[Potential Oncogenic Role of Genes in PLC Family on T-ALL Cell Lines by Suppression of Apoptosis].

OBJECTIVE: To explore the role of phospholipase C(PLC) family in the progression of acute T lymphoblastic leukemia (T-ALL). METHODS: The apoptosis of T-ALL cells was determined by Annexin V-PE/7-AAD staining after treatment of PLC inhibitor U73122 and Edelfosine. Cox regression and Kaplan-Meier were used to analyze the impact of PLC expressions on the event-free survival (EFS) of T-ALL patients. PLC expression in each subtype of T-ALL were analyzed by One-way ANOVA. The siRNA expression plasmids targeting the PLCß1, PLCγ1, PLCη1 gene were constructed, and T-ALL cells were infected with retrovirus packaging in HEK-293T cells. The mRNA and protein level were tested by RT-PCR and Western blot. RESULTS: P12-ICH and CCRF-CEM cell line were sensitive to U73122 and Edelfosine treatment, while Jurkat and MOLT4 were resistant to them. In the TARGET-ALL database, the prognosis of T-ALL patients with high expression of PLCß1, PLCγ1 and PLCη1 was poor, and PLCß1, PLCγ1, PLCη1 were unevenly distributed in T-ALL subtypes. PLCß1, PLCγ1 and PLCη1 maintained the survival of P12-ICH and CCRF-CEM cell lines, respectively, while they had no effect on the survival of MOLT4. CONCLUSION: PLCß1, PLCγ1 and PLCη1 can maintain the growth of T-ALL cell lines in vitro and promote the malignant progression of T-ALL, which are potential therapeutic targets.

[The Effect of KRAS on Proliferation and Apoptosis of T-ALL Cell Lines].

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.KRASG12D) 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.

[Application of Flow Cytometry Combined Fluorescence in Situ Hybridization to Indentify the Lymphocyte Subtypies with Epstein-Barr Virus Infection].

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.

Carbapenem-Resistant Klebsiella aerogenes Clinical Isolates from a Teaching Hospital in Southwestern China: Detailed Molecular Epidemiology, Resistance Determinants, Risk Factors and Clinical Outcomes.

PURPOSE: Little is known about the epidemiology and carbapenem-resistance determinants of carbapenem-resistant K. aerogenes (CRKA) isolated from a single medical center. The present study was initiated to characterize the molecular epidemiology and the carbapenem-resistance mechanisms of CRKA isolated during 2012-2018 from a teaching hospital in southwest China, and to investigate the risk factors and clinical outcomes of CRKA infections as well. METHODS: Pulsed-field gel electrophoresis (PFGE) was employed for epidemiological analysis. PCR amplification and DNA sequencing were used to examine the antibiotic-resistance determinants. Plasmids were extracted and characterized by PCR-based replicon typing and conjugation assays. In order to further investigate the risk factors and clinical outcomes of CRKA infections, a retrospective case-control study was also performed. RESULTS: PFGE analysis showed 32 different PFGE patterns among the 36 non-duplicated CRKA strains collected. Most of the isolates harbored multi-drug resistance (MDR) genes, including 2 (5.6%) carrying bla NDM-1, 1 (2.8%) harboring bla KPC-2, 13 (36.1%) carrying ESBL genes, 23 (63.9%) carrying ampC genes, 34 (94.4%) carrying quinolone resistance determinants (QRD) genes and 9 (25%) carrying aminoglycoside resistance determinants (ARD) genes. The outer membrane porins, OmpE35 and OmpE36, were, respectively, lost in 4 and 2 isolates. The efflux pump inhibition experiments were positive in 25 (69.4%) of the CRKA strains. Multivariate analysis indicated that hypo-albuminaemia, invasive procedures, and carbapenem exposure were independent risk factors for acquiring CRKA infections. CONCLUSION: No clonality relationship was identified among most of the 36 CRKA isolates. The over-expression of ESBLs and AmpC coupled with the efflux pumps contributed to carbapenem resistance in K. aerogenes. Additionally, this is the first report of CRKA isolate co-harboring bla NDM-1, bla CTX-M-15, bla EBC, bla ACC, acc (6')-Ib, armA, qnrD and loss of OmpE36 in China. Hypo-albuminaemia, invasive procedures and carbapenem exposure were associated with acquisition of CRKA infections.

[Clinical effect of modified Xuefuzhuyutang on senile chronic subdural hematoma after operation].

OBJECTIVE: To investigate the curative effect of modified Xuefuzhuyutang on chronic subdural hematoma (CSDH)after burr holes irrigation and drainage. METHODS: From January 2010 to April 2013,137 CSDH patients were randomly divided into two groups: 65 cases of control group and 72 cases of medicine group (modified Xuefuzhuyutang). RESULTS: Compared with the control group, the cases of total absorption of hematoma in medicine group increased significantly (P < 0.05). The cases of 50%-99%, 30%-49% and 0%-29% absorption in above two groups had no significant differences (P > 0.05). There were no significant side effects were observed in the two groups. Compared with the control group, the marked effective cases and total effective cases in medicine group were higher (P < 0.05). CONCLUSION: Modified Xuefuzhuyutang is effective in reducing the postoperative residual volume and recurrent CS-DH.