To explore the possible linkage between telomerase and acute leukemia, we detected telomerase activity expressed in 3 leukemia cell lines, 22 acute leukemia bone marrow and 6 normal bone marrow with PCR ELISA assay. Results showed that telomerase activities of three leukemia cell lines were positive with the average (1.57 +/- 0.056) U, normal bone marrow samples average was (0.085 +/- 0.081) U, telomerase value from 22 acute leukemia patients was (0.512 +/- 0.294) U. Telomerase activity is higher expressed in acute leukemia than normal samples and decreased significantly after chemotherapy (P < 0.01). The results suggested that telomerase activity was related to some malignant diseases and might be used as a marker for tumor diagnosis and therapy.

Exosome is a nano-level vesicle composed of lipid bilayer layers.It is prevalent in plasma,urine,cerebrospinal fluid and other body fluids.The microRNAs(miRNAs) wrapped in the exosomes can be taken up by the target cellsand play their role through silencing target gene as endogenous miRNAs.Exosomes can promote information exchange between organs or cells by carrying miRNA,participating in the process of angiogenesis,cell migration and communication.In this paper,the author briefly reviewed the application of exogenous maternal miRNAs in the diagnosis and treatment of cardiovascular system diseases,central nervous system diseases and tumor in the recent years.

To evaluate the use of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for treatment of acute and chronic leukemia, from March 1997 to January 2003, 21 adult patients with malignant hematopoietic diseases underwent allo-PBSCT from HLA-identical siblings (19 patients) and haplo-identical mother (one) and one B point site mismatched sibling (one). All donors were mobilized with G-CSF for 4 days and peripheral blood stem cells were collected by CS-3000 separator. The conditioning regimen included the high dose combination chemotherapy and TBI. Cyclosporine-A (CsA) plus a short course of MTX was used for GVHD prophylaxis in all patients. The results showed that after trans plantation, median time for the recovery of granuocyte > or = 0.5 x 10(9)/L and platelets > or = 20 x 10(9)/L were 12 (10 - 20) and 15 (11 - 35) days, respectively. Acute GVHD was observed in 8/17 patients (47%), of which one transplanted from HLA-haploidentical mother. Chronic GVHD occurred in 12/17 patients (70%). All of four female survivals did not show acute and chronic GVHD. Day 100 transplantation-related mortality was 14% (3/21). Relapse occurred in two patients (9.5%) who underwent allo-PBSCT in stage of non-remission at one and six months. After follow-up of 40 (15 - 70) months, 11 patients (52.4%) are still disease-free survival. These results suggested that peripheral blood stem cells produce a faster hematopoietic recovery and a lower relapse of leukemia. The rate of aGVHD is not increased when using the peripheral blood as source of stem cells; however, cGVHD continues to be a significant problem. Donors tolerated the procurement procedure without complications.
Adolescent ; Adult ; Female ; Graft vs Host Disease ; etiology ; Humans ; Leukemia ; therapy ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Transplantation, Homologous ; Treatment Outcome

OBJECTIVETo study the prevalence of hepatitis B virus (HBV) genotype in 5 cities of Fujian province and the clinical implications of distinct genotypes in HBV-related liver diseases.

METHODSHBV genotype was determined by the restriction fragment length polymorphism analysis in patients with chronic HBV infection in 5 cities of Fujian province. The relationship between HBV genotype and its clinical implications was studied by multinomal logistic regression and correspondence analysis.

RESULTSOf the 431 HBV DNA positive patients detected by PCR, 275 (63.8%) belonged to HBV genotype B, 100 (23.2%) to genotype C, 51 (11.8%) to genotype D and D-mixed genotype. Genotype A, E and F were not found. Multinomal logistic regression showed that genotype B was more prevalent in Quanzhou and Sanming cities than in Fuzhou (P = 0.002, P = 0.006), and genotype B appeared significantly more common in asymptomatic carriers (ASC), chronic hepatitis B (CHB) and severe hepatitis (SH). Genotype C was most prevalent in patients with liver cirrhosis (LC) (47.0%) than in those with ASC (14.5%) and SH (14.7%) (P = 0.009, P < 0.001). The positive rate of hepatitis B e antigen was higher in patients with genotype C than in those with genotype B and genotype D (56.0% vs. 52.4%, P = 0.008, and 56.0% vs. 30.8%, P = 0.051, respectively). By correspondence analysis, genotype D and D-mixed genotype seemed to be correlated with hepatocellular carcinoma (HCC).

CONCLUSIONS(1) The major popular genotypes of HBV were B, C and D in Fujian. (2) Data of our study suggested that the geographic distribution of genotype B and C might be different in some cities of Fujian. (3) Genotype B might have a tendency to lead to SH in younger patients with chronic hepatitis B and the development of LC might be associated with genotype C among the elder patients. (4) Genotype D appeared to associate with development of HCC, which called for further study to confirm.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; Female ; Gene Frequency ; Genotype ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length

OBJECTIVETo investigate if CYP3A5 is involved in drug resistances mechanisms of acute leukemia.

METHODSBy using RT-PCR, immunohistochemistry and MTT assay, CYP3A5 mRNA and protein were detected in leukemia cell lines and acute leukemia patients, meanwhile transcriptional regulation of CYP3A5 induced by daunorubicin was observed. A pcDNA3-CYP3A5 reconstituted plasmid and its stably transfected cell line HL-60/CYP3A5 were both established.

RESULTSCYP3A5 mRNA was detected in K562 and U937 cells, whose IC(50) values of daunorubicin were 2.1-fold higher than those of NB4 and HL-60 cells. Bone marrow CYP3A5 positive blast cell percentage at the time of diagnosis in primary drug resistance group (17.2%) was significantly higher than that of continuous complete remission (CCR) group (0.4%) and secondary drug resistance group (5.4%). In their first complete remission of the early relapsed group, the positive rate had been 23.9% as compared with that of CCR group (1.3%). Daunorubicin increased CYP3A5 mRNA level in K562/A02 and activated its transcription in HL-60/ADR. HL-60/CYP3A5 cell was significantly resistant to daunorubicin and vincristine than HL-60 cells did (3.0 and 4.0 times, respectively).

CONCLUSIONCYP3A5 expressed in leukemia cells may cause in situ metabolization of many kinds of anticancer drugs, thus led to drug resistance.

Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; genetics ; physiology ; Daunorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Leukemia ; drug therapy ; enzymology ; RNA, Messenger ; analysis ; Tumor Cells, Cultured

Objective To explore the siRNA as a new antiviral therapy,evaluate the inhibition effect of siRNA based on vector on the HBV of HepG2.2.15 cell,and observe the side effect and toxicity of siRNA vector on cells and the off-target effect of siRNA.Methods Three pairs of siRNA duplexes targeting HBV C gene were designed as double strands,and the duplex were annealed and ligated into the p-Silencer-Cmv 4.1-hygro vector.The ligation products were used to transform JM109 cells.The clones with shRNA were obtained,and the vectors were purified.After the initial identification of the vector with agarose gel and the size of the inserted sequence got examined by native polyacrylamide gel electrophoresis,furthermore the sequencing was further carried out.The recombinant plasmids were purified with ultrapure Midipreps DNA Purification System.Then HepG2.2.15 cells were transfected with the plasmid mixed with siPort XP-1.The expression of HBsAg and HBeAg were detected by immunofluorescence and Western blot,and the HBV RNA was investigated by RT-PCR.Furthermore the real-time quantitive PCR was carried out to detect the changes of HBV DNA.In order to evaluate the toxicity of the shRNA,MTT was used to examine the growth rate and curve of cells.The ELISA was performed to detect the changes of interferon-? (IFN-?).Results The Western blot showed that the HBsAg and HBeAg protein were suppressed with (81.15?0.69)%,(88.12?0.92)% respectively by vector p-C2 on the third day of post-transfection.It had the similar result indicated by immunofluorescence.And the RT-PCR showed that the specific siRNA targeting HBV C gene could markedly suppress the expression of HBV mRNA and the HBV C gene mRNA was inhibited with 96.9%.The real-time quantitive PCR showed that the specific functional siRNA could markedly suppress HBV DNA copy with two orders of magnitude,while the siRNA vector had no effect on the growth of cell showed by MTT detection.Compared with the non-transfected group and p-NC group,the IFN-? level was almost the same with siRNA p-C1,p-C2,p-C3 groups.Conclusions The siRNA based on the expression vector can suppress the expression and replication of HBV in HepG2.2.15 cell.The inhibition effect was specific and had a certain dependency on siRNA concentration.No toxicity effect was found in the study.And the drug resistance wouldn′t happen because the silence was based on the split of gene.

Exosomes are nanoscale vesicles produced and secre-ted into extracellular fluid by all cells. They mediate cell com-munication through carrying and transferring informational car-goes ( proteins, nucleic acids, lipids and so on ) to recipient cells. In central nervous system, exosomes can be released from all cell types including neurons, neural stem cells and neuroglia cells. These exosomes shuttle nucleic acids ( miRNAs, mRNAs and so on) and play an important role in nervous system devel-opment and function as well as diseases including Alzheimer's disease and drug addiction. Furthermore, the functional effects and targeting characteristics of exosomes-shuttle-RNAs suggest that exosomes-shuttle-RNAs can be diagnostic and therapeutic targets. In this review, we elaborate the effects, functions and mechanisms of exosomes-shuttle-RNAs in order to gain a new recognition of CNS development and diseases.

Objective To detect the expression of microRNA (miR)-181 a-5 p in serum exosomes of methamphetamine-dependent rats and healthy rats,and to predict target gene of miR-181a-5p and related bioinformatics analysis.Methods The conditioned place preference (CPP) model was established by methamphetamine:male SD rats were randomly divided into two groups(ten rats in each group):normal group (0.9％ NaCl),and experimental group (methamphetamine 2 mg· kg-1).The experimental group was subcutaneously injected methamphetamine at 8 am,injected saline at 4 pm for four days.The normal group was injected subcutaneously with equal volume of normal saline.In the morning,after the experimental group(normal group)were injected subcutaneously with methamphetamine(saline),they were immediately put in the white side for one hour.In the afternoon,after rats were injected subcutaneously with saline,they were immediately put in the black side for one hour.This training was carried out continuously for 4 days.After 1 day of the last subcutaneous injection of methamphetamine (saline),the conditioned place preference was measured.The residence time and movement distance in the white side were recorded within 15 main.The expression of miR-181a-5p in rats' serum exosomes was detected by RT-qPCR technique.The miR-181a-5p target gene was predicted by Target Scan,miRanda and miRBD.The target gene was analyzed by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genomes and Genes pathways (KEGG pathways)with DAVID DataBase.The target gene protein was carried out interactive analysis by STRING online analysis software.Results After CPP training,the residence time (s)and the moving distance (cm)of normal group in the white side were 202.67 ± 28.24 and 3349.26 ± 412.28 respectively;the experimental group were 460.47 ± 66.71 and 4475.97 ± 585.74 respectively.Compared with the normal group,the residence time and the moving distance of the experimental group were significandy increased in the white side(P ＜ 0.01).The expression of miR-181a-5p in serum exosomes in the normal group and the experimental group was 3.39 ± 1.79,10.23 ± 4.32 respectively.Compared with the normal group,the expression of miR-181a-5p in the experimental group had increased significantly(P ＜0.01).The 108 miR-181a-5p target genes were predicted to be rich in biological processes such as neuronal generation and differentiation,gene expression regulation and glutamate transport,and involved in the pathogenesis of protein processing in endoplasmic reticulum.MiR-181a-5p target gene encodes a complex interaction between proteins,including RAD23B,ATXN3,PRKCD,MAP2K1,NCBP1,GABRA1 and so on.They played a core position in the protein-protein interaction (PPI)network.Conclusion miR-181 a-5p may regulate the downstream target protein and participate in the development of methamphetamine addiction by regulating gene expression,glutamate transport and participate in the pathway of protein processing in endoplasmic reticulum.

This study was purposed to analyze the changes of T-cell clonality after induction of peripheral T lymphocytes by autogenous DC and cytokines in the preparation of adoptive immunotherapy for leukemias. The bone marrow and peripheral blood from 21 leukemia patients at remission stage after treatment and subjected to adoptive immunotherapy were collected. Their DCs and T-cells were stimulated with cytokines and then were mixed to activate T-cells. T-cell receptor beta variable region (TCRBV) families were amplified by RT-PCR, and genescan method and sequencing of the PCR products were used to observe the clonality changes of T-cells before and after the induction and cultivation of T-cells. The flow cytometry was used to identify CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOXP3(+) cells to disclose the ratio change of cytotoxic T lymphocytes (CTL), helper T-cells, regulatory T-cells and NK T-cells before and after induction and cultivation of T-cells. The results showed that in the 21 patients, most of the 24 TCRBV families presented as oligoclonal distribution on genescan, several families were not expressed, and only a few families remained polyclonal. TCRBV24 was found to be oligoclonal in all of the 21 patients. DNA sequence analysis of TCRBV24 revealed a common motif of VAG in CDR3 in 3 cases and a common motif of GGG in CDR3 in 2 cases. In patient 5, both TCRBV 24 and TCRBV8 contained the same motif of GGG in CDR3. The identical motif in these patients may suggest that these T-cells recognize the same antigen. The peripheral lymphocytes demonstrated recovery of clonal profile on genescan from oligoclonal profile and absence of several families before the induction and cultivation to typical polyclonal profile in all TCRBV families after the induction by DC and cytokines for 13 days. After the induction and cultivation, the number of lymphocytes increased to 3.38 +/- 1.20 times. CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOX P3(+) cells were 71.1 +/- 11.8%, 26.7 +/- 11.4%, 35.7 +/- 12.9%, 3.1 +/- 1.6% and 0.12 +/- 0.1% respectively before the induction and cultivation, and changed to 95.4 +/- 3.2% (p < 0.01), 27.0 +/- 13.1% (p > 0.01), 55.5 +/- 13.8% (p < 0.01), 9.8 +/- 6.1% (p < 0.01) and 0.22 +/- 0.18% (p < 0.01) respectively after the induction and cultivation. It is concluded that the major action of this induction and cultivation method on T-lymphocytes in vitro is the promotion of CTL and NK T-cell proliferation. In leukemic patients at the remission stage, the TCRBV profile is characterized by the oligoclonal proliferation of T-lymphocytes. Several proliferated clones may have the same motif in CDR3, suggesting the recognition of the same antigen by these lymphocyte clones. Cytokine induction and co-culture with autogenous DCs can stimulate the T-lymphocytes to recover their immunocompetence as manifested by the polyclonal profile and the proliferation of CTL and NK-T cells.
Adolescent ; Adult ; Aged ; Child ; Female ; Genes, T-Cell Receptor beta ; Humans ; Immunotherapy, Adoptive ; Leukemia ; genetics ; immunology ; therapy ; Lymphocyte Activation ; Male ; Middle Aged ; T-Lymphocytes ; chemistry ; cytology ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; T-Lymphocytes, Regulatory ; chemistry ; immunology ; Young Adult