Transplantation of human amnion epithelial cells improves learning and memory function in Alzheimer’ s disease-like pathology rat model / 中国病理生理杂志

[ ABSTRACT] AIM:To observe the treatment effect and its immune regulation of human amnion epithelial cells ( hAECs) on Alzheimer’ s disease ( AD)-like pathology rat model.METHODS: The hAECs were isolated from amnion with trypsin digestion, and the phenotype of hAECs was analyzed by flow cytometry.SD rats ( n=48) were randomly divid-ed into sham control group, model group, medium group and hAECs group.AD-like pathology rat model was induced by bilateral intraventricular injection of lipopolysaccharide (LPS).hAECs (5 ×105) were injected into the hippocampus of the AD-like pathology rats.At 2 weeks after transplantation, the animals were tested by Morris water maze to observe the function of learning and memory.The pathological change of the brain was observed by HE staining.The expression of am-yloid β-protein 42 (Aβ42) and Tau protein and the level of acetylcholine (ACh) in the injury brain were determined by immunohistochemistry.The survival and differentiation of hAECs in the hippocampus were measured by immunofluorescent technique.The percentages of lymphocyte subsets in the peripheral blood mononuclear cells were analyzed by flow cytome-try.The contents of serum cytokines were detected by cytometric bead array.RESULTS:Compared with model group and medium group, hAECs group showed shortened escape latency ( P<0.01) , increased frequency of going through the plat-form (P<0.05), reduced loss of hippocampal neurons, decreased expression of Tau protein and Aβ42 in the hippocampus (P<0.05), increased ACh level in the hippocampus (P<0.05), decreased percentages of Th1 and Th17 subsets, in-creased percentages of Th2 and Treg cells ( P<0.05) , decreased concentrations of IFN-γand IL-2 in the serum, and in-creased concentration of IL-4 ( P<0.05 ) .CONCLUSION: hAECs improve the cognitive learning and memory function and alleviate pathologic damage of hippocampus through immune regulation in AD-like pathology rats.

Therapeutic effect and immunologic regulation of human amniotic mesenchymal stem cells in rats with experimental type 1 diabetes mellitus / 中华内分泌代谢杂志

Objective To study the therapeutic effect and immunologic regulation of human amniotic mesenchymal stem cells (hAMSCs) in rats with experimental type 1 diabetes mellitus (T1DM).Methods The hAMSCs from human amnion were isolated and cultured in vitro,then phenotype was analyzed by flow cytometry.T1DM were produced by administering streptozocin to rats.The rats were divided into normal control group (n =6),T1 DM model group (n =6),medium treated group (n =6),hAMSCs transplanted group(n =6),and insulin treated group(n =6).5 × 106of hAMSCs or vehicle were administered to rats via sublingual vein.Blood glucose levels of rats were recorded weekly in the groups for six weeks by Blood Sugar Meter.At the end of 6 weeks after hAMSCs transplantation,concentrations of plasma insulin were detected by ELISA; histopathological changes of pancreas,surviwl and differentiation of transplanted hAMSCs in pancreatic tissue were studied with HE staining,and immunofluorescence staining; percentages of lymphocyte subsets in peripheral blood mononuclear cells were determined by flow cytometry ; concentrations of plasma cytokines were determined by cytometric bead array.Results After hAMSCs transplantation,blood glucose levels in rats with T1DM were decreased (P ＜ 0.01),while concentrations of plasma insulin were increased significantly (P＜0.01).At 6 weeks,cell-treated animals showed an improvement in pancreas damage ; the percentages of CD4 + IFN-γ+ (Th 1) and C D4 + interleukin (IL)-17 + (Th 17) cells were reduced (all P＜0.05),while the percentages of FoxP3-positive regulatory T cells (FoxP3 +Treg) and CD4+ IL-4+(Th2) cells were increased (all P＜0.01) ; plasma concentrations of interferon-γ,IL-2,and tumor necrosis factor-αwere decreased (all P＜0.01),but IL-4 level was increased (P＜0.05).No histological evidence of insulin producing cells from hAMSCs was seen within pancreas.Conclusions hAMSCs may reduce blood glucose and alleviate the islet damage in rats with T1 DM,which is related to their potential to up-regulate FoxP3 +Treg cells.

Preparation of cytoplasts from HL-60 cells / 生物医学工程学杂志

This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.

Analysis of larval excretory-secretory antigen and its immunodiagnosis of Angiostrongyliasis cantonensis infection / 南方医科大学学报

<p><b>OBJECTIVE</b>To analyze the diagnostic value of larval excretory-secretory antigen in Angiostrongylus cantonensis (LESA) infection.</p><p><b>METHODS</b>A.cantonensis larvae harvested from mice brain were cultured in vitro. The LESA and the adult worm antigens of A.cantonensis (AWA) were collected and analyzed using SDS-PAGE and Western blotting. Two ELISA systems were established using the two antigens (LESA-ELISA and AWA-ELISA) to detect the serum spectra from different sources.</p><p><b>RESULTS</b>SDS-PAGE and Western blotting displayed fewer protein and antigen bands for LESA than for the adult antigen. Two distinct bands of LESA (with relative molecular masses of 40 000 and 26 000) showed reactivity with the sera from patients with A. cantonensis infection. The serum levels of IgG and IgM antibodies to LESA increased at the beginning of infection in mice, reaching the peak on day 5 after infection and decreased on day 10. Compared with AWA-ELISA, LESA-ELISA showed a lower seropositive ratio in suspected patients with A.cantonensis, with also a lower cross-positive ratio in patients with schistosomiasis and clonorchis sinensis.</p><p><b>CONCLUSION</b>LESA possesses fewer antigen reaction bands than AWA. Although with a slightly lower positive ratio than AWA, LESA has a higher specificity for detecting serum antibodies in suspected cases of A.cantonensis infection, and therefore shows a potential for the diagnosis of angiostrongyliasis especially in the early stage and in current infection.</p>

Inducing differentiation of human amnion-derived mesenchymal stem cells into insulin-secreting cells in vitro / 中华内分泌代谢杂志

ObjectiveTo investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.MethodsThe hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10％ fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results( 1 ) The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.( 2 ) At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67％ ± 1.53％,75.00％:1.00％,and 74.33％ ±1.53％,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were ( 331.62 ± 1.76 ),( 330.50 ± 1.22 ),and ( 331.65 ± 0.48 ) μIU/ml,respectively,but non-induced groups were negative.(3) PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.( 4 ) Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin ( all P ＞ 0.05 ).ConclusionThe hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes.

Properties of hematopoietic stem/progenitor cells derived from human placenta tissues / 中国组织工程研究

BACKGROUND: As is well known that hematopoietic stem and progenitor cells (HSPCs) contain in bone marrow, peripheral blood, and cord blood. Recent studies found that human placenta tissue (PT) also exists in HSPCs. But so far the property and differentiation capacity of human PT-HSPCs is not yet known. Furthermore the composition of lymphocyte subpopulations and immunogenicity regarded to human PT-HSPCs are also unclear.OBJECTIVE: To verify whether there are more HSPCs in human PT than those in human umbilical cord blood (UCB), to investigate their capacities of proliferation and differentiation, and to analyze the phenotypes of lymphocyte subpopulations in human PT.DESIGN, TIME AND SETTING: Open eXperiments were performed at the Key Laboratory of Cell Engineering of Guizhou Provinee from January 2004 to December 2006.SETTING: Key Laboratory of Cell Engineering of Guizhou Province, the Affiliated Hospital of Zunyi Medical College.MATERIALS: Twelve human placenta and UCB samples through cesarean delivery were collected aseptically with the informed consents of parturients derived from Maternity Department of the Affiliated Hospital of Zunyi Medical College. The main reagents were detailed as follows: lymphocyte subpopulations analysis reagents Simultest IMK-lymphocyte Kit, CD34 absolute counting reagents Kit (Becton Dickinson); CD34 Multisort Kit, FITC conjugated CD38 monoclonal antibody, anti-FITC microbeads and MS/LS mini MACS segregating, columns (Miltenyi Biotec).METHODS: UCB samples were 1:1 diluted with RPMI-1640 containing 0.1 volume fraction of fetal bovine serum and the mononuclear cells (MNCs) were isolated on Ficoll-Histopaque by centrifugation for 30 minutes. The MNCs at the interface were collected and washed with PBS. Single cells suspension liquid of human PT was prepared by mechanical method combined with 0.25g/L collagenase digestion. After that, the placenta samples underwent the same protocol as used in UCB to isolate MNCs. The percentage of CD34+CD38-, CD34+CD38+ HSPCs and the phenotype of lymphocyte subpopulations derived from human PT-MNCs were analyzed by flow cytometry (FCM). CD34+CD38-, CD34+CD38- cell subsets isolated by magnetic-activated cell sorting (MACS) from human PT were used to carry out colony-forming culture including granulocyte/macrophage colony-forming unit (CFU-GM), burst forming unit-erythroid (BFU-E) and mixed colony-forming unit (CFU-Mix) in order to assess their capacities of hematopoietic progenitor cells' proliferation and differentiation. In parallel, UCB samples underwent the same protocols for comparison.MAIN 0UTCOME MEASURES: Percent compositions of CD34+ HSPCs, hematopoietic progenitors' lineage colony-forming capacities of CD34+ HSPCs, phenotypes and compositions of lymphocyte subpopulations both in PT and UCB.RESULTS: The percentage of CD34+ cells contained in human PT was 8.8 times higher than that of in UCB (P<0.01). The total number of lymphocytes, T cells (CD3+CD2+), B cells (CD19+), Th (CD3+CD4+) and Th/Ts ratio were apparently lower in human placenta, while the number of CD8+CD28- T suppressor cells were higher compared to UCB samples (P<0.01). Among PT, CFU-GM, BFU-E and CFU-Mix frequencies of CD34+CD38- cells subset were much higher than that of CD34+CD38- (P<0.01). Within the same phenotype of cell subsets, however, the number of each colony-forming unit was similar between PT and UCB (P 0.05).CONCLUSION: Human PT is richer in CD34+CD38-, CD34+CD38+ HSPCs and both of them have the abilities of proliferating and differentiating into CFU-GM, BFU-E and CFU-Mix. Considering that human PT have a lower lymphocyte subpopulations and higher Ts cells, human PT might be a alternative and suitable source of HSPCs for clinical transplantation.

Epidemiological Investigation of Angiostrongylus cantonensis in Jiangmen of Guangdong Province / 中国寄生虫学与寄生虫病杂志

Objective To make an epidemiological survey on Angiostrongylus cantonensis in Jiangmen City of Guangdong Province. Methods From October 2006 to November 2007, the characteristics of A. cantonensis infection were investigated in Jiangmen district in various hosts, including the third stage larva infection in the snails Achatina fulica and Pomocea canaliculata by digestion method, and the adult A. cantonensis in rats by the dissection of heart and lungs. Relevant symptoms and dietary habits in Jiangmen residents who were randomly recruited were also investigated by questionnaire, and the specific IgG and IgM antibodies against A. cantonensis in their sera were detected by ELISA. Results 695 A. fulica and 720 P. canaliculata were examined. The infection rate of third stage larva of A. cantonensis were 45.0% and 1.8% respectively, with an infectiosity of 53.74?147.30 and 5.23?8.51 respectively. Natural infection rate of A. cantonensis in all 229 rats was 4.4%. Among the 300 people surveyed, 11.3% had a history of eating raw or undercooked fish and shrimp, 5.3% directly or indirectly exposed to A. fulica or P. canaliculata. The positive rate of specific IgG antibody against A.cantonensis for serum samples among residents was 14.0% (42/300), and 5 serum samples in the 42 positive samples showed specific IgM antibody, with a positive rate of 1.7%. Conclusion Jiangmen district is an endemic area of A. cantonensis, and the local residents are under the risk of infection.

The Effect of Antitumor Immunity Elicited by Heat Shock Protein70-Associated Peptide Complexes on Hepatoma in Mice / 中国肿瘤生物治疗杂志

Objective: To observe effective of antitumor immunity elicited by heat shock protein70(HSP70) associated peptide complexes isolated from hepatoma (HCaF), a tumor model of no MHC Ⅰmolecule expression in mice. Methods: Specific active immunization and adoptive cellular immunization assay was adopted to observe the immunoprotective effect elicited by HSP70 associated peptide complexes purfied from the HCaF. Results: Mice immunized with HSP70 associated peptide complexes were protected from HCaF challenge. The immunoprotective effect in female mice was better than the male groups. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70 associated peptide complexes could be elicited immunity against HCaF challenge, and the mice free of tumor can resist repeated challenge, the maximal challenge quantities exceeded 1?10 7 HCaF cells. Mice immunized once with spleen cells pulsed by HSP70 associated peptide complexes could also be elicited certain immunity against HCaF challenge. Conclusions: HSP70 associated peptide complexes derived from the HCaF can elicit no MHC Ⅰ molecule restrictive immunoprotective effect against HCaF and could be continuously enhanced by repeated challenge with alive HCaF cells. Adoptively transferred immune spleen cells of mice which had been immunized with HSP70 associated peptide complexes could provide protection against HCaF cells challenge.

Significance of NF-?B in immunopathogenesis of Graves disease / 中国病理生理杂志

AIM:To investigate the activity of NF-?B in peripheral blood mononuclear cells (PBMCs) from patients with Graves disease (GD) and the significance in immunopathogenesis of GD.METHODS:Peripheral blood was collected from 22 untreated GD,20 treated GD with tapazole more than 1 year,and 25 healthy volunteers. PBMCs were isolated from the blood by histopaque-1077 density-gradient centrifugation. The activity of NF-?B in PBMCs was analyzed using gel electrophoretic mobility shift assay (EMSA). The contents of IL-1?,IL-6 and TNF-? were tested by radioimmunoassay.RESULTS:The activity of NF-?B in PBMCs of untreated GD group was increased remarkably,compared with that in the treated group and control (P

Tumor necrosis factor-related apoptosis-inducing ligand and tumor immunity / 中国药理学通报

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily.It is extensively expressed on different tissues and cells. TRAIL can initiate singnaling through binding to TRAIL receptors. TRAIL selectively induces apoptosis of a variety of tumor cells, but has less toxition or no damage to most normal cells. TRAIL treatment in combination with chemotherapy drugs and cytokines can induces apoptosis of tumor cells. But a tumor emerges immune escape or immune privilege by expressing TRAIL on itself. The action of TRAIL is influenced by NF-?B or type and amount of TRAIL receptors on target cell.

Modified procedure for the histochemical demonstration of myosin ATPase / 西安交通大学学报(医学版)

Objective To study the effect of self-modified Guth & Samaha's myosin-ATPase staining method on the classification of SD rat skeletal muscle fiber types.Methods 8 ?m-thick cryostat sections from rat chest muscles and biceps brachii muscles were transfected with cryostat.Myosin-ATPase staining was carried out according to the following procedure: ① Fixing sections for 5 min in fixative solution containing 40 g/L paraformaldehyde;② Rinsing slides in Tris-rinse solution and then preincubating them in alkaline preincubation solution for 15 min;③ Rinsing slides in Tris-rinse solution twice and then incubating them for 60 min in incubation solution;④ Washing slides for three times in 10 g/L CaCl2 and then placing them in 20 g/L CoCl2 for 3 min;⑤ Washing sides in distal water and then placing them in 10 g/L(NH4)2S for 3 min and;⑥ Washing slides in running tap water for 3 min,dehydrating in graded ethanol,clearing in xylene and mounting in neutral balsam.Results After being stained by modified myosin-ATPase staining method,both chest muscle and biceps brachii muscle samples from SD rats could be clearly identified as type Ⅰ fibers and type Ⅱfibers as the fibers of type Ⅰ were stained white while the fibers of type Ⅱ were stained brown.Conclusion Modified myosin-ATPase staining method is a simple and effective way for muscle fiber type classification and can be applied in skeletal muscle related study.