Ectopically expressed perforin-1 is proapoptotic in tumor cell lines by increasing caspase-3 activity and the nuclear translocation of cytochrome C.

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell's plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.

Replication-dependent γ-H2AX formation is involved in docetaxel-induced apoptosis in NSCLC A549 cells.

Docetaxel is a member of the taxane anti-microtubule class of chemotherapeutic agents, which are currently widely used in clinical cancer therapy. However, the anti-tumor mechanisms of docetaxel are not fully understood. Herein we show that docetaxel induces dose-dependent apoptosis in non-small cell lung cancer A549 cells, as detected by Annexin-V positive cells and PARP cleavage, which is via mitochondrial pathway and dependent on caspase-3 activation.

[Comparison of the effect of arsenic sulfide on apoptosis and hTERT-mRNA expression in two leukemia cell lines].

Aim: To explore the different effect and mechanism of arsenic sulfide on telomerase activity and hTERT-mRNA expression in CML cell lines-K562 and APL cell lines-NB4.

Methods: Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). The expression of hTERT-mRNA was analyzed by semi-quantitative RT-PCR.

[Expression and localization of adenovirus-mediated transcriptional factor Runx3 in malignant glioblastoma cells].

Aim: To construct the replicative deficient adenovirus Ad-Runx3 expressing Runx3, and to express it in U251 malignant glioblastoma cells.

Methods: The runx3 gene with a flag tag was amplified by PCR using pCMV5-AML2 as a template, and was confirmed by DNA sequencing. The adenovirus shuttle vector pShuttle-CMV-Runx3 was constructed by introducing runx3 DNA fragment into the sites of Kpn I and Xho I of pShuttle-CMV vector.

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFDelta1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF.

[Experimental study of IFN-alpha and IFN-gamma on reversing ATRA-resistance in MR2 cell line].

Aim: To explore the possibility and the possible mechanism of reversing ATRA-resistance in MR2 cells by using IFN-alpha and IFN-gamma in combination with all-trans retinoic acid (ATRA).

Methods: After MR2 cells(ATRA-resistance cell line) were treated with IFN-alpha, IFN-gamma and ATRA alone or IFN-alpha and IFN-gamma in combination with ATRA respectively, the cell proliferation was tested by MTT colorimetry, the cell differentiation was tested through light microscope, by NBT test and flow cytometry (FCM). The expression of promyelocytic leukemia (PML) protein was observed by indirect immunofluorescence staining.

HSP70 gene fused with Hantavirus S segment DNA significantly enhances the DNA vaccine potency against hantaviral nucleocapsid protein in vivo.

Heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of Hantaan virus (HTNV) nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), we developed a novel chimeric HTNV S-HSP70 DNA vaccine plasmid by genetically linking HSP70 gene to the full-length HTNV S segment DNA (HTNV S DNA). C57BL/6 mice were immunized with this plasmid followed by a subsequent boost with homologous recombinant protein.

Dynamic changes of apoptosis-inducing ligands and Th1/Th2 like subpopulations in Hantaan virus-induced hemorrhagic fever with renal syndrome.

The expression of the apoptosis-inducing ligands, TNF-alpha, FasL and TRAIL on peripheral blood mononuclear cells (PBMC) and the levels of their soluble form (TNF-alpha, sFasL and sTRAIL) in plasma from 40 hemorrhagic fever with renal syndrome (HFRS) patients as well as 26 healthy blood donors were determined by flow cytometry (FCM) analysis and sandwich ELISA, respectively. The status of Th1, Th2, Tc1 and Tc2 subsets in PBMC was evaluated by intracellular cytokine staining and FCM. Compared to controls, the expression of membrane bound FasL and TRAIL was up-regulated on surface of PBMC isolated from the HFRS patients, particularly on CD8+ T lymphocytes.

[Construction of TRAIL gene eukaryotic expression vector modulated by hTERT gene core promoter and its effect on apoptosis of ovarian cancer cells].

Aim: To construct the TNF-related apoptosis inducing ligand(TRAIL) gene eukaryotic expression vector modulated by human telomerase reserse transcriptase (hTERT) gene core promoter and to study its effect on apoptosis of ovarian cancer cells.

Methods: Genomic RNA was extracted from human placenta tissues and the fragment of TRAIL was obtained by RT-PCR. The amplified gene fragment was subsequently cloned into hTERTpromoter-pIRES2-EGFP vector and CMV promoter-pIRES2-EGFP vector after sequencing.

[Effect of a hypothetical gene Af116609 on multi-drug resistance of gastric cancer cells].

Objective: To investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR.

Methods: Gene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation.

[Expression and function of CD226 on NK cells activated by Superantigens].

Aim: To study the regulation of superantigen in expression and function of CD226 on NK cells.

Methods: Double fluorescent staining and flow cytometry analysis were employed to detect the expression of CD226 on NK cells from human peripheral blood mononuclear cells (PBMC) stimulated by staphylococcus enterotoxin A (SEA) or SEB. (51)Cr release assay was performed to compare the cytotoxicities of NK cells in different activation models.

[Growth inhibitory effects of recombinant granzyme B containing different N-terminal translocating peptides].

Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N-terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II-GrBa fusion protein genes were constructed by replacing N-terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280-364/358) by recombinant PCR, and then cloned into pIND inducible expression vector.

[Effect of gestrinone on growth and apoptosis in isolated ectopic endometrium cells in vitro].

Objective: To investigate the effects of gestrinone on growth and apoptosis, as well as the expression of phosphatase and tension homologue deleted on chromosome 10 (PTEN) in isolated ectopic endometrium cells in vitro and the underlying mechanisms.

Methods: Ectopic endometrium cells were cultured and exposed to gestrinone of different doses of 0, 10(-6) and 10(-4) mol/L respectively. The inhibition of the cells during 48 hours was determined by methylthiazolyl tetrazolium (MTT) assay, and the cell growth curve was made.

[The effects of rBCG expressing Der p2 in the form of lipoprotein on murine immune response].

Aim: To investigate the effects of rBCG vaccination containing foreign antigen Der p2 in the form of lipoprotein on murine immune response.

Methods: 6 to 8 weeks old and newborn BALB/c mice were vaccined intraperitoneally with 10(6) CFU rBCG or BCG. At the same time, the control group was injected with saline.

[Regulation of soluble TRAIL and membrane TRAIL in Jurkat cells by PMA].

Aim: To investigate the regulation of soluble and membrane bound TNF-related apoptosis-inducing ligand (TRAIL) in Jurkt cells by phorbol myristic acctate (PMA), and the cytotoxicity of the two forms of TRAIL.

Methods: Jurkat cells were cultured in the presence of 40 ng/mL PMA for different time. The production of sTRAIL was determined by ELISA, and expression of mTRAIL was analyzed by indirect fluorescence staining and flow cytometry analysis.

Effects of STI571 and p27 gene clone on proliferation and apoptosis of K562 cells.

Aim: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.

Methods: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.

Expression pattern of epithelial cell adhesion molecule on normal and malignant colon tissues.

Aim: To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance.

Methods: cDNA encoding Ep-CAM extracellular domain was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from excised malignant colon tissues and inserted into a glutathione S-transferase (GST)-tagged vector. Ep-CAM-GST fusion protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with glutathione-sepharose.

[Experimental research on tyrosine-kinase inhibitor STI571 and P21WAF gene clone to treat chronic myeloid leukemia].

To explore the effect of a tyrosine-kinase inhibitor STI571 and P21(WAF) gene clone on the proliferation, cycle, apoptosis of leukemia cell line K562, P21(WAF) gene was obtained by RT-PCR, and its sequence was approved to be correct, then P21-pcDNA3.1 vector was constructed and transfected into K562 cell line. After selected with G418, P21-pcDNA3.

[Preparation and characterization of nanoemulsion].

Aim: To prepare nanoemulsion-encapsulated BSA-FITC (NEBSA-FITC), study its characteristics, and measure its uptake by dendritic cells (DCs) and peritoneal macrophages.

Methods: NEBSA-FITC was prepared by a method of interfacial polymerization.The encapsulation rate, drug-carrying capacity and stability of the nanoemulsion were determined by Sephadex-G100 chromatography.

[Apoptosis-inducing ligand TRAIL can be recruited to lipid rafts].

Aim: To investigate the relationship between tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) and cell membrane microdomain lipid rafts.

Methods: The expression of TRAIL on K562 cells was detected by indirect immunofluorescence staining and flow cytometry. The lipid rafts on K562 cells were detected with FITC-labeled cholera toxin B subunit (CTx-FITC) which bound to the GM1 glycosphingolipid in lipid rafts.

[Expression of GST-PRL-2 fusion protein in prokaryotic cells and preparation of Hen egg yolk immunoglobulin (IgY) against PRL-2].

Aim: To express phosphatase 2 of regenerating liver (PRL-2) fusion protein in E.coli and to prepare specific hen egg yolk immunoglobulin(IgY) against PRL-2.

Methods: A full-length human PRL-2 gene coding sequence was cloned into expression vectors pGEX-4T-2 and pET21a, then transformed into E.

[Combinative effects of FAP-1 antisense oligonucleotide and carboplatin on apoptosis of ovarian cancer cell SKOV3].

Background & Objective: Recent studies have shown that overexpression of Fas associated phosphatase-1 (FAP-1) can been detected in human ovarian cancer cell line SKOV3, suggesting that this overexpression may play an important role in the tumorigenesis and drug resistance of ovarian cancer. This study was designed to explore the effects of fas associated phosphatase-1 antisense oligonucleotide (FAP-1 ASODN) combined with carboplatin on the apoptosis of ovarian cancer cell SKOV3.

Methods: Antisense oligonucleotide technique was used to transfect FAP-1 ASODN into SKOV3 cells.

[Using flow cytometry to detect peripheral blood eosinophils and their related molecules].

Aim: To set up an accurate and rapid method to detect the phenotype of peripheral blood eosinophils using flow cytometry.

Methods: 10(-4) mol/L of theophylline, 10(-4) of dexamethasone and 10(-8) mol/L of rhIL-5 were added to peripheral blood sampled from normal human subjects. Anti-CD16-PE mAb and FITC-labelled mAbs to eosinophil's molecules were used to perform double labeling staining.

[The influence of TLSFJM on the proportion of Th1/Th2-like cell subsets].

Aim: To explore the influence of TLSF(JM) on the proportion of alloantigen activated Th1 and Th2-like cell subsets.

Methods: TLSF(JM) or IL-4 was added to mixed lymocyte reaction(MLR) system. The influence of TLSF(JM) on the proportion of Th1 and Th2-like cell subsets was analyzed by intracellular immunofluorescence staining and FACS.

[Experimental research using human bone marrow mesenchymal stem cells as the seed cells for bone and cartilage tissue engineering].

Aim: To investigate the feasibility of human bone marrow mesenchymal stem cells(hMSCs) as the seed cells for bone and cartilage tissue engineering.

Methods: Purified hMSCs were cultured in-vitro and induced to differentiate into osteoblasts and chondrocytes. Cellular morphologies were observed under inverted and electron microscopes.