Study on inducing differentiation of human amniotic epithelial cells into insulin secreting cells in vitro / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro.</p><p><b>METHODS</b>The hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>(1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups.</p><p><b>CONCLUSION</b>hAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.</p>

CD133(+) cells derived from human placenta identified as initiating cells for LTC-IC colony formation / 中国实验血液学杂志

The aim of this study was to evaluate whether human placenta CD133(+) cells have an ability to reconstitute long-term hematopoiesis. Magnetic-activated cell sorting (MACS) was applied to enrich human placental CD133(+) cells. The isolated human placental CD133(+)cells of four different densities were established by limiting-dilution assay and primary fetal bone marrow stromal cells separated from bone marrow as feeder layer cells were co-cultured in long-term culture system so as to observe the incidence of long-term culture initiating-cells (LTC-IC) and their ability of proliferation and differentiation.The results showed that human placenta derived CD133(+) cells contained LTC-IC with frequency of 1/645 which have an ability to proliferate and differentiate into granulocyte/macrophage colony-forming units (CFU-GM) and mixed colony-forming units (CFU-Mix). In all LTC-IC positive wells, 71.43% form only CFU-GM and 28.57% display both CFU-GM and CFU-Mix formation. It is concluded that human placental CD133(+) cells possess LTC-IC with colony-forming capacity of hematopoietic early progenitor cells.

Ex vivo expansion of megakaryocyte progenitor cells for CD133(+) cells derived from human umbilical cord blood / 中国实验血液学杂志

To study the expansion potentiality of megakaryocyte progenitor cells (MPCs) derived from human umbilical cord blood CD133(+) (UCB-CD133(+)) cells and determine the optimal harvest time. UCB-CD133(+) cells were purified from mononuclear cells (MNCs) by magnetic activated cell sorting (MACS) and seeded in serum-free liquid culture medium supplemented with thrombopoietin (TPO), interleukin-3 (IL-3), and stem cell factor (SCF) to expand MPCs. At day 0, 6, 10 and 14 of culture, the total cell number was counted and the dynamic changes of CD133, CD34, and CD41 antigen expression during ex vivo expansion were analyzed by flow cytometry (FCM). At different expansion times, the CD133(+) cells were collected and cultured in collagen semisolid medium to carry out CFU-MK colony culture. The incidence of CFU-MK was calculated and the morphology of MPCs and CFU-MK were detected by immunohistochemistry and Wright-Giemsa staining. The results showed that UCB-CD133(+) cells optimally expanded at day 7 with expansion multiple of 8.2 +/- 2.2 in serum-free liquid culture systems and the total cell number was expanded by 116-fold at day 14. At 10 days, each UCB-CD133(+) cell can form 2.5 +/- 1.0, 2.6 +/- 0.5 and 20.3 +/- 5.9 cells of CD133(+)CD41(+), CD34(+)CD41(+) and CD41(+) respectively, from which the number of CD133(+)CD41(+) and CD34(+)CD41(+) cells reach the highest. UCB-CD133(+) cells both before and after expansion could form CFU-MK, the total number of CFU-MK reached the peak from cells of 10 days expansion of UCB-CD133(+) cells and the expansion multiple of CFU-MK was 59.5 +/- 11.8. Immunohistochemical results indicated that the expanded megakaryocytic cells were immature and no sign of platelet formation. It is concluded that the human UCB-CD133(+) cells have a high ability of MPC expansion, 10 days of culture can be result in optimal expansion effect.

Phenotypic characteristics of peripheral blood lymphocytes and its clinical significance in patients with coal arsenic poisoning / 中国地方病学杂志

Objective To study the change and rule of immunological function among the patients with coal arsenic poisoning in order to provide a basis for tumor risk evaluation and monitoring.Methods Seventy patients with coal arsenic poisoning aged from 24 to 71 years old(44 men,26 women,averaging 41 years old)were divided into 4 groups including 23 cases having a course less than 10 years,21 case8 lasting for 10～19 years,20 cages for more than 20 years,6 cases of cancer,and 26 healthy normal controls.Flow cytometer(FCM)was used to analyze the frequency of CD3+(total T cell),CD3+CD4+(inducer/helper T cell),CD3+CD8+(suppressor/cytotoxic T cell),CD19+(B lymphocyte),and CD56+CD16+(natural killer cell)lymphocyte subsets in the peripheral blood of the subjects and the expression rates of lymphocytic membrane surface molecules of human leucocvte antigen (HLA)-DR,CD25,CD38 were also determined by FCM.Results The pmportions of CD3+cells in periDheral blood of less than 10 years,10～19 years,more than 20 years and cancer groups were (63.76±9.32)%。(55.63± 12.97)%,(51.00±12.23)%and(49.83±,9.89)%respectively,which were significantly lower than that in control group[(68.10±8.62)%],and there was a significant difference between different groups(F=12.862,P<0.05). In less than 10 years,10～19 years,more than 20 years and cancer groups,the proportion of CD3+CD4+cells cells was (31.35±6.62)%,(28.38±8,66)%,(24.13±6.46)%and(19.17±4.96)%respectively,which wag significantly lowerthan that in control group[(34.28±7.32)%],and significant in a-group difference was found(F=10.455, P<0.05).The percentages of CD19+cells in more than 20 yeats and cancer groups[(9.00±5.32)%,(9.00± 3.29)%]were lower than that in control group and less than 10 years group[(11.80±3.43)%,(12.35±4.53)%] (P<0.05),while no statistical difference was found between other groups.The expression rates of CD25 and CD38 in lymphocytes of cancer group[(17.96 ±4.98)%,(41.38±8.54)%]were obviously higher than those in control group[(13.10±338)%,(28.60±5.51)%]and there were statistical differences between the experimental groups(P<0.05).The expression rate of HLA-DR in 10～19 years groups[(18.20±6.25)%]was significantly higher than that in control group[(10.72±7.06)%]and less than 10 years group[(11.78±5.13)%],while it was the same in more than 20 years and cancer group[(20.30±8.01)%,(21.82±10.97)%].Conclusions Reduction of cellular immune function caused by coal arsenic poisoning may be an important mechanism of skin cancer.CelMar immune function may be used as a warning signal of skin cancerization of patients with coal arsenic poisoning.

In vitro expansion of megakaryocyte progenitor cells from human placenta CD133+ cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expansion potential of megakaryocyte progenitor cells (MPC) from human placenta tissue CD133+ (PT-CD133+) cells.</p><p><b>METHODS</b>PT-CD133+ cells were purified from mononuclear cells (MNC) by magnetic activated cell sorting (MACS) and seeded in serum-free liquid culture medium supplemented with thrombopoietin (TPO), interleukin-3 (IL-3), and stem cell factor (SCF) to expand MPC. At day 7, 10 and 14, the total cell number was counted and the dynamic changes of CD133, CD34, and CD41 antigens expression during ex-vivo expansion were analyzed by flow cytometry (FCM). PT-CD133+ cells at different expansion time were collected and cultured in collagen semisolid medium for colony forming units-megakaryocyte (CFU-MK) assay.</p><p><b>RESULTS</b>PT-CD133+ cells could be optimally expanded at day 7 by 13 +/- 2 fold increase in serum-free liquid culture systems and the total cell number was expanded by 160 fold at day 14. With the expansion time going on, the expression of CD133, CD34 decreased and that of CD41 increased. The expanded megakaryocytes were immature and no sign of platelet formation. Both PT-CD133+ cells before and after expansion could form CFU-MK, the total number of CFU-MK peaked at day 10 of expansion by 54 +/- 10 fold increase.</p><p><b>CONCLUSION</b>Human PT-CD133+ cells have a high capacity of MPC expansion, 10 days culture could give rise to the maximum number of CFU-MK.</p>

Differentiation of Human Amniotic Mesenchymal Cells into Cardiomyocyte-like Cells in Vitro / 中国生物工程杂志

To explore the plasticity of human amniotic mesenchymal cells(hAMCs) into cardiomyocyte-like cells,hAMCs were isolated from human amnion with collagenase digestion.Phenotype of the isolated cells was analyzed by flow cytometry(FCM).hAMCs were treated with 5-azacytidine and basic fibroblast growth factor(bFGF) to investigate their ability of differentiation into cardiomyocytes.The induced differentiated cells were evaluated by immunofluorescence for desmin and ?-actin expression and by RT-PCR for Nkx2.5,GATA-4 and alpha-myosin heavy chain(?-MHC) mRNA expression.The results showed that,after primary culture,hAMCs could reach a confluence of 80% with swirl like growth at 6 days.The cells proliferated rapidly after passages with a 100% confluence at 3~4 d.hAMCs were positive expression of CD44 and vimentin,but negative for CK19.After induced differentiation at 8~10d,the differentiated cells have close-up arranged with long spindle-shape.At 2 weeks and 4 weeks,induced cells expressed ?-actinin and cardiac-specific transcription factor Nkx2.5.Expressions of GATA-4 and desmin can be detected but ?-MHC can not in the hAMCs both before and after the induction.In conclusion,hAMCs have the ability of differentiation into cardiomyocyte-like cells,which means that hAMCs can be regarded as candidate cells for cellular cardiomyoplasty(CCM).

Isolation and enrichment of hematopoietic stem/progenitor cells from human placenta tissue / 中国实验血液学杂志

The aim of this study was to establish the standard protocols for isolating and enriching hematopoietic stem/progenitor cells (HSPC) from human placenta tissue (PT). Single-cell suspension from of human PT was prepared by mechanical method combined with collagenase digestion. Mononucleated cells (MNC) derived from PT were separated by hydroxyethyl starch (6% HES), then the three cell subsets of different immunophenotypes (CD34(-), CD34(+)CD38(-), CD34(+)CD38(+)) contained in MNC were isolated by Magnetic Activated Cell Sorting (MACS). The cell immunophenotype of each sorting steps was analyzed by flow cytometer (FCM). The cell enrichment and recovery rate of each sorting step were calculated. The results showed that MNC could be harvested up to (12.30 +/- 3.51) x 10(8) from a single-cell suspension of human PT by mechanical method and collagenase digestion, no significant difference existed as compared with umbilical cord blood (UCB) initial sample [(8.86 +/- 5.38) x 10(8)], but the percentage of CD34(+) cells in MNC of human PT was (3.93 +/- 2.31)%, much higher than that in UCB [(0.44 +/- 0.29)%] (P < 0.001). recovery rate of MNC and CD34(+) cells from PT after separation with 6% HES were (45.3 +/- 11.7)% and (51.1 +/- 9.8)%, respectively. After MNC being sorted by MACS, the enrichment and recovery rate of CD34(+) cells in CD34(+) group were (73.4 +/- 14.1)% and (52.7 +/- 11.7)% respectively. It is concluded that the protocols established here for isolating and enriching hematopoietic stem/progenitor cells from human placenta can acquire HSPC with high abundance, enrichment and viability and may be a useful reference of isolating methods for future related study.

Expressions of homing-related adhesion molecules in hematopoietic stem/progenitor cells derived from human placenta, umbilical cord arterial and venous blood / 中国实验血液学杂志

The aim of this study was to evaluate the homing capabilities of hematopoietic stem/progenitor cells (HSPCs) derived from human placenta tissues (PT). Single cell suspension of human PT was prepared by mechanical method. The expression levels of homing-related adhesion molecules (HRAM) including CD11a, CD49d, CD44, CD49e, CD62L and CD54 on CD34(+) cells and the percentages of CD34(+) cells and their subpopulations in nucleated cells (NC) from fresh human PT, umbilical cord arterial blood (UCAB) and umbilical cord venous blood (UCVB) were detected by using flow cytometry. The results showed that the percentage of CD34(+) cells and CD34(+)CD38(-) cells in placenta were higher than those in UCAB and UCVB. There were no significant difference in percentage of HSPC between UCAB and UCVB. Placenta-derived CD34(+) cells strongly expressed CD11a, CD49d, CD44, CD49e and CD54, among which expression levels of CD49e and CD54 on placenta-derived CD34(+) cells were significantly higher than those on UCAB and UCVB-derived CD34(+) cells. While the percentage of CD34(+)CD62L(+) cells in placenta was only lower than that in UCVB. It is concluded that human placenta is rich in HSPC. Moreover, the expression levels of most HRAM in CD34(+) cells from PT are higher than those from UCAB and UCVB or are close to them. It suggested that HSPCs derived from PT might have stronger homing capabilities than those from UCB.

Hematopoietic stem/progenitor cells and phenotypes of lymphocyte subpopulations in human placenta / 中华血液学杂志

<p><b>OBJECTIVE</b>To study whether human placenta contains hematopoietic stem/progenitor cells (HSPCs), and analyze phenotypes of lymphocyte subpopulations in the placenta.</p><p><b>METHODS</b>Nucleated cells from fresh human placenta were analyzed for phenotypes of HSPCs and lymphocyte subpopulations by flow cytometry (FCM). And CD(34)(+) cells were sorted from human placenta nucleated cells by FCM or MiniMACS.</p><p><b>RESULTS</b>(1) CD(34)(+) cells, CD(34)(+)/CD(38)(+) cells, and CD(34)(+)/CD(38)(-) cells from a human placenta were 8.8, 4.6 and 11.9 times higher than those from umbilical cord blood (UCB), respectively. (2) The yields and purity of CD(34)(+) cells isolated from human placenta by FCM sorting system were (63.05 +/- 10.14)% and (86.39 +/- 11.27)%, respectively. (3) Lymphocytes, T cells (CD(3)(+)/CD(2)(+)), B cells (CD(19)(+)), Th cells (CD(3)(+), CD(4)(+)), and Th/Ts ratio in the placenta tissue were apparently lower than those in the UCB, while the CD(8)(+)/CD(28)(-) T suppressor cells were higher in the placenta than in the UCB.</p><p><b>CONCLUSIONS</b>Human placenta is rich in HSPCs, and has important hematopoietic function in ontogeny. It is probable that human placenta would be graft resource for HSPCs transplantation. CD(8)(+)/CD(28)(-) T suppressor cells might play an important role in feto-maternal immunologic tolerance.</p>

Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj).</p><p><b>METHODS</b>A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients.</p><p><b>RESULTS</b>Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07 x 10(6) individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%.</p><p><b>CONCLUSIONS</b>The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.</p>

Optimization of Technique Conditions for Decolorization and Extract of Exopolysaccharide by Cordyceps jiangxiensis / 微生物学通报

The technique conditions of decolonization of fermentation broth were successively optimized using single factor assay and orthogonal layout method in Cordyceps jiangxiensis. The optimal condition of decolorization was investigated to be 3g/100mL active carbon, 5 min absorption time, pH5 of fermented broth and 25℃absorption temperature. Under the optimal condition, the maximum decolorization rate of fermented broth reached 89. 6% , simultaneously 10. 7% consuming rate of exopolysaccahride was minimum. Subsequently, the extract condition of exopolysaccharide of C. jiangxiensis was further optimized by orthogonal layout design. The maximum exopolysaccharide production was 0. 38 g/L under the optimal condition, i. e. firstly fermented filtrate decolorized and deproteined was concentrated to 1/7 of its total volume, secondly concentration broth was mixed with four times its volume of absolute ethanol and stirred vigorously, lastly precipitation of exopolysaccharide proceeded at 4℃for 16 hrs and the exopolysaccharide collected by centrifugal ion and dryness.