OBJECTIVE: To investigate the function of RAS protein on the progression of the T-ALL cell lines in vitro. METHODS: The DNA of the T-ALL cells was purified then amplified the coding regions of three RAS genes (KRAS, NRAS, HRAS) by PCR reaction. After T-A cloning, the coding regions of KRAS, NRAS and HRAS were sequenced by Sanger Sequencing. The siRNA oligonucleotides were cloned into the pSEH-361 vector, which were then packaged into retroviral together with pAMPHO and pVSVG in the HEK-293T cells. The T-ALL cells were infected with the retrovirus. The gene expressions were detected by qRT-PCR and Western blot. The T-ALL cells were stained with Annexin V-PE/7-AAD and the apoptotic cells were detected by flow cytometry. The T-ALL cells were stained with Hoechst 33258, and the cell cycle distribution was determined by flow cytometry. The expression of cleaved-Caspase 3 was stained with antibody and observed with fluorescence microscope. RESULTS: For RAS genes, beside the Loucy and the P12-ICH cells harbored KRAS c.6187G>A (p.KRAS^G12D) homozygous mutant, no missense mutation of RAS was found in other T-ALL cells genome. The pan RAS inhibitor compound 3144 showed toxicity to all tested T-ALL cells, except PEER (IC₅₀=47.916 μmol/L). Similarly, Tipifarnib induced apoptosis of multiple T-ALL cell lines except for the PEER cells (IC₅₀=94.2265 μmol/L). After KRAS knock-down, the T-ALL cells showed significant apoptosis and an arrested cell cycle. CONCLUSION The KRAS protein is vital for the progression of the T-ALL cells in vitro, it is a potential therapeutic target for T-ALL patients.
Apoptosis ; Cell Line ; Cell Proliferation ; Humans ; Mutation ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Proto-Oncogene Proteins p21(ras)/genetics*

OBJECTIVE: To establish the technique that take the advantages of flow cytometry combined fluorescence in situ hybridization (Flow-FISH) to identify the Epstein-Barr virus(EBV) infected lymphocyte subtypies in patients' peripheral blood sample. METHODS: Peripheral Blood monocyte from 9 patients with EBV infection enrolled at Children's Hospital in Chongqing Medical University were isolated by Ficoll-paque centrifugal separation. The expressions of EBER1, EBER2 in cell were detected by qRT-PCR. The surface markers of cell were detected by Flow cytometry after staining with their antibodies. The cell was treated Fix-Permeabilization Buffer before hybridization with fluorescent labeled probe at 37 ℃ overnight. The cell status, surface markers and targeted mRNA are detected by flow cytometry and fluorescence microscope. RESULTS: It was optimized that the Fix-Permeabilization Buffer and recipe with 0.2% Tween-20 were picked out as providing a good cell integrity and high resolution of surface markers. Hybridization with 20% formamide and 7% dextran sulfate at 37 ℃ overnight is the optimal hybridization condition as a good hybridization effect, a detectable cell integrity and a high resolution of cell markers under flow cytometry detection. Finally, upon the established Flow-FISH method, lymphocyte subpopulations of the EBV⁺ cells from cell lines and blood samples of patients were identified successfully. CONCLUSION A Flow-FISH technology is established, which can be applied in the identification of EBV infected cell subtypes. This research provides a foundmental for its application in clinical test in EBV⁺ related proliferative diseases.
Epstein-Barr Virus Infections ; Flow Cytometry/methods* ; Herpesvirus 4, Human ; Humans ; In Situ Hybridization, Fluorescence/methods* ; Lymphocyte Subsets

OBJECTIVE: To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province. METHODS: The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017. RESULTS: The clinical data of 1 442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (r=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (r=0.196, P<0.01). CONCLUSIONS Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.
Anti-Bacterial Agents ; Birth Weight ; Humans ; Infant ; Infant, Extremely Low Birth Weight ; Infant, Newborn ; Intensive Care Units, Neonatal ; Surveys and Questionnaires

We review the representatives literatures on chronic osteomyelitis, sum up the new insights in recent years into diagnostic options and treatment regimens, analyze the advantages and disadvantages of various diagnostic approaches and treatment strategies, and propose areas of interest to make current diagnostic and treatment strategies more specific.

Objective To observe the expression of silent information regulator 1(SIRT1)in esophageal squamous cell carcinoma,and its relationship with invasion and metastasis of esophageal squamous cell carcinoma and its effect on survival and prognosis.Methods The clinical and pathological data of 104 patients with esophageal cancer who underwent radical esophagectomy at the Affiliated Tumor Hospital of Xinjiang Medical University from January to December 2009 were retrospectively reviewed. Immunohistochemical method (EnVision method) was used to detect the expression of SIRT1 in cancer tissues and adjacent tissues. The clinicopathological features of different SIRT1 expressions were compared. The factors affecting the prognosis of the patients with esophageal cancer were analyzed.Results The positive expression rate of SIRT1 protein was significantly higher in esophageal squamous cell carcinoma tissues than that in adjacent tissues(61.54% vs. 29.81%, χ2=21.100, P<0.05). The expression of SIRT1 in esophageal squamous cell carcinoma was significantly correlated with vascular infiltration(VI),infiltration depth(pT),lymph node metastasis(pN)and clinical stage(pTNM)(P<0.05).Univariate survival analysis showed that the overall survival time(OS)was significantly lower in patients with SIRT1 positive expression than that of patients with negative expression(Log-rank χ2=10.065,P<0.05).SIRT1 positive expression, lymph node metastasis,deep tumor infiltration,late clinical stage and vascular infiltration showed poor prognosis(P<0.05). Cox multivariate regression analysis showed that SIRT1 positive expression, lymph node metastasis, depth of invasion and clinical stage were independent prognostic factors for esophageal squamous cell carcinoma. The prognosis of patients with deep tumor invasion, lymph node metastasis, late clinical stage and high expression of SIRT1 was worse. Conclusion SIRT1 is highly expressed in esophageal squamous cell carcinoma, and which is closely related to the invasion and metastasis of esophageal squamous cell carcinoma and has an effect on the prognosis of patients.

BACKGROUND: Inducing factors are currently used as a main method for the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocytes. OBJECTIVE: To investigate the collaborative stimulation of transforming growth factor beta1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) to induce the directed differentiation of BMSCs to chondrocytes, and to explore the best inductive effect. METHODS: Rat BMSCs were isolated, cultured and purified using adherent culture. Then, different inducing factors were added in the induction medium: TGF-β1+IGF-1 group, TGF-β1 group, IGF-1 group, and control group without growth factors. Immunofluorescence was carried out at 21 days of induction. The expression of collagen type Ⅱ was evaluated by immuncytochemical staining at 7, 14 and 21 days of induction. RESULTS AND CONCLUSION: (1) Immunofluorescence detection of the TGF-β1+IGF-1 and TGF-β1 groups showed highly expressed collagen type Ⅱ (brown red-stained cytoplasm), while negatively expressed collagen type Ⅱ in the other two groups. (2) Findings from the immuncytochemical staining showed that the expression of collagen type Ⅱ was stronger in the TGF-β1+IGF-1 group than the TGF-β1 group (P < 0.01), and the expression was gradually enhanced with time. Meanwhile, there was also no expression of collagen type Ⅱ in the IGF-1 and control groups. To conclude, the combination of TGF-β1 and IGF-1 can achieve the better inductive effect on the chondrogenic differentiation of BMSCs in vitro.

Objective: To explore the effect of the AXL expression on the chemosensitivity of prostate cancer PC-3 and DU145 cells to docetaxel and possible mechanisms. METHODS: Using Western blot, we examined the expressions of the AXL protein, p-AXL and Gas6 in the docetaxel-resistant PC-3 (PC-3-DR) and DU145 (DU145-DR) cells stimulated with gradually increased concentrations of docetaxel. We transfected the PC-3 and DU145 cells with negative NC ShRNA and AXL-ShRNA, respectively, which were confirmed to be effective, detected the proliferation, apoptosis and cycle distribution of the cells by CCK8, MTT and flow cytometry after treated with the AXL-inhibitor MP470 and/or docetaxel, and determined the expression of the ABCB1 protein in the PC-3-DR and DU145-DR cells after intervention with the AXL-inhibitor R428 and/or docetaxel. RESULTS: The expression of the AXL protein in the PC-3 and DU145 cells was significantly increased after docetaxel treatment (P <0.05). The expressions AXL and p-AXL were remarkably higher (P <0.05) while that of Gas6 markedly lower (P <0.05) in the PC-3 and DU145 than in the PC-3-DR and DU145-DR cells. The inhibitory effect of docetaxel on the proliferation and its enhancing effect on the apoptosis of the PC-3 and DU145 cells were significantly decreased at 48 hours after AXL transfection (P <0.05). MP470 obviously suppressed the growth and promoted the apoptosis of the PC-3-DR and DU145-DR cells, with a higher percentage of the cells in the G2/M phase when combined with docetaxel than used alone (P <0.05). R428 markedly reduced the expression of ABCB1 in the PC-3-DR and DU145-DR cells, even more significantly in combination with docetaxel than used alone (P <0.05). CONCLUSIONS The elevated expression of AXL enhances the docetaxel-resistance of PC-3 and DU145 prostate cancer cells and AXL intervention improves their chemosensitivity to docetaxel, which may be associated with the increased cell apoptosis in the G2/M phase and decreased expression of ABCB1.
ATP Binding Cassette Transporter, Subfamily B, Member 1 ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Count ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Docetaxel ; Drug Resistance, Neoplasm ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Male ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Proto-Oncogene Proteins ; drug effects ; genetics ; metabolism ; Pyrimidines ; pharmacology ; RNA, Small Interfering ; Receptor Protein-Tyrosine Kinases ; drug effects ; genetics ; metabolism ; Taxoids ; pharmacology

This article studied the chemical constituents from the aerial part of Vitis thunbergii var. taiwaniana. The 60% ethanol extract was eluted with 95% ethanol though HP-20 macroporous adsorption resin column. 12 compounds, including (1) betulinic acid, (2)2, 2, 2'-bis (4-hydroxyphenyl) propane bis (2, 3-epoxypropyl) ether, (3) eriodictyol, (4) trans-ε-viniferin, (5) (+)-cis-ε-viniferin, (6) kobophenol A, (7) ampelopsin A, (8) nepalensinol B, (9) cis-miyabenol C, (10) cis-vitisin B, (11) cis-gnetin H and (12) (+)-hopeaphenol, were separated by using normal phase silica gel, ODS, Sephdadex LH-20 column chromatographies and semi-preparative or preparative HPLC. Compounds 2, 5, 6, 8, 9, 10, 11 were separated from the genus Vitis for the first time and compounds 3, 7, 12 were separated from Vitis thunbergii var. taiwaniana for the first time. At a concentration of 50 μmol · L(-1), compound 6, 7 and 11 showed strong cytotoxicity against MCF-7 cell lines with the inhibition rate of 66.58%, 57.16%, 52.84%, respectively.
Antineoplastic Agents, Phytogenic ; pharmacology ; Humans ; MCF-7 Cells ; Plant Extracts ; analysis ; pharmacology ; Vitis ; chemistry

OBJECTIVETo investigate the feasibility, effectiveness and practicability of transurethral enucleation plus pneumocystostomy rotary cut (TUE + PCRC) for large benign prostatic hyperplasia (BPH).

METHODSWe performed TUE + PCRC for 26 BPH patients aged 62 - 85 years with the prostate volume of 80 - 165 ml. We conducted transurethral enucleation of the hyperplastic prostate glands and pushed them into the bladder, followed by bladder puncture for pneumo-cystostomy rotary cut.

RESULTSAll the surgical procedures were successfully accomplished, with the mean surgical time of 41 (32 - 54) minutes and intraoperative blood loss < 60 ml in all the cases. Twenty-three of the patients were followed up for 2 - 8 months, which revealed no stricture of the urethra or any other severe complications. Compared with the preoperative baseline, significant improvement was achieved in the IPSS (6.5 +/- 2.2 vs 26.2 +/- 2.4), QOL (1.4 +/- 0.9 vs 4.6 +/- 1.2) and Qmax ([5.8 +/- 1.0 ] vs [19.6 +/- 2.8] ml/s) of the patients after surgery (P < 0.01).

CONCLUSIONTUE + PCRC, with its advantages of short operation time and less severe complications, is a safe and effective approach to the management of large BPH.

OBJECTIVETo investigate the efficacy of surgical management for pyloric stenosis induced by gastrointestinal chemical burn in children.

METHODSClinical data of 11 children with pyloric stenosis induced by gastrointestinal chemical burn were analyzed retrospectively. After the failure of medicine, intervention of low balloon expansion and stent placement, they underwent pylorectomy and gastroduodenostomy. The body weight, height, serum albumin, hemoglobin, transferrin were compared between 1 day before and 3 months after operation.

RESULTSThere were 10 males and 1 female with a mean age of 4.5 years old. The main cause of serious pyloric stenosis was the wrong intake of hydrochloric acid. Lesions involved the esophagus and stomach in the early stage, and 4 weeks later, the lesion mainly involved the pylorus, which resulted in scarring pyloric stenosis and complete pyloric obstruction. Pylorectomy and gastroduodenostomy was successfully performed. The mean operative time was (134±26) min. The estimated blood loss was (5±2) ml. The postoperative length of stay was (10±3) d. There were no surgical complications. During the follow-up of 3 months, all the patients resumed regular diet. The height, body weight, and intelligence appeared to be normal. They showed significant improvement in weight, serum albumin, globulin, hemoglobin, transferrin at 3 months after the surgery(P<0.05). Six months after surgery, the anastomosis was shown to be nornal in barium follow through exam with no signs of stricture of ulcer.

CONCLUSIONPylorectomy and gastroduodenostomy is an effective management for pyloric stenosis induced by gastrointestinal chemical burn in children, whose short-term efficacy is good.