OBJECTIVETo observe the inhibitory effect of gefitineb on the proliferation and its inducing effect on the apoptosis of mouse I-10 Leydig testicular cancer cells in vitro.

METHODSWe treated I-10 Leydig testicular cancer cells of mice with gefitineb at 0, 1.25, 2.5, 5, 10, 20, and 40 µmol/L. Then we determined the inhibitory effect of gefitineb on the growth of the cells by MTT, detected their early and late apoptosis by Annexin V-FITC/propidium iodide double staining and Hoechst 33258 nuclear staining, respectively, and observed the expressions of apoptosis-related proteins Bcl-2, Bax and caspase 3/9 by Western blot.

RESULTSCompared with the blank control group, gefitineb significantly inhibited the proliferation of the I-10 cells at 10 and 20 µmol/L (P < 0.05). The survival rate of the cells was (32.4 ± 2.8)% (P < 0.01) and their early and late apoptosis rates were (26.7 ± 4.2)% and (59.33 ± 10.2)% in the 40 µmol/L group, significantly different from those in the control (P < 0.05 and P <0.01). In comparison with the blank control group, gefitineb at 10, 20, and 40 µmol/L increased the expression of pro-apoptotic protein Bax by (41.9 ± 7.1), (60.1 ± 9.8), and (69.0 ± 11.3)% (all P < 0.05), decreased that of apoptosis-inhibitory protein Bcl-2 by (50.3 ± 8.9), (63.9 ± 6.9), and (88.7 ± 13.9)% (all P < 0.05), and elevated that of the cleft proteins caspase-3 by (69.0 ± 6.9)% (P < 0.05), (71.5 ± 8.1)% (P < 0.05), and (110.9 ± 14.2)% (P < 0.01) and caspase-9 by (51.8 ± 4.9), (54.7 ± 6.7), and (43.8 ± 11.8)% (all P < 0.05).

CONCLUSIONGefitineb can increase the cytotoxicity of I-10 Leydig testicular cancer cells of mice and induce their apoptosis via the mitochondria-mediated apoptosis signaling pathway.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Proliferation ; drug effects ; Cell Survival ; Leydig Cell Tumor ; drug therapy ; metabolism ; pathology ; Male ; Mice ; Neoplasm Proteins ; metabolism ; Neoplasms, Germ Cell and Embryonal ; drug therapy ; metabolism ; pathology ; Quinazolines ; pharmacology ; Testicular Neoplasms ; drug therapy ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism

Objective To evaluate the value and clinical application of cardiac structure Z scores in children with preoperative tetralogy of Fallot (TOF) by echocardiography. Methods Considering the result of 31 patients who underwent echocardiography in Shenzhen Children’s Hospital before TOF repair from January 2011 to September 2013, the median age was 10 months (range:3 days to 14 years). Other 31 normal children were selected as normal control group with age-and sex-matched, the median age was 10 months (range: 6 days to 13 years). Echocardiographic measurements included the diameter of right ventricular outflow tract (RVOT), pulmonary valve diameter (PVD), left pulmonary artery diameter (LPAD), right pulmonary artery diameter (RPAD), pulmonary valve peak velocity (PV-Vmax), tricuspid annulus diameter (TVD), mitral annulus diameter (MAD), left ventricular end-diastolic volume (LVEDV) and left ventricular mass (LVmass). Shenzhen Children’s Hospital pediatric echocardiography normal reference values and the regression equation are applied to obtain the forecast average, and using the Z-score formula into the standard value (Z=[M-y]/ MSE ). Results There was no significant difference in body surface area between the TOF group and control group (P>0.05). Compared with control group, the true value of RVOT reduced significantly in TOF group, its Z-score showed negative values increase (-4.87±1.86 vs 0.33±0.93, t=-13.90, P<0.05). PVD, LPAD and RPAD reduced significantly in TOF group, those Z scores showed a negative value increase (-4.15±2.93 vs 0.11±0.85, t=-7.76;-1.35±2.38 vs 0.66±1.17, t=-4.24;-1.47±2.03 vs 0.59±1.04, t=-4.94;all P<0.05). While PV-Vmax increased significantly, and it’s Z score showed a positive value increase (8.38±0.19 vs 0.24±0.98, t=29.50, P<0.05). TVD of TOF patients was slightly larger than the control group (P=0.10), but the Z score was significant greater than the control group (1.00±1.17 vs 0.29±0.52, t=3.06, P<0.05). MV-D, LVEDV and LVmass of TOF patients were slightly decreased than the control group (all P>0.05), but the Z scores of TOF were significantly reduced than the control group (0.25±0.78 vs 0.76±0.65, t=-2.75;-0.92±1.94 vs 0.03±1.01, t=-2.41;-0.83±1.59 vs 0.67±0.69, t=-4.71; all P<0.05). The Z scores of RVOT, PVD, LPAD and RPAD had no significant difference between transannular patch surgery and pulmonary valve-sparing repair patients (-4.89±2.03 vs -4.84±1.67, t=-0.08; -4.73±3.49 vs -3.34±1.75, t=-1.31; -1.88±2.54 vs -0.62±2.00, t=-1.49;-1.89±1.90 vs-0.84±2.15, t=-1.41;all P>0.05). For the Z scores of PV-Vmax, there were no significant difference between transannular patch surgery and pulmonary valve-sparing repair (8.58±1.20 vs 8.12±1.16, t=1.07, P>0.05). Conclusions The Z scores value of cardiac structure can be used in quantitative analysis of RVOT obstruction and development of left ventricular backward of TOF. The Z score of transannular patch surgery is more lower than pulmonary valve-sparing repair patients. The normalized Z score by body surface area correction is important for the preoperative diagnosis and the surgical planning.

Objective To investigate the pathogenesis of gallbladder chaotic dynamics after partial gastrec-tomy. Methods 140 cases operated by partial gastrectomy after 6 months were randomly examined, they in-cluded one group of 40 cases by Billroth type and the other 100 cases by Eiselsberg type. The ultrasonograph was applied to evaluate the function of gallbladder dynamics and radio-immunity method to determine the content of CCK at the time of having no food and 30 minutes after med. Results BV and RV of Eiselsberg group were bigger than Billroth Ⅰ obviously P<0.05. Gallbladder contraction rate displayed not well obvi-ously as too. The plasm level of CCK had no manifested distinctions in empty stomach cases of the 2 groups, but the increasing gradient plasm level of CCK in Billroth Ⅰ exceeded Eiselsberg type. Conclusions The pathogenesis of gallbladder chaotic dynamics after partial gastrectomy was correlated with the alterative type of partial gastrectomy, the reduction of CCK plasm level, the damage of anterior vagal trunk hepatic branches during the operations and so on led to the disorder of gallbladder emptying and induced cholecystolithiasis.

Measles and rubella virus cause fever rash diseases that are uneasy to differentiate clinically from each other. Specific primers and fluorescence-labeled probes were designed, and a multiplex Real-time RT-PCR with an internal control was developed to simultaneously identify the measles and rubella virus. The multiplex Real-time RT-PCR assay was specific and no false positive or false negative results were found. The sensitivity of the assay was 0.1TCID50/mL and 1TCID50/mL for measles and rubella virus respectively. Analysis with 0.1-10(3)TCID50/mL measles or rubella virus samples demonstrated high validity and reproducibility with the coefficient of variability(CV) of below 0.9% for both measles and rubella virus. Using ribonuclease P (RNase P) as internal false negative control, the developed multiplex Real-time RT-PCR assay is suitable for rapid clinical diagnosis of measles and rubella virus.
Fluorescent Dyes ; Humans ; Measles ; diagnosis ; virology ; Measles virus ; genetics ; RNA, Viral ; genetics ; isolation & purification ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Ribonuclease P ; genetics ; Rubella ; diagnosis ; virology ; Rubella virus ; genetics ; Sensitivity and Specificity

OBJECTIVETo study the relationship between influenza epidemic and genetic characteristic on HA and NA regions of influenza virus subtype A3 isolates of Zhejiang province in the recent years.

METHODSRNA of 25 influenza virus subtype A3 isolates, circulated in Zhejiang province during 1998 to 2005, was extracted. HA1 and NA regions were amplified and sequenced. All the sequence data were analyzed using BioEdit.

RESULTSHA1 and NA regions of all the isolates belonged to 987nt and 1362nt, encoding protein of 329 and 454 amino acids respectively. Isolates shared amino acid homology of 90.9%-99.3% and 95.2%-99.5% on HA1 and NA regions, while divergence on HA1 was greater than that on NA region. During a period of 8 years, 30 amino acids on HA1 region were substituted and 14 of which refer to 4 antigenic determinant sites. Meanwhile,21 amino acids on NA region were substituted and 5 of which referred to 3 antigenic determinant sites. Significant divergences, both in HA1 and NA, were observed among isolates in 1998 and 2002, showing that they belonged to absolutely different branches. Additionally, influenza virus subtype A3 isolates identified in recent years, with 11 N-linked glyeosylation sites in HA1 region, had 5 sites more than early A/Aichi/2/68 strain. Since 1998,3 sites had been inserted in epidemic strains, indicating the accelerated trend of glyeosylation sites were increasing.

CONCLUSIONThere is a correlation between antigenic drift of influenza virus subtype A3 and the two epidemics in Zhejiang province in 1998 and 2002.

Amino Acid Sequence ; Antigens, Viral ; genetics ; China ; Epitopes ; genetics ; Evolution, Molecular ; Genetic Drift ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Humans ; Influenza A virus ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; Molecular Sequence Data ; Neuraminidase ; genetics ; RNA, Viral ; genetics ; Sequence Analysis, RNA ; Sequence Homology, Amino Acid

Objective To determine whether real-time three-dimensional transesophageal echocardiography(RT-3D TEE) is an accurate non-invasive technique for defining the morphology of atrial septal defects(ASD). Methods In 20 patients with secundum ASD, mean age 42.2 years (7 male, 13 female) ,live three dimensional surgical views of ASD were acquired,qualitative morphology of ASD such as the shape, presence of fenestrations and the defect margins were noted during the cardiac cycle. The measurements obtained from 2-dimensional transesophageal echocardiography(2D-TEE) and RT-3D TEE were compared to those obtained from stretched balloon diameter(SBD) or surgery. Measurements of the size and area change of ASD were validated during the cardiac cycle by RT-3D TEE,stepwise multiple linear regression analysis was performed to test the correlation between the maximum change ratios of area and diameter of ASD and age, atrial septal length, ASD shunting velocity, the maximum diameter of ASD and right ventricular systolic pressure. Results ① The morphology of ASD was circle-like or elliptical in systole,irregular or elliptical in diastole. ②The area and diameter of ASD measured by RT-3D TEE was minimal in isovolumetric contraction phase, maximal in isovolumetric diastole phase. The correlation coefficient of maximum diameter measured by 3D with SBD was 0.962 , greater than that by 2D and SBD (0.820). ③The change ratio of area and maximum diameter of ASD acquired by RT-3D-TEE was 11.48%-71.12% and 2.80%-43.87% respectively,and the correlation coefficient of them was 0.921. Conclusions RT-3D TEE using live 3D-Zoom mode accurately displayed the varying morphology,dimensions and spatial relations of ASD. RT-3D TEE can offer visualization morphological changing of ASD in different periods of cardiac cycle,providing more information for percutanous catheter intervention and open heart surgery.

Objective To explore the effect of transplantation of mesenchymal stem cells (MSCs) transfected with the human receptor activity modifying protein 1 (hRAMP1) gene on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) after carotid balloon angioplasty was performed in rabbits with carotid atherosclerosis.Methods Density gradient centrifugation and adherent culture were carried out to obtain MSCs,which were then transinfected with an adenovirus vector carrying the hRAMP1 gene or an empty adenovirus vector.A rabbit model of atherosclerotic stenosis and balloon angioplasty was successfully established.Results were randomly divided into three groups:the hRAMP1-MSCs group,theadipose-derived MSCs (Ad-MSCs) group and the control group.MSCs were transinfected with Ad-EGFP-hRAMP1,Ad-EGFP or PBS by transplantation into the injured carotid arteries.Homing and differentiation were assessed with MSCs harvested at 7 d.With MSCs collected at 28 d,Western blotting was used to measure the expression of the hRAMP1 target gene in the carotid artery; the neointima and media area in the injured carotid arteries were estimated; carotid artery morphology was examined with H&E staining; and the proliferation and apoptosis of VSMCs were determined by immunohistochemistry and TUNEL.Results The expression of CD31 and EGFP was found in proliferating neointima lesions at 7d in the hRAMP1-MSCs group and the Ad-MSCs group.At 28d of MSC transplantation,the level of RAMP1 significantly increased in the hRAMP1-MSCs group,compared with the Ad-MSCs and control groups [(63.0±4.9) vs.(28.3±2.5) and (27.2±7.2),all P＜0.05],but there was no differencein the RAMP1 level between the Ad MSCs group and the control group (P＞0.05).Positive expression of the α-smooth muscle antibody (α-SMA) was found in all three groups at 28 d of MSC transplantation.The thickness of the hyperplastic neointima significantly decreased in the hRAMP1-MSCs group,compared with the other two groups (P＜0.05),and was lower in the Ad-MSCs group than in the control group (P＜0.05).The expression of proliferating cell nuclear antigen (PCNA) was lower in the hRAMP1-MSCs group than in the Ad-MSCs and control groups at 28d of MSC transplantation (P ＜0.05),while the PCNA level was lower in the Ad-MSCs group than in the control group (P＜ 0.05).The VSMC apoptosis rate significantly increased in the hRAMP1-MSCs group,compared with the Ad MSCs and control groups (P＜0.05),and was the lowest in the control group (P＜0.05).Conclusions Gene-modified stem cell therapy can effectively inhibit vascular intimal hyperplasia,thereby reducing restenosis after angioplasty.

Objective To evaluate the biological characteristics of hip joint in infants with developmental dislocation of the hip (DDH).Methods From January 2013 to June 2016,30 patients (age from 1 to 8 months,25 females and 5 males) who were diagnosed as DDH,underwent ultrasound examination in Shenzhen Children's Hospital,in these patients 20 cases were treated surgically,and 10 cases were treated with non-surgical treatment.There were 34 hips (26 unilateral dislocation and 4 bilateral dislocation) dislocation in 60 hips.All the patients were confirmed by X-ray,magnetic resonance imaging (MRI) examination or operation.All the patients were examined by ultrasound through the coronal and transverse plane of the hip joint.The α angle,femoral head coverage ratio by acetabulum (FHC),femoral head length and width,distance from pubis to femoral head (P-H) and distance from ischium to femoral head (I-H) were measured.The dislocation joints were compared with contralateral joints.Results The α angle in the hip dislocation group was smaller than the contralateral group [(50.5±3.75)° vs (64.8±3.38) °],and there was significant difference between the two groups (t=-15.181,P ＜ 0.001).The FHC,femoral head length and width in the hip dislocation group were all smaller than the contralateral group [(23.4t17.63)％ vs (64.3±6.45)％,(0.98 ±0.15) cm vs (1.19 ±0.11) cm,(1.38±0.21) cm vs (1.61 ±0.16) cm],and there were significant differences between the two groups (t=-12.469,-6.034,-4.568,all P ＜ 0.001).The P-H and I-H in the hip dislocation group were larger than the contralateral group [(0.97±0.45) cm vs (0.27±0.05) cm,(0.75±0.30) cm vs (0.17±0.05) cm],and there were significant differences between the two groups (t=8.805,10.696,both P ＜ 0.001).The α angle,femoral head length and width in bilateral dislocation of hip group were slightly smaller than the unilateral dislocation of hip group [(50.3±2.75)° vs (51.3±4.77)°,(0.90 ±0.15) cm vs (0.97 ±0.12) cm,(1.25±0.20) cm vs (1.37 ±0.17) cm],but there were no significant differences between the two groups.The P-H and I-H in bilateral dislocation of hip group were slightly larger than the unilateral dislocation of hip group [(0.97 ± 0.49) cm vs (0.80±0.31) cm,(0.92±0.26) cm vs (0.68±0.18) cm],but there were no significant differences between the two groups.The value of acetabular index in the ultrasound group was slightly larger than the X-ray group [(33.13 ± 7.82)° vs (31.20 ± 8.31)°],and there were no significant differences between the two groups.Conclusions The characteristics of DDH includes acetabulum and femoral head dysplasia,and femoral head and acetabulum position relationship abnormalities.Quantitative ultrasonography of the hip can be used to quantitatively evaluate the structural features of DDH,and it is helpful to the early diagnosis and follow-up of DDH.

Objective To investigate the drug resistance genes of extended-spectrum beta- lactamase-producing bacteria in 49 strains.Methods Extended-spectrum ?-lactamase -producing strains were detected by the disc diffusion test.The techniques of polymerase chain reaction,sequence analysis, pulsed-field gel electrophoresis were used to analyze the genotype and homology of extended-spectrum ?- lactamase-producing strains.Results The incidence of ESBL-producing strains from E.coli,K pneumoniae,K oxytoca,was 20% in 2000,and 40% in 2003.Among the 49 ESBLs producers the most common genotype was CTX-M-14( n=33).The others were CTX-M-3,CTX-M-9,CTX-M-12,CTX-M-15, CTX-M-24 and SHVSa.Both two CTX-M subtypes,CTX-M-3 and CTX-M-14,were detected in one strain. However,4 ESBL-producing strains confirmed by phenotype remained untyped.The results showed that the ESBLs producers were not closely related,except for two strains of E.coli and two strains of K.pneumoniae which were homgenic respectively.Concolusions The incidence of ESBL-producing strains increases with years.The most common genotype of ESBLs is CTX-M.There is no evidence for epidemiologic spread of ESBL-producers by pulsed-field gel electrophoresis.

PRAS40 (proline-rich Akt substrate 40 kD) associates with mammalian target of rapamycin complex 1(mTORC1), serine 183 site (Ser183) of PRAS40 can be phosphorylated by mTORC1. To prepare the phosphorylated PRAS40 (Ser183) antibody, We chosen 10-amino acid including Ser183 as antigen peptide through antigenicity and hydrophobicity analysis, hinged on keyhole limpet hemocyanin (KLH), and used the KLH-peptide to immunize rabbits. After antibody serum titer detection by enzyme linked immunosorbent assay (ELISA), the antibody was purified with rProtein A sepharose fast flow and dephosphorylated antigen membrane. The antibody titrate reached 1:10 000 after purification and its special property was enhanced with absorption treatment of dephosphorylated antigen membrane. In addition, we used rabbit anti-PRAS40 antibody and the phosphorylated PRAS40 (Ser183) antibody to detect PRAS40 expression in several cell lines, including the normal cells HL7702, HEK293, tumor cells HepG2, A549 and S180. There were no quite difference among these cells; otherwise, we observed the decreased phosphorylation level of Ser183 after amino acid withdrawal treatment. Therefore, the polyclonal phosphorylated PRAS40 (Ser183) antibody was specific to PRAS40 (Ser183) site and could be used for the function study of PRAS40.
Adaptor Proteins, Signal Transducing ; Animals ; Antibodies ; analysis ; Cell Line, Tumor ; Humans ; Male ; Mechanistic Target of Rapamycin Complex 1 ; Multiprotein Complexes ; Phosphoproteins ; chemistry ; immunology ; Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rabbits ; Serine ; metabolism ; TOR Serine-Threonine Kinases