Objective To observe the effect of astragaloside IV (A) and SB203580 antagonize cadmium (Cd) toxicity on expression of associated protein and blood-testis barrier(BTB) in rats and to study the protective mechanism of A on it.Methods Totally 21 SD male rats were randomly divided into 7 groups, 3 rats per group:Cd [ intraperitoneally injected with 0.1%CdCl2,1mg/(kg?d)],Cd+A [at the above dose of CdCl2,at the same time with A,10mg/(kg?d)], and Cd +SB203580 [at the above dose of CdCl2,at the same time with SB203580,100μg/(kg?d)], each of the above groups was further divided into continuous five and ten days treatment groups .The control group was intraperitoneally injected with equal dosage of normal saline .The testes were studied by light , electron microscopy , immunohistochemistry and Western blotting .Results In the control group ,irregular and lightly stained nuclei of Sertoli cell ( Sc) in seminiferous tubules were observed by HE staining .A continuous electron density line of tight junction ( TJ) and normal ultrastructure of BTB were observed .After Cd treatment ,the vesicular formation in the Sc was observed .The ultrastructural damage of Sc and TJ was observed .Compared with the corresponding time point of Cd group ,these were weakened in morphology of testis and ultrastructure of TJ after Cd +A or Cd +SB203580 treatment .The positive products of zonula occludens-1 ( ZO-1 ) and claudin-11 were localized mainly in the base of the seminiferous tubule .After Cd treatment , the average absorbance (AA) of ZO-1 and Claudin-11 was decreased significantly compared with that of the control group (P<0.05).After Cd +A or Cd +SB203580 treatment,AA of ZO-1 and Claudin-11 were increased significantly compared with that of the Cd group(P<0.05),though lower than that of the control group .The result of Western blotting showed that phosphorylation-p38MAPK in Cd group was increased significantly compared with that of the control group (P<0.05).After Cd +A or Cd+SB203580 treatment, it was decreased significantly compared with that of the Cd group (P<0.05).Conclusion Cd decreases ZO-1 and Claudin-11 expression and damages ultrastructure of TJ in BTB , asⅣhas protective effect on it , and is related to inhibiting activation of p 38 MAPK pathway .

Objective:To evaluate the short-term prognostic value of model for end-stage liver disease (MELD) combined with high density lipoprotein-cholesterol (HDL-C) in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF).Methods:From December 2015 to December 2018, 182 patients with HBV-ACLF who were treated in Henan Provincial People′s Hospital were included. Prognosis and clinical data including HDL-C, total bilirubin, international standardized ratio (INR), creatinine of patients within 24 hours after admission were collected and analyzed retrospectively.The values of MELD were calculated. The binary logistic regression analysis was used to analyze the independent risk factors affecting 90-day mortality in HBV-ACLF patients.The receiver operator characteristic curve (ROC) and MedCalc 15.2 software were used to assess the predictive value of MELD, HDL-C and MELD-HDL-C model for prognosis. Kaplan-Meier survival curve was performed to analyze the prognosis of patients in different groups.Results:Sixty patients were divided into the death group and 122 patients were divided into the survival group according to the prognosis during hospitalization and 90 days after discharge. The MELD score of patients in the survival group was 21(19, 24), which was significantly lower than that in the death group (29(25, 34)), and the HDL-C value of patients in the survival group was significantly higher than that in the death group (0.3 (0.1, 0.6) mmol/L vs 0.2(0.1, 0.5) mmol/L). The differences were both statistically significant ( Z=-6.290 and -4.087, respectively, both P<0.01). Multivariate logistic regression analysis showed that MELD score and HDL-C value were the independent risk factors for 90-day mortality in patients with HBV-ACLF(odds ratio ( OR)=1.432, 95% confidence interval ( CI)1.271-1.613; OR=0.584, 95% CI 0.487-0.700, respectively; both P<0.01). Areas under the ROC of MELD, HDL-C and MELD-HDL-C scoring models were 0.775, 0.782 and 0.878, respectively. MELD-HDL-C scoring model was superior to both MELD and HDL-C , and the differences were both statistically significant ( Z=3.944 and 3.104, respectively, both P<0.01). When the MELD-HDL-C Youden′s index was set at 0.72, the optimal threshold was 24.69. Patients with MELD-HDL-C score≥24.69 had lower survival rate than patients with MELD-HDL-C score<24.69, and the difference was statistically significant ( χ2=142.900, P<0.01). Conclusion:MELD, HDL-C and MELD-HDL-C scoring systems could predict the short-term prognosis in patients with HBV-ACLF, and the predictive value of MELD-HDL-C has the superiority.

Objective To investigate the toxic effect of cadmium (Cd) on the ultrastructure ,expression of related protein and the signal molecule phosphrylated P38 mitogen-actived protein kinase(P-P38MAPK) of primary cultured rat sertoli cell(Sc) ,and the protective effect of astragaloside (A ) on it .Methods The primary cultured rat Sc were divided into the control group ,Cd (50 mol/L)group and Cd(50 mol/L) plus A(10 mg/L) group ,they were used for the electron microscope observation and the im-munohistochemistry detection of vimentin ,E-cadherin ,-catenin and P-P38MAPK .Results The Sc ultrastructural changes included that the swelled mitochondria ,abundant lipid droplets and dilated endoplasmic reticulum were found in the Cd group .Further ,apop-tosis occurred in some Sc .However these ultrastructure changes above mentioned were slighter in the Cd plus A group ;the immu-nohistochemistry showed that the positive products of vimentin ,E-cadherin and-catenin were obviously decreased in the Cd group (P<0 .05) ,and those in the Cd plus A group were higher compared with the Cd group(P<0 .05);the expression of P-P38MAPK in cytoplasm was increased in the Cd group ,and showed the trend to move from cytoplasm to nucleus ,meanwhile ,the positive prod-ucts expression in the Cd plus A group was lower than that in the Cd group (P<0 .05) .Conclusion Cadmium can cause the injury of the Sc ultrastructure ,damage of cytoskeletal protein and fibronectin ,and increase of P-P38MAPK level ;astragaloside can antago-nize the toxicity of cadmium on Sc ,the protective effect maybe related with the decrease of P-P38MAPK in Sc .

Objective To investigate the virological response in hepatitis C virus (HCV)genotype 1b relapsers after 48 weeks of peginterferon/ribavirin (peg-IFN/RBV)combination retreatment,and to explore the predictive value of interleukin (IL )-28B rs12978960 genetic polymorphismon virological response.Methods From 2012 to 2014,genotype 1b chronic hepatitis C (CHC)relapsers in He′nan Provincial People′s Hospital were retreated with combined peg-IFN/RBV for 48 weeks and followed up for 24 weeks off-treatment.Host IL-28B genetic polymorphism was detected.Predictive factors associated with virological response and sustained virological response (SVR)were analyzed.Independent-samples t test was conducted in continuous variables,whileχ2 test or Fisher exact probability test was conducted in counts data.Results A total of 61 patients finished 48 weeks of peg-IFN/RBV combination therapy and were further followed up for 24 weeks off-treatment.Mean age was (46.7 ±12.4)years.Thirty-seven patients (60.7%)were male and 49 were rs12978960 CC genotype.After 48 weeks of retreatment with peg-IFN/RBV and 24 weeks of off-treatment follow-up,40 patients (65 .6%)achieved SVR.Rapid virological response (RVR)and SVR of younger patients were both significantly higher than those of older patients (100.0% vs 67.4% and 85 .0% vs 47.6%,respectively;both P =0.006).IL-28B rs12978960 genotype was predictive to RVR and SVR.Patients with RVR and SVR had higher carriage rates of IL-28B rs12978960 CC genotype compared with those without RVR and SVR (both P <0.05 ).Patients with CC genotype had higher rates of RVR (34.1 % vs 0;χ2 = 10.625 ,P =0.006 ),end-of-treatment virological response (84.1 % vs 70.6%;χ2 =5 .563,P =0.039 )and SVR (77.3% vs 35 .3%;χ2 =9.572,P =0.007)than those with CT/TT genotype.However,there were no statistical differences of extended RVR (34.1 % vs 29.4%;χ2 =0.122,P =0.809)and early virological response (79.5 % vs 82.3%;χ2 =0.612,P =0.964).Conclusions Retreatment with antiviral therapy is necessary in CHC patients with genotype 1b. IL-28B rs12978960 genetic polymorphism is predictive to the SVR of retreatment,especially for patients without RVR,which will provide individualized treatment and optimize the treatment strategy.

In this paper, five different methods were carried out for DNA extraction directly from soil. The result shows that all five methods could generate DNA with more than 15 kb in size. They were subsequently used as templates for PCR amplification with success, using primers of the bacterial 16S rRNA gene and Shiva-1 gene encoding an antibacterial peptide. However, method 5 is more suitable for DNA extraction directly from a small amount of soil sample as it produced a good yield of DNA in high integrity with reliable reproducibility.

Leptin, the product of obese gene, is a secreting protein that exerts multiple biological functions by binding to its receptor. Leptin regulates nutrient intake and metabolism, and is secreted from adipocytes, which occupy most of the bone marrow cavity and constitute the microenvironment. Leptin not only plays an important role in the control of the proliferation and differentiation of normal primitive hematopoietic cells, but it also stimulates the growth and viability of leukemic cells. Leukemic cells of some patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia, and chronic myeloid leukemia also express the leptin receptor. Furthermore, leptin also stimulates leukemic cell growth in vivo by promoting angiogenesis. These findings suggest the possibility that leptin and its receptor play roles in the pathophysiology of leukemia, and blockage of leptin binding to its receptor might have potential therapeutic benefits in the treatment of certain leukemias. This review discusses the biological characteristics of leptin and its receptor, the relation of leptin and its receptor with normal hematopoiesis, the relation of leptin and its receptor with AML and so on.
Animals ; Hematopoietic System ; Humans ; Leptin ; physiology ; Leukemia, Myeloid, Acute ; Receptors, Leptin ; physiology

Long-tem culture initiating cells(LTC-IC), and in vitro assay of hematopoietic stem/progenitor cells, still represent a heterogeneous population in terms of proliferative capacity and sensitivity to different growth factors. Human umbilical cord (CB) is rich of hematopoietic progenitor cells measured by clonogenic assays and stem cells capable of reconstituting the marrow after transplantation. The influence of culture conditions on the in vitro behavior of LTC-IC from CB was evaluated. First, by using IRES sequence, FL and TPO cDNA were recombined with retroviral vector pLXSN by gene recombination technology. The recombinant plasmid pLFSN, pLTSN, pLFTSN were transfected into human stromal cell line HFCL. Then, LTC-IC were evaluated in long term cultures, comparing five types of stromal feeder layers: human bone marrow stromal cell, human stromal cell line HFCL, and stromal cell lines HDF tranfected with FL gene, HLT transfected with TPO gene or HFT co-transfected with FL and TPO genes. The results were demonstrated that after 8 weeks of coculture, three types of stromal cell lines that supported the maintenance of CFU-C for up to 3 weeks in vitro were identified. However, cocultivation of human bone marrow stromal cell and CB CD34(+) cells on HFT, CFU-C production continued up to 6 weeks or longer on these stroma. The absolute LTC-IC frequency in CD34(+) cells on human bone marrow stromal cell (2.65 +/- 0.76/1 000 cells) was no significant difference with on HFT (3.65 +/- 0.58/1 000 cells). Thus, HFT acts by direct contact to maintain the phenotype and function of the most primitive and quiescent human progenitors. Furthermore, HFT cell line was selected as the optimal one for supporting long-term culture feeder. It was concluded that LTC-IC progenitors from cord blood maintain growing upon the FL/TPO gene-modified stromal feeder layers in vitro.

As main component of fetal liver hematopoietic microenvironment, different stromal cells may play distinct roles in the regulation of hematopoietic stem cell self-renewal, proliferation and differentiation. It is a unique approach to establish stromal cell lines for analyzing the interaction of hematopoietic cells with the stroma on the clonal level to dissect the function of hematopoietic microenvironment. In this study two immortal stromal cell lines-A4, B3 were established from mouse embryonic day 12.5 fetal liver by transfection of pSV3 neo plasmid. A4 exhibited a fibroblast-like morphology, 25 hours population doubling time as well as high levels of CD29, CD44, UEA-1 and low levels of CD105 expression. In contrast B3 displayed an epithelium-like morphology, 37 hours population doubling time along with high levels of CD105 and low levels of UEA-1 expression. In addition no or low levels of CD31, CD34, CD45 and CD144 expression were found in the two cell lines. These results indicate that A4, B3 are two discriminating cell lines in terms of morphological characters, growth behaviors and surface molecular expression types. Next functional assays using Limited-Diluted Assay(LDA) and Bulk-LTC-IC were done: both stromal cell lines had similar ability to maintain the survival proportion of inoculated mouse bone marrow-derived Long-Term Culture-Initiating Cells (LTC-ICs), and they could also support LTC-ICs expansion up to 4 weeks by co-culture in vitro. More strikingly, B3 could expand the absolute number of LTC-ICs over 13-fold at week 4 than that of week 0, and the ability of B3 to expand absolute number of LTC-ICs was over 8-fold of that of A4. Proportions of LTC-ICs in proliferating cell populations in two long-term culture systems was similar at week 4, no matter with or without extra cytokines. Further study indicated that the ability of LTC-ICs to yield CFCs was held as the number 6( +/- 1.2) after 4 weeks co-culture. Extra cytokines-SCF + IL-3 + IL-6 + Epo had no influence in the maintenance and expansion of LTC-ICs, but expanded the absolute number of CFCs and proliferating cell populations, and maintained similar proportions of LTC-ICs and CFCs in the end in two culture systems at week 4. Take together, these results implicate that B3 may act as an important functional component in embryonic day 12.5 fetal liver microenvironment to effectively expand primitive hematopoietic cells, yield more committed hematopoietic progenitor cells and mature hematopoietic cells to meet the need of quick development especially in the early phase of embryonic development. Alternatively A4 can probably function as being a structural element. Moreover the function of hematopoiesis-associated cytokines employed in this investigation was not to expand LTC-ICs, but to modulate the limited-proliferation and differentiation of CFCs. The maintenance and proliferation of LTC-ICs may depend on the type of the stromal cell as well as the interaction between stromal cells and LTC-ICs. It is suggested that B3 with cytokines including SCF, IL-3, IL-6, EPO in vitro can mimic embryonic day 12.5 hematopoietic microenvironment to investigate the mechanism of interaction between hematopoietic microenvironment and hematopoietic cells in clonal level.
Animals ; Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; Cell Culture Techniques ; methods ; Female ; Fetus ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Hyaluronan Receptors ; metabolism ; Integrin beta1 ; metabolism ; Interleukin-3 ; metabolism ; Interleukin-6 ; metabolism ; Leukocyte Common Antigens ; metabolism ; Liver ; embryology ; Male ; Mice ; Mice, Inbred C57BL ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Stromal Cells ; cytology ; metabolism

This study was aimed to investigate the effect of RPMI 1640 and IMDM on the development of human peripheral blood monocyte-derived dendritic cells. Under the same cytokines and culture conditions, the different medium types were tested, and the morphology of mature and immature dendritic cells was observed by microscopy, the cell phenotype and endocytosis ability were detected by flow cytometry. Furthermore, the immunoregulatory function of various DC was analyzed by mixed lymphocyte reaction (MLR), the expression of cytokine in culture supernatant of MLR system was also analyzed by Bio-plex technology. The results showed that there were no difference in morphology, CD14, CD83 expression and endocytosis ability between IMDM-cultured DC and RPMI-1640 medium-cultured DC, but there was a lower expression of CD1a in IMDM-cultured DC. Moreover, DC cultured with IMDM displayed a significant reduction in stimulating T cell proliferation, and highly expressed IL-6, IL-8 and IL-10, but low expressed IL-12. It is concluded that the different cultural mediums can induce DC with different functions and DC cultured with IMDM may correlated with induction of immune tolerance. The results of this study will provide a new idea for DC clinical application.
Cell Differentiation ; Cells, Cultured ; Culture Media ; pharmacology ; Cytokines ; Dendritic Cells ; cytology ; Flow Cytometry ; Humans ; Leukocytes, Mononuclear ; cytology ; Lymphocyte Culture Test, Mixed ; Monocytes ; cytology

OBJECTIVETo construct a retroviral-mediated vector of FLT3 ligand (FL) and express it in human bone marrow stromal cells.

METHODSFL cDNA was inserted into the retroviral vector pLXIN by gene recombination technology. The recombinant plasmid pLFIN was transferred into retrovirus packaging cell line PA317 by lipofectamine, and the positive clones were selected by G418. The mRNA expression in human stromal cells and integration of genome DNA were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and genomic DNA-PCR. The expression of FL protein and its biological activities in the culture were investigated by ELISA and mouse bone marrow CFU-GM assay.

RESULTSThe recombinant plasmid pLFIN was successfully constructed. In genome of these transfected target cells, Neo gene and FL gene were integrated, FL mRNA was transcripted and FL protein was expressed at 4.35 ng/ml/24 h. The specific activities of FL in the culture indicated that human bone marrow stromal cells transfected with FL could significantly express FL in vitro.

CONCLUSIONThe retroviral-mediated FL gene was expressed in bone marrow stromal cells and the biological activities of FL were detectable in the supernatant of the transfected cells. These results provide a basis for studies on hematopoietic regulation by gene transfected bone marrow stromal cells.

3T3 Cells ; Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Line ; Colony-Forming Units Assay ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Membrane Proteins ; genetics ; metabolism ; Mice ; RNA, Messenger ; genetics ; metabolism ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stromal Cells ; cytology ; metabolism ; Transfection