Objective:To explore the effects and mechanism of rat epidermal stem cells (ESCs) that were treated with exogenous vascular endothelial growth factor (VEGF) on the healing of deep partial-thickness burn wounds in rats.Methods:ESCs were isolated and cultured from the trunk skin of a 3-month-old female Sprague-Dawley (SD) rat. The third passage of cultured cells in the logarithmic growth phase was used in experiments (1)-(3). (1) The cells were routinely cultured in keratinocytes-specified serum-free medium (K-SFM) (the same routine culture condition below). The morphology of cells cultured for 3 and 5 days was observed under the inverted optical microscope. (2) After 24 hours in routine culture, the expression of cell surface markers CD44, CD45, CD11b, and CD11c was detected by flow cytometer, with triplicate samples. (3) Four batches of cells were collected, and each batch was divided into VEGF group or blank control group according to the random number table. The cells in blank control group were routinely cultured, while the cells in VEGF group were cultured in K-SFM containing VEGF in the final mass concentration of 10 ng/mL. The protein expressions of cytokeratin 19 (CK19) and CK10 in cells cultured for 10 days were detected by Western blotting. The Nanog mRNA expression in cells cultured for 0 (immediately), 2, 4, 6, 8, and 10 day (s) was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The absorbance value was detected with cell counting kit-8 in cells cultured for 2, 4, 6, 8, and 10 days. The clone number of more than 50 cells was observed and counted under the optical microscope in cells cultured for 10 days, and the cell colony formation rate was calculated. Three samples at each time point was analysed. (4) Thirty-six 3-month-old SD rats (either male or female) were used for the study, and two deep partial-thickness burn wounds (10 mm in diameter) were created in each rat by pressing a 100 ℃ electric iron plate on symmetric dorsal side. According to the random number table, the injured rats were divided into VEGF+ ESCs group, ESCs alone group, and blank control group, with 12 rats and 24 wounds in each group. From 0 (immediately) to 2 day (s) after injury, 20 μL phosphate buffer solution (PBS) was injected into each wound in the three groups in one time, once a day, with the solution in VEGF+ ESCs group containing 0.8×10 6 cells/mL of ESCs treated by 10 ng/mL VEGF for 10 days, the solution in ESCs alone group containing 0.8×10 6 cells/mL of ESCs without any treatment, and the solution in blank control group being PBS only. On post first injection day (PFID) 0 (immediately), 3, 7, and 14, three rats from each group were taken respectively according to the random number table for wound healing assessment, and the wound healing rates on PFID 3, 7, and 14 were calculated. The mice at each time point were sacrificed with wound tissue harvested for histology, and the skin structure was observed by hematoxylin-eosin staining. Data were statistically analyzed with independent sample t test, analysis of variance for factorial design, least significant difference test, and Bonferroni correction. Results:(1) By day 3 in culture, cells distributed in slowly-growing clusters. By day 5, the clusters were large and round, in which the cells mainly with large and round nuclei and little cytoplasm were observed. The above results were consistent with the morphological characteristics of ESCs. (2) The positive expression rate of CD44 was (94.3±1.2) %, and the expressions of CD45, CD11b, and CD11c were negative. The cells were confirmed as ESCs. (3) Compared with those of blank control group, the protein expression of CK19 in the cells of VEGF group was significantly increased after 10 days in culture ( t=3.756, P<0.05), while the protein expression of CK10 was significantly decreased ( t=3.149, P<0.05). Compared with those of blank control group, the Nanog mRNA expression in the cells cultured for 0 and 2 day (s) and absorbance values of the cells cultured for 2 and 4 day (s) were not significantly changed in VEGF group ( t=0.58, 0.77, 0.53, 3.02, P>0.05), while the Nanog mRNA expression in the cells cultured for 4, 6, 8, and 10 days and absorbance values of the cells cultured for 6, 8, and 10 days were significantly increased in VEGF group ( t=6.34, 5.00, 5.58, 4.61, 5.65, 10.78, 15.51, P<0.01). After 10 days in culture, the cell colony-forming rate in VEGF group was (56.4±1.3) %, significantly higher than (31.5±1.3) % of blank control group ( t=13.96, P<0.01). (4) The burn wounds of rats in the three groups were confined to the superficial dermis of the skin on PFID 0. On PFID 3, normal skin tissue at wound margins slightly contracted in the rats of VEGF+ ESCs group, which was earlier than that in the other two groups. On PFID 7, the newly generated epidermis covered most parts of the rat wounds in VEGF+ ESCs group, and some of the epithelium crawled around the rat wounds in ESCs alone group, but no obvious epithelialization was observed in the rat wounds in blank control group. On PFID 14, the rat wounds in VEGF+ ESCs group were basically healed, while some parts of the rat wounds were unhealed in ESCs alone group, and most parts of the rat wounds were unhealed in blank control group. On PFID 3, the wound healing rates of rats in the three groups were similar ( P>0.05). On PFID 7 and 14, the wound healing rates of rats in ESCs alone group, respectively (26.0±2.0) % and (64.4±4.7) %, were obviously higher than (12.4±1.1) % and (29.1±3.3) % of blank control group ( P<0.01), all of which were obviously lower than (41.0±2.4) % and (91.3±3.5) % of VEGF+ ESCs group ( P<0.01). On PFID 3, infiltration of a large number of inflammatory cells were observed in the rat wounds in VEGF+ ESCs group, which was earlier than those in the other two groups. On PFID 7, a large number of endothelial cells were observed in the rat wounds in VEGF+ ESCs group, while proliferation of a few endothelial cells were observed in the rat wounds in ESCs alone group, and a large number of inflammatory cells infiltrated the rat wounds in blank control group. On PFID 14, the newly generated epidermal cells covered nearly all the rat wounds in VEGF+ ESCs group and most parts of the rat wounds in ESCs alone group, while a large number of endothelial cells were observed and the newly generated epidermal cells covered some parts of the rat wounds in blank control group. Conclusions:ESCs of rats treated with exogenous VEGF can promote the healing of deep partial-thickness burn wounds in rats, which may be related to VEGF′s roles in promoting the proliferation of ESCs and reducing its differentiation level, so as to maintain the potency of stem cells.

Objective To study the clinical features of patients with scrub typhus and provide scientific basis for clinical diagnosis and treatment. Methods Clinical data of patients with scrub typhus in the First Affiliated Hospital of Kunming Medical University from January 2016 to December 2016 were collected. Epidemiological data, clinical manifestations, laboratory findings, image examination results, treatment and outcome were retrospectively analyzed. Results The clinical manifestations included 59 cases (100.0％) with fever, 44 cases (74.6％) with headache, 39 cases (66.1％) with chills, 34 cases (57.6％) with muscle and joint pain, 29 cases (49.2％) with prostration, 49 cases (83.1％) with eschar or ulcer, 42 cases (71.2％) with lymphadenectasis, 23 cases (39.0％) with hepatosplenomegaly. Laboratory test results: 51 cases (86.4％) had normal or elevated white blood cell count, 50 cases of eosinophil reduced (84.7％), 27 cases of blood platelet (PLT) reduced (45.8％), 33 cases of albumin reduced (55.9％), 50 cases of alanine aminotransferase (ALT) increased (84.7％), 48 cases of aspartate aminotransferase (AST) increased (81.4％), and 56.1％ (23/41) of the patients with triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3) and free thyroxine (FT4) levels significantly lowered, with predominantly free FT3 reduction (82.6％,19/23); C reactive protein (CRP), procalcitonin (PCT), erythrocyte sedimentation rate (ESR) and ferritin were increased in 93.9％(46/49), 35.4％ (17/48), 64.9％ (24/37), and 83.8％ (31/37) of the patients, and 95.6％(43/45) was accompanied with chest radiographic abnormalities. Tetracycline, doxycycline and azithromycin treatment were all effective. Conclusions The clinical manifestations of patients with scrub typhus, involving multisystem, are diverse and thyroid hormones decrease is observed. Early diagnosis and treatment is the key to improve the prognosis of patients with scrub typhus.

Objective To observe effects of the electroacupuncture combined with music therapy on cerebral neurometabolites measured with magnetic resonance spectroscopy in patients with post-stroke depression.Methods From January, 2013 to December, 2017, 60 patients with post-stroke depression were randomly divided into drug group (n=30) and electroacupuncture music group (n=30). Another 20 cases of normal control group were recruited. The concentration of N-acetylaspartate (NAA), creatine (Cr) and choline (Cho) in the bilateral prefrontal white matter was measured with magnetic resonance spectroscopy before and after eight-week treatment in three groups. The clinical efficiency was defined as the different of Hamilton Depression Rating Scale before and after treatment.Results There was no significant difference in the effective rate between two treatment groups (Z=-0.145, P>0.05). Before treatment, the Cho/Cr was lower in the electroacupuncture music group and the drug group than in the normal control group (t>3.093, P<0.05). After treatment, the Cho/Cr increased in both treatment groups (t>2.219, P<0.05), and no significant difference was found between them (P>0.05).Conclusion The choline level may be the neurobiochemical basis for glial cell or myelin sheath function and integrity in white matter of patients with post- stroke depression. The regulation of glial cell and myelin function in prefrontal white matter may be one of the action targets of electroacupuncture combined with music therapy.

OBJECTIVETo observe the changes in expression of microRNA-203 and P63 in human epidermal stem cells and KCs, and to investigate their effects and significance in the epidermal proliferation and differentiation.

METHODS(1) Five normal foreskin tissue specimens were collected from 5 patients by circumcision in Department of Urinary Surgery of the First Affiliated Hospital of Nanchang University from March to June in 2013. Then single cell suspension was obtained by separating epidermis with trypsin digestion method. The cells were divided into quick adherent cells and non-quick adherent cells by type IV collagen differential adherent method. The biological characteristics of cells were observed by inverted phase contrast microscope immediately after isolation and on post culture day (PCD) 3. The expression of CD29, keratin 19, keratin 1, and keratin 10 was identified by immunocytochemical staining. The expression of microRNA-203 and mRNA of P63 was determined by real-time fluorescent quantitative RT-PCR. The protein expression of P63 was determined by Western blotting. Data were processed with t test and Pearson correlation analysis.

RESULTS(1) Immediately after isolation, quick adherent cells were small, round, and dispersed uniformly. On PCD 3, the cells adhered firmly, and they grew in clones. Immediately after isolation, non-quick adherent cells appeared in different shapes and sizes, and dispersed unevenly. On PCD 3, the cells adhered precariously and did not show clonal growth. Quick adherent cells showed positive expression of CD29 and keratin 19, while non-quick adherent cells showed positive expression of keratin 1 and keratin 10. Quick adherent cells were identified as epidermal stem cells, and non-quick adherent cells were identified as KCs. (2)The expression level of microRNA-203 in epidermal stem cells (0.74 ± 0.20) was lower than that in KCs (3.66 ± 0.34, t =16.582, P <0.001). The mRNA expression level of P63 in epidermal stem cells (4. 16 ± 0.28) was higher than that in KCs (2.90 ± 0.39, t =5. 850, P =0.001). The protein expression level of P63 in epidermal stem cells (1.42 ± 0.05) was higher than that in KCs (0.73 ± 0.03, t =26.460, P <0. 001). (3) The expression level of microRNA-203 was in significantly negative correlation with the expression levels of mRNA and protein of P63 (with r values respectively - 0. 94 and -0.98 , P values below 0.05).

CONCLUSIONSThe expression levels of microRNA-203 and P63 in human epidermal stem cells and KCs were significantly different, which might be related to the different characteristics of proliferation and differentiation of the cells.

Cell Differentiation ; Cells, Cultured ; Epidermis ; cytology ; growth & development ; Epithelial Cells ; cytology ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Humans ; Integrin beta1 ; Keratin-10 ; genetics ; metabolism ; Keratin-19 ; genetics ; metabolism ; Keratinocytes ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; metabolism ; Stem Cells ; cytology ; metabolism

Objective: To investigate how fatty liver was developed in ventromedial hypothalamus(VMH)-lesioned obese rats. Methods: Two groups of rats were prepared: (1)VMH-lesioned obese rats, and (2)sham VMH-lesioned rats. One week after VMH lesions, livers of all rats were isolated for morphological observation and for determination of microsomal triglyceride transfer protein(MTP), phosphatidate phyosphohydrolase (PAP), malic enzyme (ME), and glucose-6-phosphate dehydrogenase(G6PDH). Results: Triglyceride contents in livers of VMH-lesoned obese rats increased significantly, and were about 1.8-fold of control group. Activities of ME, G6PDH and PAP in the livers were also enhanced markedly compared to their controls. Many lipid droplets in cytoplasm of hepatocytes from VMH-lesioned obese rats were observed, while there was no similar finding in hepatocytes of control rats. MTP activity in livers of VMH-lesioned obese rats was higher than that in livers of sham-operated non-obese rats [0.201?0.013 vs. 0.175?0.014 ?g/(mg protein?h),[WTBX]P0.05). Conclusion: Hepatic triglyceride production and activity of MTP were increased in VMH-lesioned obese rats, but magnitude of the latter did not exceed the former. This resulted in hepatic triglyceride accumulation in spite of increase in transport of triglyceride out of liver by MTP. This may contribute to the development of fatty liver in VMH-lesioned obese rats.

Objective: To investigate the effects of green tea polysaccharides (TPs) on glucose metabolism and the activity of peroxisome proliferator-activated receptor gamma (PPAR-?) in KKAy type 2 diabetic mice. Methods: Glucose tolerance test, fasting and postprandial glucose, gluconeogenesis, and insulin sensitivity were investigated in type 2 diabetic mice with orally administered TPs at the dose of 500mg/kg for 4-10 w. Effect of TPs on activity of PPAR-? was tested in vitro. Results: TPs could not only improve glucose tolerance, but also reduce fasting and postprandial blood glucose. In addition, TPs could inhibit gluconeogenesis and enhance insulin sensitivity in KKAy diabetic mice. TPs had also an effect of activating of PPAR-? with dose-response. Conclusion: TPs have beneficial effect of lowering blood glucose in KKAy type 2 diabetic mice, which may be induced by enhancing insulin sensitivity by activating of PPAR-?.