OBJECTIVETo study the effects of CD133 suppression by lentivirus-mediated RNA interference (RNAi) on the proliferation and chemosensitivity of CD133(+) cancer stem cells (CSCs) sorted from HepG2 cell line.

METHODSCD133(+) and CD133- cells were sorted from HepG2 cell line by flow cytometry, and the expression of CD133 before and after cell sorting were detected. The stem cell property of sorted CD133(+) cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo. Lentivirus-mediated short hairpin RNA (shRNA) targeting CD133 were transfected into CD133(+) cells, and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting, respectively. Before and after the transfection, the proliferative ability of CD133(+) cells was evaluated by colony formation assay, and the cell growth inhibition rate and apoptosis following cisplatin exposure were detected using CCK-8 assay and flow cytometry.

RESULTSThe sorted CD133(+) cells showed a high purity of (88.74∓3.19)%, as compared with the purity of (3.36∓1.80)% before cell sorting. CD133(+) cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133- cells. CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133(+) cells (P<0.01), resulting also in a significantly lowered cell proliferative ability (P<0.01) and an increased growth inhibition rate (P<0.01) and obviously increased cell apoptosis (P<0.05) after cisplatin exposure.

CONCLUSIONLentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133(+) liver cancer stem cells and increases their chemosensitivity to cisplatin.

AC133 Antigen ; Antigens, CD ; genetics ; Cell Proliferation ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Glycoproteins ; genetics ; Hep G2 Cells ; Humans ; Lentivirus ; Neoplastic Stem Cells ; cytology ; drug effects ; Peptides ; genetics ; RNA Interference

Intracellular pH (pHi) has an important influence on the metabolic activity of cells or cellular processes. The intracellular pH (pHi) of long-chain alpha,omega-dicarboxylic acid-producing Candida tropicalis was determined by fluorescence technique using a pH-sensitive fluorescent probe 5(6)-carboxyfluorescein diacetate. Optimal loading conditions of the fluorescent probe into the cells were experimentally determined. Effects of extracellular pH and carbon sources for growth on pHi in the cell grown in a flask were studied; the results indicated that extracellular pH has a slight influence on pHi, whereas carbon sources such as sucrose, glucose, acetic acid, and n-tridecane showed moderate influences. Further work on the relationship between the cell growth activity and pHi was carried out in a 5 L bioreactor. The time course of specific rates of the cell growth, glucose consumption, CO2 production, and pH gradients across cell plasma membrane were plotted, where the cell growth was improved by the higher pHi at 8 - 12 h. The measured pHi values were varied from 5.72 to 6.15 at medium pH 6.0 in which glucose and sodium acetate were used together as carbon source. The investigation of pHi can be helpful for understanding its effects on the kinetics of the metabolic steps involved in the synthesis rate of alpha,omega-dicarboxylic acid and alpha,omega-dicarboxylic acid transport across plasma membranes.
Candida tropicalis ; growth & development ; metabolism ; Culture Media ; Culture Techniques ; methods ; Dicarboxylic Acids ; metabolism ; Hydrogen-Ion Concentration ; Microscopy, Fluorescence

To investigate the correlation between methylation and expression of multidrug resistance (mdr1) gene, restriction endonuclease HpaII combined with competitive PCR technique was used to quantitatively detect the methylation status of two CCGG sites located at -110 and -50 bp (region I and II) up to the transcription start site in mdr1 promoter in 54 AL and 9 MM patients. Semi-quantitative RT-PCR was used to detect the expression level of mdr1 gene. The results showed that inverse correlation between methylation rate of either region or total methylation rate and expression of mdr1 gene was observed. The correlation in the region I (r = -0.64) was closer than that in the region II (r = -0.4). High expression rate of mdr1 ascended significantly in low methylation group (n = 36) (P < 0.001). In comparison with chemotherapy sensitive group (n = 8), the methylation rate in refractory AL patients (n = 16) was lower (P = 0.05) in the region I, P < 0.05 in the region II and total regions. Comparing with the untreated patients (n = 36), the methylation rate in the region I and total methylation rate were lower in the patients with chemotherapy (n = 14) (P < 0.05). The methylation rate in the region II was also decreased after chemotherapy, however, no statistical significance was shown (P > 0.05). Increased mdr1 expression level accompanying with decreased methylation rate after chemotherapy was found, although no significant difference was shown (P = 0.06). It is concluded that the expression level of mdr1 gene was associated with the methylation status of CCGG in -110 and -50 bp upstream to the transcription start site, especially the -110 site. In both the patients treated with chemotherapy and the refractory patients, the methylation level of mdr1 gene decreased relatively. The rising expression of mdr1 gene after chemotherapy was associated with the decrease of methylation level.
DNA Methylation ; Genes, MDR ; Hematologic Neoplasms ; drug therapy ; genetics ; Humans ; Reverse Transcriptase Polymerase Chain Reaction

Objective To explore the feasibility of open visiting among mothers' of very-low-birth-weight(VLBW) infants and observe effects.Methods Totally 74 eligible VLBW infants were recruited,those who were visited and cared daily by mothers were assigned into the experimental group(n=36) and those who were not visited by mothers were assigned into the control group(n=38).The experimental group received open visiting as well as individualized developmental nursing care,and the control group only received individualized developmental nursing care.Relevant regulations and procedures were designed,and clinical pathways of mothers' involvement in nursing care were developed and implemented.Incidence of infection,time to reach full oral feeding,weight gaining during hospitalization,rate of breast-feeding during hospitalization and 30 days after discharge,hospitalization time and rate of readmission 30 days after discharge were recorded and compared.Results There was no significant difference in incidence of infection between two groups (P＞0.05).Time to reach full oral feeding in the experimental group was earlier than that of the control group,weight gaining during hospitalization in the experimental group was faster than that of the control group,rate of breast-feeding 30 days after discharge was higher in the experimental group,and the differences were significant (P＜0.05).Hospitalization time in the experimental group was shorter than that of the control group,rate of readmission 30 days after discharge in the experimental group was lower,and the differences were significant (P＜0.05).Conclusion Mother's open visiting for VLBW infants in NICU does not increase the incidence of infection,but can shorten time to reach full oral feeding,promote weight gaining,increase rate of breast-feeding 30 days after discharge,reduce hospitalization time,and decrease rate of readmission 30 days after discharge.

OBJECTIVETo analyze epidemiological features of severe acute respiratory syndrome (SARS) in Haidian district, Beijing.

METHODSEach SARS case was interviewed by trained investigator using standardized questionnaire followed a descriptive analysis.

RESULTSFour hundred and three SARS cases were identified and 27 of them died from March 18 and May 31, 2003. The incidence rate of SARS was 18.0/100,000 with case fatality rate as 6.7% in Haidian district, Beijing. Seventy-four percent of patients were adults with higher risk in age group of 20 - 29 year. SARS patients were scattered around in 32 out of 33 streets and villages in this district. The disease appeared to be sporadic but the case of outbreaks in family or university only seen in three streets. The course of SARS epidemic in this district could be divided into three phases: initial-which last for days, peak-21 days and then rapid decline-for 26 days. Number of patients having had a history of close contact to other SARS were gradually decreasing along with the process of the epidemics (trend chi(2) = 8.800, P = 0.003). Seventy-two point seven percent of the SARS cases had been exposed to the injection in the hospital settings. When the epidemics came to a rapid decline, 85.7% of the patients diagnosed during that period could be traced down to have had the history of contacting SARS cases within their own families. The distribution of occupation was also showed significantly different in the three respective stages (chi(2) = 36.41, P < 0.01). Among the patients who could not be identified as having confirmed contact history, 26.6% having had outward activities and 47.6% of them visited hospitals, especially during the peak stage.

CONCLUSIONThe intensity of SARS epidemic among the residents of Haidian district was recognized as similar to the other parts of Beijing. Nosocomial infection in hospital settings was most important cause responsible for the transmission of SARS in this district.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; China ; epidemiology ; Contact Tracing ; Cross Infection ; transmission ; Family Health ; Female ; Humans ; Incidence ; Male ; Middle Aged ; Severe Acute Respiratory Syndrome ; epidemiology ; mortality ; transmission ; Surveys and Questionnaires

OBJECTIVETo study the effect of polyamine biosynthesis inhibition on growth characteristics of human lung carcinoma cells and its correlation with the expression of human lung carcinoma associated antigen ALT-04ag gene.

METHODSThe gene expression was detected by RT-PCR and immunocytochemical tests. The cell growth characteristics were studied by cell growth curves, morphological observation, FCM analysis and DNA electrophoresis.

RESULTSHuman lung squamous carcinoma cells L78 treated with 5 mmol/L alpha-difluoromethylornithine (DFMO) for 5 days showed significant growth inhibition and apoptosis induction. The mRNA and protein expressions of ALT-04ag gene in the cells were downregulated, while these changes resulted from DFMO treatment were prevented by provision of DFMO along with exogenous putrescine.

CONCLUSIONThe effect of polyamine biosynthesis inhibition induced by DFMO restrains the growth characteristics and promotes apoptosis of human lung carcinoma L78 cells, which is associated with down regulation of ALT-04ag gene expression.

Antigens, Neoplasm ; biosynthesis ; genetics ; Apoptosis ; Carcinoma, Squamous Cell ; pathology ; Cell Division ; Down-Regulation ; Eflornithine ; pharmacology ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; pathology ; Oncogene Proteins ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured

Objective Little is known about the effect of RNAi on mitochondrial apoptotic pathways. This study aims to explore the effects of the Survivin shRNA-APC double-gene on colon cancer mitochondrial apoptosis pathway-related factors survivin,cytochrome C (Cytc),second mitochondria-derived activator of caspases (Smac),and cysteine aspartic acid specific protease 9 (Caspase-9) as well as on the apoptosis of colon cancer transplanted tumor (CCTT) cells. Methods Thirty nude mice were randomly divided into five groups of equal number,Survivin shRNA-APC double-gene,survivin shRNA,APC,empty vector and blank transfection. The CCTT model was established in the nude mice by subcutaneous injection of the colon cancer cell strains stably transfected with the Survivin shRNA-APC double-gene,survivin shRNA,APC,an empty vector and HT-29,respectively,into the mid-posterior part of the left armpit of the nude mice. The rate of tumor growth inhibition was calculated by measuring the volume and weight of the CCTTs in the nude mice. The mRNA and protein expressions of survivin,Cytc,Smac and Caspase-9 in the tumor tissue were detected by real time PCR and immunohistochemistry,respectively,and the apoptosis rate of the CCTT cells was detected by TUNEL. Results The model of CCTT was successfully established in the nude mice. Com-pared with the empty vector and blank transfection groups,the mice in the double-gene,survivin shRNA and APC groups showed sig-nificantly decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). In comparison with the survivin shRNA and APC groups,the double-gene group exhibited remarkably decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). The mRNA and pro-tein expressions of survivin were significantly lower while those of Cytc,Smac and Caspase-9 markedly higher in the double-gene,sur-vivin shRNA and APC groups than in the empty vector and blank transfection groups (P<0.05),the former even lower (P<0.05) and the latter even higher in the double-gene than in the survivin shRNA and APC groups (P<0.05). The apoptosis rate of the CCTT cells was significantly increased in the double-gene ([56.78±3.04]%),survivin shRNA ([33.61±2.02]%) and APC groups ([30.16± 1.72]%) as compared with the empty vector ([10.05±0.42]%) and blank transfection groups ([9.87±0.30])% (P<0.05),even higher in the double-gene group than in the survivin shRNA and APC groups (P<0.05). Conclusion The Survivin shRNA-APC double-gene may induce apoptosis of colon cancer transplanted tumor cells by down-regulating the expression of the apoptosis inhibitor survivin,upregulating the expressions of Cytc,Smac and Caspase-9,and suppressing the growth of the colon transplanted tumor,with more significant abilities than a single gene in regulating apoptosis-related factors,inducing cell apoptosis and inhibiting the growth of the transplanted tumor.

The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.
China ; Humans ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Molecular Sequence Data ; Phylogeny

OBJECTIVEProgressive pseudorheumatoid dysplasia (PPD) (MIM#208230) is a rare autosomal recessive disease of cartilage homeostasis characterized by axial and peripheral skeletal dysplasia. Analysis of WISP3 (Wnt1-inducible signaling pathway protein 3, MIM#603400) gene mutation can confirm the clinical and radiographic diagnosis for PPD. This study aimed to recognize PPD based on clinical manifestations and imaging characteristics of bones, and to investigate the mutations of WISP3 gene in three patients with PPD.

METHODThree male patients (9 - 16 years old) from three unrelated Chinese families, who presented with joint pain, swelling, deformities and motion limitation, were referred to this study. PPD was diagnosed on the basis of the clinical manifestations, imaging characteristics of bones and laboratory evaluation. All five exons and their exon/intron boundaries of the WISP3 gene were amplified by polymerase chain reaction (PCR) from the peripheral blood DNA of three PPD family members, and mutation analysis was performed by bidirectional DNA sequencing.

RESULT(1) Three patients were diagnosed as PPD by characteristic evidences: all patients presented with non-inflammatory multiple joints swelling and stiffness including joints in hand and feet as they age. Radiographs showed platyspondyly, ovoid or wedged anterior end-plate of vertebral bodies, coxa vara, widened epiphyses or metaphyses including capital femoral, metacarpophalangeal, interphalangeal joints and metatarsals. Normal laboratory values were found for the erythrocyte sedimentation rate and C-reactive protein, rheumatoid factors, antinuclear antibodies etc. (2) The three different mutations of WISP3 gene were identified in three patients with PPD, including two small insert mutations (c.624_625insA, c.866_867insA), one was deletion mutation (c.729_735delGAGAAAA). The types of mutation of two alleles in three patients were c.624_625insA/c.729_735delGAGAAAA, c.624_625insA/c.866_867insA and c.866_867 insA/c.866_867insA, respectively. These mutations were found in exon 4 and exon 5 of WISP3 gene, accounting for 50%(3/6) respectively. All three different mutations were novel variations, and none of 3 novel variations was found in the 50 controls.

CONCLUSIONThe characteristic evidences of PPD were non-inflammatory multiple enlarged joints (including hand and feet), limited movement, normal laboratory values such as rheumatoid factors. It is essential for making diagnosis to carefully examine the entire skeleton including spine. The characteristics of bone imaging are platyspondyly, widened epiphyses or metaphyses including large and small joints and narrow joint spaces. Three different novel variations of WISP3 gene were identified in three PPD patients, they are c.624_625insA, c.866_867insA and c.729_735delGAGAAAA. Each of novel mutations is insert or deletion mutation.

Adolescent ; Arthropathy, Neurogenic ; diagnosis ; genetics ; CCN Intercellular Signaling Proteins ; Child ; Humans ; INDEL Mutation ; Insulin-Like Growth Factor Binding Proteins ; genetics ; Joint Diseases ; congenital ; Male ; Molecular Sequence Data

The management of postoperative leaks into the mediastinum after esophagectomy remains a challenge. We describe our clinical management of this complication through endoscopic transluminal drainage. Between 2008 and 2011, 4 patients with esophageal squamous cell carcinoma (ESCC) who underwent McKeown-type esophagectomy with two-field lymphadenectomy experienced complicated anastomotic fistulae in the presence of superior mediastinal sepsis. All 4 patients underwent endoscopic transluminal drainage, and all survived. The mean healing period was 50 days (range, 31 to 58 days), the mean stay in the intensive care unit was 7.3 days (range, 1 to 18 days), and the mean hospital stay was 64.5 days (range, 49 to 70 days). Endoscopically guided transluminal drainage should be considered for ESCC patients with superior mediastinal fistulae after esophagectomy.
Aged ; Carcinoma, Squamous Cell ; surgery ; Drainage ; Endoscopy ; Esophageal Fistula ; etiology ; therapy ; Esophageal Neoplasms ; surgery ; Esophagectomy ; adverse effects ; Humans ; Lymph Node Excision ; Male ; Mediastinum ; Middle Aged ; Sepsis ; etiology ; therapy