Objective To study the effect of siRNA targeting survivin and tumor necrosis factor related apoptosis-inducing ligand on the proliferation and apoptosis of hepatocellular carcinoma ( HCC) cells. Methods siRNA eukaryotic expression vector was constructed and the effects of soluble tumor necrosis factor related apoptosis-inducing ligand ( sTRAIL) on HCC cells were observed. Results The recombinant plasmid Psilence ( + )-survivin was successfully constructed. Survivin mRNA expression inhibition ratio reached 73% by RT-PCR. Observed by MTT method sTRAIL failed to inhibit the survival rate of HepG2、HepG2/Silence( - ) cells at 12 h、24 h、48 h when compared to control groups. With the survivin gene being inhibited, the survival rate of HepG2/Silence( + ) cells(0. 518?0. 017) decreased in 12 h compared to control groups (0. 741?0. 005 ) and reached the lowest level in 48 h ( P

With patients' general situation, medication use, occurrence time of adverse drug reaction/event (ADR/ADE), clinical manifestations and prognosis as reference items, a retrospective study was made for 315 cases with ADR/ADE induced by Gastrodin in Chongqing from January 2008 to June 2014, in order to analyze the characteristics of ADR/ADE and provide reference for rational clinical medication. The results showed that among the 315 cases with ADR/ADE, 143 cases (45.4%) were males and 172 cases (54.6%) were females, most of them (74.9%) were aged above 45; 60 cases (19.0%) with ADE were caused by off-label indications and 66 cases (21.0%) with ADE were caused by over dosage; ADR/ADE cases induced by intravenous drip mainly happened within 30 min (85.5%), ADR/ADE cases induced by oral administration mainly happened within 2 h (74.4%), and all of ADR/ ADE cases induced by intramuscular injection happened within 10 min. Totally 593 ADR/ADE cases were reported, which were mainly damages in gastrointestinal system, skin and its adnexa; And 61.9% of ADR/ADE cases were newly reported. It is suggested that medical workers shall learn about the regularity and characteristics of ADR/ADE induced by gastrodin, apply it in clinic with standards, pay close attention to changes of patients' situations and attach importance to the monitoring of ADR/ADE, so as to enhance the safety of medication.
Adolescent ; Adult ; Aged ; Benzyl Alcohols ; administration & dosage ; adverse effects ; Child ; Child, Preschool ; China ; epidemiology ; Drug-Related Side Effects and Adverse Reactions ; epidemiology ; Drugs, Chinese Herbal ; administration & dosage ; adverse effects ; Female ; Gastrointestinal Tract ; drug effects ; Glucosides ; administration & dosage ; adverse effects ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Skin ; drug effects ; Young Adult

To improve ethanol production in Saccharomyces cerevisiae,an integration plasmid pUPGKAT with PGK promoter(phosphoglycerate kinase promoter),adh1 gene(the coding sequences of alcohol dehydrogenaseⅠ) and CYC1 terminator(Cytochrome c transcription terminator) was constructed.Firstly,a fusion fragment composed of PGK promoter and adh1 gene was generated by over lap extension PCR and ligated into pUG6 resulting in plasmid pUPGKA.Subsequently,CYC1 termi nator was amplified from pSH65 by PCR and ligated to the SpeⅠand SacⅡrestriction site of pUPGKA.To integrate PGK-adh1-CYC1 into S.cerevisiae genome,pUPGKAT was digested by TthⅢⅠand the lin-earized plasmid was used to transform S.cerevisiae YS2-△adh2(adh2 disrupted strain) by lithium acetate method.The yeast mutant YS2-△adh2-adh1 which had the adh1 gene placed under the PGK promoter and harbored the adh2 deletion was constructed.Anaerobic fermentation showed overexpression of adh1 by PGK promoter resulted in a 8.84% higher ethanol production compared to YS2-△adh2.

Red in vivo recombination is a new kind of genetic engineering technique based on homologous recombination. In this work, plasmid pKD46 which expresses Red recombination proteins is transferred into Escherichia coli strain DH5?.The kanamycine resistant gene is generated by PCR by using primers with homology to hisDCB gene of E.coli chromosome. Thus, the hisDCB gene was replaced with kanamycine resistant gene by the plasmid recombination system, then the resistant gene was eliminated by a helper plasmid encoding the FLP recombinase. At last, a E.coli histidine auxotroph which is sensitive to kanamycine was got. The results indicate that Red in vivo recombination is a convenient method to construct auxotrophs.

Objective To discuss the effect of psychological nursing on critically ill patients in department of thoracic surgery.Methods Totals of 56 critically ill patients after thoracic surgery were randomly divided into theexperimental group (29 cases) and the control group (27 cases).The control group received conventional nursing care while the experimental group received psychological nursing in addition.SF-36 was used before and after nursing in both groups,and incidence rates of complications and medicaldisputes were investigated and compared.Results There was no statistically significant difference in SF-60 score between two groups before nursing.After nursing,the score of role physical,physical function,body pain,vitality,social function,role emotional,psychological health and total health in SF-36 scale was respectively (87.3 ± 1.2),(84.8 ±1.0),(85.2 ±0.7),(83.2 ±0.6),(85.1 ±1.2),(74.8 ±1.0),(86.4 ±0.5) and (79.2 ±l.2)in the experimental group,higher than (86.1 ± 1.1),(78.9 ± 1.2),(74.2 ± 1.1),(73.4 ± 0.9),(79.4 ± 1.4),(65.9 ± 1.4),(77.3 ± 0.9) and(73.6 ± 1.3)in the control group,and the differences were statistically significant (t =21.051,28.903,37.812,20.962,30.873,26.761,31.080,19.050,respectively; P ＜ 0.05).The incidence rate of complications including postoperative blooding,infection and heart disorder,and medical disputes was respectively 3.4％,6.9％,6.9％ and 3.4％ in the experimental group,lower than 7.4％,11.1％,14.8％ and 29.6％ in the control group,and the differences were statistically significant (x2 =2.10,2.76,3.42,4.78,respectively; P ＜ 0.05).Conclusions Psychological nursing should fit the needs of patients' conditions,and its application can reduce the incidence of complications and disputes,so as to guarantee patients' nursing quality.

Objective To investigate the correlation of peripheral blood neutrophil-to-lymphocyte ratio(NLR)and standardized serum uric acid(SUA),i.e.,the ratio of SUA to serum creatinine(SUA/Scr),with the traditional Chinese medicine(TCM)syndrome types of early diabetic kidney disease(EDKD)from the pathogenies of deficiency-dampness-heat-stasis,thus to provide an objective basis for the precise TCM treatment of EDKD patients.Methods A retrospective study was conducted.The clinical data collection was carried out in 197 EDKD patients,including 42 cases of qi and yin deficiency syndrome(qi-yin deficiency group),44 cases of qi and yin deficiency complicated with dampness syndrome(qi-yin deficiency with dampness group),53 cases of qi and yin deficiency complicated with heat syndrome(qi-yin deficiency with heat group),and 58 cases of qi and yin deficiency complicated with stasis syndrome(qi-yin deficiency with stasis group).The differences in lipid metabolism indicators,renal function indicators,NLR,and standardized SUA of EDKD patients with various TCM syndromes were compared,and then the correlation of NLR and standardized SUA with the TCM syndromes of EDKD was investigated.Results(1)There were no statistically significant differences in the gender and glycosylated hemoglobin(HbA1c)level among EDKD patients with different TCM syndromes(P＞0.05).The age of qi-yin deficiency with stasis group was older than that of qi-yin deficiency group(P＜0.05),and the duration of EDKD in qi-yin deficiency with stasis group was longer than that in the other syndrome groups(P＜0.01).The body mass index(BMI)presented an ascending order in the qi-yin deficiency group,qi-yin deficiency with stasis group,qi-yin deficiency with heat group,and qi-yin deficiency with dampness group(P＜0.05).(2)The total cholesterol(TC)level in qi-yin deficiency group was lower than that in qi-yin deficiency with dampness group,and qi-yin deficiency with stasis group(P＜0.05),the triglyceride(TG)level in qi-yin deficiency group was lower than that in qi-yin deficiency with stasis group(P＜0.05),Scr level in qi-yin deficiency with heat group was higher than that in qi-yin deficiency group and qi-yin deficiency with dampness group(P＜0.05),the estimated glomerular filtration rate(eGFR)level in qi-yin deficiency group was higher than that in qi-yin deficiency with heat group and qi-yin deficiency with stasis group(P＜0.05),the NLR level in qi-yin deficiency with heat group was higher than that in the other three syndrome groups(P＜0.05),and the level of standardized SUA in qi-yin deficiency with stasis group was lower than that in qi-yin deficiency with dampness group and qi-yin deficiency with heat group(P＜0.05).(3)The results of the non-ordinal multinomial logistic regression analysis showed that BMI and SUA were the influencing factors for qi-yin deficiency with dampness syndrome;BMI,SUA,and NLR were the influencing factors for qi-yin deficiency with heat syndrome;BMI,TC,and standardized SUA were the influencing factors for qi-yin deficiency with stasis syndrome.The differences were all statistically significant(P＜0.05 or P＜0.01).Conclusion NLR and standardized SUA can be used as the reference indicators for the differentiation of TCM syndromes of EDKD.The elevated level of NLR may accelerate the development of qi-yin deficiency with heat syndrome,and the decreased level of standardized SUA may accelerate the development of qi-yin deficiency with stasis syndrome.

OBJECTIVETo observe the expression change of signal regulatory protein alpha1 (SIRPalpha1) in autoimmune hepatitis (AIH) and approach the relationship between SIRPalpha1 and the extent of inflammation.

METHODSImmunohistochemistry is used to detect the expression of SIRPalpha1 in the paraffin section preparations of 33 AIH and 10 normal hepatic tissue.

RESULTSSIRPalpha1 is positive or weakly positive expressed in AIH. The staining is localized in the cytoplasm of Kupffer cells in the hepatic sinusoid with focal distribution. It is negative in normal hepatic tissue. In light AIH, it is negative or weakly positive expressed with a 36.4 percent of the positive rate (4/11). The positive or strong positive expression is found in the moderate AIH with an 84.2 percent of the positive rate(16/19). There is statistical significance between both light AIH, moderate AIH and severe AIH (P less than 0.001) and moderate AIH and light AIH (P less than 0.001). There is no statistical significance between both light AIH and severe AIH (P = 0.145 ) and moderate AIH and severe AIH (P = 0.084).

CONCLUSIONSAs a negative regulatory factor, the expression of SIRPalpha1 in hepatic sinusoid Kupffer cells is some associated with the extent of AIH.

Adolescent ; Adult ; Aged ; Antigens, Differentiation ; metabolism ; Cell Communication ; Child ; Female ; Hepatitis, Autoimmune ; metabolism ; pathology ; Hepatocytes ; metabolism ; pathology ; Humans ; Kupffer Cells ; metabolism ; pathology ; Male ; Middle Aged ; Receptors, Immunologic ; metabolism ; Young Adult

OBJECTIVEThe aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.

METHODSCLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells.

RESULTSQuantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.

CONCLUSIONSThe C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

ADP Ribose Transferases ; metabolism ; physiology ; Apoptosis ; Bacterial Toxins ; metabolism ; Cell Line, Tumor ; Claudin-3 ; Claudin-4 ; Claudins ; genetics ; metabolism ; Enterotoxins ; metabolism ; physiology ; Exotoxins ; metabolism ; physiology ; Female ; Humans ; Immunotoxins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Recombinant Fusion Proteins ; metabolism ; physiology ; Virulence Factors ; metabolism ; physiology

BACKGROUNDHepatocellular carcinoma tends to present at a late clinical stage with poor prognosis. Therefore, it is urgent to explore and develop a simple, rapid diagnostic method, which has high sensitivity and specificity for hepatocellular carcinoma at an early stage. In this study, the serum proteins in patients with hepatocellular carcinoma or liver cirrhosis and in normal controls were analysed. Surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF-MS) spectrometry was used to fingerprint serum protein using the protein chip technique and explore the value of the fingerprint, coupled with artificial neural network, to diagnose hepatocellular carcinoma.

METHODSOf the 106 serum samples obtained, 52 were from patients with hepatocellular carcinoma, 22 from patients with liver cirrhosis and 32 from healthy volunteers. The samples were randomly assigned into a training group (n = 70, 35 patients with hepatocellular carcinoma, 14 with liver cirrhosis, and 21 normal controls) and a testing group (n = 36, 17 patients with hepatocellular carcinoma, 8 with liver cirrhosis, and 11 normal controls). An artificial neural network was trained on data from 70 individuals in the training group to develop an artificial neural network diagnostic model and this model was tested. The 36 sera in the testing group were analysed with blind prediction by using the same flowchart and procedure of data collection. The 36 serum protein spectra were clustered with the preset clustering method and the same mass/charge (M/Z) peak values as those in the training group. Matrix transfer was performed after data were output. Then the data were input into the previously built artificial neural network model to get the prediction value. The M/Z peaks of the samples with more than 2000 M/Z were normalized with biomarker wizard of ProteinChip Software version 3.1 for noise filtering. The first threshold for noise filtering was set at 5, and the second was set at 2. The 10% was the minimum threshold for clustering. The statistical analysis of the data of serum protein mass spectrum was performed in the groups (normal vs. hepatocellular carcinoma, and liver cirrhosis vs. hepatocellular carcinoma) with the t test.

RESULTSComparison between the groups of hepatocellular carcinoma and normal control: The mass spectra from 56 samples (hepatocellular carcinoma and normal controls) in the training group were analysed and 241 peaks were obtained. In addition, 21 peaks from them were used for comparison between the groups of hepatocellular carcinoma and normal controls (P < 0.01). Only 2 peaks at 3015 M/Z and 5900 M/Z were selected with significant difference (P < 10 (-9)). A model was developed based on these two proteins with different M/Z. It was confirmed that this artificial neural network model can be used for comparison between the groups of hepatocellular carcinoma and normal controls. The sensitivity was 100% (17/17), and the specificity was 100% (11/11). Comparison between the groups of hepatocellular carcinoma and liver cirrhosis: The mass spectra from 49 samples in the training group (including patients with hepatocellular carcinoma and liver cirrhosis) were analysed and 208 peaks were obtained. In addition, 21 peaks from them were used for comparison between the groups of hepatocellular carcinoma and liver cirrhosis (P < 0.01). Only 2 peaks at 7759 M/Z, 13134 M/Z were selected with significant difference (P < 10 (-9)). A model was developed based on these two proteins with different M/Z. It was confirmed that this artificial neural network model can be used for comparison between the groups of hepatocellular carcinoma and liver cirrhosis. The sensitivity was 88.2% (15/17), and the specificity was 100% (8/8).

CONCLUSIONSThe specific biomarkers selected with the SELDI technology could be used for early diagnosis of hepatocellular carcinoma.

Adult ; Aged ; Aged, 80 and over ; Blood Proteins ; analysis ; Carcinoma, Hepatocellular ; blood ; diagnosis ; Female ; Humans ; Liver Cirrhosis ; blood ; Liver Neoplasms ; blood ; diagnosis ; Male ; Middle Aged ; Neural Networks (Computer) ; Peptide Mapping ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; alpha-Fetoproteins ; analysis

OBJECTIVETo gain an insight into large genomic deletions in mismatch repair genes MSH2 and MLH1 in Chinese hereditary nonpolyposis colorectal cancer (HNPCC) patients in order to improve genetic detections of HNPCC kindreds.

METHODSFourteen peripheral blood DNA samples were obtained from 14 unrelated HNPCC patients, and fluorescent labeled quantitative multiplex PCR was used to detect large genomic deletions in MSH2 and MLH1 genes.

RESULTSOne of the fourteen probands, a man, was found to have MSH2 exon 1-7 deletions. His cancer-distressed son was also found to have the mutations. Additionally, three normal members of the family had the same mutations.

CONCLUSIONLarge genomic deletions which mainly present to MSH2 account for 20% of general pathological sequence changes of MSH2 and MLH1 genes in Chinese HNPCC patients, and large genomic detections of mismatch repair genes should be included in the regular genetic detections of Chinese HNPCC kindreds.

Adaptor Proteins, Signal Transducing ; genetics ; Asian Continental Ancestry Group ; genetics ; China ; Colorectal Neoplasms, Hereditary Nonpolyposis ; ethnology ; genetics ; pathology ; Female ; Humans ; Male ; MutL Protein Homolog 1 ; MutS Homolog 2 Protein ; genetics ; Nuclear Proteins ; genetics ; Pedigree ; Polymerase Chain Reaction ; Sequence Deletion