Objective To construct the recombinant adeno-associated virus vector(rAAV) expressing the human tissue kallikrein(HK)gene and to detect the expression of interested gene in human umbilical vein endothelial cells(HUVEC)which were infected with different titers of rAAV. Methods The HK gene was cloned into the pAAV-MCS and co-transfected AAV-293 cells with other two plasmids(the pAAV-RC,and pHelper)by lipofectamine.The recombinant AAV(rAAV HK)particles was harvested and its viral titer was measured.HUVEC were infected with different titers of rAAV-HK particles.Seventy-two hours later,the expression of the interested gene was detected by RT-PCR and ELISA.Results The AAV expression system of human tissue kallikrein gene was successfully constructed.The viral titer of recombinant AAV reached 6.2?10~7 particles/ml. When compared with the control group,the transcription of HK gene in the group which was infected with rAAV-HK increased significantly[(0.59?0.12)vs(0.26?0.05)(P＜0.05)],and the expression of HK in the HUVEC was three times more than that in the control[(120.1?40.9)vs (30.8?12.8)](P＜0.001).The transcription of eNOSmRNA in the infected HUVEC was higher than that of the control[(1.19?0.28)vs(0.66?0.11)](P＜0.05).The transcription of caspase-3 mRNA was lower than that of the control[(0.30?0.25)vs(0.67?0.27)](P＜0.05).And there was no obvious variation happened in the transcription of VEGF,ET-1,TR-B,bradykinin B1 receptor and Bradykinin B2 receptor.Conclusions When co-transfected AAV-293 cells with three plasmids (pAAV-HK,pAAV-RC,pHelper),the HK gene expression is significantly and stably increased. Introducing HK gene into HUVEC can improve endothelial transfection efficiency.

OBJECTIVETo study whether hematologic malignancy patients with anemia have a lower erythropoietin (EPO) response.

METHODSSerum EPO levels were detected by ELISA in patients with hematologic malignancies and with iron deficiency anemia (IDA). Eighty patients with hematologic malignancies, including 13 multiple myeloma (MM), 7 chronic lymphocytic leukemia (CLL) and 60 non-Hodgkin's lymphoma (NHL) were studied. Thirty of them had anemia(21 NHL,6 MM and 3 CLL). Twenty patients with IDA were studied as the control.

RESULTSHematologic malignancy patients with anemia had higher EPO levels [(97.8 +/- 183.9) IU/L] than those with normal Hb values [(27.8 +/- 85.4) IU/L; P <0.01]. In patients with IDA, serum EPO response was inversely correlated with Hb level (r= -0.5, P <0.05) , but no such inverse correlation was found in the hematologic malignancy patients with anemia (r = -0.14). After corrected for Hb level, the serum EPO levels were significantly lower in anemic patients with hematologic malignancies than in IDA patients (P = 0.032) , indicating a decreased EPO response in the former group.

CONCLUSIONAnemia associated with hematologic malignancy might result from an inappropriately low EPO response. EPO treatment for these patients may be beneficial.

Adolescent ; Adult ; Aged ; Anemia, Iron-Deficiency ; blood ; complications ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; blood ; Female ; Hematologic Neoplasms ; blood ; complications ; Hemoglobins ; metabolism ; Humans ; Male ; Middle Aged ; Prospective Studies

Objective:To construct HIV-1 pseudovirus containing enhanced green fluorescent protein ( EGFP ) gene. To understand the interaction between the virus and the cells. Methods: HIV-1 pseudovirus containing EGFP gene was constructed by lentiviral packaging systems, and its EGFP gene was amplified using RT-PCR. The level of genomic integration and transcription of HIV-1 pseudovirus containing EGFP gene were detected on iDCs infected with HIV-1 pseudovirus. At the same time, research on expression of the EGFP gene in iDCs infected with HIV-1 pseudovirus was performed. Results:The EGFP gene of HIV-1 pseudovirus was detected through RT-PCR. The EGFP gene was identified in iDCs infected with HIV-1 pseudovirus through PCR and RT-PCR. The EGFP was observed in iDCs infected with HIV-1 pseudovirus under fluorescence microscopy. Conclusion: HIV-1 pseudovirus containing EGFP gene has been successfully produced. The HIV-1 pseudovirus that we constructed can infect iDCs,then its RNA can integrate into the genome of iDCs in the way of reverse transcription,and the EGFP gene could express in the iDCs after infected with HIV-1 pseudovirus.

OBJECTIVETissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene delivery on the proliferation of vascular smooth muscle cells of SHR (VSMCs(SHR)) induced by platelet derived growth factor-BB (PDGF-BB).

METHODSPrimary VSMCs(SHR) were isolated and cultured from thoracic aorta of male SHR. The VSMCs(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazolium (MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27(Kip1) and p21(Cip1). The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCs(SHR).

RESULTSProliferation of VSMCs(SHR) induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3% (cell counting, n = 3, P < 0.01), 30.2% (MTT, n = 3, P < 0.01) and 36.4% (peak stunning rate of cell-cycle in phase G(0)/G(1)). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27(Kip1) and p21(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n = 3, P < 0.001, respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCs(SHR).

CONCLUSIONThe hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCs(SHR) through Bradykinin B2 receptor and up-regulate expression of p27(Kip1) and p2l(Cip1).

Animals ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Humans ; Kallikreins ; genetics ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Rats ; Rats, Inbred SHR ; Recombination, Genetic

OBJECTIVETo investigate the effects of human tissue kallikrein 1(Ad-hKLK1) gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats (SHRs).

METHODSCarotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups: Sham-operated (n = 6); Angioplasty (phosphate buffered solution 50 microl, n = 8); Vector virus (control virus, 1 x 10(9) IU in 50 microl, n = 8) and Ad-hKLK1(Ad-hKLK1, 1 x 10(9) IU in 50 microl, n = 8). Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor (B1R and B2R) expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p2lCip1 were determined by Western blot analysis.

RESULTSWall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38.8%in Ad-hKLK1 treated SHRs compared to angioplasty group (all P < 0.001). The expression of p27Kip1 and p2lCip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats (all P < 0.001). The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats (P < 0.05) while mRNA expression of B1R was similar between the 2 groups.

CONCLUSIONhKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p2lCip1 signaling pathways in carotid arteries of SHRs after balloon injury.

Angioplasty, Balloon ; adverse effects ; Animals ; Carotid Artery, Common ; pathology ; Gene Transfer Techniques ; Humans ; Male ; Neointima ; etiology ; Rats ; Rats, Inbred SHR ; Tissue Kallikreins ; genetics

BACKGROUND: Acute liver failure (ALF) caused by viral and non-viral hepatitis is often accompanied with severe metabolic disorders, the accumulation of toxic substances and continuous release and accumulation of a large number of endogenous toxins and inflammatory mediators. The present study aimed to investigate the effects of various combined non-biological artificial liver treatments for patients with acute liver failure (ALF) complicated by multiple organ dysfunction syndrome (MODS). METHODS: Thirty-one patients with mid- or late-stage liver failure complicated by MODS (score 4) were randomly divided into three treatment groups: plasmapheresis (PE) combined with hemoperfusion (HP) and continuous venovenous hemodiafiltration (CVVHDF), PE+CVVHDF, and HP+CVVHDF, respectively. Heart rate (HR) before and after treatment, mean arterial pressure (MAP), respiratory index (PaO2/FiO2), hepatic function, platelet count, and blood coagulation were determined. RESULTS: Signifi cant improvement was observed in HR, MAP, PaO2/FiO2, total bilirubin (TBIL) and alanine aminotransferase (ALT) levels after treatment (P<0.05). TBIL and ALT decreased more signifi cantly after treatment in the PE+CVVHDF and PE+HP+CVVHDF groups (P<0.01). Prothrombin time (PT) and albumin were signifi cantly improved only in the PE+CVVHDF and PE+HP+CVVHDF groups (P<0.05). TBIL decreased more significantly in the PE+HP+CVVHDF group than in the HP+CVVHDF and PE+CVVHDF groups (P<0.05). The survival rate of the patients was 58.1％ (18/31), viral survival rate 36.4％ (4/11), and non-viral survival rate 70％ (14/20). CONCLUSION: Liver function was relatively improved after treatment, but PE+HP+CVVHDF was more efficient for the removal of toxic metabolites, especially bilirubin. The survival rate was significantly higher in the patients with non-viral liver failure than in those with viral liver failure.

Adolescent ; Adult ; Aged ; Anemia ; drug therapy ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; blood ; therapeutic use ; Female ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; complications ; Lymphoma, Non-Hodgkin ; complications ; Male ; Middle Aged ; Multiple Myeloma ; complications

OBJECTIVETo analyze possible difference in bronchial asthma between ethnic and geographic groups and explore its correlates among Uygur and Han adults in Turpan Prefecture, Xinjiang.

METHODSOne hundred and sixty-six clinically diagnosed asthmatic patients at Turpan Prefecture Hospital, Xinjiang, 86 of Uygur and 80 of Han ethnic, and 166 1:1 matched controls from ophthalmological outpatient department at the same hospital were recruited into the study. Interview with questionnaire was conducted and serum levels of eosinophilic cation protein (S-ECP), total IgE (T-IgE) and specific IgE (S-IgE) were measured for all of the participants to study related factors for asthma with univariate and multivariate conditional logistic regression analyses.

RESULTSBronchial infection (OR(U) = 5.111, 95%CI: 1.203 - 21.710; OR(H) = 2.498, 95%CI: 1.471 - 5.069), family history of asthma (OR(U) = 3.078, 95%CI: 1.812 - 5.188; OR(H) = 2.711, 95%CI: 1.010 - 6.176), personal allergy history (OR(U) = 2.083, 95%CI: 1.043 - 4.162; OR(H) = 3.998, 95%CI: 1.739 - 9.198), weather change (OR(U) = 2.218, 95%CI: 1.199 - 3.778; OR(H) = 1.733, 95%CI: 1.004 - 2.994) and positive S-IgE (OR(U) = 1.592, 95%CI: 1.018 - 2.491; OR(H) = 3.858, 95%CI: 2.246 - 8.507) correlated with asthma in patients of both Uygur and Han ethnic. Percentage of asthma attack induced by respiratory infection [59.30% (51/86)] and weather change [36.05% (31/86)] in Uygur patients was significantly higher than that in Han ethnic [42.50% (34/80) and 21.25% (17/80), respectively], but percentage of those with personal allergy history [48.75% (39/80)] and positive S-IgE [52.50% (42/80)] in Han ethnic was significantly higher than that in Uygur [32.56% (28/86) and 30.23% (26/86), respectively]. Levels of S-ECP and T-IgE in patients with moderate and severe asthma of both Uygur and Han ethnic [(S-ECP(U) = 7.95 +/- 3.98) microg/L, S-ECP(H) = (11.21 +/- 4.74) microg/L, T- IgE(U) = (72.23 +/- 45.92) kU/L, T-IgE(H) = (108.81 +/- 64.07) kU/L, respectively]were significantly higher than those in controls of the same ethnic [S- ECP(U) = (1.94 +/- 1.16) microg/L, S-ECP(H) = (2.07 +/- 1.63) microg/L, T-IgE(U) = (46.19 +/- 32.47) kU/L, T-IgE(H) = (50.97 +/- 38.51) kU/L; t values were 8.96, 10.52, 2.81, 4.97, P < 0.01], higher in Han ethnic than those in Uygur (t values were 3.01, 2.68, P < 0.01).

CONCLUSIONBronchial infection, family asthma history, personal allergy history, weather change and positive S-IgE all were important correlates of asthma in Turpan Prefecture, Xinjiang. Levels of S-ECP and T-IgE in patients with moderate and severe asthma increased during its attacks, higher in Han ethnic than those in Uygur. Genetic and environmental factors may be involved in occurrence and development of asthma.

Adult ; Asthma ; blood ; epidemiology ; ethnology ; Causality ; China ; epidemiology ; Climate ; Environmental Exposure ; Eosinophil Cationic Protein ; blood ; Female ; Humans ; Immunoglobulin E ; blood ; Logistic Models ; Male ; Pedigree ; Surveys and Questionnaires

We systematically reviewed the results of the studies on expression regulation, biological functions, and clinical prognostic significance of CASP8AP2 gene. At present, the studies showed that the expression of CASP8AP2 gene was regulated by Homeobox proteins and DNA methylation, and could be silenced by miRNA-210. This protein was involved in apoptosis mediated by FAS and TNFα, NF-κB activation mediated by TNFα, regulation of gene expression induced by glucocorticoid and mineralocorticoid receptor, comprising Cajal body and histone locus body, transcription of replication-dependent histone, 3' end processing of histone, regulation of S phase progression, in addition to functioning as coactivator of transcription factors c-Myb and p73 to activating many genes' expression. On the other hand, low expression of CASP8AP2 gene was associated with relapse in childhood ALL. The deletion of this gene was related to the poor prognosis of children with T-ALL and T lymphoblastic lymphoma. Furthermore, 3 SNPs in this gene were possibly correlated with genesis of diffuse large B cell lymphoma and childhood leukemia. In conclusions, CASP8AP2 was a multifunctional protein. It could function to regulate cell proliferation, apoptosis, and gene expression. In childhood hematological malignancies, CASP8AP2 was a promising molecular marker with prognostic significance. Some SNPs were possibly correlated with leukemo- and lymphomogenesis.
Apoptosis ; Apoptosis Regulatory Proteins ; Calcium-Binding Proteins ; DNA Methylation ; Gene Expression ; Gene Expression Regulation ; Histones ; Humans ; NF-kappa B ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Prognosis ; Recurrence

Stem cell transplantation holds a promising future for central nervous system repair. Current challenges, however, include spatially and temporally defined cell differentiation and maturation, plus the integration of transplanted neural cells into host circuits. Here we discuss the potential advantages of neuromodulation-based stem cell therapy, which can improve the viability and proliferation of stem cells, guide migration to the repair site, orchestrate the differentiation process, and promote the integration of neural circuitry for functional rehabilitation. All these advantages of neuromodulation make it one potentially valuable tool for further improving the efficiency of stem cell transplantation.