverted microscope after dopa-staining, cell viability evaluated by MTT assay, the adhesion to AM examined by hematoxylin and eosin (HE) staining protocol. Results The density of melanocytes was 1543.1±13.3 cells per mm2 and 857.4±101.7 cells per mm2 in skin sheets obtained from the forearm flexure and lower abdomen of the volunteer, respectively. A skin sheet of about 25.1 mm2 from approximately two blister roof was required to ensure the success of primary culture of melanocytes within 1 month. After culture on fresh or cryopreserved AM for 4, 8, and 12 days, most melanocytes were bi-polar with extended slender dendrites compared with those cultured in common cell culture medium. HE staining showed that melancytes adhered and were evenly distributed on the basement membrane of AM. MTT assay showed that the AM inhibited the proliferation of melanocytes, and no statistical difference was observed in the inhibitory effect between fresh AM and cryopreserved AM (P＞ 0.05). Conclusions Enriched with melanocyes, flexural forearm is a preferable donor site to offer skin sheets for primary culture of melanocytes. Human AM could improve the adhesive growth and dendrite development of melanocytes, and may serve as a promising bioscaffold for in vitro expansion of melanocytes.

Methods: This paper introduces the concept of “order parameters” and proposes a method for establishing an order parameter model of gas discharge visualization (GDV) based on the principle of “mastering both permanence and change (MBPC)”. The method involved the following three steps. First, average luminous intensity () and average area () of the GDV images were calculated to construct the phase space, and the score of the health questionnaire was calculated as the health deviation index (H). Second, the k-means++ clustering method was employed to identify subclasses with the same health characteristics based on the data samples, and to statistically determine the symptom-specific frequencies of the subclasses. Third, the distance (d) between each sample and the “ideal health state”, which determined in the phase space of each subclass, was calculated as an order parameter describing the health imbalance, and a linear mapping was established between the d and the H. Further, the health implications of GDV signals were explored by analyzing subclass symptom profiles. We also compare the mean square error (MSE) with classification methods based on age, gender, and body mass index (BMI) indices to verify that the phase space possesses the ability to portray the health status of the human body. Results: This study preliminarily tested the reliability of the order parameter model on data samples provided by 20 participants. Based on the discovered linear law, the current model can use d calculated by measuring the GDV signal to predict H (R2 > 0.77). Combined with the symptom profiles of the subclasses, we explain the classification basis of the phase space based on the pattern identification. Compared with common classification methods based on age, gender, BMI, etc., the MSE of phase space-based classification was reduced by an order of magnitude. Conclusion In this study, the GDV order parameter model based on MBPC can identify subclasses and characterize individual health levels, and explore the TCM health meanings of the GDV signals by using subjective-objective methods, which holds significance for establishing mathematical models from TCM diagnosis principles to interpret human body signals.

Objective To construct a lentiviral vector containing mir-7-3 gene and green fluorescent protein (GFP) gene,and to detect the expression of mir-7-3 gene in U251 cells.Methods The fragments containing all the mir-7-3 gene were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP,which was transfected together with the packaging plasmids into 293T cells by CaC12.The supernatant was collected,concentrated,identified,and was transfected to U251 cells of gliomas.Fluorescent microscopy was used to observe the fluorescence in the 293T cell,and real time RT-PCR was used to examine the relative contents of mir-7-3 in U251 cells.Results Electrophores was shown that the sequence of the RT-PCR product was consistent with the data of mir-7-3 by DNA sequence analysis,indicating that the mir-7-3 gene was successfully cloned,and strong green fluorescence was observed by fluorescent microscopy.The supernatant of lentivirus-transfected 293T cells effectively infected U251 cells and the relative content of mir-7-3 was observed in the transfected U251 cells.Conclusion It is concluded that the lentiviral vector containing mir-7-3 gene was constructed successfully,which provides a basis for further study of mir-7-3 function.

Objective To evaluate the MUC1 promoter's role in driving gene expression in pancreatic cancer and its therapeutic significance.Methods Two plasmids were made.The plasmid pEGFP-MUC1N1 contained MUC1 promoter fragment connected to the pEGFP-N1 vector with the EGFG reporter gene.The pShuttle-MUC1-EGFP plasmid contained MUC1 promoter fragment and EGFP reporter gene connected to pShuttle plasmid.Lipofectamine 2000 was used to transfect the two plasmids into cells of MUC1-positive human pancreatic cell line Panc-1 and MUC1-negative human cervical carcinoma Hela.Fluorescence microscopy and flow cytometry compared the specificity and activity of the MUC1 promoter and CMV promoter.Results Reporter gene EGFP-positive cells 48 hours after transfection with pEGFP-MUC1-N1 and pShuttleMUC1-EGFP plasmid were 69.6％ and 63.6％ respectively,in Panc-1 cells,and 4.2％ and 3.7％ respectively,in Hela cells.Conclusions MUC1 promoter can drive reporter gene activity in MUC1-positive tumor cells targeting functional expression.There is potentially a use of targeted therapy in pancreatic cancer at the genetic level.

Objective: To explore the effect of single-nostril transsphenoidal approach on pituitary adenoma. Methods: We retrospectively analyzed 46 cases of pituitary tumors treated with single-nostril transsphenoidal approach and the effects and complications of surgery.Dunng the surgery,a nasal speculum was inserted through right nostril slowly towards the anterior wall of sphenoid sinus.A nasal mucosa incision of about 1.5cm was made in the right nasal cavity at the level of the middle nasal turbinate.With a fracture of the bony septum,a space was developed between the bilateral nasal mucosa and bony septum to the sphenoid sinus.Then,the face of the sphenoid sinus was exposed.The remainder of the bony septum,the anterior sphenoid sinus wall,and the sphenoid mucosa were removed.The antenor sphenoidotomy should be less than 1.5cm wide.After confirming the tumor by dural puncture,a cross incision of dura was made and the tumor was removed.The saddle was usually Collapsed and visible after total tumor removal.When the tumor was resected,sevaral gelatin sponges were stuffed into the Surgical cavity to stop bleeding. Results: Thirty-four cases had total resection and 12 cases had subtotal resection.No deaths or disability occurred.Hormone levels in almost all patients were improved.Seventeen cases had a sign of diabetes insipidus.Electrolyte disturbance occuwed in 5 cases.NO postoperative cerebrospinal fluid rhinorrhea was observed. Conclusion: Single-nostril transsphenoidal approach has many advantages in treating pituitary adenomas such as simplified approach,brief technology and high security.

AIMTo design and synthesize compounds with insulin-sensitizing activity.

METHODSUsing association principle of drug design, ten title compounds were designed and synthesized on the basis of known compounds with insulin-sensitizing activity, and their insulin-sensitizing activity were evaluated on 3T3-L1 pre-adipocyte cells.

RESULTSOne of the synthesized compounds showed strong insulin-sensitizing activity in vitro.

CONCLUSIONThis compound may possess good sugar-lowering activity, and will be chosen for further hypoglycemic evaluation in vivo.

3T3-L1 Cells ; metabolism ; Adipocytes ; drug effects ; Animals ; Drug Design ; Hypoglycemic Agents ; chemical synthesis ; pharmacology ; Indoles ; chemical synthesis ; pharmacology ; Insulin ; pharmacology ; Mice ; Triglycerides ; metabolism

Objective To determine the fluoride contents in dried chilies in Southwest China to provide the basis for the prevention of the fluoride contamination in chili.Methods The dried chili samples collected from the markets and farmers in 76 counties of 9 regions in Southwest China.Their dehydration methods and storage time were investigated.These dried chilies were classified by Bailey'Criteria.The total fhorine content in chili were determined with ion wlective electrodes,fluoride forms with acid-soluble ultrasonic and water-soluble ultrasonic methods,Based on the differences of chili variety,edible part,dehydration method,storage time and fluoride form, a systemic statistics of the fluoride content in dried chili Was established.Results Theere were 296 dried chili samples collected from 76 counties of 9 regions.The geometric mean of fluoride content in dried chili was 19.6 mg/kg;The dried chili samples were classified into 4 types:cherry chili,corn chili,long chili and cluster chili; their ranges of fluoride content in cherry chili,corn chili,long chili and cluster chili Were 1.7～233.4,3.4～ 367.3,2.0～380.3,3.9～104.0 mg/kg,respectively,and the high to low sequence of fluoride content was cherry chili(25.9 mg/kg),long chili(20.3 mg/kg),corn chili(19.5 mg/kg) and cluster chili(15.3 mg/kg).The waler- soluble fluoride content in dried chili about 27.9 mg/kg occupies 77.5%(15.2/19.6)of total fluoride content and the acid-soluble fluoride content about 33.5 mg/kg reached as high as 93.0%(1 8.2/19.6).The high to low sequence of fluoride content in dried chili Was the sun-dried stored less than 1 year(10.9 mg/kg),the sun-dried stored more than 1 year(13.7 mg/kg),the fumace-dried stored less than 1 year(21.4 mg/kg),the fumace-dried stored more than 1 year(53.9 rag/ks).Conclusions The research shows that inappropriate dehydration method and storage time are the two main reasons leading to fluoride contamination in chili.

Objective To investigate the distribution of fluoride content in fresh chili in southwestern China and provide the fluoride background content for the confirming fluoride contamination discrimination value for fresh chili.Methods The method of analyzing fluoride in food as stipulated in GB/T 5009.18-2003 was adopted to determinate fluoride content in chilies.175 fresh chili samples were collected from 76 counties in southwestern China.Based on the origin and types of the chilies,we systemically analyzed the fluoride content in fresh chilies.Results In fresh chilies directly sampled from farms,the averaging content was up to 8.9 mg/kg(dry weight)and the fluoride content in more than 95.54% of fresh chili samples was less than 24.7 mg/kg(dry weight).Conclusions The current fluoride content standard for vegetables as stipulated in GB 2762-2005(≤1.0 mg/kg)is unsuitable for chili,therefore it is essential to draw a new value for discriminating fluoride contamination in fresh chili.

Lung cancer is the leading cause of death in malignant tumors. Surgery is one of the main treatments for early stage lung cancer. Idiopathic pulmonary fibrosis (IPF), one of the most common interstitial lung disease, is a chronic, progressive lung disease, characterized by insidious onset and progressive deterioration. It is reported that there are common pathways in idiopathic pulmonary fibrosis and lung cancer, and patients with IPF have a higher risk of lung cancer than the general population, whose prevalence was 3.34 times than that of the general population. The incidence of acute exacerbations of IPF and postoperative mortality was significantly higher in lung cancer patients complicated with IPF than those without IPF. The long-term outcomes of patients with IPF are dramatically worse than those without IPF. It was concluded that lobectomy has been still the standard surgical procedure for such patients, but sublobectomy should be taken as potential alternative choice for patients with high risk after preoperative evaluation. Moreover, further studies should be conducted on the prevention and treatment of acute exacerbation of IPF after surgery.

AIM To evaluate anti-gastric cancer effects and benefits of the combination therapy of celastrol and curcumin in vitro and in vivo.METHODS Curcumin,celastrol and curcumin + celastrol applied to BGC823 gastric cancer cells had their impact on the cells growth and apoptosis in vitro checked by CCK-8 assay and flow cytometry of AnnexinV-FITC/PI;and their in vivo anti-tumor effect,systemic toxicity,and influence to the survival time evaluated by using BGC823-bearing gastric tumor mouse models induced by subcutaneous implantation.RESULTS IC50 of curcumin,celastrol and curcumin + celastrol on BGC823 cell were found to be at (57.47 ± 5.22),(2.11 ±0.07) and (0.22 ±0.01) μmol/L,respectively.Curcumin + celastrol achieved a most optimal BGC823 cell apoptosis rate of (73.07 ±3.82)％ at concentrations of (10 mol/L +2 mol/L);and they left a positive impact on the BGC823 gastric tumor-bearing mice,given a (64.87 ±4.16)％ inhibitory rate,and a 50.5-day median survival time.Additionally,the combination therapy demonstrated its less side effects in terms of the body weight,spleen and liver of the nude mice,and significantly improved serum concentrations of TNF-α and IL-6.CONCLUSION Combination of curcumin and celastrol shows significantly synergistic anti-gastric cancer effects in vitro and in vivo.