In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Doxycycline ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo investigate the expression of miR-9 in B lymphocytes, B cell lymphoma and classical Hodgkin's lymphoma (cHL) cell lines and its significance.

METHODSCD19(+) B lymphocytes were sorted from normal lymph node by magnetic beads. Total cellular micro-RNA was extracted from cHL cell line L428, B cell lymphoma cell lines Ly1 and Ly10 (diffuse large B cell lymphoma), Raji cells (Burkitt's lymphoma) and CD19(+) B lymphocytes, respectively. These micro-RNAs were separately transformed into cDNA by reverse transcription. The expression levels of miR-9 were measured by fluorescence quantitative PCR. In situ hybridization was used to detect the expression of miR-9 in cell lines.

RESULTSThe expression of miR-9 was high in L428 cells (104.44 ± 1.61), and low in cell lines of B cell lymphoma (Ly1: 2.17 ± 0.38; Ly10: 1 ± 0.015; Raji: 2.65 ± 0.89), and extremely low in CD19(+) B lymphocytes (0.0026 ± 0.00040). Compared with that in the other cell lines, the expression of miR-9 in L428 cells was statistically significant (P < 0.05). miR-9 localized in the cytoplasm diffusely and strongly in L428, but scattered and slightly with some prominent distribution around the nuclear membranes in Ly1 and Ly10, and only weakly in Raji.

CONCLUSIONSmiR-9 highly expressed in cHL cell line and might be a molecular marker for diagnosis and treatment of cHL.

B-Lymphocytes ; metabolism ; Cell Line, Tumor ; Cell Lineage ; Hodgkin Disease ; metabolism ; pathology ; Humans ; Lymphoma, B-Cell ; metabolism ; pathology ; MicroRNAs ; metabolism

OBJECTIVETo study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.

METHODSThe cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.

RESULTSThe plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.

CONCLUSIONThe antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.

Animals ; Central Nervous System ; embryology ; Cloning, Molecular ; Digoxigenin ; chemistry ; Gene Expression Regulation, Developmental ; Oligonucleotide Probes ; RNA Probes ; Uridine Triphosphate ; chemistry ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

OBJECTIVETo investigate the effects of murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of yolk sac hematopoietic progenitors.

METHODSThe serum-free mBMEC-CM was obtained from subcultures of murine endothelial cell line derived from bone marrow which was established in our laboratory. The murine yolk sacs were harvested on day 8.5 postcoitus (pc) and incubated with 0.1% collagenase in 10% fetal calf serum at 37 degrees C for 40 minutes. Yolk sac cells were incubated in tissue culture dishes at 37 degrees C for 1 hour. Nonadherent cells were collected for semisolid culture assay of granulocyte-macrophage colony forming unit (CFU-GM) and high proliferative potential-colony forming cell (HPP-CFC) after being cultured in DMEM with 10% mBMEC-CM and 10% FBS for 24 hours. The number of CFU-GM and HPP-CFC was counted at day 7 and day 14 respectively.

RESULTSThe growth of CFU-GM and HPP-CFC was supported by mBMEC-CM with GM-CSF. mBMEC-CM could induce the proliferation and differentiation of yolk sac hematopoietic stem cells and progenitors in liquid culture system. The percentages of CFU-GM and HPP-CFC compared with the 0 hour control were (119.5 +/- 5.7)% and (130.8 +/- 9.8)% respectively after 24 hours liquid culture (P < 0.05). The expansion effects of mBMEC-CM on CFU-GM and HPP-CFC were enhanced by compounded with flt3 ligand (FL) and thrombopoietin (TPO). The percentages of CFU-GM and HPP-CFC compared with the 0 hour control were (132.0 +/- 6.2)% and (176.9 +/- 12.8)% respectively after 24 hours liquid culture (P < 0.01).

CONCLUSIONMurine bone marrow endothelial cell conditioned medium could support the growth and proliferation of yolk sac hematopoitic stem cells and progenitors, and this promoting effect was further enhanced by addition of FL and TPO.

Animals ; Bone Marrow Cells ; cytology ; Cell Division ; Cells, Cultured ; Culture Media, Serum-Free ; Endothelium ; cytology ; Female ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Male ; Mice ; Yolk Sac ; cytology

OBJECTIVETo construct a eukaryotic expression vector of CD99 gene for transfection into Hodgkin lymphoma L428 cells.

METHODSThe full-length cDNA of CD99 gene was amplified from Jurkat cells by RT-PCR and cloned into the pcDNA3.1(+) vector and transfected into L428 cell line using Lipofextamine 2000. The sequence of CD99 mRNA in the transfected cells was confirmed by restriction endonuclease digestion and DNA sequencing, and the expression of CD99 protein was identified using immunocytochemistry.

RESULTSA gene fragment of 558 bp was amplified from the transfected cells and the sequence was verified by DNA sequencing. Immunocytochemistry identified the presence of CD99 expression in the transfected cells.

CONCLUSIONA eukaryotic expression vector pcDNA3.1(+)-CD99 is successfully constructed and stably expressed in L428 cell line.

12E7 Antigen ; Antigens, CD ; biosynthesis ; genetics ; Base Sequence ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cell Line, Tumor ; Cloning, Molecular ; Genetic Vectors ; genetics ; Hodgkin Disease ; metabolism ; pathology ; Humans ; Jurkat Cells ; Molecular Sequence Data ; Transfection

This study was to investigate the antiviral effects of a hot water soluble extract S-03 isolated from Isatis indigotica root on different subtypes of influenza A and B viruses in MDCK cell cultures, using plaque reduction, immunofluorescence and hemo-agglutination inhibition (HAD) assays. Chemical analysis of the extract S-03 showed that it contained high proportion of polysaccharides. The antiviral effects in vitro showed that the S-03 had no effect on different influenza viruses if the drug was used before virus adsorption, but S-03 showed obvious activities against influenza viruses if treatment after virus adsorption or direct reaction of drug and virus before virus adsorption. Hemagglutination inhibition assay showed that S-03 inhibited HA activities of different human influenza viruses (inhibition concentration ranged from 3.12 to 25 mg/mL), avain influenza viruses (inhibition concentration ranged from 25 to 50 mg/mL). The antiviral effects of S-03 on different influenza A and B viruses in vitro might be through the inhibition of the HA to prevent infection.
Animals ; Cells, Cultured ; Dogs ; Fluorescent Antibody Technique ; Hemagglutination Inhibition Tests ; Influenza A virus ; drug effects ; Influenza B virus ; drug effects ; Isatis ; chemistry ; Plant Extracts ; pharmacology ; Plant Roots

A retrospective study was performed to explore the relationship between molecular subtypes and clinicopathological features of breast cancer in Chinese women. Six hundred and twenty-eight Chinese women with breast cancer were classified into four molecular subtypes according to their estrogen receptor (ER), progesterone receptor (PR) and Her-2 status. The prevalence rate of each molecular subtype was analyzed. Relationship between the subtypes and clinicopathologic features was determined. The distribution of molecular subtypes was as follows: luminal A 46.5%, luminal B 17.0%, basal 21.5%, HER2/neu 15.0%. The subtypes had no significant difference under different menopausal status. However, in the age-specific groups, the age group of ≤35 years was more likely to get basal cell-like cancer (36.9%). Statistically significant differences were found among molecular subtypes by age, nuclear grade, tumor size, lymph node (LN) metastasis, tumor stage by American Joint Committee on Cancer (AJCC), radiotherapy but not by chemotherapy, types of surgery. After adjusting for several relative confounding factors, the basal subtype more likely had lower nodal involvement in both the incidence of LN metastasis (≥1 positive LN) and incidence of high-volume LN metastasis (≥4 positive LN). The HER2/neu subtype had higher nodal involvement in the incidence of high-volume LN metastases. After adjusting for relative confounding factors, the HER2/neu subtype more likely had higher AJCC tumor stages. It was suggested that there existed close relationship between molecular subtypes and clinicopathological features of breast cancer. In addition, the breast cancer subtypes have been proven to be an independent predictor of LN involvement and AJCC tumor stage. These findings are very important for understanding the occurrence, development, prognosis and treatment of breast cancer in Chinese population.
Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; classification ; epidemiology ; metabolism ; pathology ; China ; epidemiology ; Female ; Humans ; Middle Aged ; Molecular Diagnostic Techniques ; statistics & numerical data ; Prevalence ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

Objective To explore the possibility of rat adipose-derived stem cells (ADSCs) to differentiate into oligodendrocyte precursor cells(OPCs) and find an effective way to treat demyelinating disease. Methods ADSCs from the inguinal region of SD rats were isolated, digested with collagenase type I and trypsin, collagenase type I digestion method as control, counted and compared; Cultured in vitro and observed the growth characteristics. After ADSCs subcultured 3 times of passages, CD29, CD90 and CD45 were detected by flow cytometry; After differentiation into adipocyte, the cells were identified by the staining of oil red 0; After differentiation into OPCs by stem cell differentiation medium and OPCs induced differentiation medium, the expression of a-N-acetylneuraminic acid a-2, 8-sialyltransferase I (A2B5) and NG2 was detected by immunofluorescent staining. Results The number of ADSCs in the combined enzyme group was higher than the collagenase type 1 group (P < 0 . 05, re = 7); ADSCs grew in a long shuttle type and their morphology tended to be stable after passage. The surface marker CD29, CD90 were positive, and CD45 was negative. After adipogenic induction, oil red 0 staining showed red lipid droplets of varying sizes in the cells. After OPCs induction, immunofluorescence detection showed that positive reaction of cell surface fluorescence was seen with antibody to A2B5 and NG2,(87. 03±0. 94)% expressed A2B5, (90. 07±0. 96) % expressed NG2. After cultured for 3 days, immunof'luorescence detection showed that positive reaction of cell surface fluorescence was seen with antibody to myelin basic protein (MBP). Conclusion ADSCs are obtained by combined enzyme digestion and the cells are much more than collagenase alone and can be induced to OPCs in vitro.

BACKGROUNDChromosomal abnormalities have been shown to play an important prognostic role in multiple myeloma (MM). Interphase fluorescence in situ hybridization (i-FISH) has been much more effective to identify cytogenetic aberrations in MM than conventional cytogenetic technique (CC). To clearly determine the cytogenetic features of Chinese MM patients and identify their prognostic implications, we designed a multicenter study based on i-FISH including 672 patients from 52 hospitals in China.

METHODSAll 672 patients were systematically screened for the following genomic aberrations: del(13q), IgH rearrangement, del(p53) and 1q21 amplifications.

RESULTSThe analysis showed that the chromosomal changes were detected in 22.1% patients by CC and in 82.3% patients by i-FISH. The most common abnormalities by CC were chromosome 1 aberrations (48.4%), -13/13q- (37.6%), hyperdiploidy (36.6%), hypodiploidy (30.1%) and IgH rearrangements (23.7%). The most frequent abnormalities by FISH was del(13q), which was found in 60.4% patients, whereas IgH rearrangement, 1q21 amplification and p53 deletions were detected in 57.6%, 49.0% and 34.7% cases, respectively. By statistical analysis, -13/13q- by CC was associated with low level of platelet (P = 0.015), hyperdiploidy was associated with low level of serum albumin (P = 0.028), and IgH rearrangement by FISH was associated with high level of β2 microglobulin (P = 0.019). Moreover, 1q21 amplification and del(p53) by FISH conferred a high incidence of progressive disease (PD) after initial therapy. Metaphase detection of IgH rearrangements and chromosome 1 aberrations concurrently was associated with a short progression free survival (PFS) (P = 0.036). No significant prognostic implications of other cytogenetic abnormalities were found associated with overall survival and PFS.

CONCLUSIONSChinese MM patients had similar cytogenetic abnormalities compared with the previous reported studies. However, the prognostic significance of FISH aberrations were not clearly determined and further study is required.

Adult ; China ; Chromosome Aberrations ; Chromosomes, Human, Pair 1 ; genetics ; Cytogenetic Analysis ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Male ; Middle Aged ; Multiple Myeloma ; genetics ; pathology

Adult ; China ; Cross-Sectional Studies ; Epidemiologic Methods ; Epidemiology ; education ; Female ; Humans ; Male