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BACKGROUND@#Cancer stem-like cells (CSCs) are a small subset of cells in tumors that exhibit self-renewal and differentiation properties. CSCs play a vital role in tumor formation, progression, relapse, and therapeutic resistance. B7-H3, an immunoregulatory protein, has many protumor functions. However, little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer (GC) stemness. Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism.@*METHODS@#GC stemness influenced by B7-H3 was detected both in vitro and in vivo . The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction, Western blotting, and flow cytometry. Sphere formation assay was used to detect the sphere-forming ability. The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments. The signaling pathway (Protein kinase B [Akt]/Nuclear factor erythroid 2-related factor 2 [Nrf2] pathway) of B7-H3 on the regulation of glutathione (GSH) metabolism was examined by Western blotting assay. Multi-color immunohistochemistry (mIHC) was used to detect the expression of B7-H3, cluster of differentiation 44 (CD44), and Nrf2 on human GC tissues. Student's t -test was used to compare the difference between two groups. Pearson correlation analysis was used to analyze the relationship between two molecules. The Kaplan-Meier method was used for survival analysis.@*RESULTS@#B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo . Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells, which was further confirmed by the experimental results. Meanwhile, stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism. Furthermore, Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway, and inhibition of AKT signaling pathway could suppress not only GSH metabolism but also GC stemness. mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2. Importantly, GC patients with high expression of B7-H3, CD44, and Nrf2 had worse prognosis ( P = 0.02).@*CONCLUSIONS@#B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway. Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.
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Humans , Cell Line, Tumor , Neoplasm Recurrence, Local , NF-E2-Related Factor 2/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Stomach NeoplasmsABSTRACT
Objective:To investigate the effects of B7-H3 molecule on clear cell renal cell carcinoma (786-O) metastasis.Methods:Lentiviral transfection method was used to construct 786-O cells stably expressing low level of B7-H3 (shB7-H3 group) and a negative control cell line (shNC group). RT-qPCR, flow cytometry and Western blot were used to assess the efficiency of lentiviral transfection. CCK-8 method was used to detect the proliferation of 786-O cells in the two groups. Flow cytometry was performed to detect the changes in cell cycle. Cell scratch test and Transwell assay were used to detect the differences in cell migration and invasion. Western blot was used to detect the expression of marker proteins in the process of epithelial-mesenchymal transition (epithelial-mesenchymal transition, EMT). Changes in the expression of chemokines and their receptors were analyzed by flow cytometry and RT-qPCR. Effects of anti-CCL4 antibody on cell migration and invasion were analyzed by Transwell assay.Results:Flow cytometry showed that 786-O cells highly expressed B7-H3 molecules and the lentiviral transfection method successfully constructed the cell line with lower expression of B7-H3 (786-O-shB7-H3) and control cell line (786-O-shNC). B7-H3 molecule had no significant effect on the proliferation of 786-O cells. No significant difference in cell cycle was found between the two groups. Compared with 786-O-shNC cells, the migration and invasion ability of 786-O-shB7-H3 cells was suppressed. Moreover, the expression of EMT-related marker proteins (fibronectin and N-cadherin) was reduced and the expression of E-cadherin was increased in 786-O-shB7-H3 cells. The expression of CCL4 and its receptor CCR5 in the shB7-H3 group was lower than that in the shNC group. After intervention with anti-CCL4 antibody, the migration and invasion ability of 786-O-shNC cells was reduced, while that of 786-O-shB7-H3 cells had no significant change.Conclusions:Knocking down the expression of B7-H3 molecule had no significant effect on the proliferation of 786-O cells, but could affect the EMT process of 786-O cells and reduce tumor migration and invasion ability, thereby inhibiting tumor progression.
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Objective To investigate the effects of costimulatory molecule B7-H3 on the prolifera-tion and invasion of human non-small cell lung cancer cell line A549. Methods Flow cytometry was used to detect the expression of B7-H3 at protein level on A549 cells. B7-H3-targeting siRNA was transfected into A549 by lentivirus to construct B7-H3-A549 cells, which were identified with Western blot and qPCR. Differences in proliferation between B7-H3-A549 and B7-H3+A549 cells were analyzed by CCK8 assay. Flow cytometry was performed to detect the changes in apoptosis and cell cycle after AnnexinⅤ-PE/propidi-um iodide ( PI) staining. Transwell assay was used to evaluate the migration and invasion of B7-H3-A549 and B 7-H 3+ A 549 cells . Expression of apoptosis-related proteins was detected by Western blot . Results (1) B7-H3 was highly expressed on A549 cells. A stable B7-H3-A549 cell line and its control cell line B7-H3+A549 were successfully prepared. (2) A549 cell proliferation was significantly reduced after knocking down B7-H3 expression. (3) The percentage of early apoptotic cells in B7-H3-A549 cell group was higher than that in B7-H3+A549 cell group, but no significant difference in the percentages of cells undergoing late apoptosis was found between the two groups. B7-H3-A549 cells were arrested at the G0/G1 phase of cell cy-cle. (4) Compared with B7-H3+A549 cells, B7-H3-A549 cells showed suppressed migration and invasion. (5) Enhanced expression of Bad and Caspase-3 and decreased expression of Bcl-2, P-AKT and MMP-9 were detected in B7-H3-A549 cells as compared with those in B7-H3+A549 cells, but no significant difference in the total AKT was observed. Conclusions Knocking down the expression of B7-H3 molecule in A549 cells could inhibit cell proliferation and invasion, induce cell cycle arrest at G0/G1 phase and promote cell apoptosis.
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Objective@#To understand effects of timing and duration of separation experiences from parents on emotion regulation of left-behind preschoolers,and to provide the reference for phychological instruction and intervention among the special groups of children.@*Methods@#Children’s emotion regulation strategy and the relevant information among 1 373 left-behind preschool children from Funan county in Fuyang.Qianshan county in Anqing,Changfeng county and Fexi county inFeixi were investigated.@*Results@#Children with left-behind experiences younger than 18 months old tend to use less cognitive restructuring (P=0.03) and alternative action strategies (P=0.00) than non-left behind children. Children separated from father less than 47 months (median) and 36 months (median) from mother tend to use less cognitive restructuring (P=0.00) and alternative action strategies (P=0.00) than non-left behind children.@*Conclusion@#Separation experiences from parents younger than 18 months old exert severe damage on children’s emotional regulation. With the duration of separation increases, children show resilience of emotion regulation, which might be a protective factor for negative emotion due to parent-child separation.
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Objective To explore the role of B7-H3 in the Tie2 expressing monocytes (TEMs) mediated angiogenesis of clear cell renal cell carcinoma (ccRCC).Methods Level of B7-H3 expression on TEMs surface was detected by flow cytometry in ccRCC tissues and normal renal tissues,which were obtained from April 2016 to August 2016 from 20 patients.Microvessel density (MVD) labeled by CD34 in high B7-H3 + TEMs group and low B7-H3 + TEMs group was detected by immunohistochemical examination in ccRCC specimens.B7-H3 + TEMs and B7-H3-TEMs were co-cultured with the 786-O cell lines,and B7-H3 + TEMs and B7-H3-TEMs culture supernatants were collected as conditioned medium,then the effect of B7-H3 + TEMs on angiogenesis was tested by tubule formation assay and mouse aortic ring assay.Results Flow cytometry showed that the frequency of B7-H3 expression on TEMs in ccRCC was (45.10 ± 17.78)％,and the frequency of B7-H3 expression in normal kidney tissues was (10.28 ± 4.28) ％.The frequency of B7-H3 expression on TEMs was significantly higher than that in normal renal tissues (P ＜ 0.001).The MVD of high B7-H3 + TEMs group (103.81 ± 29.28) was higher than that of low B7-H3 + TEMs group (76.55 ± 20.80) (P =0.027).The results of tube formation assay showed that the number of tubule formation of HUVEC in B7-H3 +TEMs group(55.25 ± 11.48) was significantly greater than that of B7-H3-TEMs group (31.34 ± 8.45) and blank control group (25.00 ± 6.74) (P ＜ 0.001).The results of mouse aortic ring assay showed that the number of neovascularization in B7-H3 + TEMs group(77.35 ± 18.47) was significantly greater than that of B7-H3-TEMs group (39.42 ± 8.29) and blank control group (28.79 ± 7.63) (P ＜0.001).Conclusions B7-H3 + TEMs can promote angiogenesis in ccRCC,and might act as an effective target in anti-angiogenic therapy for ccRCC.
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Objective To study the impacts of B7-H3 molecule on the proliferation of T lymphocytes in different activation conditions and on the secretion of relevant cytokines.Methods Peripheral blood samples were collected from healthy subjects to separate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation.T lymphocytes were isolated from some of the PBMCs and purified with T Cell Enrichment Kit.PBMC and purified T lymphocytes were activated by anti-CD3/CD28 monoclonal antibodies (McAb) in vitro.Flow cytometry analysis was used to detect the expression of CD28 and CTLA-4 on T lymphocytes at different time points for further analyzing the activation states of T lymphocytes.On this basis, human B7-H3-Fc fusion protein was added into the mixed co-cultivation system on days 0, 1, 2, 3 and 4, and Hu-Fc fusion protein was used as isotype control.CCK-8 method was performed to detect the proliferation of T lymphocytes in each group.ELISA method was used to detect the secretion of cytokines (IL-2, IL-10 and IFN-γ) and to analyze the immune responses induced by stimulating T lymphocytes at different states of activation with B7-H3.Results B7-H3 molecule significantly inhibited the quiescent T lymphocytes from secreting IL-2 and IL-10, but had no significant impact on IFN-γ secretion.Moreover, it significantly promoted the activated T lymphocytes to secret IL-2 and IFN-γ, but had no obvious impact on IL-10 secretion.Results of the cell proliferation assay showed that B7-H3 molecule inhibited the in vitro proliferation of T lymphocytes in the PBMC, but had no obvious impact on purified T lymphocytes.ConclusionThe regulatory effects of B7-H3 molecule on the immune functions of T lymphocytes vary with the activation states of T lymphocytes.
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Objective To study on the expression and clinical significance of programmed death ligant 1 (PD-1) in the development of colorectal cancer.Methods Flow cytometry (FCM) was used to detect and analyse the positive expression rate of PD-1 on CD3+ T cells in peripheral blood samples of 88 colorectal cancer patients and 74 healthy volunteers,and in sixpairs of tissue specimens (colorectal cancer tumor and adjacent normal tissues).Colon cancer tumor-bearing mice models were constructed by subcutaneous implantation of murine colon cancer cell line CT26,and the changes of positive expression rate of PD-1 on CD3+ T cells in spleens and transplanted tumor tissues were also detected by FCM in different time points during five,eight,11,14,17,20 days after the model was constructed.Finally,the experimental results were analyzed by t test or variance analysis.Results The positive expression rate of PD-1 on CD3+ T cells in peripheral blood of colorectal cancer patients was significantly higher than that in healthy volunteers ((29.25 ± 9.37) ％ vs (19.35 ± 7.37) ％,t =7.375,P＜ 0.01),and the positive expression rates were significantly increased in colorectal cancer patients with lower degree of differentiation,lymph node metastasis and higher Duke's stage.The positive expression rate of PD-1 on CD3+T cells was significantly higher in colorectal cancer tissues compared to adjacent normal tissues ((52.38±12.28)％ vs (24.72±4.78)％,t=5.143,P＜0.01).In the colon cancer tumor-bearing mice models,along with the growth of transplanted tumors,PD-1 positive expression rates on the CD3+T cells of tumor-bearing mice spleens and transplanted tumors showed a gradually rising tendency.From the eighth day to the 20th day after subcutaneous transplanted colon cancer cells,the average positive expression rate of PD-1 on the CD3+T cells of tumor-bearing mice spleens rose from (52.83±6.17)％ to (77.30± 6.84) ％,and the average positive expression rate of transplanted tumors rose from (60.43 ± 2.77)％ to (90.47±4.31) ％.Conclusion There is a correlation between the expression of PD-1 on mature T cell surface and the development of colorectal cancer,and the high specific expression of PD-1 may promote the invasion and metastasis of colorectal cancer.
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Objective To explore the clinical significance of B7 family homology factor-3 (B7-H3),an expression membrane type of myeloid-derived suppressor cell (MDSC),in patients with acute pancreatitis (AP).Methods A total of 63 patients with AP initially treated in the Emergency Department at the First Affiliated Hospital of Soochow University from January,2014 to December,2015 were selected.Of them,25 suffered from mild AP (MAP),20 had moderate AP (MSAP) and 18 had severe AP (SAP).Another 20 healthy subjects with matching age and gender served as the control group.All patients with AP conformed to the diagnostic criteria of Guidelines or Diagnosis and Treatment of Acute Pancreatitis set in 2013 in China.Patients with other underlying diseases that might influence the clinical outcomes were excluded,including those with tumors,autoimmune diseases,viral infections,trauma and other disorders.A flowcytometer was used to detect the expression rate of MDSC in peripheral venous blood and the expression of B7-H3 on MDSC membrane.The continuous monitoring was carried out for 24 h,48 h and 72 h in patients with AP.Results Compared with healthy subjects,the MDSC cells in patient groups 24 hours after AP onset increased notably (P ＜0.01) especially the highest increase in the SAP group,followed by the MSAP group and the lowest in the MAP group.There were significant differences in pairwise comparisons (P ＜ 0.05).From successive observation of each group,there was no significant difference in MDSC between the MAP group and the MSAP group 24 hours,48 hours and 72 hours after AP onset.However,MDSC reached its peak 48 hours after AP onset,but it declined 72 hours after AP onset in the SAP group (P ＜ 0.05).B7-H3 expressed significantly 24 hours after AP onset,but there was no expression of B7-H3 in the healthy group.Meanwhile,B7-H3 was expressed most highly in the SAP group,followed by the MSAP group and lowest in the MAP group.There were significant differences in expression of B7-H3 found in pairwise comparisons (P ＜ 0.05).The successive observation showed that there was no significant difference in B7-H3 expression between the MAP group and the MSAP group 24 hours,48 hours and 72 hours after AP onset.However,there was a trend of increase in B7-H3 expression as time prolonged found among 24 hours,48 hours and 72 hours after AP onset in the SAP group (P ＜ 0.05).Conclusions The expressions of MDSC and B7-H3 were high in AP,and there were significant differences in both expressions among MAP,MSAP and SAP groups.These phenomena offer clues in further understanding about the immunological disorders during AP giving better guidelines for clinical practice.
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Objective To investigate the potential relationship between neck circumference and obesity related indexes and metabolic disorders associated with insulin resistance.Methods A random cluster sampling method was used to identify study population among the 4 412 60 -70 years old permanent residents in Renqiu region.Face to face health questionnaire,physical examination,laboratory tests were used.According to the gender group,the correlation between neck circumference and obesity related indexes and metabolic disorders associated with insulin resistance were analyzed.Results Comparing neck circumference and waist circumference,waist height ratio, and body mass index(BMI) of man and woman respondents,the differences were statistically significant.Neck circum-ference and waist circumference,waist height ratio,and BMI had positive correlation(male:r =0.752,0.695 and 0.761.W:r =0.707,0.655,0.721,all P <0.01).Increased trends of neck circumference,waist circumference,waist height ratio and BMI coincided with increased trend of thypertension,diabetes,hyperlipidemia and hyperinsulinemia and hyperlipidemia,and no gender differences.With the increase of the neck circumference,the incidence of above mentioned diseases also increased accordingly.Conclusion Neck circumference was associated with obesity related indexes and metabolic disorders associated with insulin resistance.Neck circumference measurement can be used as an effective indicator of central obesity,and had great significance for early prediction and prevention of metabolic disorders associated with clinical insulin resistance.
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Objective To investigate the feasibility of using leukocytes that were filtered out by LeukoReduction System ( LRS) to replace conventional human peripheral blood leukocytes in experimental researches and to comparatively analyze the differences between them in vitro biological functions and pheno-types of T cells. Methods Mononuclear cells were isolated from LRS-separated leukocytes and whole blood sample that collected from the same person by using Ficoll. Fluorescence-activated cell sorting ( FACS) was performed to analyze the phenotypes of T cells. CD3+T cells were sorted out by using magnetic beads. The T cells that were collected by using two different ways were incubated with anti-CD3 and anti-CD28 antibodies and IL-2 in vitro for 10 days. Several assays including cell counting, FACS and cytometric beads array ( CBA) were performed to comparatively analyze the differences in biological functions and phenotypes of T cells that were isolated by different methods. Results The phenotypes of T cells isolated from LRS filter and whole blood sample were highly similar at the initial stage. The sorting rate of CD3+T cells form LRS filter reached a high level and met the requirements for experimental researches. No statistically significant differ-ences in cell count, phenotype, expression of costimulatory molecules and cytokine secretion were observed between T cells isolated from LRS filter and whole blood sample. Conclusion This study suggested that the T cells isolated from LRS filter could be used as an alternative to whole blood T cells for fundamental resear-ches since they were similar in cell vitality, phenotype and biological functions. It provided a new way to solve the problem of blood shortage in clinic and scientific research.
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Objective To investigate the level of subpopulations of myeloid-derived suppressor cells (MDSCs) in elderly tumor-bearing mice versus elderly tumor-free mice,and to study the difference in immune suppressive functions between different subpopulations and their mechanisms.Methods A total of 20 healthy C57BL/6 elderly mice(aged 18-20 months) were randomly chosen to establish Lewis lung cancer models.The amount of monocytic-MDSCs (MO-MDSCs) and polymorphonuclear granulocytic-MDSCs(PMN-MDSCs) in tumor-free and tumor-bearing elderly mice was evaluated by using flow cytometry.MO-MDSCs and PMN-MDSCs were separated with Magnetic-Activated Cell Sorting (MACS) MicroBeads and their morphological characteristics were observed after May-Grunwald-Giemsa staining.The effects of MO-MDSCs and PMN-MDSCs on the proliferation of T cells were determined by Brdu-enzyme-linked immunosorbent assay(ELISA).And the immune suppressive mediators secreted by the subpopulations were detected by Real-time polymerase chain reaction(PCR).Results Compared to the tumor-free group,the proportion of MO-MDSCs in the spleen of tumorbearing group were increased [(12.44± 1.20) ％ vs.(38.42±3.66) ％,t=5.67,P＜0.001],while PMN-MDSCs were not [(10.34±0.68) ％ vs.(12.18±1.27) ％,t=2.21,P=0.09].The result of Brdu-ELISA showed that MO-MDSCs could suppress the proliferation of T cells [(0.30 ± 0.18) vs.(3.38±0.96),t=8.33,P＜0.001],while PMN MDSCs could not [(2.69±0.45)vs.(3.38±0.96),t =1.72,P=0.11].The result of PCR showed that as compared with PMN-MDSCs,Mo-MDSCs had the increased expression levels of arginase-1 (ARG-1),inducible nitric oxide synthase (iNOS),interleukin-10 (IL-10),interferon-γ(IFN-γ) (t =4.31,8.89,1.70,3.13,respectively,P ＜ 0.01 or 0.05),while the expression levels of interleukin-13 (IL-13),transforming growth factor-β(TGF-β) had no differences (t=4.94 and 2.75,P =0.39 and0.47).Conclusions MO-MDSCs are significantly increased in elderly Lewis lung cancer mice models.MO-MDSCs could mediate lung tumor evasion by suppressing the proliferation of T cells through highly expressing ARG-1,iNOS,IL-10 and IFN-γ.
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Background:The abnormal expression of costimulatory molecules is closely related to immune escape of tumor cells. Programmed death-1(PD-1)is an important negative costimulatory molecule,and can induce and maintain the immune tolerance of tumor cells by binding with related ligands,thus promotes the development and progress of tumor. Aims:To investigate the expression of peripheral blood PD-1 and its clinical significance in patients with colorectal cancer. Methods:Eighty-eight patients with colorectal cancer from March 2015 to September 2015 at the First Affiliated Hospital of Soochow University were enrolled,and 16 healthy volunteers were served as controls. Level of soluble PD-1(sPD-1)was determined by ELISA. The expression of PD-l on CD3 + T cells in peripheral blood was measured by flow cytometry. Results:Level of sPD-1 and expression of PD-l on CD3 + T cells in peripheral blood in colorectal cancer patients were significantly higher than those in controls(P ﹤ 0. 05). Level of sPD-1 in colorectal cancer patients was closely related to tumor differentiation,lymph node metastasis and Dukes’stage( P ﹤ 0. 05),but not related to gender,age and tumor location(P ﹥ 0. 05). Conclusions:Peripheral blood PD-1 is highly expressed in patients with colorectal cancer,and is positively correlated with tumor stage and metastasis. The detection of PD-1 is helpful to estimate the progress of colorectal cancer,and it may become a new tumor marker or a target for anti-tumor targeted therapy.
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Objective To explore the immune property and possible mechanism of the CD71+ CD235a+ nucleated erythroid cells from peripheral blood in elderly,as compared with those of healthy young.Methods Peripheral blood obtained by venipuncture from healthy young(n=59,mean age=28 years) and elderly(n=78,mean age=68 years)donors were measured by flow cytometry to evaluate the frequency of the CD71+ CD235a+ cells in peripheral blood mononuclear cells (PBMC).In vitro assays,CD71+ CD235a+ cells were sorted by flow cytometry and T cells were sorted using CD4+T cell isolation kit,then the T cell proliferation assays were conducted by following groups:T cell + CFSE group;T cell +CFSE co-cultured with CD71+ CD235a+ cells from the young donors;T cell + CFSE co-cultured with CD71+ CD235a+ cells from the older donors.Real-time PCR were used to identify the expression of the immune cytokine secreted by CD71+ CD235a+ cells.Results (1)the results showed a significant increase in the percentage of CD71+ CD235a+ cells in the elderly compared with the young [(9.93± 2.95)％ vs (1.96 ± 1.16)％,t =3.37,P ＜ 0.001];(2)the CD71+ CD235a+ cells from the older donors could suppress the proliferation of the T cells (t =2.91,P＜ 0.05)while from the young group we could not observe this phenomenon(t =0.387,P＞0.05).(3)The results of Real-time PCR revealed that,compared with CD71+ CD235a+ cells from the young,CD71+ CD235a+ cells from the elderly expressed higher levels of Arg-2 (t =9.04,P＜0.01),IL-1β,IL-6 and TGF-β(t =4.51,5.46,6.92,all P＜0.05).Conclusions There are higher frequencies of CD71+ CD235a+ cells in aged samples than the young.And the CD71+CD235a cells from the elderly could secrete more Arg-2,IL-1β,IL-6 and TGF-β to suppress the T cell proliferation.
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Objective To study characteristics and immune mechanisms of CD11b+ GR-1-myeloid-derived suppressor cells (CD11b+ GR 1+ MDSCs) in elderly mice,as compared with those of healthy young mice.Methods Totally 20 healthy C57BL/6 young mice (aged 1-2 months) and 20 elderly mice (aged over 18 months) were randomly chosen and splenetic CD11b+ GR-1+ MDSCs were sorted with the MDSCs Isolation Kit.In vitro assays,the effects of young and elderly CD1 1b+ GR 1+ MDSCs on the proliferation of T cells were determined by Brdu Elisa.Transwell co-culture and real-timePCR were used to identify the mechanisms of different immune suppressive functions of CD11b+GR 1+ MDSCs sorted from young mice and elderly mice.Results Compared with young MDSCs,elderly MDSCs could evidently suppress the proliferation of T cells (t=8.67,P＜0.001),and this function could be reversed by trans-well co-culture (t=6.93,P＜0.001).The results of realtime PCR revealed that,compared with young MDSCs,elderly MDSCs expressed higher levels of arginase-1 (ARG-1),inducible nitric oxide synthase (iNOS),reactive oxygen species (ROS),interleukin 10 (IL-10),IL13 and transforming growth factor (TGF)-β (t=9.04,4.86,7.04,6.92,4.51,5.46,respectively,P＜0.05 or P＜0.01).Conclusions CD11b+GR-1+MDSCs sorted from healthy elderly mice can evidently suppress the proliferation of T cells through cell-cell contact and secretion of suppressive medium.
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Objective To investigate the clinical value of sB7-H3 in predicting the severity of acute pancreatitis at early stage. Methods By using the double antibody sandwich ELISA method, the level of plasma sB7-H3 was measured at 24h after onset of abdominal pain in 75 patients with acute pancreatitis (MAP30, MSAP20, SAP25), and 20 healthy persons were enrolled in the controlgroup.The sensitivity and specificity correlations of sB7-H3 in acute pancreatitis with severity degree , as well as with the clinical detection index , were also evaluated. Results The level of plasma sB7-H3 at 24 h in the AP group was significantly higher than that in the healthy control ( HC) group (t = 3.925, P = 0.0002), however, no significant difference was found between the MAP groep and the HC group (P>0.05). The levels of plasma sB7-H3 in the MSAP and the SAP group were significantly higher than that in the HC group (P<0.05和P<0.001)or the MAP group (P<0.05 和P<0.01);The level of plasma sB7-H3 in the SAP group was also markedly higher than that in the MSAP group (P < 0.01). sB7-H3 had a linear positive correlation with LDH、hs-CRP、WBC(P<0.05). ALB had a linear negative correlation with and Ca (S)(P<0.05). By the cutoff of sB7-H3, the sensitivity and specificity to judgethe above moderate pancreatitis were 88.9%and 83.3%,and to judgethe SAP were 96%and 96%. Conclusion sB7-H3 has important clinical value to judge the severity of acute pancreatitis at early time with high sensitivity and specificity , with a linear correlation with the clinical severity index.
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Objective To evaluate the effects of B7-H3 gene transfection on 18F-FDG uptake and 18F-FLT uptake in prostate cancer cells.Methods The absorption (A) values of untransfected prostate cancer(RM1) cells and B7-H3 gene-transfected RM1 (RM1-B7-H3) cells were detected at different culturing time points (0.5,1,2,3,4 and 5 d) with cell counting kit-8 (CCK-8) test.Cell cycle phase distribution of RM1 and RM1-B7-H3 cells was measured with flow cytometry.18F-FDG uptake of RM1 and RM1-B7-H3 cells was measured with γcounter and calculated under different conditions:5× 104-5× 106 cells; 0-11.0 mmol/L glucose; 20-120 min incubation in 37 ℃.18F-FLT uptake of RM1 and RM1-B7-H3 cells was measured in 1×106 cells under incubation for 100 min at 37 ℃.After administering anti-B7-H3 monoclonal antibody 4H7,18F-FDG uptake of RM1-B7-H3 cells was measured.The data were analyzed using one-way analysis of variance and two-sample t test.Results The A values of RM1-B7-H3 cells after being incubated for 1,2 and 3 d were higher than those of RM1 cells(1.59±0.23,2.26±0.15 and 2.01±0.60 vs 1.22±0.14,1.10± 0.09 and 1.04±0.15,t=3.923,19.228,4.467,all P＜0.01).There was no statistical significance between the 2 groups at other time points (t=-0.094,0.858,2.000,all P＞0.05).The ratios of RM1-B7-H3 cells in G1,S and G2/M phases were(32.96±2.56) ％,(39.11 ±2.57) ％ and (27.94±0.21) ％,respectively.The ratio of S phase in RM1-B7-H3 cells was higher than that in RM1 cells ((32.76±1.90)％,t=3.442,P＜ 0.05).18F-FDG uptake of the both cell lines decreased with the increase of glucose concentrations,while the uptake went up with the increase of cell number and incubation time.With the cell number of 1.0× 106,incubation time of 100 min and temperature of 37 ℃,the 18F-FDG uptake of RM1-B7-H3 and RM1 cells was (55.07±3.99)％ vs (44.16±3.60)％ (t=4.977,P＜0.01) ; and 18F-FLT uptake of RM1-B7-H3 and RM1 cells was (5.25±0.81)％ vs (3.33±0.64)％ (t=4.567,P＜0.01).After treated with antibody 4H7,18F-FDG uptake of RM1-B7-H3 cells ((45.36±2.92) ％) was lower than that of untreated group (F=10.001,P＜ 0.01).Conclusion B7-H3 gene transfection may promote the metabolism and proliferation of prostate cancer cells,and thereby increase the 18F-FDG uptake and 18F-FLT uptake.
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Objective To explore the expression of negative costimulatory molecule B7-H1 of myeloid derived suppressor cells(MDSCs) and its roles in age-related immunosuppressive functions.Methods 36 C57BL/6 mice were divided into 2 groups:the elderly group(12-month-old,n=18) and the young group(4-8-week-old,n=18).The expressive level of negative costimulatory molecules of MDSCs in the two groups was measured by flow cytometry(FCM).Besides,MDSCs were sorted by using MDSC isolation kit and T cells were obtained by grinding lymph nodes.To explore the influence of B7-H1 on the MDSCs' immunosuppressive functions,T cell proliferation and intervention assay was conducted using the five following groups:T cells group,T cells + CFSE group [T cells with carboxyfluorescein diacetate succinimidyl ester(CFSE)],T cells +-CFSE cocultured with MDSCs from the young group,T cells + CFSE cocultured with MDSCs from the elderly group,and T cells + CFSE cocultured with MDSCs from elderly group and anti-B7-H1 blocking antibody.The effect of B7-H1 on T cell proliferation in MDSCs were observed.Results The expression level of B7-H1 of the MDSCs was significantly increased in the elderly group as compared with the young group(t=3.27,P＜0.05).T cell proliferation assay revealed that T cells were remarkably suppressed by MDSCs in elderly group as compared to the youth group(t=5.42,P＜0.05).The suppression of T cells by MDSCs was dramatically reversed by anti-B7-H1 blocking antibody in elderly group(t=8.28,P＜0.05).Conclusions B7-H1,a molecule expressed on the surface of MDSCs,plays a key role in the age-related immunosuppressive function of MDSCs.
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Objective To explore the immunomodulatory effects of 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) in the treatment of experimental colitis induced by dextran sulfate sodium (DSS) in mice.Methods According to random number table,thirty BALB/c mice were randomly divided into control group,model group,low dose,moderate dose,and high dose intervention group.Mice of model group,low dose,moderate dose and high dose intervention group drank 5％ DSS solution for seven days to create colitis model.On the 1st,3rd,5th,7th day,the mice of low dose,moderate dose and high dose intervention group were intraperitoneal injected with low,moderate,and high dose of 1,25(OH)2D3 (50,100 and 200 ng/each mouse,respectively).Mice of control group and model group were intraperitoneal injected with sterile soybean oil as control.The observed indicators included disease activity index (DAI) and colonic histopathological score (HPS).On the 8th day,all mice were sacrificed.The expression of interferon (IFN)-γ,interleukin (IL-17) and IL-21 in mice colon tissues and spleens at mRNA and protein level were measured by reverse transcription-polymerose chain reaction (RT-PCR) and flow cytometry,respectively.The data were analyzed by one way ANOVA.LSD-test or Tamhane test were performed for comparison in groups.Results Compared with the control group,the DAI and colitis HPS of mice in the model group significantly increased (0.33±0.52 vs 7.33±1.03,0.17±0.41 vs 12.00±0.63).Compared with the model group,the DAI and colonic HPS of intervention groups treated with 1,25 (OH)2 D3 declined with varying degrees (2.83 ± 0.40,2.83±0.75,2.33±0.52 and 10.83±0.98,7.50±0.84,6.67±0.52,LSD-t=0.39 and 0.41,all P＜0.01).The expression of IFN-γ,IL-17 and IL-21 of the model group were significantly higher than those of the control group.The expressions of IFN-γ,IL-17 and IL-21 of intervention group were signifiantly lower than that of the model group (mRNA:LSD-t =0.12,0.13,0.09; protein:F =20.61,22.46,4.80,all P＜0.01).Conclusion 1,25 (OH)2D3 might have a direct role on T-cell phenotype,down-regulate effective cytokines IFN-γ,IL-17 and IL-21 and then play an interventional role.
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Objective To investigate the effect of B7-H3 on human hepatocellular carcinoma cell line HepG2 mediating regulation on human peripheral blood CD8+T cell activation,cell cycle and secretion of IL-17.Methods The expression of the B7-H3 on HepG2 cells was detected by RT-PCR and FCM respectively.B7-H3 was silenced by PGPU6/GFP/neo-B7-H3shRNA plasmid via cathodolyte liposome transfection method.CD8+T cells were sorted from healthy human peripheral blood with immunomagetic beads.The effect of HepG2 cells on activation,cell cycle and cytokine secretion of CD8+T cells which was stimulated by PHA or PMA respectively were analyzed by FCM.Results B7-H3 was highly expressed on HepG2 cells,and PGPU6/GFP/neo-B7-H3shRNA plasmid could effectively block down its expression.Otherwise,HepG2 cells could inhibit the expression of CD69,the early activation phenotype of T cell,blockade B7-H3 on HepG2 cells could significantly attenuate the inhibitory effects.Likewise,blockade B7-H3 on HepG2 cells apparently reversed the inhibitory effects of HepG2 cells on CD8+T cell cycle through down-regulating the cell number in G0/G1 phase and up-regulating the cell number in S phase;Moreover,HepG2 cells caused a sharp increase of IL-17 which was secreted by CD8+T cells and the level of IL-17 was further up-regulated after blocking down B7-H3.Conclusion HepG2 cells highly expressed B7-H3 that could promote the inhibitory the effect of HepG2 on expression of CD69 and cell cycle of CD8+T cells.HepG2 cells were able to up-regulate the level of IL-17 secreted by CD8+T cells,in which B7-H3 played an inhibitory role.
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Objective To investigate the value of B7-H3 in expressed prostatic secretions (EPS) in differential diagnosis of patients with inflammatory elevation of PSA in t-PSA gray zone (4-10 ng/ml). Methods One hundred and sixteen patients from the ages of 19 to 80 years (mean, 40 years) were stu-died. In the group there were 91 chronic prostatitis (CP) patients (mean age 31 years, 19-49 years), including 11 chronic bacterial prostatitis (type II) patients, 26 inflammatory nonbacterial prostatitis (IIIA) patients and 54 noninflammatory nonbacterial prostatitis (IIIB) patients. Transrectal ultrsound guided prostate biopsy was performed on 25 patients (mean age 71 years, 62-80 years) with t-PSA in gray zone (7.21±2.60 ng/ml). Five had positive results, Gleason score was 6 in two cases, 7 in two cases and 8 in one case. Twenty patients had negative results, of whom 11 patients had inflammatory cell infiltration. EPS was collected by transrectal massage, and Enzyme-linked immunosorbent assays (ELISA) were performed for B7-H3 detection. In addition, 11 normal male controls with a mean age of 30 years (24-46 years) were recruited into the study. Volunteers were excluded if they had a history of genitourinary symptoms or surgery.Results The EPS B7-H3 levels of controls, II, IIIA, IIIB groups were 49.81±11.54, 19.33±13.90, 17.67±15.76, 25.14±13.44 ng/ml, respectively. The levels of EPS B7-H3 in positive biopsy, noninflammatory negative biopsy and inflammatory negative biopsy groups were 26.30±16.32, 30.23±18.42, 10.11±5.42 ng/ml, respectively. The highest levels were found in the control group (P0.05). The EPS B7-H3 levels in the inflammatory negative biopsy group were statistically lower than in positive biopsy and noninflammatory biopsy groups (P0.05). Receiver operating curve (AUC=0.883, P=0.001) utilizing EPS B7-H3 levels≤16.24 ng/ml identified patients with inflammatory elevation of PSA with a sensitivity of 78.6% and a specificity of 81.8% from patients with t-PSA in gray zone. Conclusion The EPS B7-H3 detection provides a new way for differential diagnosis of patients with inflammatory elevation of PSA in t-PSA gray zone resulting in a reduction of unnecessary prostate biopsy.