Objective To observe the interferon-γ(IFN-γ),interhukin-2(IL-2)levels of Th1 cytokine and IL-4、IL-10 levels of Th2 cytokine in serum and culture supernatants of splenic cells of the rats in the prevention of experimentaI autoimmune uveoretinitis(EAU)by oral tolerance. Methods 72 Lewis rats were randomly divided into EAU group,oral tolerance group(which including 10 μg、100 μg、1 mg、10 mg of S antigen group respectively)and control group,12 rats in each group.The animal model of EAU was induced by immunization with S antigen(50 μg)and Freund's complete adjuvant.Oral tolerance 10 μg、100μg、1 mg and 10 mg group were fed with 1 ml mixture of 10 μg、100μg、l mg、10 mg S antigen and 1 mg trypsin inhibitor respectively by intuhation,once the other day,totally 7 times,and then induced EAU according to above methods;control group was fed with 1 ml mixture of phosphate buffered saline and 1 mg trypsin inhibitor,once the other day,totally 7 times,and then induced EAU.The clinical manifestation of EAU in the eye were recorded,the eyeballs were enucleated at the peak of EAU,followed by pathological grading.Meanwhile the serum was colleced;splentic cells were separated and cultured to collect the supernatant.Cytokine levels of IFN-γ,IL-2,IL-4 and IL-10 in serum.cultured supernatant of splenic cells were determined by enzyme-linked immunosorbent assay(ELISA). Results Compared with EAU and control group,the levels of IFN-γ and IL-2(Th1 eytokine)in the serum in 100 μg and 1 mg group were decreased while the levels of IL-4 and IL-10(Th2 eytokine)were inereased,the diffefences were statistically significant(F=51.9,68.8,35.7,7.5,P＜0.01).Compared the levels of Th1 and Th2 cytokines in the serum in 10 μg,10 mg group with EAU and control group.the differences were not statistically significant.In 100 gg、1 mg group,the levels of IFN-γ and IL-2(Th1 cytokine)in the culture supernatant of splenic cells were decreased while the Ievels of IL-4 and IL-10(Th2 cytokine)were increased,compared with EAU and control group,the differences were statistically significant(F=57.1,15.6,33.1,167.7,P＜0.01).Compared the levels of Th1 and Th2 eytokine in the culture supernatant of splenic cells in 10 μg、10 mg groups with EAU and control group,the difference are not statistically significant. Conclusions In the process to prevent EAU by oral intake,the levels of IFN-γ and IL-2 (Th1 cytokine)were decrease while the levels of IL-4 and IL-10(Th2 cytokine).Oral administration with tOO high or low dose of the antigen can not prevent EAU as well as the cytokine levels do not change obviously.Cytokines has played an important role in the prevention of EAU.

Objective To observe the efficacy of the anti-tumor necrosis factor-α monoclonal antibody(TNF-α MCAb)in the treatment of experimental autoimmune uveoretinitis(EAU). Methods EAU animal models were induced by interphotoreceptor retinoid-binding protein(IRBP)R16 peptide with immunization.The rats were divided into 2 groups according to the injection times.TNF-α MCAh was administered intravenously on day 6 or 4,6 and 8 post-immunization respectively,and then to observe the clinical expression by slit-lamp microscope.Meanwhile,take the rats which did not accept TNF-α MCAb as conttol group.Delayed type hypersensitivity(DTH)responses were measured on day 13 postimmunization of IRBP R16;the rats were killed on day 14 post-immunization of IRBP R16,and then enucleated the eyes for histopathological examination.To detect the cytokine level of IFN-γ,IL-4 in serum and IFN-γ in aqueous humor by enzyme-liked immunosorbent assay(ELISA)on day 14 postinjection.The hyperplasia responses of antigen specific lymphocyte of draining lymph node cells were detected. Results The TNF-αMCAb group had mitigated ocular inflammation and decreased pathological grades compared with the control group;the IFN-γ concentrations in aqueous humor and serum were decreased,IL-4 was increased in serum;DTH responses were decreased;the hyperplasia responses of draining lymphocytes to IRBP R16 peptide were decreased,all the differences were statistically significant(P＜0.01).The rats accepted TNF-α MCAb thrice had much better curative effect than the rats injected once(P＜0.05). Conclusions Injection of TNF-α MCAb can inhibit ocular inflammation and specific immune cells of EAU remarkably and change the Th1/Th2 balance.Many times injections of TNF-α MCAb were more effective than once.

Neonatal inherited metabolic diseases are a group of metabolic disorders caused by singe gene defect to cause a series of clinical symptoms.Neonatal dried blood spots have the advantages of simple preparation,safety,good stability,and show strong practicability in different screening methods for inherited metabolic diseases.With the development of screening methods,more and more diseases could be diagnosed by screening.The emergence of tandem mass spectrometry and molecular biological techniques promote the newborn screening and automation for inherited metabolic disease effectively.Inherited metabolic diseases induce great harm to the newborn,which could cause not only system organs damage,but also lead to death.Therefore,early screening is important for patients' prognosis.

Objective To compare large dose or conventional dose of budesonide/formoterol fumarate powder in treatment of stable chronic obstructive pulmonary disease(COPD).Methods 120 patients with stable COPD from March 2015 to March 2016 in Jiaxing Second hospital were selected,were treated with budesonide/formoterol,and were divided into the conventional dose group and high dose groupaccording to different doses,60 cases in each group.Pulmonary function tests,CAT Chinese version scale assessment and 6 min walking distance(6MWD)test were performed before and after treatment,and adverse reactions and acute attack were observed.Results FEV1/FVC and FEV1％were significantly improved after treatment in the two groups,and those in the high dose group were significantly better than those in the conventional dose group,the difference was statistically significant(P<0.05),CAT score and 6MWD were significantly improved after treatment in the two groups,and those in the high dose group were significantly higher than those in the conventional dose group,the difference was statistically significant(P<0.05),adverse reactions such as palpitations,tachycardiain the high dose group were significantly higher than those in the conventional dose group,while the acute attack rate was significantly lower than that of the conventional dose group,the difference was statistically significant(P<0.05).Conclusion High dose of budesonide/formoterol inhalation in the treatment of patients with stable COPD powder can improve the pulmonary function and health status,but adverse reactions increase.

Objective To explore the clinical value and significance of the chronic obstructive pulmonary disease (COPD) assessment test (CAT) in patients with acute exacerbation of COPD (AECOPD).Methods A prospective observational study was conducted,AECOPD patients were admitted to the Department of Respiration Medicine in Jiaxing Second Hospital from February 2011 to July 2016 were enrolled,and they all underwent CAT assessment test and lung function examination.The patients were assigned to Ⅱ,Ⅲ and Ⅳ grade groups according to the lung function level,and the difference of forced expiratory end volume in 1 second (FEV1) and FEV1/forced vital capacity (FVC) ratio were compared among the three groups;the patients were also assigned into 2,3 and 4 grade groups according to CAT scores,and the difference of length of stay in the hospital and hospitalization expenses were compared among the three groups.Spearman correlation analysis was used to analyze the correlations between the CAT score and lung function indexes.Results One hundred and thirty-five patients with AECOPD were accepted and all of their clinical data were analyzed in the study.Their mean FEV1 was (0.42±0.16) L,FEV1/FVC was 0.44 ±0.13 and CAT score was 26.64 ± 5.43.The correlation analyses showed:there were significant negative correlations between CAT score and FEV1,FEV1/FVC (r value was-0.691,-0.728,both P ＜ 0.001).With the elevation of lung function grade,FEV1 and FEV1/FVC were decreased gradually (1Ⅱ,Ⅲ,Ⅳ grade groups were 0.62±0.07,0.40±0.06,0.25±0.03 and 0.64±0.01,0.40±0.00,0.33±0.06 respectively),while CAT score raised gradually (20.03 ± 3.36,28.30 ± 3.31,30.18±3.86,all P ＜ 0.01);with the CAT score getting higher and higher,hospitalization expenses (yuan) and the length of stay in hospital (day) of patients with AECOPD were increased significantly (2,3,4 grade groups:expense were 6214.09±1396.16,8339.31±1866.46,9600.97±4339.87,and length of stay in hospital were 7.54± 1.62,9.52±2.21,14.85 ±5.62,respectively,all P ＜ 0.01).Conclusion CAT is a reliable tool to measure the life quality of AECOPD patients and has certain relevance to the severity and prognosis of the disease.

Objective To determine the distribution of IL-4, IL-17, IFN-γ in the lacrimal gland lesions and submaxillary lymph nodes of MRL/Ipr mice-a model for idiopathic dacryoadenitis, and investi-gate the possible role of T-belper cells in the disease. Methods Frozen sections of lacrimal glands and sub-maxillary lymph nodes from MRL/lpr and BALB/c mice age 3 months were stained with antibodies to the cy-tokines IL-4, IL-17 and IFN-γ, then calculated the numbers or proportions of positive cells respectively,double staining for CD4 and each cytokine was taken in lymph nodes of MRL/Ipr mice additionally. Results The major cytokine in lacrimal gland lesions is IL-4, which contrasts to little IL-17 or IFN-γ/stained(P <0.05), the numbers of cells staining for each cytokine in submaxillary lymph nodes elevate in MRL/lpr mice, and the expressions of IL-4 and IL-17 are greater than IFN-γ significantly. Conclusion Th2 may play an important role in the development of local dacryoadenitis and systemic abnormality in MRL/lpr mice,the role of IL-17 maybe different in lacrimal gland lesions and lymph nodes which need more research.

Objective To investigate the approaches and effect of Ilizarov external fixators combined with limited operation in treatment of posttraumatic clubfoot in children.Methods The study involved 40 cases (43 feet) of posttraumatic clubfoot treated with Ilizarov external fixators combined with limited operation including soft-tissue releases (26 cases,28 feet) or osteotomies (14 cases,15 feet)from January 2006 to June 2012.Scoring system of intemational clubfoot study group (ICFSG) including aspects of functional assessment (36 points),morphology (12 points) and radiological assessment (12points) were employed before and after surgery.Results All the cases were available to the follow-up of 0.5-6 years.ICFSG score was excellent in 28 feet,good in 10,fair in four and poor in one foot,with excellent-good rate of 88％.This suggested the good correction of posttraumatic clubfoot,satisfactory weight-bearing exercise and a low relapse rate.Conclusions Conservative open operation brings large trauma and poor results in treatment of posttraumatic clubfeet in children,while Ilizarov invasive distraction technique is probable to offset those weak points.Therefore,Ilizarov external fixators combined with limited operation is an effective treatment.

Objective To investigate the clinical efficacy of three-dimensional (3D) and two-dimensional (2D) laparoscopic surgeries in the treatment of Todani type Ⅰ choledochal cyst.Methods The retrospective cohort study was conducted.The clinical data of 59 patients with Todani type Ⅰ choledochal cyst who were admitted to the People's Hospital of Hunan Province between January 2013 and January 2016 were collected.Thirty patients undergoing 2D laparoscopic surgery between January 2013 and June 2014 were allocated into the 2D group and 29 patients undergoing 3D laparoscopic surgery between July 2014 and January 2016 were allocated into the 3D group.There were the same Trocar placement and surgical procedure in the 2 groups,and surgical procedure completely followed the treatment principle of Todani type Ⅰ choledochal cyst.Observation indicators included (Ⅰ) surgical situations:conversion to open surgery,operation time,volume of intraoperative blood loss,(2) postoperative situations:postoperative complications,(3) follow-up.Patients were followed up by outpatient examination or telephone interview to detect postoperative recovery up to April 30,2016.Measurement data with skewed distribution were presented as M (range) and analyzed using the Mann-Whitney U test.Count data were compared by Fisher exact probability.Results (1) Surgical situations:patients in the 2 groups underwent laparoscopic choledochal cystectomy + Roux-en-Y hepaticojejunostomy.Two patients in the 2D group received conversion to open surgery and patients in the 3D group received the successful surgery without conversion to open surgery.Rate of conversion to open surgery in the 2D and 3D groups were 6.7％ (2/30) and 0,respectively,with no statistically significant difference (P ＞ 0.05).Operation time in the 2D and 3D groups were 285 minutes (range,240-390 minutes) and 190 minutes (range,140-215 minutes),with a statistically significant difference (U =40.0,P ＜ 0.05).Volume of intraoperative blood loss in the 2D and 3D groups were 50 mL (range,10-300mL) and 45 mL (range,20-250 mL),with no statistically significant difference (U =1 018.5,P ＞ 0.05).(2)Postoperative situations:patients in the 2 groups had good recovery,without occurrence of severe complications in Clavien-Dindo≥ Ⅲ stage.Four and 1 patients in the 2D and 3D groups were complicated with bile leakage (in Ⅱ stage of Clavien-Dindo) and 1 and 1 were complicated with upper gastrointestinal hemorrhage (in]][stage of Clavien-Dindo),respectively,with no statistically significant difference (P ＞ 0.05).Overall incidence of complications in the 2D and 3D groups were 16.7％ (5/30) and 10.3％ (3/29),with no statistically significant difference (P ＞ 0.05).All the patients were cured by conservative treatment.(3) Follow-up:59 patients were followed up for 5-36 months,with good recovery and without occurrence of reflux cholangitis,hepatic and intestinal anastomosis stenosis and reoperation.Conclusions 3D and 2D laparoscopic surgeries are safe and effective for Todani type Ⅰ choledochal cyst.Compared with 2D laparoscopic surgery,3D laparoscopic surgery can reduce the operation time and not increase the complications,and it should be discreetly promoted based on the experiences of surgeons.

Aim To investigate the anticancer effect of a new xanthono-pyridine derivative N, N '-( 7-oxo-7H-chromeno[3,2-h] quinoline-5,9-diyl)-bis(2-( pyrroli-din-1-yl)acetamide) (XP-16) on human lung carcino-ma cell line A549 and the potential mechanism. Meth-ods Antiproliferative effect of XP-16 on A549 cells was evaluated by MTT assay, morphological examina-tion and colonial assay. Apoptosis detection was car-ried out using Hoechst 33258 and PI double-dyeing method. Intracellular Ca2+ concentration ( [ Ca2+] i ) and mitochondria membrane potential were detected by fluorospectrophotometer. A549 cells treated with XP-16 were collected for Bad and metallothionein 1 A ( MT-1 A ) transcript analysis by real-time reverse tran-scriptase-polymerase chain reaction ( qRT-PCR) . Re-sults XP-16 inhibited A549 cell proliferation in dose-and time-dependent manner. Typical apoptotic mor-
phology such as chromatin aggregation and nuclear fragmentation was observed in A549 cells treated with XP-16 for 24 h, and the apoptosis was showed in a dose-dependent manner. After treated with XP-16, [ Ca2+] i and mitochondria membrane potential of A549 cells were decreased, and relative mRNA level of Bad and MT-1A was up-regulated. Conclusions XP-16 has anticancer effect on A549 cells through apoptosis, which might be associated with decreasing intracellular Ca2+ concentration and mitochondria membrane poten-tial. Up-regulation of MT-1A expression might be the result of decreased [ Ca2+] i .

Objective To establish a rapid and accurate method, and to determine the density of mice and rats sperm with enzyme-labeled instrument.Methods ①The optimal wavelengths and the regression equation set up: After six Kunming mice and six Sprague-Dawley rats were sacrificed, the left epididymis was separated and fully cut up in phosphate buffer saline.With water bath, the sperm were fully dissociated.Using the enzyme-labeled instrument to detect the wavelength absorbance respectively under different wavelength and fitting absorbance curve.The best wavelength will be the most close to 1 of the correlation coefficient ( R2 ) and the standard deviation of minimum.After ten Kunming mice and ten Sprague-Dawley rats were sacrificed,the sperm suspension of different concentration gradient were got.The regression equation of the sperm density and absorbance was established by using enzyme-labeled instrument and haemocytometer.②The test of sperm absorbance stability: Mice and rats,six respectively,were used to make the sperm suspension.Samples were put in room temperature (25℃) or 37℃water bath continued,and after water bath about 0,20,30,40,50, 60 min,the change of absorbance was recorded.③The regression equation verification:The mice were administrated orally with 20% ethanol solution for 30 days to make oligospermia.In order to verify the new method, two different method were used to get the sample sperm.Results The optimal absorbancy-sperm density curve could be established at 380 nm.The means of KM mice sperm count ( x1 ) and absorbance ( y1 ) are showed to be the linear function,and the linear regression equation is y1 =2 ×10 -9 x1 +0.0648, R2 =0.9743.The means of SD rat sperm count ( x2 ) and absorbance (y2) are showed to be the linear function,and the linear regression equation is y2 =5 ×10 -9x2 +0.0621,R2 =0.9940.SD rat sperm suspension liquid after 60 min in water bath, absorbance value at 0 min significantly decreased(P<0.05), but at room temperature after 40 min significantly increased ( P<0.05); KM mice sperm suspension in the water bath and under the condition of normal temperature after 50 min, compared with the 0 min, absorbance value increased significantly(P<0.05).Compared with control group, sperm density of ethanol oligozoospermia group by enzyme standard detector and standard curve calculation were significantly decreased ( P <0.05 );compared with absorbancy-sperm density equation, determination of ethanol oligozoospermia group of sperm density by cell counting plate method had not significant difference.The results suggested absorbancy-sperm density equation could effectively detect the reduction of the mice sperm in oligospermia.Conclusion Using enzyme-labeled instrument to set up the curve of absorbancy-sperm density equation can estimate the sperm density of mice and rats rapidly and exactly.