A multicenter comparison study on the quantitative detection of bcr-abl (P210) transcript levels in China / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the comparability of bcr-abl (P210) transcript levels detected in different hospitals.</p><p><b>METHODS</b>Ten hospitals in China took part in the four times of sample exchange and comparisons from April, 2010 to August, 2011. The exchange samples were prepared by Peking University People's Hospital. Firstly, the BCR-ABL (P210)(+) cells from a newly diagnosed chronic myeloid leukemia patient were 10-fold serially diluted by BCR-ABL (P210)(-) cells and they covered 4 magnitudes. Then, TRIzol reagents were thoroughly mixed with cells in each tube. Every 12 samples (three samples per magnitude) were sent to the other 9 hospitals. The cell number of each sample was 8×10(6). The detection of bcr-abl transcript levels by real-time quantitative PCR were performed in every hospital according to their own protocols. Conversion factors (CF) were calculated using regression equation.</p><p><b>RESULTS</b>Differences in bcr-abl transcript levels did exist among results of 10 hospitals in each comparison. In general, the results of the most of hospitals were in line with the dilutions of cells. CF of every hospital fluctuated. Three hospitals had relatively stable CF, and their ranges were 2.8 - 5.2, 1.2 - 2.8 and 2.2 - 6.8, respectively; two hospitals had unstable CF with ranges 0.76 - 7.0 and 2.1 - 18.7; three hospitals couldn't be calculated CF one or two times because of the significant deviation of the results from the actually bcr-abl transcript levels, and their ranges of CF which could be calculated were 1.9 - 19.2, 3.6 - 7.6 and 0.18 - 14.7; One hospital only had two CF (3.3 and 5.0) because of the replacement of an important reagent during the period of comparisons.</p><p><b>CONCLUSIONS</b>Comparability of bcr-abl (P210) transcript levels between different hospitals could be achieved through CF which acquired by sample exchange and comparison. The stable and reliable detection system is the premise to acquire correct CF.</p>

Effect of interleukin 21 and/or interleukin 12 on the antitumor activity of peripheral blood mononuclear cells in patients with endometrial cancer / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the role of interleukin (IL) 21 alone, IL12 alone, and IL21 plus IL12 for inducing the antitumor activity of peripheral blood mononuclear cells (PBMCs) in patients with endometrial cancer.</p><p><b>METHODS</b>PBMCs were isolated from peripheral blood in patients with endometrial cancer in vitro, and kept the culture with low-level IL2. IL2-stimulated PBMCs were cocultured under different conditions (with anti-IL21 antibody, IL21 alone, IL12 alone, or IL21 plus IL12) for 72 h. The cytotoxicity of PBMCs was then examined by lactate dehydrogenase(LDH) released assay. CD4(+) CD25(+) FOXP3(+) regulatory (Treg) cell and CD4(+) IL17A(+) T-helper (Th17) cell proportion were determined with flow cytometry. Cell proliferation and apoptosis were measured by cell counting kit-8(CCK-8)assay and flow cytometry, respectively.</p><p><b>RESULTS</b>In comparison to control group, both IL21 and IL12 significantly enhanced the cytotoxicity of PBMCs. The IL21 plus IL12 group had superior effect to IL21 alone and IL12 alone. IL21 and IL12 significantly decreased the percentages of Treg cells and the rate of PBMCs apoptosis. IL21 or IL12 had no significant effect on the differentiation of Th17 cells and the proliferation of PBMCs.</p><p><b>CONCLUSIONS</b>IL21 and IL12 can enhance the cytotoxicity of PBMCs in patients with endometrial cancer, which can be further strengthened with treatment of IL21 plus IL12. Such effects may be achieved by inhibiting the differentiation of Treg cells and the apoptosis of PBMCs, but not by the differentiation of Th17 cell.</p>

Effect of CD44 gene silence on multi-drug resistance reversal and biologic activity in K562/A02 cells / 中国实验血液学杂志

This study was aimed to investigate the effect of CD44 gene silence on the drug resistance and biologic activity of human multidrug resistant leukemia cell line K562/A02. The oligonucleotides of CD44 gene were designed according to related data of GenBank, double-stranded DNA was produced by annealing, and was inserted into pGCsilencerU6/Neo/GFP vector. The resultant recombinant plasmid pGCsiRNA-CD44 was transfected into K562/A02 cell line. Expressions of CD44, mdr-1 and blc-2 mRNA were assayed by real time RT-PCR. The 50% inhibitory concentration (IC50) of doxorubicin (ADM) for K562/A02 cell line was determined by MTT method. Cell cycle was determined by flow cytometry. The morphology of apoptotic cells was examined by Hochst 33258 staining. The results indicated that the siRNA eukaryotic plasmid directing at CD44 gene could effectively silence the CD44 gene of K562/A02 cells; as compared with control group, the CD44 expression in K562/A02 cells transfected with 4 pGCsiRNA-CD44 plasmids was obviously inhibited, while the inhibition of CD44 expression in cells transfected with siCD44-1 was strongest. After being transfected with pGCsiRNA-CD44, the expression of CD44 mRNA in K562/A02 cells reduced by 64.1% (p<0.05), at the same time the expression of mdr-1 and bcl-2 mRNA in pGCsiRNA-CD44-transfected K562/A02 cells reduced by 25.6% and 50.8% respectively. IC50 of K562/A02 cells after transfection decreased to (8.77+/-1.63) microg/ml and was obviously lower than that of control (17.97+/-1.61) microg/ml (p<0.01). After transfection for 48 hours, the ratio of K562/A02 cells in G0/G1 increased by 10.7%, and the cells displayed karyopyknosis, nuclear margination and apoptotic bodies. It is concluded that the siRNA plasmid specifically targeting CD44 gene can remarkably down-regulate the expression of CD44 gene, inhibit K562/A02 cell proliferation, induce its apoptosis and effectively reverse the multidrug resistance of K562/A02 cells.

Chromosomal aberrations in chronic lymphocytic leukemia by interphase fluorescence in situ hybridization and their association with clinical features / 中国实验血液学杂志

This study was aimed to investigate the common chromosomal aberrations in chronic lymphocytic leukemia (CLL) and to explore the relationship between these chromosomal aberrations and clinical features of CLL. Sequence-specific DNA probes (D13S25, RB1, p53, ATM) and one centromeric probe CSP12 were applied to detect del(13q14), del(17p13), del(11q22-q23) and trisomy 12 by using interphase fluorescence in situ hybridization (I-FISH). 9 CLL patients with negative conventional cytogenetics or without mitotic figure were enrolled in this study. The threshold was established using 10 controls without hematopoietic malignancies. The results indicated that compared with the established threshold, all of the 9 CLL patients showed cytogenetic abnormalities. The detection using p53 and D13S25 showed positive in 7 cases, positive was observed in 5 cases by using ATM and in 4 cases by using both RB1 and CSP12. There was significant correlation between the ATM and the hemoglobin level of the patients. In addition, the elevated probability of gaining bulky lymphadenopathy was found in ATM positive patients. It is concluded that the I-FISH is a more rapid and sensitive technique for analysis of chromosome aberrations in CLL. A large series study with long-term follow-up is needed to reveal the role of cytogenetic abnormalities in the determination of CLL prognosis.

Synergistic effect of DNA methylation inhibitor and histone deacetylase inhibitor on RASSF1A gene expression in U266 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of DNA methylation in combination with histone deacetylase inhibitor on transcription regulation of Ras associated domain family gene 1(RASSF1A) tumor suppressor gene and the molecular biological behaviors in U266 cells.</p><p><b>METHODS</b>The U266 cells were treated with different doses of 5-Aza-2'-deoxycytidine (5-Aza-CdR) and Valproate (VPA) each alone or in combination. Methylation-specific PCR (MSP) was used to detect CpG island methylation in RASSF1A promoter. Quantitative real-time reverse transcription polymerase chain reaction (RQ-PCR) was used to examine the expression of RASSF1A gene in U266 cells. MTT was used for cell proliferation. Cell apoptosis and cell cycle were analyzed by flow cytometry.</p><p><b>RESULTS</b>The methylation of RASSF1A gene promoter was detected in U266 cells, while there was little RASSF1A gene expressing in the control group. The demethylation effect could be detected in the 5-Aza-CdR treated and combined treatment groups but no in the VPA group. The expression level of RASSF1A was induced by 5-Aza-CdR in a concentration-dependent manner while VPA had no such effect. The expression level of RASSF1A mRNA was increased significantly in the combined treatment group. Higher growth inhibition and apoptosis effects were found in 5-Aza-CdR and VPA combination group than that in 5-Aza-CdR or VPA alone group (P < 0.05). After treatment with 5-Aza-CdR or VPA alone for 72 h, more cells were arrested in G(0)/G(1) phase as conpared with control group (P < 0.05), and even more cells were so arrested in combined treatment group (P < 0.05).</p><p><b>CONCLUSION</b>DNA methylation and histone deacetylase inhibitor can synergistically induce demethylation of the RASSF1A gene, re-express RASSF1A gene silenced in U266 cells, inhibit the proliferation of U266 cells and induce cell apoptosis.</p>

Study of a humanized anti-platelet GP VI single chain Fv phage antibody / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the anti-platelet GPVI single chain Fv phage antibody which can inhibit the aggregation function of platelet by using phage antibody library technology.</p><p><b>METHODS</b>ITP patients with anti-platelet GPVI autoantibody that could inhibit the aggregation function of platelet were screened by MAIPA assay and platelet aggregation test. The gene fragments of heavy chain and light chain variable region (VH and VL) of immunoglobulin were amplified by RT-PCR from peripheral blood lymphocytes mRNA of the screened patients. The VH and and VL fragments were linked through a DNA linker encoding the peptide (Gly4Ser)3 to construct single chain Fv (ScFv) gene. The ScFv gene was digested with SfiI/NotI restriction enzymes and cloned into the pHEN2 phage display vector, then electrically transformed to E. coli TG1. The TG1 containing ScFv-pHEN2 was rescued by helper phage M13K07 to produce ScFv phage antibody. The anti-platelet GPVI phage ScFv antibody was enriched and purified. The effect of the phage antibody on platelet aggregation function was studied.</p><p><b>RESULTS</b>Of 806 chronic ITP patients, 11 (1.36%) were positive for anti-platelet GPVI autoantibody and 2 (0.24%) patients'plasma significantly inhibited the collagen induced platelet aggregation. The length of VH and VL fragments was about 380 to 400 bp, and were successfully formed ScFv fragments of about 800 bp by DNA linker. After cloning ScFv to phagemid vector pHEN2 and transforming ScFv-pHEN2 to TG1, 4.1x10(7) clones were obtained. After M13K07 rescue, 2.62x10(10) cfu/ml ScFv phage antibodies were produced. The purified anti-platelet GPVI ScFv phage antibody inhibited the collagen induced platelet aggregation.</p><p><b>CONCLUSION</b>Anti-platelet GPVI ScFv phage antibody produced by phage antibody library technology can inhibit the aggregation function of platelet.</p>

The role of interleukin-18 and interleukin-18 receptor in predominant Th1 immune response of patients with immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the role of interleukin (IL)-18 and IL-18 receptor (IL-18R) in the predominant Th1 type cytokine response in patients with immune thrombocytopenia (ITP).</p><p><b>METHODS</b>Fifteen patients with active phase ITP, eighteen in remission and thirteen healthy controls were enrolled in this study. T-bet and GATA-3 mRNA levels in peripheral blood mononucleated cells (PBMNC) were measured by reverse transcriptase polymerase chain reaction (RT-PCR); the plasma IL-18 level by enzyme linked immunosorbent assay (ELISA), the expression of IL-18R on CD3(+) lymphocytes and total lymphocytes by flow cytometry(FCM).</p><p><b>RESULTS</b>The T-bet mRNA levels in patients with active phase ITP was 3.572 fold as much as that in the controls (P < 0.05), while the GATA-3 mRNA levels were 0.378 fold of that in controls (P < 0.05). The levels of plasma IL-18 and IL-18R on CD3(+) lymphocytes were significantly increased in active phase ITP than in remission phase and controls. There was no difference in ratio of T-bet/GATA-3 between remitted ITP and controls and so was for T-bet mRNA, GATA-3 mRNA, plasma IL-18 and IL-18R on CD3(+) lymphocytes.</p><p><b>CONCLUSION</b>ITP as a disease of Th1-dominant response there is an unbalance between T-bet and GATA-3 in its active phase; IL-18 and IL-18R being upregulated.</p>

N-ras and fms gene mutation in idiopathic thrombocytopenic purpura and myelodysplasia / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the pathogenesis of idiopathic thrombocytopenic purpura (ITP) and improve the differential diagnosis from myelodysplastic syndromes (MDS).</p><p><b>METHODS</b>Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was performed to detect the point mutation of codon 12,13 in N-ras gene and codon 301, 969 in fms gene in adult and aged ITP and MDS patients.</p><p><b>RESULTS</b>In 25 ITP patients, N-ras mutation and fms mutation were detected in one each (4%). Mutations were found in 3 of 8 MDS patients: two (25%) with N-ras mutation and one (12.5%) with fms mutation.</p><p><b>CONCLUSIONS</b>Patients with N-ras or fms gene mutation diagnosed as MDS rather than ITP.</p>

Notch signaling in human breast cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the role of Notch signaling in human breast cancers, the expression of Notch1 and its ligand JAG1 in human breast cancers and their relationships with clinical stages of breast cancers were analyzed.</p><p><b>METHODS</b>RT-PCR was used to detect the expression of Notch1 and JAG1 in 62 breast cancer specimens and 22 normal breast tissues at the margin of tumor sections, and the statistical difference of expression rates and standardized coefficient between the two groups were analyzed. To compare the expression intensity of Notch1 and JAG1 at different development stages of the illness and at different stages with or without axillary node metastasis.</p><p><b>RESULTS</b>The expression rate and standardized coefficient of Notch1 in human breast cancers were significantly higher than those of normal breast tissues at the margin of tumor sections. The expression rate of JAG1 in human breast cancers was 15%, while JAG1 was not detected in normal breast tissues at the margin of tumor sections. The standardized coefficient of Notch1 in cases with axillary node metastasis was significantly higher than that in cases without axillary node metastasis. The standardized coefficient of Notch1 at stage I was significantly lower than that at stage II, and stage II was significantly higher than stage III. There was no statistically significant difference between stage I and stage III.</p><p><b>CONCLUSION</b>Notch1 and JAG1 are highly expressed in human breast cancers, indicating that the aberrant expression and activation of Notch1 may be related with tumorigenesis of human breast cancer. Notch1 may play different roles at different developmentl stages of human breast cancer.</p>

Killing effect of double suicide genes mediated by retroviral vector on k562 cells / 中国实验血液学杂志

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.

Reversal of multi-drug resistance in K562/A02 cells by two short hairpin RNAs (shRNA) of mdr1 / 中华血液学杂志

<p><b>OBJECTIVE</b>To construct two recombinant plasmids of mdr1 and mcl1 shRNA, and to investigate their reversal effect on drug resistance in K562 adriamycin resistant cell lines (K562/A02).</p><p><b>METHODS</b>Two oligonucleotides of mdr1 and mcl1 gene were designed referring to that of GenBank, double-stranded DNA was derived through annealing, and cloned into pRNAT vector digested by two restricted endoenzymes. K562/A02 cells were transfected with the recombinant plasmids. The mdr1 mRNA expression and its protein product P-glycoprotein (P-gp) were detected by RT-PCR and flow cytometry. The expression of mcl1 gene was detected by RT-PCR. 50% inhibition concentration (IC50) of adriamycin (ADM) on K562/A02 cells was determined by MTT method. Cells apoptosis was analyzed by flow cytometry.</p><p><b>RESULTS</b>Comparing with K562/A02 cells, the shRNA of mdrl or mcl1 gene in vitro can remarkably increase the sensitivity of K562/A02 to adriamycin, down-regulate mdr1 or mcl1 gene expression, increase the K562/A02 cells apoptosis rates induced by adriamycin. Cotransfection of mdrl and mcl1 genes shRNA can also down-regulate the expression of their gene, more remarkably increase the sensitivity and apoptosis of K562/ A02 to adriamycin.</p><p><b>CONCLUSION</b>Transfection of mdrl or mcl1 gene shRNA can promote the sensitivity of K562/A02 to adriamycin and cotransfection of the two shRNA can more remarkably do so. The mel1 gene might be involved in adriamycin resistant in K562/A02 cells.</p>

Experimental study on immunotherapy with dendritic cell in leukemic mice model / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the feasibility and efficiency of immunotherapy with dendritic cell (DC) in leukemic mice model after allogeneic bone marrow transplantation (allo-BMT).</p><p><b>METHODS</b>Mature DC were expanded from mice bone marrow mononuclear cells (MNC) by adding mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) and interleukin-4 (mIL-4). Three days later they were pulsed with frozen thawing L7212 leukemia-related antigen. Mice bearing leukemia received allo-BMT at d 0, and then were divided into control group (A), T cells group (B) and DC + T cells group (C) to receive respective immune therapy at d 14. The survival rate, survival time, occurrence of graft-versus-host disease (GVHD), cytotoxicity of spleen cells and serum cytokine level were observed. The survivors in each group were rechallenged with L7212 cells to observe the immune response to the leukemia.</p><p><b>RESULTS</b>Mature DC were successfully induced from bone marrow MNC. In groups B and C, the relapse rates were 30% and 0%, while the long term survival rates after BMT was 30% and 70% respectively. Both of the differences were statistically significant (P < 0.05). However, the incidence of GVHD in these two groups were similar. The mean survival times were (32.95 +/- 13.29) days and (41.15 +/- 13.88) days, respectively (P < 0.01). MTT assay indicated that spleen cells from group C had specific killing activity to L7212 cells. Enzyme-labeled immunosorbent assay (ELISA) showed that the serum IL-2 level in group C was (419.75 +/- 26.66) pg/ml, being significantly higher than that in the other two groups (P < 0.01). When the survivors were rechallenged with L7212 cells, there was difference between the survival rates of groups C and B (85.7% vs 33.3%, P < 0.05).</p><p><b>CONCLUSION</b>Immunotherapy with leukemia related antigen-pulsed DC in combination with donor lymphocyte infusions is an effective approach to reinforce GVL effect and decrease relapse after allo-BMT.</p>

The immunoreactivity of IgG and its fragments from ITP patients and their effects on platelet aggregation function / 中华血液学杂志

<p><b>OBJECTIVE</b>To prepare ITP plasma IgG and its F(ab')2 fragments and investigate their immunoreactivity to platelet GPIIb/IIIa and/or GPIb/IX and their effects on platelet aggregation function.</p><p><b>METHODS</b>The ITP patients having inhibitory autoantibody to the platelet aggregation were selected by modified MAIPA and platelet aggregation test with turbidimetry. Plasma IgG and its F(ab')2 fragments were prepared by streptococcal protein A affinity column and pepsin digestion. The immunoreactivity and the effects on platelet aggregation function of the whole antibody and its fragments were detected by modified MAIPA and platelet aggregation test, respectively.</p><p><b>RESULTS</b>(1) Anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were detected in 34 of 68 (53.6%) ITP patients' plasmas and that from 5 patients significantly inhibited the platelet aggregation induced by ADP or ristocetin. (2) By using protein A column combined with protease digestion, pure IgG and its F(ab')2 fragments were successfully obtained. (3) The purified IgG and its F(ab')2 fragments retained the ability to bind to their respective glycoproteins and inhibited the platelet aggregation function, whereas the IgG depleted plasma lost the ability of binding to the platelet GPs.</p><p><b>CONCLUSIONS</b>F(ab')2 fragment of the IgG antibody is a functional fragment, which not only has the binding ability to the platelet GPs but also inhibits the platelet aggregation function in a dose-dependent manner.</p>

Spatial genetic structure of two HIV-I-resistant polymorphisms (CCR2-64 I and SDF1-3'A) alleles in population of Shandong Province, China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the spatial genetic structure of two HIV-I-resistant polymorphisms (CCR2-64 I and SDF1-3'A) alleles in the population of Shandong Province, China.</p><p><b>METHODS</b>Using the techniques of spatial stratified sampling and spatial statistics, the spatial genetic structure of the locus (CCR2-64 I and SDF1-3'A), which was shown to be important co-receptor for HIV infection, was quantified from the populations of 36 sampled counties of Shandong Province, and a total of 3147 and 3172 samples were taken for testing CCR2-64I and SDF1-3'A respectively from individuals without known history of HIV-I infection and AIDS symptoms.</p><p><b>RESULTS</b>There were significantly spatial genetic structures of the two alleles at different spatial distance classes on the scale of populations, but on the scale of individuals, no spatial structure was found in either the whole area of Shandong Province or the area of each sampled county. Although the change of frequencies of the two alleles with geographic locations in Shandong Province both showed gradual increase trends, their changing directions were inverse. The frequency of CCR2-64I allele gradually increased from the southwest to the northeast, while the frequency of SDF1-3'A allele gradually increased from the northeast to the southwest. However the RH to AIDS of combined types of their different genotypes did not represent obvious geographic diversity on the whole area of the Province.</p><p><b>CONCLUSION</b>The frequency of allele usually has some spatial genetic structures or spatial autocorrelation with different spatial distance classes, but the genotypes of individuals have random distribution in the same geographic area. Evaluating spatial distribution of the genetic susceptibility of HIV (AIDS) to CCR2-64I and SDF1-3'A alleles, should focus on the frequencies of combined genotypes of CCR2 and SDF1 based on the two-locus genotypes of each individual rather than the frequencies of CCR2-64I and SDF1-3'A alleles.</p>

Inhibition of multidrug resistance related P-gp expression in human neuroblastoma by antisense peptide nucleic acid / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the efficiency of a peptide nucleic acid (PNA) delivery system by using liposome via PNA-DNA hybrids and to test the inhibitive action of antisense PNA on expression of multidrug resistance (MDR) related P-glycoprotein (P-gp) in human neuroblastoma cell line SK-N-SH.</p><p><b>METHODS</b>Two antisense PNAs were designed targeting at MDR-1 mRNA and then combined with partially complement DNAs respectively. The hybrids were delivered into cells using cationic liposome. The transfection efficiency, expression of P-gp and MDR-1 mRNA, intracellular adarimycin (ADM) were measured by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), and high performance liquid chromatography (HPLC).</p><p><b>RESULTS</b>Transfection of PNA increased the cell average fluorescence intensity significantly and the extent of increase was dependent on the concentration of PNA. After being transfected by both PNAs, P-gp expression of SK-N-SH cells decreased significantly and the intracellular ADM level was increased by about 3 times. The level of MDR-1 mRNA expression slightly decreased after transfection, but no statistical significance was observed.</p><p><b>CONCLUSIONS</b>PNA can be delivered into tumor cells in form of PNA-DNA hybrids by cationic liposome. Properly designed antisense PNA can inhibit MDR related P-gp expression of SK-N-SH cells efficiently and specifically.</p>

Function of Delta4 gene and its effects on 32D cell differentiation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Notch activation leads to transcriptional suppression of lineage-specific genes, inhibiting differentiation in response to inductive signals. The Notch signal system contains three parts: Notch molecules, Notch ligands and effectors. Delta4 is a newly-discovered Notch ligand which has received the attention of few detailed studies. This study sought to explore the biological function of Delta4 and observe its effects on 32D cell differentiation.</p><p><b>METHODS</b>Delta4-expressing vector pTracer.CMV.Delta4.FLAG was constructed using molecular biological techniques. CHO cells stably transfected with pTracer.CMV.Delta4.FLAG were confirmed to have a Delta4 protein band via Western blotting. High-expression Delta4-CHO clones were selected for the following functional studies. Notch1-CHO and Notch2-CHO were used as host cells. After transiently transfecting with transition protein 1 (TP1), Delta4 activity was compared in both cell lines by means of luciferase analysis. CHO cells were incubated with Notch1-32D cells that had been transfected with Notch1 and were observed for granulocyte colony-stimulating factor (G-CSF)-induced differentiation. Jagged2-CHO and Delta4-CHO cells transfected with the Notch ligands Jagged2 and Delta4, respectively, were incubated with Notch1-32D cells to observed inhibition of Notch on G-CSF-induced differentiation.</p><p><b>RESULTS</b>The vector pTracer.CMV.Delta4.FLAG was constructed successfully. CHO cells were stably transfected with the vector pTracer.CMV.Delta4.FLAG. Two CHO cell lines expressing Delta4 at high levels were selected for use in the study. Delta4 was found to induce signal activity via both Notch1 and Notch2 and the induction of signaling activity was stronger in Notch2 cells than in Notch1 cells. Compared with other Notch ligands, Delta4 was slightly weaker than Jagged2, but stronger than Delta1 and Jagged1 in terms of Notch1 ligands. In terms of Notch2, Delta4 had a strong signaling activity, but was weaker than Delta1, Jagged1, and Jagged2. Jagged2 could inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 could not.</p><p><b>CONCLUSIONS</b>Delta4 induces both Notch1 and Notch2 activity and is a ligand for both of them. The effect of Delta4 is stronger on Notch2 than that on Notch1. Jagged2 can inhibit Notch1-32D cell differentiation induced by G-CSF, but Delta4 cannot.</p>

Application of pseudotyped lentivirus-mediated double suicide genes to allogeneic bone marrow transplantation in mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the feasibility and efficiency of cytosine deaminase (CD)/thymidine kinase (TK) gene-modified donor T cells used in allogeneic bone marrow transplantation (allo-BMT) as an approach to mitigate GVHD without compromising engraftment.</p><p><b>METHODS</b>The pseudotyped lentivirus vectors containing CD and TK double suicide genes were transfected with lipofectine to donor T cells. Lethally irradiated 615 leukemia mice were transplanted with BALB/c bone marrow plus CD(+)TK(+)T cells. GVHD prophylaxis was by administration of ganciclovir (GCV) and 5-Fluoride cytosine (5-FC).</p><p><b>RESULTS</b>The pseudotyped lentivirus-mediated gene transfer system could efficiently transfer CD and TK double suicide genes into donor T cells. Administration of GCV and 5-FC to the mice could markedly potentiate the CFU-S and CFU-GM yields and raise the number of peripheral white blood cells. 1 x 10(7) CD(+)TK(+) allogeneic T cells caused GVHD of a similar magnitude and time course to that of fresh, naive T cells after allo-BMT. Administration of GCV and 5-FC in mice received CD(+)TK(+)T cells reduced the severity of GVHD and resulted in significantly longer survival as compared with non-administration mice, and the effect was stronger than that of administration of GCV or 5-FC alone.</p><p><b>CONCLUSION</b>Administration of CD + TK gene-modified donor T cells to recipient in allo-BMT might be an approach to mitigate GVHD without compromising alloengraftment.</p>

Establishment of lentivirus-mediated system of double suicide genes and its killing effects on K562 cells / 中国实验血液学杂志

To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus-mediated gene transfer system could transfer CD and TK double suicide genes into K562 cells with high efficiency and it had strong killing effects when giving 5-FC and/or GCV. The cytotoxic effects of double suicide genes were superior to that of single suicide gene. The lentivirus-mediated double suicide gene transfer system is a high-efficiency gene transfer vector.

Treatment of ovarian cancer cell line Skov3 with HSV-tk/GCV under the control of human telomerase reverse transcriptase gene promoter / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the in vitro effect of HSV-tk/GCV using a hTERT promoter-driven vector system on Skov3 ovarian cancer cells.</p><p><b>METHODS</b>An expression vector (pBTdel-279-tk) containing tk gene under the hTERT promoter was constructed by molecular biological methods, and then was transfected into Skov3 ovarian cancer cells, normal ovarian epithelial cells (NOEC) and human embryonic lung fibroblast by cationic liposome. Following the transfection with tk, GCV was added, and MTT and flow cytometry methods were applied to investigate its antitumor effect. RT-PCR was used to detect the tk gene in ovarian cancer cells and normal cells after the transfection of pcDNA3-tk or pBTdel-279-tk.</p><p><b>RESULTS</b>pBTdel-279-tk/GCV system induced apoptosis in hTERT-positive ovarian cancer cells, but not in hTERT-negative normal ovarian epithelial cells and fibroblasts. The hTERT promoter system was almost as efficient in inducing cancer cell death as the CMV promoter. tk gene was expressed in Skov3 cells and NOEC after pcDNA3-tk transfection, while positive was only in ovarian cancer cells after pBTdel-279-tk transfection.</p><p><b>CONCLUSION</b>The telomerasespecific transfer of the tk gene under the hTERT promoter is a novel targeting approach for the treatment of ovarian cancer and may lead to an effective and specific gene therapy.</p>