Objective Aim to set up the method of glycoprotein B genotype of human cytomegalovirus (HCMV) analysis by using nested polymerase chain reaction(nPCR) and restriction fragment length polymorphism(RFLP). Methods Eleven blood samples and twenty-three urine samples were obtained from thirty-four HCMV-infected patients. A fragment of gB gene was amplified by nPCR. HCMV gB genotyping was carried out by RFLP, and the amplified DNA fragments were verified by DNA sequencing. Results Of the 34 patients, gB type Ⅰ was found in 13 patients, gB type Ⅱ in 12 patients, gB type Ⅲ in 9 patients, and none had the gB type Ⅳ sequence. The similarities of PCR products of HCMV gB Ⅰ, Ⅱ and Ⅲ amplified compared with the sequences of prototype strains in GenBank were 98.1%～99.6%, 98.9%～100%, 97.3%～98.9%. Conclusions The nPCR assay developed in this study was sensitive and specific for detection of HCMV, and RFLP analysis of HCMV gB genotype was definite and reliable.

Objective To analyze and summarize the clinical features of delayed occurrence of senile asthma,to add the knowledge and understanding of the disease. Methods To retrospectively analyze clinical data of the cases of senile asthma treated in our hospital,all the cases were divided into groups of early onset senile asthma and delayed occurrence senile asthma,according to the age of first onset.Then related indexes were analyzed,to summarize the clinical features of group of delayed occurrence of senile asthma. Results There were 28 cases of delayed occurrence of senile asthma,accounting for 34.6% of total cases.Compared to early onset group,there was no significant difference (all P＞0.05) between two groups for the following items such as age,allergic history and positive rate of family's history,disease causes,clinical symptoms,basic diseases and complications,proportion of severe cases,rate of misdiagnosis and mistreatment,proportion of standardized treatment and un-standardized treatment,prognosis of diseases and mortality.Both groups had low rate of knowledge and application on PEF monitoring equipment and ACT score.The period of misdiagnosis and mistreatment for delayed occurrence group was shorter than the early onset group (P＜0.05＝; the seasonal nature and day and night pattern was significant in delayed occurrence group (P＜0.05＝. Conclusion Late onset elderly asthma had the features such as shorter course of the disease,relatively obvious onset rule during day and night,and obvious symptoms during night,which are different from that of early onset group.

Objective To investigate the clinical characteristics and prognosis of patients with systemic lupus erythematosus (SLE) complicated with acute pancreatitis (AP) .Methods From January 1999 to December 2013 ,the clinical data of 16 patients with SLE complicated with AP among the total 2 526 cases of SLE was collected .A retrospective analysis was performed and the clinical data of patients was classified and documented ,which included general information ,past history ,clinical symptoms , laboratory findings ,imaging findings ,treatment and outcome .The rank sum test was performed for analysis of non‐normal distributed measurement data ,and the Fisher′s exact test was used for count data analysis .Results The incidence of SLE complicated with AP was 0 .63% (16/2 526) .Among them ,ten patients were mild acute pancreatitis (MAP) and six patients were severe acute pancreatitis (SAP) .All patients were treated with fasting ,gastrointestinal decompression ,nutritional support ,anti‐acid ,anti‐inflammatory ,glucocorticoid and somatostatin and so on . Six patients were cured , seven patients improved and three patients died (two SLE complicated with SAP ,one SLE complicated with MAP) . Compared with the SLE patients complicated with SAP ,the SLE patients complicated with MAP were more easily to have lupus nephritis(6/6 versus 5/10 ,Fisher′s exact test) ,hematological system injuries (6/6 versus 5/10 ,Fisher′s exact test) ,liver injuries (5/6 versus 0/10 ,Fisher′s exact test) ,more organs involved (mean 7 versus 3 ,Z= -3 .225) and higher SLE disease active indexes (DAI) score (mean 13 .5 versus 6 .5 ,Z= -2 .876);the differences were statistically significant (all P<0 .05) .Compared with the cured and improved SLE patients complicated with AP ,lupus encephalopathy (2/3 versus 1/13 ,Fisher′s exact test) ,more organs involved (mean 7 versus 5 ,Z= -2 .276) and higher SLE DAI score (mean 21 versus 12 ,Z= -2 .195) was more common in dead SLE patients complicated with AP;the differences were statistically significant (all P< 0 .05) .Conclusions SLE patients complicated with SAP are more easily to get lupus nephritis ,hematological system injuries ,liver injuries ,activity of SLE and multiple‐organ systems involved . The prognosis of SLE patients complicated with AP was poor in those with activity of SLE ,multiple‐organ involved and lupus encephalopathy .

Objective:This study aimed to observe the synergistic effect of a new tumor vaccine combined with metronomic che-motherapy in vivo on breast cancer. This study was also conducted to investigate the mechanism of this combination. Methods:Balb/c mice inoculated with 4T1 mouse breast cancer cell were used as tumor models. High-mobility group nucleosome-binding protein 1 (HMGN1) gene was used to transfect 4T1 cell lines as cancer vaccines. After 4T1 cell was inoculated, the mice were randomized into four groups:normal saline (NS);metronomic gemcitabine (GEM) alone;cancer vaccine alone;and combination therapy group. Tumor growth and potential toxicities of these regimens were observed. The Foxp3 expression of regulatory T cells (Tregs) was detected by western blot and immunohistochemical staining. The microvessel density (MVD) of the tumor was also detected by immunohistochemi-cal staining. Results:The tumor volume of the mice was significantly lower in the combination group than in the MET group or cancer vaccine group (P<0.05). This result exhibited a higher significant difference than the tumor volume of the mice in the NS group (P<0.01). Foxp3 expression was significantly lower in the mice treated with GEM (combination or MET group). MVD was significantly lower in these two groups than in the cancer vaccine group or NS group (P<0.05). Furthermore, adverse reactions slightly occurred in each group. Conclusion: The combination of cancer vaccines and metronomic GEM is a very active and well-tolerated regimen for breast cancer in mice.

Objective To investigate the infection rate of Legionella pneumophila(LP)in children younger than 5 years with lower respiratory infections by ELISA and PCR.Method Serum LP-IgM and IgG were measured by ELISA,and LP DNA in sputum or throat swab were detected by PCR in 300 children less than 5 years with lower respiratory infections.The data were analyzed by chi-square test and the consistency of the two methods was analyzea by NcNemar test.Results Serum antibody detected by ELISA was positive in 17 cases(5.67%),including 10 positive of IgM and 7 positiile of lgG.In these 17 eases,11 were males and 6 were females with similar positive rates(5.76%vs 5.5%).Four cases(2.53%)were positive in children aged from 27 days to 1 year,while 7(7.37%)and 6 cases(12.77%)were positive in children aged 1-3 years and 3-5 years,respectively.Seven cases(5.19%)found in the winter and spring seasons and 10 cases(6.06%)in summer and autumn seasons.Eleven children(11.83%)were from urban area and only 6(2.9%)from countryside.DNA in sputum or throat swab detected by PCR was positive in 16 cases(5.33%),included 10 males and 6 females with similar positive rates(5.24%vs 5.5%).Five cases(3.16%)were positive in children aged from 27 days to 1 year,while 6(6.32%)and 5 cases(10.64%)were positive in children aged 1-3 years and 3-5 years,respectively.Three cases(2.22%)found in the winter and spring seasons and 13 cases(7.88%)in sunmmr and alltumll seasons.Nine children(9.68%)were from urban area and only 7(3.38%)from countryside.McNemar test was used to compare the data of ELISA and PCR results with a Qm of 0.037 and a Pr of 0.8474.Conclusions LP might contribute partly to the lower respiratory infections in children less than 5 years.The infection rate of LP increases with age increasing.Urban children have a higher infection rate than those living in countryside.Both methods have a good consistency.

AIM:To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells.METHODS:Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene.The mRNA expression of Wip1 was measured by real-time PCR.The protein level of Wip1 was detected by Western blotting.The viability of RKO colon cancer cells was measured by MTS assay.The cell apoptosis and cell cycle were analyzed by flow cytometry.RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels.The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression.The viability of RKO colon cancer cells was decreased from (89.4 ±6.6)%to (74.7 ±3.9)%af-ter treated with 5-fluorouracil (P<0.05) and decreased from (77.9 ±2.4)%to (66.7 ±2.9)%after treated with oxali-platin ( P<0.05 ) .The cell apoptotic rate was increased from ( 7.7 ±0.5 )% to ( 12.3 ±3.2 )% and from ( 14.7 ± 2.1)% to (34.0 ±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05).CONCLUSION:Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.

Objective To explore the efficacy and adverse reactions of diminishing scheme of cefaclor sustained re-lease tablets in the treatment of recurrent urinary tract infection(RUTI).Methods 60 RUTI patients in a hospital were divided into treatment group(n=30)and control group (n=30),patients in treatment group were treated with diminishing scheme of cefaclor sustained release tablets,patients in control group were treated with diminishing scheme of levofloxacin tablets,clinical therapeutic efficacy and adverse reactions of two groups were observed. Results The curative rate in treatment group was higher than control group ([80.00%,n =24]vs [53.33%,n =16])(χ2 =4.80,P =0.028).The incidence of RUTI in treatment group was lower than control group ([6.67%,n=2]vs [26.67%,n=8])(χ2 =4.32,P =0.038).Incidence of adverse reactions in treatment group was lower than control group (16.67% vs 50.00%)(χ2 =7.50,P =0.006).Conclusion The diminishing scheme of cefaclor sus-tained release tablets in the treatment of RUTI has good curative efficacy,low recurrence rate,fewer adverse reac-tions,and can be used for the treatment of recurrence of RUTI.

AIM:To investigate the role of side population ( SP) cells in multidrug resistance of colon cancer cells and microRNA biomarkers of SP cells in colon cancer cells .METHODS:SP cells in colon cancer cells were sorted by flow cytometry .The cell viability was measured by MTT method .MicroRNA expression profiles were detected by mi-croRNA chip.MicroRNA expression was verified by real-time PCR.RESULTS:The ratios of SP cells in HCT-15, HT-29 and LoVo colon cancer cell lines were 16.75%, 13.02%and 9.52%, respectively.In all 3 colon cancer cell lines, IC50 of the antitumor drugs including 5-fluorouracil , oxaliplatin and adriamycin for the SP cells were significantly higher than those for non-SP cells (P<0.05).MicroRNA profiling showed that miR-5000-3p, miR-5009-3p and miR-552 were all up-regulated in the SP cells of all 3 colon cancer cell lines .This result was verified by real-time PCR.CONCLUSION:miR-5000-3p, miR-5009-3p and miR-552 are all up-regulated in the SP cells of colon cancer cell lines , and may be the poten-tial microRNA biomarkers of SP cells in colon cancer .

[ ABSTRACT] AIM:To study the process of promoting mouse embryonic stem cells ( ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells.METHODS:The cells were divided into different groups based on the culture medium:ESC group ( serum-free medium +LIF) , natural differentiation group ( serum-free medium) and activin A group ( serum-free medium +50μg/L activin A).The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation.The proportion of CXCR4 +cells was detected by flow cytometry.The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot.RE-SULTS:The proportion of CXCR4 +cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was ob-served at 5 d.Among the 3 groups, the proportion of CXCR4 +cells at 5 d was the highest in activin A group.The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry.The pro-tein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed.The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CX-CR4 were gradually increased with the highest level at 5 d.It was highest in the cells in activin A group.CONCLUSION:The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation.Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.

AIM:To investigate the effect of microRNA-429 (miR-429) on the expression of occludin (Ocln) in intestinal epithelial cells ( IECs) and intestinal epithelial barrier function in diabetic mice.METHODS:Diabetes mel-litus mouse model was induced by intraperitoneal injection of streptozocin.The expression of miR-429 in IECs of diabetic mice was inhibited by antagomiRNA-429 injected via tail vein.The expression of miR-429 and mRNA expression of Ocln were detected by real-time PCR.The protein expression of Ocln was determined by Western blotting and immunohistochem-istry.The urinary lactulose/mannitol ratio was measured by gas chromatography.The plasma LPS concentrations in the mice were measured by chromogenic end-point TAL kit.RESULTS:The results of real-time PCR confirmed that the ex-pression of miR-429 in IECs of diabetic mice was remarkably inhibited by tail-vein administration of antagomiRNA-429, and resumed to similar level of normal mice on the 6th day after the administration.After suppressing the level of miR-429, the expression of Ocln in IECs of diabetic mice increased significantly (P<0.05), while the urinary lactulose/mannitol ra-tio and the plasma LPS concentration decreased obviously ( P<0.05 ) .CONCLUSION:AntagomiRNA-429 effectively suppresses miR-429 expression in IECs of diabetic mice, and then enhances the expression of Ocln and partially resumes the intestinal epithelial barrier function.