Objective To investigate the biological activities of a conditioned medium for human dermal papilla. Methods Culture medium of the lower passage human dermal papilla cells was collected as the conditioned medium. The growth pattern and the growth curve of the higher passage human dermal papilla cells cultured with conditioned medium were observed in vitro. And the morphology of the co-culture of the higher passage human dermal papilla cells and the lower passage human dermal papilla cells was observed. Results The higher passage human dermal papilla cells, which was cultured with conditioned medium from the lower passage human dermal papilla cells, showed aggregative growth pattern. And the growth curve of the higher passage human dermal papilla cells was much better than that in the control groups (P

OBJECTIVE:To provide reference for promoting the development of related legislation work on ensuring pediatric drug use in China. METHODS:The legislation process of pediatric drug use in US,the effects of current Best Pharmaceuticals for Children Act and Pediatric Research Equity Act on pediatric drug use in US were intrduced and analyzed,suggestions on guarantee-ing the accessibility and safety on pediatric drug use in China were put forward. RESULTS & CONCLUSIONS:The policy6-month period of pediatric exclusive protectionin Best Pharmaceuticals for Children Act has motivated the enthusiasm of phar-maceutical companies developing pediatric studies,while it affects the marketing of generic drugs. Pediatric Research Equity Act has achieved remarkable effect in terms of ensuring the safety of pediatric drug use by forcing pharmaceutical companies to carry out pediatrics assessments,while there is no enough force for the marketed drugs that has not been widely used for children. En-couragement and enforcement are useful methods for promoting development of pediatric drug use. It is suggested to take measures like modifingone drug two formsin Prescription Management Regulations,accelerating approvals of related pediatric drugs on the premise that safety is guaranteed,implementing tax preference for related pharmaceutical companies that produced pediatric drugs,promoting the entry of drug reimbursement list and empowering CFDA to force pharmaceutical companies to implement pedi-atric studies to promote the accessibility and safety of pediatric drugs.

ObjectiveTo evaluate the effects of intranasal administration of recombinant interleukin-17A(rIL-17A) on the expressions of β-Defensin-2(Defb2) and macrophage inflammatory protein(MIP) in pneumococcal pneumonia murine models.MethodsTwenty-four BALB/c mice were divided randomly into normal control,pneumococcal pneumonia,and rIL-17A intervention groups ( n =8 ).Before intranasal (i.n) infection with Streptococcus pneumoniae,the mouse was treated with PBS or rIL-17Ai.n respectively.The mRNA levels of Defb2,MIP-1α and MIP-2β expression in lung tissue were detected by real-time quantity PCR.The numbers of bacteria and leukocytes in bronchoalveolar lavage fluid (BALF) were counted as well.And the concentrations of MIP-1α,MIP-2β,IFN-γ and IL-4 in BALF and in supematants of spleen cells and mediastinal lymph node cells were assayed by ELISA.Changes in lung tissue histopathology were observed with HE staining through light microscope.ResultsNeutrophil and macrophage numbers are higher in BALF of rIL-17A group,while the numbers of bacteria were lower,when compared with those in pneumonia group( P＜0.01 ).The expression of Defb2 and MIP-1α mRNA were up-regulated in lung after rIL17A treatment(P＜0.01 ).When compared with rIL-17A non-treated mice,rIL-17A treated mice secretedhigher levels of MIP-1α in lymph node cell culture supernatants( P＜0.01 ),higher levels of MIP-2β were observed in spleen cell and lymph node cell culture supernatants( P＜0.01 ),higher levels of IFN-T were detected in BALF( P ＜ 0.01 ) and culture supernatants of spleen cell ( P ＜ 0.01 ) and lymph node cell ( P ＜0.05),and higher levels of IL-4 were detected in BALF and spleen cell culture supernatant(P＜0.01 ).Comparative analysis have not detect a significant irflammatory cell increases in rIL-17A treated mice lung tissue; however the histopathological lesions were decreased.ConclusionIntranasal inoculation of rIL-17A can promote pulmonary neutrophil and macrophage recruitment and bacterium clearance,Intranasal inoculation of riL-17enhances the host defense against Streptococcus pneumoniae infection partly through increasing the expression levels of defensins,MIP,IFN-T and IL-4 etc.

Objective To express and purify mouse interleukin 17A(mIL-17A) in E. coli and to analyze its ability of stimulating macrophage inflammatory factors expression. Methods The coding gene of mouse mIL-17A mature protein was amplified from mouse spleen cells by RT-PCR. PCR product was cloned into the prokaryotic expressing vector pET28a, and the resulting recombinant plasmid pET28a/mIL-17a was then transformed into the host E. coli strain BL21(DE3) for expression. The mIL-17A protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by the Ni-NTA affinity chromatography, and was further tested on the stimulation of cytokine and chemokine of RAW264.7 cells by ELISA and real-time quantity PCR in vitro. Results The mIL-17A with bioactivity was over-expressed and purified successfully, and the results of real-time PCR and ELISA showed that recombinant mIL-17A stimulated macrophage mRNA upregulation of IL-6, defensin β2 and Cxcl3 and secretion of defensin β2, Ccl3, Cxcl3,IFN-γ, IL-6 and IL-4. Interestingly, these effects could be blocked by the addition of anti-IL-17A neutralizing antibody partly. After treatment with mIL-17, 74. 87-fold of defensin β2 mRNA expression was increased comparing with that of untreated cells( P ＜0.01 ), while blocking with anti-IL-17A antibody the increase was only 5.4-fold(P ＜ 0.01 ). Conclusion The recombinant mIL-17A has a strong stimulation on secretion of cytokine and chemokine of macrophage, that maybe result to the enhancement of anti-infection ability of macrophage.

Objective To investigate the influence of the hematoma involving the aortic arch in endovascular aortic repair of complicated type B intramural aortic hematoma. Methods A total of 69 patients[58men; mean age(58.1±8.9)years; range 38-77]underwent endovascular repair between February 2011 and June 2015 were retrospectively reviewed. Patients with hematoma involving about the left subclavian artery level were categorized as group A(n=28) and patients without hematoma involvement to the aortic arch were categorized as group B (n=41). Results All the patients were treated with coverd aortic stents. The success rate was 97.1％ with complete isolation of lesion in 67 patients. The average follow-up period was(19.6±14.1)months. During perioperative period, no procedure related deaths was recorded. Perioperative complications include paraplegia in 1case(1.4％) in group B and stent graft-induced new entry in 2 cases(2.9％) in group A. During the follow-up period 1 case in group A within 1 month and another 1 case in group B within 1 year developed new entries at proximal end of stents. 1 case (1.4％) in group B had asymptomatic type Ⅰ endoleak 2 years after TEVAR. Conclusions Type B aortic intramural hematoma with arch involvement is not a risk factor of stent-induced new entry in perioperative period after endovascular treatment and further studies are needed. Strict control of blood pressure is essential for the prevention of stent-related complications.

Objective:To explorethe the clinical manifestations, treatment and prognosis of anastomotic pseudoaneurysm after renal transplantation caused by infection.Methods:Clinical data of 1 recipient with pseudoaneurysm after renal transplantation due to Pseudomonas aeruginosa infection were retrospectively analysed and combined with a literature review. Results:At Month 2 post-transplantation, the recipient developed right lower abdominal pain, and contrast-enhanced ultrasound examination showed a pseudoaneurysm at the artery anastomosis. Anti-infection and anti-rejection therapy had no obvious effect, and therefore next surgical exploration was performed. A size4.0 cm×3.5cm pseudoaneurysm was found intraoperatively at the graft renal artery anastomosis.After graft was evaluated as having no preservation value, the transplanted kidney and pseudoaneurysm were resected. Bacterial culture indicated Pseudomonas aeruginosa infection.The recipient recovered well and waited for next transplantation. Conclusions:Pseudoaneurysm of transplanted kidney is a very rare complication after renal transplantation, and caused by infection of Pseudomonas aeruginosa is more rarer, It has not been reported in mainland China.This type of recipient has the characteristics of high graft inactivation rate and high mortality rate. Timely surgical resection can effectively prevent the deterioration of disease.

BACKGROUND:Human amniotic epithelial cells have some properties of stem cells, which can be induced to differentiate into corneal epithelial cells, but cannot be tracedin vitro. OBJECTIVE:To investigate the feasibility and infection efficiency of adenovirus vector carrying enhanced green fluorescent protein (EGFP) into the human amniotic epithelial cells. METHODS:The adenovirus vectors carrying EGFP was transferred into human amniotic epithelial cells culturedin vitro. After cultured and amplified, the morphology difference between transfected and non-transfected human amniotic epithelial cels was observed. The transfected human amniotic epithelial cells were observed under fluorescence microscope, and the cell cycle and the expression rate of EGFP in transfected human amniotic epithelial cells were detected by flow cytometry. RESULTS AND CONCLUSION:No obvious difference in the cell morphology was found between transfected human amniotic epithelial cells and normal human amniotic epithelial cells cultured in vitro. Flow cytometry analysis revealed that the EGFP positive rate was highest and reached up to 99.01% at 48 hours after transient transfection. The cell cycle of human amniotic epithelial cells transfected by the adenovirus vector was slowed a bit. To conclude, the adenovirus vector is a good medium of transfecting EGFP into human amniotic epithelial cells, and makes it more convenient to observe the further transformation of human amniotic epithelial celsin vitro.

AIM:To investigate the effects of resveratrol ( Res) on the proliferation of ARPE-19 cells and to ex-plore the possible mechanisms.METHODS:After ARPE-19 cells were treated with Res at concentrations of 0, 50, 100, 150, 200 and 300 μmol/L for 24 h, 48 h and 72 h, the effects of Res on the proliferation of the cells were tested by CCK-8 assay.The ARPE-19 cells were treated with Res at concentrations of 0, 100, 150 and 200 μmol/L for 48 h.The effects of Res on the cell cycle and apoptosis were detected by flow cytometry with Annexin V-FITC/PI staining.The protein expression of proliferating cell nuclear antigen (PCNA) was detected by immunofluorescent assay.The mRNA expression of PCNA, P21 and P27 was determined by real-time PCR.RESULTS:The results of CCK-8 assay showed that Res inhibited the prolifera-tion of ARPE-19 cells in a time-and dose-dependent manner.The treatment with Res for 48 h resulted in an arrest of cell cycle at S phase without increasing cell apoptosis.Res inhibited the protein expression of PCNA in ARPE-19 cells.The re-sults of real-time PCR showed that Res increased the mRNA expression of P21 and P27, and decreased the mRNA expres-sion of PCNA.CONCLUSION: Res inhibits the proliferation of ARPE-19 cells and induces the cell cycle arrest at S phase.The mechanism may be related to up-regulation of P21 and P27, and down-regulation of PCNA.

Objective To investigate the location and proliferation of human hair follicle stem cells. Methods The expression of keratin 19(K19) in the human hair follicles on the occiput was detected by immunohistochemical staining, and the positive area was located. The follicle epithelium containing the K19-positive area above the hair bulb were cultured with or without mesenchymes (dermal papilla cells, DPCs) at the air-liquid interface of the collagen gel. When the proliferating colonies formed, the distance from the colony to the bottom of hair follicles was measured and the relationship between the K19-positive position and the proliferation colonies was analyzed. The ultrastructure of the proliferation colony was observed by transmission electron microscopy. Results Two portions were found positive for K19 in the outer root sheath of the human hair follicles, the upper one being at the bulge region and the lower one at the outer root sheath above the hair bulb. The proliferating colony formed only when the epithelium of the hair follicle was co-cultured with mesenchymes cells. The statistical analysis suggested the lower portion positive for K19 and the proliferation colonies in the culture were traced to locate at the same site of the human hair follicles. Under electron microscopy, juvenile cells, mature keratinocytes and apoptotic cells were found in the proliferating colonies. Conclusions The human hair follicles may contain two distinct reservoirs for stem cells, which locate in the bulge area and in the region of the outer root sheath above the hair bulb, respectively. Mesenchymes are needed for the proliferation of stem cells. New stem cells, mature keratinocytes and apoptotic cells may be three endings of the stem cells′ proliferation.

Objective Toll-like receptor ( TLR) gene family is the most important pathogen recognition receptor and animal experiment have found TLR4 mice is inclined to infect aspergillosis ( IA) .The study was to investigate the variation of TLR4 gene in Chinese Han nationality and its relation with the susceptibility of IA. Methods 25 patients diagnosed with proven IA from June 2011 to December 2012 in our hospital were enrolled, among which 17 were males.Their average age was 52.4 ±12.3, and 12 pa-tients had underlying diseases, the others had no underlying diseases.The control group consisted of 103 normal persons, among which 70 were males.Their average age was 56.0 ±17.2.All of the subjects were Chinese Han population.DNA was extracted from periph-eral blood.Polymerase chain reaction ( PCR) was applied to amplify the coding sequence of TLR4 gene followed by sequencing.The sequencing result was compared with TLR4 coding sequence in NCBI GenBank along with the analysis on amino acid change caused by genetic mutation and its effect on protein function.Comparison analysis was made on genetic mutation rate between IA group and con-trol group. Results Two missense mutations,TLR4 219 C>G and 1420 C>T, were identified in TLR4.The prediction result of protein structure showed 219 C>G resulted in the change of functional area for TLR4 to recognize pathogen and 1420 C>T caused no change in domains.The variation rate of TLR4 in IA group was 8%( 2/25 ) and both the patients had no underlying diseases. No mutation has been founded in control group and the difference between two groups was of statistical significance (P=0.037). Conclusion Two missense mutations (219 C>G and 1420 C>T) have been detec-ted in encoding area of TLR4 gene of IA patients.Variation in the cod-ing region of TLR4 gene may increase the susceptibility to IA.