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Objective]To discuss the effect of sciatic nerve injury on the expressions of tumor necrosis factor-alpha(TNF-α), interleukin-1β(IL-1β)and interleukin-10(IL-10)in anterior cingulate cortex(ACC),and further to explain their roles resided in the development of neuropathic pain.[Method]With use of the methods of behavioral test,western blot and immunohistochemistry, we examine the effects of spared sciatic nerve injury(SNI)on the expressions of TNF-α,IL-1β,and IL-10 in ACC,and observe the effect of the neutralizing antibody of TNF-α,IL-1β on the rat mechanical allodynia.[Result]In present experiment ,SNI increased the protein levels of TNF-α,IL-10,but not IL-1β in ACC. Increased TNF-α-IR and IL-10-IR in ACC is located in neurons ,but not astrocytes and microglia at 7 d following L5-VRT. Pre-treatment with anti-TNFα antibody but not anti-IL-1βantibody into ACC significantly increased the rat paw withdrawal threshold to von Frey hairs.[Conclusion]These data suggested that the increased TNF-αin ACC neurons might be responsible for the development of neuropathic pain.
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[Objective] To establish an effective and stable method to induce hematopoietic cells from embryonic stem(ES) cells,the phenotype and function of ES-derived hematopoietic cells induced by stromal cell conditioned medium (SCCM) of yolk sac (YS),fetal liver (FL) or bone marrow (BM) were analyzed and compared.[Methods] 10% of YS-SCCM,FL-SCCM or BM-SCCM was added to culture system for differentiation of ES cells.Flow cytometric analysis was used to identify expression of Flk1,Integrin α4,Sca-1,and CD34.Colony analysis was used to identify the quantity of high proliferative potential colony-forming cells (HPP-CFC) in differentiated ES cells.The yield of CFU-S (colony-forming unit-spleen) was also analyzed by transplanting ES cell derivatives into lethally irradiated mice.[Results] Expression of Flk1,Integrin α4,Sca-1,and CD34 could be tested on induced EB cells.The percentage of Flk-1+,Integrin α4+ and Sca-1+ cells induced by were 3.03%,2.9%,and 13.74%,respectively,which are greater than other groups.The percentage of CD34+ cells induced by BMSC-CM was 1.07% which was greater than other groups.The yields of HPP-CFC from hematopoietic cells induced by FLSC-CM or BMSC-CM were 7.4 /105 cells (P ＜ 0.01) and 5.8 /105 cells (P ＜ 0.05) which were greater than the yields of control group.The yields of CFU-S from hematopoietic cells induced by FLSC-CM or BMSC-CM were 8.5/5 × 105 cells and 6.75/5 × 105 cells which were also greater than the yields of control group (P ＜ 0.001).[Conclusion] Both YS-SCCM,FL-SCCM,and BM-SCCM could promote hematopoietic differentiation of ESE14.1 cells.Hematopoietic differentiation induced by FL-SCCM or BM-SCCM is more effective,which generates hematopoietic progenitor cells with normal function.Application of FL-SCCM generates more primitive hematopoietic progenitor cells than that of BM-SCCM.
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AIM: To purify murine yolk sac endothelial cells (mYS-EC) and investigate the cytokines mRNA expression in mYS-EC. METHODS: The murine yolk sacs were digested with 0.1% collagenase, resuspended in DMEM and counted after digestion and centrifugation. The yolk sac adherent cells were cultured in DMEM containing 15% FBS with 10% mBMEC-CM or 5?g/L VEGF, ECGF and bFGF. The phagocytose function and expression of vWF were evaluated via particle phagocytosis and immunohistochemistry method. Atlas cDNA expression array was used for analysis of cytokine expression in mYS-EC. RESULTS: Colonies consisting of pure yolk sac endothelial cells were obtained in liquid culture system containing 15% FBS and 10% mBMEC-CM or 5?g/L VEGF, ECGF and bFGF. For complete purification of the endothelial cells, subsequent passage was also necessary. Cellular cord formed during passage culture. The endothelial cells were round or oval sharp in morphology, positive in phagocytosis and factor VIII related antigen (von Willebrand's Factor,vWF). The mRNA expressions of cytokines, such as TGF-?2, TNF-?, IFN-?, FL, BMP-4, MIP-1?, BMP-2A, FLT2, endothelin 2, thymosin ?10, IL-6, IL-13, IL-9, SCYA5 and ACBP were detected in mYS-ECs. CONCLUSION: mYS-EC was purified and expanded in vitro . The mRNA expression of 15 kinds of cytokines was detected in mYS-ECs by Atlas arrays.
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AIM: To investigate the biological characterics of human second-trimester fetal cord blood mesenchymal stem cells (MSC) and its application prospects in utero gene transfer/therapy (IUGT). METHODS: Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FACScan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Adenovirus vector deliver green fluorescent protein gene (Ad-GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluorescence analysis was conducted to determine the presence of double-positive CD105+/CD166+ cells in different organs of rats. MSC were subcutaneous injected into the human-nonobese diabetes/severe combined immunodeficiency disease (NOD/SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESULTS: MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA-DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56 32%?3 28%). The MSC were located at different organs after injected into the newborn rats and didn't have carcinogenicity in vivo. CONCLUSION: Human second-trimester fetal cord blood MSC is an promising target cells in fetal IUGT.
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AIM: To explore the potential of human bone marrow mesenchymal stem cells (hMSCs) differentiating into hematopoietic cells with murine fetal liver stromal cell-conditioned medium (FLSC-CM) in vitro. METHODS: 12.5-14.5 days post coitus (dpc ) of KM mice were used for the preparation of fetal liver stromal cell-conditioned medium (FLSC-CM) and embryonic fibroblast feeder layer (FD). Culture-expanded hMSCs were directly contacted with FLSC-CM, FD, and the combination of human interleukin-6 (IL-6) or stem cell factor (SCF), respectively. Seven days later, the non-adherent cells were collected and characterized by morphology, immunophenotypes, and colony forming unit-granulocyte/macrophage culture assay. RESULTS: The number of nonadherent cells derived from hMSCs cultured with FLSC-CM was increased remarkably than those with either FD or cytokines. The non-adhered cells with the morphology of monocyte-or small lymphocyte-like cells were positive for human CD34, CD45 and had the capacity to form the hematopoietic progenitor colonies in methylcellulose cultures containing recombined human granulocyte/macrophage-colony stimulating factor (rhGM-CSF). CONCLUSION: hMSCs were successfully induced toward their differentiation into CD34+CD45+ hematopoietic progenitors after being cultivated with FLSC-CM. This study suggests that hMSCs have the hematopoietic differentiation potential in vitro. [