OBJECTIVETo construct a convenient, reliable and visual model of hair follicle development to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling.

METHODSAn open chamber was transplanted into the nude mice dorsal skin, dermal and epidermal cells isolated from newborn C57BL/6 mice skin were mixed at a specific ratio and then injected into the chamber together, 1 week after transplantation, the chamber was removed, and then, hair formation and regeneration after hair plucking was observed.

RESULTS1 week after cells implantation, the wound was moist without apparent contraction and among that pink and translucent tissue was formed. 2 weeks after implantation, the wound healed completely. 3 weeks after implantation, black hair grew from the skin was observed. 4 weeks after implantation, thick and black hair grew from the skin vertically. Completely developed structure of hair follicle was observed with paraffin section and HE staining. 1 week after plucking, new hair had regrown. The ratio of cell component was varied, whereas the other component was fixed at 1 x 10(7) cells. When the number of epidermal cells was reduced to 1 x 10(6) cells, the efficiency of hair follicle reconstitution was mostly unchanged. On the other hand, the density of newly formed hair was diminished considerably by reducing the number of dermal cells to 5 x 10(6) cells or lower. Neither epidermal cells nor dermal cells transplanted alone formed hair follicle.

CONCLUSIONSNewborn mice skin cells transplanted by chamber method can construct a complete model of hair follicle development, which can be used to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling.

Animals ; Cells, Cultured ; Hair ; physiology ; Hair Follicle ; physiology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Regeneration ; Skin ; cytology

OBJECTIVE: To observe the effect of heat shock factor 1 (HSF1) on heat stress-induced apoptosis in Raw264.7 macrophages. METHODS: Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-HSF1 were exposed to heat stress (42.5 degrees C +/- 0.5 degrees C) for 1 h and recovered at 37 degrees C for 6, 9, 12, and 24 h respectively. Flow cytometry (FCM), Hoechst 33258 staining and DNA ladder assays were performed to assess the apoptosis. RESULTS: After heat stress, FCM showed that apoptotic cells were increased significantly and reached the peak at 9 h in Raw 264.7 cells transfected with pcDNA3.1, and were characterized with classical morphologic changes including apoptotic body and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly at 6, 9, and 12 h after the heat stress. But the overexpression of HSF1 could reduce the number of apoptotic cells and inhibit DNA fragmentation. CONCLUSION HSF1 can inhibit heat stress-induced apoptosis in Raw264.7 macrophages.
Animals ; Apoptosis ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; pharmacology ; Heat Shock Transcription Factors ; Heat-Shock Response ; Macrophages ; cytology ; Mice ; Rats ; Transcription Factors ; pharmacology ; Transfection

OBJECTIVETo investigate the value of five-repetition sit-to-stand test (5STS) in clinical evaluation of elderly patients with chronic obstructive pulmonary disease (COPD).

METHODSFifty-one patients with COPD and 20 healthy individuals were enrolled in this study. All the participants underwent 5STS, pulmonary function examination, and 6 min walking test (6MWT) and were evaluated for severity of dyspnea (by mMRC) and BODE index during the tests.

RESULTSAll the participants completed 5STS test with a good reproducibility of the time used for 3 sessions of the test (P<0.001). The mean time used by COPD patients for 5STS was significantly longer than that by healthy individuals (12.93±3.11s vs 0.72±0.71 s, P=0.002). The results of 5STS showed a significant negative correlation with those of 6MWT in the case group and control group with correlation coefficients of -0.611 and -0.682, respectively. The results of 5STS were negatively correlated with FEV1%Pre and body mass index (P<0.05) but positively with mMRC and BODE index in COPD patients (P<0.05).

CONCLUSION5STS is a simple and reproducible test to evaluate the patients' exercise capacity and the severity of COPD, and is well correlated with the current methods for clinical evaluation of COPD.

Body Mass Index ; Case-Control Studies ; Dyspnea ; Exercise Test ; Humans ; Pulmonary Disease, Chronic Obstructive ; diagnosis ; Reproducibility of Results ; Respiratory Function Tests ; Walking

OBJECTIVETo investigate the effects of platelet-rich plasma (PRP) on the proliferation of dermal papilla cells (DPCs) and hair follicle regeneration.

METHODSPRP was prepared using the double-spin method and applied to DPCs. The proliferative effect of activated PRP on DPCs was measured using MTT assay. To understand the influence of activated PRP on the hair-inductive capacity of DPCs, freshly isolated epidermal cells and DPCs of passage 4 were resuspended, mixed with various concentrations of a PRP (0%, 5% or 10%) and were then transferred to a grafting chamber, which was implanted onto the dorsal skin of nude mice. The chambers were removed 1 week after grafting and HF formation was monitored for 4 weeks; the graft site was harvested and processed for histological examination.

RESULTSActivated PRP increased the proliferation benefited the aggregative growth of DPCs. There are significant difference in the yield of hair follicles compared with 10% PRP (344 +/- 27) with 0% PRP (288 +/- 35) in the area of reconstituted skin (P < 0.05). The areas treated with PRP demonstrated an increase in hair follicles density of 19.4%. Ten percent PRP (18 +/- 1) d also can significantly shorten the time of hair formation, compared with 0% PRP (20 +/- 1) d (P < 0.05).

CONCLUSIONSThere is a considerable effect of PRP on the time of hair formation and the yield of hair follicles reconstitution.

Animals ; Cell Proliferation ; Cells, Cultured ; Female ; Hair Follicle ; cytology ; growth & development ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Nude ; Platelet-Rich Plasma ; Regeneration ; Skin ; cytology ; Skin, Artificial

OBJECTIVE: To clarify the effect of nucleolin on the proliferation and apoptosis in C2C12 cells. METHODS: After inhibiting the expression of nucleolin using antisense oligonucleotides, the cellular proliferation was determined by MTT, and the apoptosis was detected by flow cytometry (FCM) assays and DNA ladder assays. RESULTS: After being transfected with antisense oligonucleotides for 24 hours, Western blotting showed that the expression of nucleolin was repressed significantly. In cells treated with antisense oligonucleotides, the cellular proliferation was obviously inhibited; the apoptotic cell increased significantly; and the "DNA ladder" was clearly observed. But the sense and random oligonucleotides had no effect on the cellular proliferation and apoptosis. CONCLUSION The down-regulation of nucleolin can inhibit the cellular proliferation and initiate the apoptosis in C2C12cells.
Animals ; Apoptosis ; physiology ; Cell Proliferation ; Cells, Cultured ; Down-Regulation ; Mice ; Myoblasts ; cytology ; Myocytes, Cardiac ; cytology ; Oligonucleotides, Antisense ; Phosphoproteins ; biosynthesis ; genetics ; RNA-Binding Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVE: To determine the characteristics of a novel gene Mip5 (GenBank accession number AY553870) and its expression under physiological and pathological conditions. METHODS: The characteristics of Mip5 were analyzed by bioinformatic programs including BLAST, spidey, psort, ClustalW and so on. RT-PCR was performed to detect Mip5 expression. RESULTS Bioinformatic analysis showed that Mip5 gene lied in the 13th chromosome and contained 8 exons and 7 introns, its open reading frame contained 909 bp and its protein production was 302 amino acid residues including 6 kelth domains. Under normal conditions, MIP5 expressed abundantly in the heart, brain and kidney, but its expression could not be detected in the liver and muscle. Expression of Mip5 gene was increased significantly after ischemia-reperfusion compared with the sham groups, and reached its peak at 3 h and recovered at 12 h after the reperfusion. Conclusion Mip5 gene is a novel gene containing a putative open reading frame of 302 amino acids residues and may play an important role in rat cardiomyocytes suffering ischemia processing.
Amino Acid Sequence ; Animals ; Base Sequence ; Chromosomes, Human, Pair 13 ; genetics ; DNA, Complementary ; genetics ; Humans ; Male ; Molecular Sequence Data ; Myocardial Ischemia ; genetics ; Myocardial Reperfusion Injury ; genetics ; Open Reading Frames ; genetics ; Rats

OBJECTIVE: To establish immortalized embryonic fibroblast lines in heat shock transcription factor 1 (HSF1) HSF1-/- and HSF1+/+ mice and to provide experimental models to study the function of HSF1. METHODS: A mammalian expression vector (pSV3neo) containing the SV40 large T antigen was used to transfect the HSF1-/- and HSF1+/+ mouse embryonic fibroblast using Lipofectamine 2000. Colonies were screened by G418 and expanded to immortalized cell lines. PCR was used to detect the integration of the large T antigen with genome in the mouse embryonic fibroblast. Expression of SV40 large T antigen gene in expanded cells was identified by RT-PCR. HSP70 expression was examined by Western blot in the embryonic fibroblast lines. RESULTS: The stable growth and serial propagation were observed in the HSF1-/- and HSF1+/+ cell lines for six months. The mRNA of SV40 T antigen gene expressed in the two cell lines. HSP70 expression could not be induced in the heat-treated HSF1-/- mouse embryo fibroblasts. CONCLUSION The immortalized cells of HSF1+/+ and HSF1-/- mouse embryo fibroblasts are successfully established.
Animals ; Antigens, Polyomavirus Transforming ; pharmacology ; Cell Line ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; Female ; Fibroblasts ; cytology ; Heat Shock Transcription Factors ; Male ; Mice ; Mice, Knockout ; Transcription Factors ; genetics

Objective:To explore the influence of human amniotic mesenchymal stem cells (hAMSCs) on the in vivo and in vitro regulation of macrophage phenotypes and inflammatory factors associated with wound healing of full-thickness skin wounds in mice.Methods:Fresh amniotic membrane discarded from full-term delivery by 5 healthy pregnant women in the Department of Obstetrics and Gynecology of the Affiliated Hospital of Zunyi Medical University was used for the isolation and culture of hAMSCs by enzyme digestion method. The third passage of cells was used for identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of hAMSCs surface markers. Ten C57BL/6 mice (all male, aged 6 to 8 weeks, the same gender and age below) were selected for extracting mouse peritoneal macrophages by intraperitoneal lavage, and M1-type macrophages were induced by Dulbecco′s modified eagle medium (DMEM) medium containing interferon-γ. The M1-type macrophages were divided into hAMSCs+ macrophage group and macrophage alone group. Then 1×10 4 hAMSCs/per well of fourth passage were added to macrophage in hAMSCs+ macrophage group and cultured in 2 mL DMEM medium for routine culture. In macrophage alone group, each well was only added with 2 mL DMEM medium for routine culture. On day 1 and 7 in culture, the content of interleukin-12 (IL-12), arginase 1, and IL-10 in the cell culture supernatant of the 2 groups were detected by enzyme-linked immunosorbent assay with sample number of 6/per group. (2) Full-thickness skin wound model was reproduced in the back of 56 C57BL/6 mice, which were divided into hAMSCs group and phosphate buffer solution (PBS) group using the random number table, with 28 mice in each group. Mice in hAMSCs group were subcutaneously injected with 100 μL of cell suspension containing 1×10 7 hAMSCs per mL in PBS suspension along the wound edge. While mice in PBS group were only subcutaneously injected with 100 μL PBS along the wound edge. On post injection day (PID) 1, 3, 7, and 14, 7 mice in the two groups were sacrificed respectively. Histopathological observation was performed with hematoxylin-eosin staining. The expressions of macrophage surface markers [CD68 and inducible nitric oxide synthase (iNOS) double positive cells and CD68 and arginase 1 double positive] in the wounds were detected by immunofluorescent staining. The mRNA expressions of IL-10, macrophage inflammatory protein 1α (MIP-1α), and MIP-2 in the wounds were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with analysis of variance for factorial design, t test, and Bonferroni correction. Results:(1) On day 1 in culture, the content of IL-12 and arginase 1 in the cell culture supernatant of the two groups were similar ( t=0.448, 0.536, P>0.05), and the content of IL-10 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group ( t=14.722, P<0.01). On day 7 in culture, the content of IL-12 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group ( t=13.226, P<0.01), and the content of arginase 1 and IL-10 was significantly higher than that in macrophage alone group ( t=30.172, 31.406, P<0.01). (2) On PID 1, a large number of inflammatory cells infiltration were observed in the skin wounds of both groups. On PID 3, the inflammatory cells infiltration in the skin wounds increased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 7, the inflammatory cells infiltration in the wounds decreased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 14, no obvious inflammatory cells infiltration was observed in the wounds in the two groups. (3) On PID 1 and 14, the percentages of CD68 and iNOS double positive cells and CD68 and arginase 1 double positive cells in the wounds were similar in the two groups ( t1 d=0.134, 0.693, t14 d=1.146, 2.585, P>0.05). On PID 3 and 7, the percentages of CD68 and iNOS double positive cells in the wounds in hAMSCs group were significantly lower than those of PBS group ( t=6.396, 4.787, P<0.01), while the percentages of CD68 and arginase 1 double positive cells were significantly higher than those of PBS group ( t=3.928, 4.473, P<0.01). (4) On PID 1, the mRNA expressions of IL-10 in the wounds of mice in the two groups were similar ( t=2.005, P>0.05). On PID 3, 7, and 14, the mRNA expressions of IL-10 in the wounds of mice in hAMSCs group were significantly higher than those of PBS group ( t=7.758, 124.355, 80.823, P<0.01). On PID 1, 3, 7, and 14, the mRNA expressions of MIP-1α and MIP-2 in the wounds of mice in hAMSCs group (0.341±0.212, 0.648±0.004, 0.611±0.106, 0.763±0.049, 1.377±0.099, 1.841±0.042, 1.181±0.035, 0.553±0.028) were significantly lower than those of PBS group (3.853±0.035, 6.914±0.163, 3.648±0.113, 2.250±0.046, 11.119±0.495, 8.634±0.092, 5.722±0.021, 4.862±0.036, t=43.198, 101.904, 51.845, 58.231, 51.074, 177.501, 291.752, 251.614, P<0.01). Conclusions:hAMSCs demonstrates biological effects of promoting the transformation of M1-type macrophages into M2-type macrophages in full-thickness skin wounds of mice. They can up-regulate the expression of anti-inflammatory and anti-fibrotic factor IL-10, and down-regulate the expression of important inflammation mediated factors MIP-1α and MIP-2.

Objective:To investigate the clinical effects of modified fascia flap from cutaneous branch of dorsal metacarpal artery in repairing the wound at the proximal and middle finger segments.Methods:From January 2017 to September 2018, 12 patients with wounds at the proximal and middle finger segments were admitted to the Affiliated Hospital of Zunyi Medical University, including 8 males and 4 females, aged 35-70 years. The areas of wounds ranged from 3.4 cm×2.4 cm to 6.5 cm×4.0 cm. The modified fascia flaps from cutaneous branch of dorsal metacarpal artery were resected to repair the wounds, with the size ranging from 3.5 cm×2.5 cm to 6.7 cm×4.1 cm. The flap donor sites of 5 patients were repaired with direct intermittent suture, the flap donor sites of 4 patients were repaired with full-thickness skin grafts from ipsilateral medial forearm, and the flap donor sites of 3 patients were repaired with wrist pedicled flaps. The survival of the flaps was recorded. Healing of donor site and recipient site was followed. The hand functions were evaluated with trial standard for the evaluation of the functions of the upper limbs of the Hand Surgery Society of the Chinese Medical Association.Results:All the flaps survived in 12 cases. During 3 to 12 months of follow-up, the flaps recovered satisfactorily in texture and shape. The donor sites of 11 patients were healed, and the skin graft edge area was partially necrotic in the other patient but healed later after dressing change. The distances of two-point discrimination of the patients ranged from 5.6 to 9.0 mm. Hand functions were evaluated as excellent in 5 cases, good in 4 cases, and fair in 3 cases.Conclusions:Modified fascia flap from cutaneous branch of dorsal metacarpal artery for repairing the wounds at the proximal and middle finger segments has reliable blood supply. The operation is simple and safe with short course of treatment, which is worthy of clinical promotion.

Carcinoma, Non-Small-Cell Lung ; diagnosis ; metabolism ; China ; Consensus ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lung Neoplasms ; diagnosis ; metabolism ; Oncogene Proteins, Fusion ; metabolism ; Polymerase Chain Reaction ; Receptor Protein-Tyrosine Kinases ; metabolism