Objective To discuss the cause and treatment strategy for open laryngotracheal trauma.Methods The clinical data of 38 cases with open laryngotracheal trauma admitted from 1998 to 2012 were collected and analyzed.Of them,one patient died in emergency department despite energetic resuscitation,37 were hospitalized and given advanced treatment after life support measures in emergency department.They were treated with tracheotomy before or during operation.Of them,32 patients were regularly followed up for imaging studies.Result Except 1 patient died of exsanguination from carotid artery rupture,the other 37 survived after successful treatment.Twenty patients were treated with debridement and suture of the wounds and laryngoplasty (20/37,54.1％),8 patients were operated with laryngoplasty plus intraluminal stents implanted (8/37,21.6％) ; the rest 9 patients (9/37,24.3％) were separately given placement of nickel-titanium shape memory alloy stent (n =2),laryngofissure with rubber gloves throat models implanted and laryngoplasty (n =3),linear silicone tube implanted and laryngoplasty with trans-cervical approach (n =1) and tracheoesophageal fistula neoplasty with laryngoplasty and laryngofissure (n =3).The operated patients were followed-up for 0.5 to 3 years after discharge.Of them,35 patients (35/37,94.6％) had successful decannulation,breathing smoothly and swallowing normally,23 patients (23/37,62.2％) had almost normal pronunciation,12 patients (12/37,32.4％) had different degrees in hoarseness of voice,and 2 patients (2/37,5.4％) were referred to other hospital because of failure in extubation with severe stenosis of laryngotrachea.Conclusions When the patients with open laryngotracheal trauma were treated,the essential strategy was to prevent shock,hemorrhage,and asphyxia.In the case of patent respiratory tract and stable vital signs,laryngotracheal reconstruction should be carried out as soon as possible to prevent complications,thereby obtaining good therapeutic effect.

Induced pluripotent stem cells (iPSCs) can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical research. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel approach to generating iPSCs. In this study, a new combination of small-molecule compounds (SMs) (sodium butyrate, A-83-01, CHIR99021, Y-27632) under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts (MEFs) in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs. The resulting cell lines resembled mouse embryonic stem (ES) cells with respect to proliferation rate, morphology, pluripotency-associated markers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcinogenicity and the use of exogenous reprogramming factors.

In order to reaction the quality present situation, problems on the current quality of animal sources of drugs are summed up by using test data analysis, literature search and marketing research. This paper can also help the improvement of the quality management, the revision of the relevant department policy system and the improvement of standards.
Animals ; China ; Medicine, Chinese Traditional ; standards ; Pharmaceutical Preparations ; analysis ; standards ; Quality Control

ObjectiveTo evaluate the early cellular immune responses to three kinds of hepatitis B surface antigen (HBsAg) in the immunized mice.MethodsAt day 4,the levels of IFN-γand IL-2 secreted by CD4+ and CD8+T cells which selected from splenic mononuclear cells (MNC) of the vaccinated mice were detected by enzyme-linked immunospot methods (ELISPOT) after stimulation in vitro with HBsAg MHC class Ⅰ peptide S28-39 of HBsAg or recombinant hepatitis B surface antigen(rHBsAg).ResultsAfter selected by MACs,the purity of CD3+/CD4+ and CD3+/CD8+T cell was more than 90％.The positive rate of IFN-γsecreted by CD4+T cells induced by HBsAg derived from Hansenula polymorpha(rHP) was higher than that of HBsAg derived from CHO cell (rCHO).Levels of IFN-γ secreted by CD8+T cells and IL-2 secreted by CD4+T cells induced by rHP antigen were significantly higher than those of rCHO( P＜0.05 ).Meanwhile,levels of IFN-γsecreted by CD4+T cells and CD8+T cells induced by rHP were also significantly higher than those of plasma HBsAg(pHB) (P＜0.05).ConclusionAt day 4,the cellular immune responses induced by HBsAg could be detected.But the immune responses induced by the three kinds of HBsAg are different in levels.According to early cellular immune response intensity,the rHP HBsAg are superior to the rCHO and pHB,in accordance with the high protection rate interrupting the mother-infant transmission immunized by rHP vaccine in clinical trial.It provides scientific basis for necessity of timely birth dose of HB vaccine and kind of HB vaccine for high risk newborn infants vaccinated.

Objective To evaluate the influence of gestational diabetes mellitus (GDM) on maternal and perinatal outcomes in twin pregnancies. Methods We retrospectively analyzed the clinical features of both twin and singleton pregnancies, which delivered in Guangzhou Women and Children's Medical Center between January 1, 2012 and December 31, 2013. The twin pregnancies were divided into two groups:those with (GDM-T, n=51) and without GDM (non-GDM-T, n=130), which were matched by maternal age and delivery time (within one month) in a ratio of 1∶2 among singleton pregnancies with (GDM-S, n=102) and without GDM (non-GDM-S, n=102), respectively. The differences of adverse maternal and perinatal outcomes among these four groups were examined. The overall assessment of pregnancy outcomes was completed using Delphi method. Statistical analysis was performed with one-way analysis of variance, t test, Kruskal-Wallis test, rank test, Chi-square test or Fisher's exact test. Results (1) When compared to GDM-S and non-GDM-S group respectively, less women conceived with the help of assisted reproductive technology, higher proportion of women underwent and gestational age at delivery tend to be earlier in GDM-T and non-GDM-T group (all P<0.01). In oral glucose tolerance test,the fasting blood glucose level of GDM-T group was higher than the other three groups (F=21.716, P<0.01), the glucose levels at 1 and 2 h were higher than non-GDM-T and non-GDM-s respectively (both P<0.01), but no significant difference was found when compared with GDM-S group (P>0.01). Similarly, no significant difference was found in prenatal glycosylated hemoglobin value between GDM-T and GDM-S group (P>0.01). (2) There was no significant difference in the incidences of hypertensive disorders of pregnancy, anemia, premature rupture of membranes, oligohydramnios, placental abruption, postpartum hemorrhage, asphyxia neonatorum, small for gestational age, hypoglycemia of newborn, hyperbilirubinemia of newborn and perinatal death between GDM-T group and the other three groups(all P>0.01). Higher incidences of hypertensive disorders of pregnancy and postpartum hemorrhage were shown in the GDM-T group than in the GDM-S and non-GDM-S groups, respectively (both P<0.01). The incidences of preterm birth in GDM-T and non-GDM-T group were both higher than that in GDM-S and non-GDM-S, respectively [54.9%(66/102), 53.8%(140/260), 5.0%(10/102) and 3.0%(6/102), all P<0.01], while no significant difference was found between GDM-T and non-GDM-T group (P>0.01). (3) The overall assessment of pregnancy outcomes did not show any difference between GDM-T group and the other three groups (χ2=6.707, P>0.01). However, the score for fetal outcomes in the GDM-T group was higher than in the GDM-S and non-GDM-S group, but lower than in non-GDM-T group [M(Q)=1.0(2.3), 0.0(3.0), 0.0(0.0), 1.0(2.8) score, χ2=122.818, P<0.01]. Conclusions GDM does not increase the risk of adverse pregnant outcomes in twin pregnancies.

Objective To detect the impact of reactive oxygen species ( ROS) on mitochondrial morphology and distri-bution during mitosis.Methods A viral vector in which the fluorescence gene was specifically under the control of mito-chondrial promoter was constructed and confirmed through DNA sequencing and Western blotting.After transfecting HeLa s3 cell with packaged virus, the HeLa s3-COX4tp-EGFP cell line stably expressing the mitochondrial fluorescence signal was obtained.With immunofluorescent staining, the impact of ROS on the morphology and distribution of mitochondria dur-ing mitosis was inspected.Result The cell line constantly expressing mitochondrial fluorescence signals was successfully constructed.Meanwhile,it was found that H2 O2 treatment could significantly change the morphology and distribution of mi-tochondria during mitosis by confocal microscopy.Conclusion Our study demonstrates that ROS can affect the morphology and distribution of mitochondria during mitosis.This research help study the relationship between the mitochondrial function and the regulation of mitosis in the future.

Objective To explore the effect and mechanism of salvianolic acid B on bronchial epithelial cell apoptosis induced by cigarette smoke extract.Methods Human bronchial epithelial cells 16HBE were divided into control group,model group(induced by cigarette smoke extract),experimental low-dose group(induced by cigarette smoke extract+11 μmol·L-1 salvianolic acid B treatment),and experimental medium-dose group(cigarette smoke extract Induction+22 μmol·L-1 salvianolic acid B treatment),experimental high-dose group(cigarette smoke extract induction+44 μmol·L-1 salvianolic acid B treatment),experimental high-dose+Compound C group(cigarette smoke extract induction+44 μmol·L-1 Salvianolic acid B+AMPK signaling pathway inhibitor Compound C treatment).Cell counting kit-8(CCK-8)assay was used to detect proliferation;Western blot was used to detect the phosphorylated adenosine monophosphate activates protein kinase(p-AMPK),CCAAT/enhancer-binding protein C/EBP(CHOP),activating transcription factor 4(ATF4)protein expression;PI single staining was used to detect cell cycle;Annexin V-FITC/PI double staining was used to detect apoptosis,and spectrophotometry was used to detect caspase-3 activity.Results The cell proliferation activity(OD value)in the bronchial epithelial cells of the control group,model group,experimental low-dose group,experimental medium-dose group,experimental high-dose group,and experimental high-dose+Compound C group were 0.86±0.07,0.38±0.03,0.45±0.03,0.54±0.04,0.68±0.03 and 0.42±0.04;the expression levels of p-AMPK/AMPK protein were 0.41±0.03,0.13±0.03,0.20±0.02,0.28±0.04,0.36±0.04 and 0.22±0.02;G0/G1 phase were(54.40±5.84)％,(82.93±4.50)％,(75.45±4.73)％,(67.41±2.70)％,(59.15±3.73)％and(69.80±6.59)％;apoptosis rate were(3.21±0.49)％,(24.90±3.35)％,(20.56±1.73)％,(13.55±1.68)％,(9.20±1.07)％and(18.04±1.79)％.Compared experimental low-dose group,experimental medium-dose group,experimental high-dose group with model group,the difference of above indicators were all statistically significant(all P＜0.05);compared the experimental high-dose+Compound C group with the experimental high-dose group,the difference of above indicators were all statistically significant(all P＜0.05).Conclusion Salvianolic acid B affects endoplasmic reticulum stress by activating AMPK signaling to reduce bronchial epithelial cell apoptosis induced by cigarette smoke extract.

The data collected from 34 type 2 diabetic patients receiving intensive insulin therapy for six years showed that the yearly mean HbA1C was less than 7.0％,and none of the patients showed severe hypoglycemia,occurrence or evident progression of retinopathy or nephropathy,and the islet β cell function gained improvement.The DQOL score,used to evaluate the quality of patients' life had no significant change during the observation ( P ＞0.05 ).It is satisfactory and safe to maintain long-term glycemic control with prolonged intensive insulin therapy in patients with type 2 diabetes,and that such therapy does not induce untoward influence on the quality of diabetic patients life.

Objective To explore the optimal timing of operation for experimental obstructive jaundice in a dog model. Method A dog model of bile duct stricture (BDS) was established. Dogs were divided into (n = 12 in each group) 6 groups, ie control, BDS days 5, 10, 15, 20, and 30. In each dog,the morphology and local histopathology of the bile duct, and the liver function in different periods were observed. At the time of surgery biopsy was taken and Roux-en-Y hepaticojejunostomy performed. Surgical complications and survival were evaluated. Result After bile duct obstruction, the proximal bile duct dilated continuously. The diameter of bile duct was 15.6 ± 1.7 mm at the 10th day. The injury bile ductshowed the acute inflammation change. In the early time (in 10 days), inflammatory cells increased in the tissues, mucous edema aggravated, the wall was edematous thickening, it was most severe ( WBC counting 54 ±6) in the 5th day. In the later period (10 -30 days), inflammatory cells reduced, bile duct wall became fibrosis, which was most obvious in the 15th day (42 ± 7 vs 54 ± 6, P ＜ 0.05 ). During the development of jaundice, serum bilirubin reached the highest level in the early period ( BDS days 5 group),then presented a platform time, and then rised extremely at the last stage of the experiment ( BDS day 30 group) . Changes of ALT and AST paralleled that of bilirubin before the 20th day of obstruction and then plummeted. BDS was repaired successfully in 57 dogs. Ten dogs died postoperatively due to bile leakage within 10 days, 3 dogs in BDS days 5 group (3/11), 4 in BDS days 10 group (4/12), one each in other groups. Postoperatively 13 BDS dogs died of malnutrition and organ failure within 3 months, including one each in days 5 and days 10 group, two each in days 15 and days 20 group, and 7 in days 30 group (P＜0. 05). Conclusion Considering the changes of morphology, physical function and result of follow up.The period between 10 and 20 days after acute bile duct injury is optimal for surgical repair.

Objective To investigate the synergistic therapy effects of B and T lymphocyte attenuator(BTLA) extracellular domain in combination with heat shock protein 70 (HSP70)-TC-1 antigen peptide complex on the mouse model of cervical cancer and the related immunological mechanisms. Methods(1)Detecting the BTLA and herpesvirus entry mediator (HVEM) gene expression in the tumor microenvironment after C57BL/6 mice were inoculated with TC-1 tumor cells by realtime PCR; BTLA,HVEM expression on tumor infiltrating lymphocytes cell surface were detected by flow cytometry (fluorescence intensity). (2) According to different treatments, tumor-bearing mice were divided into 5 groups, which was injected with pcDNA3. 1 (empty vector plasmid as control), psBTLA (vector plasmid which expresses BTLA extracellular domain), HSP70 (HSP70-TC-1 cell peptide complex), HSP70 +pcDNA3.1 or HSP70 + psBTLA, respectively. The weight of tumor was recorded. The expression of immunoregulatory genes in tumor microenvironment were detected. The change of lymphocyte amount and cytotoxicity were detected too; lymphocyte proliferation activity was measured by tritium thymidine incorporation assay; the concentration of interleukin (IL) 2 and interferon-γ(IFN-γ) in supernatants of spleen lymphocyte were measured by enzyme-linked immunosorbent assay (ELISA). Results (1) BTLA gene expression was gradually increased after tumor cells inoculation. The highest expression level was 2. 83 + 0. 35 at 14th day, which had statistical significance difference with the 7th day expression of 1.66±0. 25 (P < 0. 05). While HVEM mRNA expression did not change significantly (P > 0. 05). The 7th and 14th day after TC-1 cells inoculation, the average fluorescence intensity of BTLA expression on the surface of tumor infiltrating lymphocytes was 33.5 and 51.8, respectively, in which there was statistically significant difference (P <0. 05); while the difference of HVEM expression was not statistically significant (57. 2 vs 49. 3 ,P >0. 05). (2)The 28th day after inoculation, tumor inhibition rate of HSP70 + psBTLA group was 88%, which was significantly higher than other treatment groups (P <0. 05). The 28th day after TC-1 cells inoculation, combination therapy not only promoted IFN-γ and IL-2 gene (3. 12 + 0.71,3.20 + 0. 62)expression but also reduced transforming growth factor-β (TGF-β), Foxp3 and IL-10 expression (0. 25±0. 03,0. 19 +0. 03,0. 31 +0. 04;P <0. 05). It also promoted CD8+ T lymphocyte infiltration(52 +6)/high power field, cytotoxicity (65.5±2.4) %, proliferation (15.0 × 103 cpm) and cytokine IL-2 , IFN-γsecretion(824±51), (1096±112) pg/ml, which were all significantly higher than other groups (P <0. 05). Conclusion The effect of immunotherapy on tumor can be augmented by the combination of psBTLA which expresses extracellular domain of BTLA and HSP70-TC-1 tumor antigen peptide complex,which could improve the expression of the related immunoregulatory genes to establish a much better microenvironment in favor of anti-tumor immune response against the mice model of the cervix carcinoma.