Objective To investigate the apoptosis of T lymphocytes,the expression of Bcl-2, Fas on the peripheral blood T lymphocytes in end stage renal disease patients;and to explore the characteristics of Th1 /Th2 profile and the influence of dialysis membranes with different permeability on the apoptosis of T lymphocytes of maintenance hemodialysis patients.Methods The study included 10 non-dialyszed (ND)patients,45 maintenance hemodialysis patients with cellulose acetate (CA) membranes(13),low-flux polusulfone(PS-LF) membranes(16),high-flux polusulfone (PS-HF) membranes (16) and 8 healthy volunteers (C).The apoptosis of T lymphocytes,expression of Bcl-2,Fas on peripheral blood T lymphocytes cultured with phytohemagglutinin (PHA) stimulation for 24 hours were measured by flow cytometry and immunohistochemical.ELISA was performed for detecting the levels of IFN-?and IL-4 in culture supematants.Results In ESRD patients,the apoptosis of T lymphocytes was greater than that of group C.Group CA was greater than group PS-HF and group PS-LF (P＜0.05).The expression of Bcl-2 on T lymphocytes in ESRD patients was lower than that of group C (P＜0.05).There was negative correlation between the T lymphocytes apoptosis and Bcl-2. The expression of Fas on T lymphocytes in ESRD patients was greater than that of group C (P＜0.05), and it was positive correlated with T lymphocytes apoptosis.The level of IFN-?of ESRD patients was decreased significantly compared with that in group C (P＜0.05),and there was negative correlation between T lymphocytes apoptosis and IFN-?.IL-4 was increased in ESRD patients (P＜0.05) and it was positive correlated with T lymphocytes apoptosis.Conclusions The accelerated apoptosis of T lymphocytes in ESRD patients may be related to the expression of Bcl-2 and Fas of T lymphocytes.ESRD patients show a suppressed secretion of IFN-?and an increased secretion of IL-4. T lymphocytes apoptosis of maintenance hemodialysis patients is influenced not only by the biocompatibility but also by the permeability of the dialysis membrane.

OBJECTIVETo screen the exon12 mutation of pot1 gene in cultured human carcinoma cell strains (lines).

METHODSThe chromosomal DNA was extracted from 27 cultured carcinoma cell strains (lines). The exon 12 of pot1 gene was amplified by PCR, and the product was purified and screened. The screening results were compared with the data of GenBank and NCBI and the exon 12 mutations in cultured human carcinoma cell strains (lines) analyzed.

RESULTSThe exon12 sequence of pot1 could be specifically amplified using the designed primers. Direct sequence analysis of the PCR products after purification showed that 4 of the 5 carcinoma cell lines of the female genital system such as Hela and HO8910-PM cells shared the same transition (G17722-->C) in exon12 of human pot1 gene resulting in a conversion of G1385-->C in the cDNA and amino acid change of Leu454-->Phe in the translated polypeptide. The rest of the 23 cell strains (lines) from different origins showed no such mutation.

CONCLUSIONThe exon12 (17,722 bp) is a mutant region specific for female genital system tumor.

Amino Acid Sequence ; Base Sequence ; Cell Line, Tumor ; DNA Mutational Analysis ; Exons ; genetics ; Female ; HeLa Cells ; Humans ; K562 Cells ; Molecular Sequence Data ; Neoplasms ; genetics ; pathology ; Point Mutation ; Telomere-Binding Proteins ; genetics

OBJECTIVETo evaluate the possible vascular effects of an environment carcinogen benzo(a)pyrene (BaP).

METHODSThe cytotoxicit of BaP and rat liver S9 (0.25 mg/mL)-activated BaP were examined by MTT assay. Thoracic aortic rings were dissected from Sprague-Dawley rats. Contraction of aortic rings was induced by 60 mmol/L KCl or 10(-6) mol/L phenylephrine (PE) in an ex-vivo perfusion system after BaP (100 μmol/L) incubation for 6 h. [Ca(2+)](i) was measured using Fluo-4/AM. For in-vivo treatment, rats were injected with BaP for 4 weeks (10 mg/kg, weekly, i.p.).

RESULTSBaP (1-500 μm) did not significantly affect cell viability; S9-activated BaP stimulated cell proliferation. BaP did not affect the contractile function of endothelium-intact or -denuded aortic rings. BaP did not affect ATP-induced ([Ca(2+)](i)) increases in human umbilical vein endothelial cells. In BaP-treated rats, heart rate and the number of circulating inflammatory cells were not affected. Body weight decreased while blood pressure increased significantly. The maximum aortic contractile responses to PE and KCl and the maximum aortic relaxation response to acetylcholine were significantly decreased by 25.0%, 34.2%, and 10.4%, respectively.

CONCLUSIONThese results suggest, in accordance with its DNA-damaging properties, that metabolic activation is a prerequisite for BaP-induced cardiovascular toxicity.

Animals ; Aorta ; drug effects ; Benzo(a)pyrene ; pharmacology ; Calcium ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Humans ; Male ; Rats ; Vasoconstriction ; drug effects

To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34+ cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 10(6) CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen (DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.
Bioreactors ; Cell Culture Techniques ; methods ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVETo construct hu-PBL/SCID chimeras and to investigate the development of lymphoma and oncogenicity of the Epstein-Barr virus (EBV).

METHODSHuman peripheral blood lymphocytes (PBLs) were isolated from healthy adult donors and transplanted intraperitoneally into severe combined immunodeficient (SCID) mice. Mice with hu-PBL engraftment from healthy EBV seronegative donors were injected intraperitoneally with EBV-containing supernatant from suspension culture of B95-8 cell line (active infection), whereas mice receiving lymphocytes from healthy EBV seropositive donors were not re-infected with B95-8 derived EBV (latent infection). Pathological examination and molecular analysis were performed on experimental animals and induced neoplasms.

RESULTSIn the early stage of this experiment, 12 mice died of acute graft-versus-host disease, mortality was 34.3% (12/35 mice) with an average life span of 17.5 days. In 19 survival hu-PBL/SCID chimeric recipients from 12 healthy donors, tumor incidence was 84.2% (16/19 mice). The average survival time of tumor-bearing mice was 65.5 days. EBV-related neoplasms in SCID mice were nodular tumors with aggressive and fatal features. Histological morphology of tumors exhibited diffuse large cell lymphomas. Immunohistochemistry revealed that LCA (CD45) and L26 (CD20) were positive, but both PS1 (CD3) and UCHL-1 (CD45RO) were negative, and EBV products ZEBRA, LMP1, and EBNA2 were expressed in a small number of tumor cells. EB virus particles were seen in the nuclei of some tumor cells by electron microscopy, and EBV DNA could be amplified in the tumor tissues by PCR. In situ hybridization indicated that the nuclei of tumor cells contained human-specific Alu sequence.

CONCLUSIONSEBV-induced tumors were human B-cell malignant lymphomas. We obtained direct causative evidence dealing with EBV-associated tumor deriving from normal human cells.

Adult ; Animals ; Antigens, CD20 ; metabolism ; Chimera ; Epstein-Barr Virus Infections ; immunology ; virology ; Graft vs Host Disease ; prevention & control ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Leukocyte Common Antigens ; metabolism ; Leukocyte Transfusion ; methods ; Lymphoma, B-Cell ; immunology ; virology ; Lysosomal-Associated Membrane Protein 1 ; metabolism ; Mice ; Mice, SCID

OBJECTIVETo study the expression of neonatal Fc receptor in podocytes in human nephritis and immune-induced rat nephritis models: anti-Thy1.1 nephritis and Heymann nephritis.

METHODSThirty-nine cases of renal biopsies were enrolled from September 2009 to February 2010, including 8 cases of minimal change disease, 4 cases of focal segmental glomerulosclerosis, 9 cases of membranous nephropathy, 12 cases of IgA nephropathy and 6 cases of lupus nephritis. Five normal kidney tissue samples adjacent to renal clear-cell carcinoma were served as normal controls. Laser capture microdissection and real-time RT-PCR were used to assess the expression level of FcRn mRNA in glomeruli of various glomerulonephritides, and immunohistochemistry (IHC) of FcRn by SuperVision method was performed. In addition, rat models of mesangial proliferative nephritis (anti-Thy1.1 nephritis) and passive membranous nephropathy (Heymann nephritis) were established and FcRn was examined in renal tissues by IHC.

RESULTSThe FcRn mRNA level in lupus nephritis was statistically higher than that of normal controls (P < 0.05). FcRn protein expression by IHC was seen in lupus nephritis (6/6), membranous nephropathy (6/9) and IgA nephropathy (7/12), significantly higher than that of normal controls (0/5), P < 0.05. Minimal change disease and focal segmental glomerular sclerosis showed minimal or none expression of FcRn (1/8, 0/4 respectively) and not statistically difference from that of normal controls. Furthermore, FcRn expression in podocytes was detected in rat anti-Thy1.1 (3/5) and Heymann nephritis models (2/7) but was not detected in normal controls.

CONCLUSIONSExpression of FcRn in podocytes was up-regulated in immune-induced human nephritis and rat nephritis models of anti-Thy1.1 nephritis and Heymann nephritis. FcRn may play a role in the development of immune-induced glomerulonephritis.

Animals ; Glomerulonephritis, IGA ; metabolism ; pathology ; Glomerulonephritis, Membranous ; metabolism ; pathology ; Glomerulosclerosis, Focal Segmental ; metabolism ; pathology ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Humans ; Laser Capture Microdissection ; Lupus Nephritis ; metabolism ; pathology ; Male ; Nephritis ; genetics ; immunology ; metabolism ; pathology ; Nephrosis, Lipoid ; metabolism ; pathology ; Podocytes ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Fc ; genetics ; metabolism ; Thy-1 Antigens ; immunology ; metabolism ; Up-Regulation

Adult ; Altitude ; Craniotomy ; methods ; Follow-Up Studies ; Humans ; Male ; Young Adult

Adolescent ; Adult ; Aged ; Female ; Hepatitis B, Chronic ; therapy ; Humans ; Male ; Middle Aged ; Plasma Exchange ; methods ; Severity of Illness Index ; Sorption Detoxification ; methods ; Treatment Outcome

Objective:To explore the effect of vascular endothelial cells (EC) in the repair of long sciatic nerve defect in rats using acellular nerve allograft (ANA).Methods:Totally 80 female Sprague.Dawley rats were used in this study, of which 20 rats were sacrificed for the harvest of bilateral sciatic nerves as acellular nerve allograft and prepared according to Hudson's method.The remaining 60 rats were transected on the right sciatic nerve a 1.5 cm defect and evenly divided into three groups, ANG for autologous nerve graft using flipped sciatic nerve, ANA for acellular nerve graft alone, and ANA+EC for ANA loaded with EC.At 1, 2, 4 and 12 weeks after operation, 5 rats from each group were selected for tests including sciatic functional index (SFI), LDR, nerve conduction velocity recovery rate (NCVRR), complex muscle action potential recovery rate (CMAPRR), max traction force recovery rate (MTFRR), gastrocnemius muscle wet weight recovery rate (MWRR) and microvessel density proliferation rate (MVDPR).The nerve fibers, myelin thickness, G ratio and electron microscopic examination were performed at 12 weeks after operation.Results:At early stage after surgery, ANA+EC group showed an increase in SFI and MVDPR compared to ANA group (P<0.05).At later stage after surgery, ANA+EC group showed an increase in CMAPRR, MTFRR, MWRR compared to ANA group (P<0.05).ANA+EC also exhibited more similar morphology to ANG in the long term.Conclusions:In the treatment of long sciatic nerve defect rat model, muscle function is superior in the short term when using ANA+EC compared to using ANA alone.In the long term, the amount and quality of nerve fibers in ANA+EC is more comparable to that in ANG.Thus indicates the possible effect of improvement of nerve regeneration in vascular endothelial cells.

OBJECTIVETo create a standard mini-swine model of chronic ischemic myocardium by endoscopy for the research of gene transfer and stem cell.

METHODSTwenty-three male China experimental minipigs were used, aged from 8 to 11 months with a mean of (9.3 +/- 1.8) months and weighed from 20 to 30 kg with a mean of (29.3 +/- 4.3) kg. The myocardial ischemia was established by gradual occlusion of the left circumflex coronary artery (LCX) with an Ameroid constrictor. The Ameroid constrictor was implanted around LCX by endoscopy. Selective coronary angiography, electrocardiogram and Echo-Doppler study were performed perioperatively to evaluate the degree of stenosis.

RESULTSChronic ischemic myocardial models were successfully generated in 20 of 23 swine by full-endoscopy. Ameroid constrictors were placed at the LCX accurately. Three swine died of anesthetic accident, cardiac arrhythmia at secondary coronary angiography, and pulmonary infection within 6 weeks after operation respectively. Operation time was 25 to 65 min with a mean of (46 +/- 9) min. The blood loss was 30 to 60 ml with a mean of (55 +/- 12) ml. Six weeks later, coronary angiography revealed the total occlusion and partial stenosis (> 85%) of the LCX occurred in 7 and 13 swine respectively. Cardiac systolic and diastolic dysfunction were found in all swine. The ejection fraction value was (65.0 +/- 6.3)% before operation and (41.0 +/- 9.3)% after operation (P = 0.008). The fractional shortening value was (36.2 +/- 4.3)% before operation and (34.2 +/- 2.3)% after operation (P = 0.027).

CONCLUSIONThe endoscopic surgery is a less invasive way to create a standard mini-swine model of chronic ischemic myocardium with effective results.

Animals ; Disease Models, Animal ; Feasibility Studies ; Male ; Myocardial Ischemia ; Swine ; Swine, Miniature ; Thoracoscopes