Effects of oxymatrine on lymphocyte proliferation and the quantity of regulatory T cells

AIM: To analyze the effects of oxymatrine (OMT) on the quantity of murine regulatory T cells (Tr cells) in the peripheral blood and mouse lymphocyte proliferation stimulated by Con A, and to probe into the immunological mechanism that OMT treats allergic contact dermatitis (ACD).METHODS: An ACD mouse model stimulated by dinitrofluorobenzene (DNFB) was established. Different dosages of OMT, PBS and hydrocortisone (HCT) were intraperitoneally injected (IP) into the mice. Blood samples were collected at〖JP+2〗 1 d, 7 d, 14 d, 21 d and 28 d, then the T cells were isolated and marked with anti-CD3, anti-CD4, anti-CD25 three-colored immune fluorescence antibody to detect the quantity of CD4+CD25+ T cells with flow cytometry. The fluorescence intensity changes of lymphocytes which were isolated from mouses lymph node and co-stimulated by polyclonal stimulator Con A and OMT were examined by carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining and flow cytometry. RESULTS: OMT at concentrations of 500, 125 and 31 mg/L had the ability to restrain the proliferation of lymphocytes from lymph node in a dose dependent manner. However, OMT at concentrations of 16, 8, 4 and 2 mg/L promoted the proliferation of T lymphocytes from lymph node, but was not obviously dependent on its concentration. Intraperitoneal injection of OMT increased the numbers of CD4+CD25+T cell in peripheral blood obviously (P<0.01). CONCLUSION: The effects of OMT on the proliferation of T lymphocytes from mouses lymph node cells are observed, OMT also increases the CD4+CD25+T cells in the peripheral blood, implying that OMT is a kind of immunoregulator with dual effects.     

Inhibitory effects of genistein on mouse allergic contact dermatitis

AIM: To investigate the inhibitory effects of genistein (Gen) on mouse allergic contact dermatitis (ACD). METHODS: The animal model of ACD was induced by DNFB. The effects of different doses of Gen on mouse ear swelling，body weight, histopathological changes in mouse ear skin, thymus index and spleen index were observed. RESULTS: All groups of Gen inhibited mouse ear swelling induced by DNFB significantly. The infiltration of inflammatory cells and thymus index were also reduced. However, the increase in mouse body weight was not affected. Low dose of Gen increased spleen index, high dose of Gen decreased spleen index. CONCLUSION: Genistein has significant inhibitory effects on mouse ACD induced by DNFB.     

Effects of probucol on the proliferation of rat vascular smooth muscle cells stimulated by basic fibroblast growth factor and hydrogen peroxide

AIM: To investigate the effect of probucol on proliferation of rat vascular smooth muscle cells(VSMC) stimulated by basic fibroblast growth factor (bFGF) and/or hydrogen peroxide(H₂O₂). METHODS: Effects of probucol on VSMC proliferation and DNA synthesis stimulated by bFGF and/or H₂O₂ were observed by means of MTT test, cell number count and [3H]-TdR incorporation. RESULTS: ①Probucol significantly inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, with dosage-dependent manner. Cell number, A value and [3H]-TdR incorporation in group probucol+bFGF and group probucol+H₂O₂ were reduced by 40.0%, 39.1%, 45.5% and 46.9%, 45.0%, 39.5%, respectively, compared with group bFGF and group H₂O₂ (P＜0.05, P＜0.01, respectively). ②Pretreatment of VSMC with probucol for 24 h prior to bFGF and/or H₂O₂ stimulation exhibited significant inhibiton of VSMC proliferation and DNA synthesis, but after prestimulation by bFGF and/or H₂O₂ for 24 h, probucol had no influence on VSMC proliferation and DNA synthesis (P＞0.05). CONCLUSION: Probucol dramatically inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, but had no inhibitory effect on the cell proliferation prestimulated by bFGF and /or H₂O₂.     

The inhibitory effect of quercetin on in vitro activation of T lymphocytes

AIM:To investigate the inhibitory effect of quercetin on in vitro activation of T lymphocytes by polyclonal activators with CD69 expression as an activation marker.METHODS:After being separated from lymphoid nodes of a C57BL/6 mouse, the lymphocytes were exposed to polyclonal activators (PDB or Con A) with or without quercetin. Then they were harvested at 2 h, 6 h and 24 h, respectively. The expressional rates of CD69 on T lymphocytes were assessed by two-color immunofluorescent staining and flow cytometry, and the inhibitory rates of quercetin at different time points were estimated.RESULTS:Quercetin had no effect on the expressional rate of CD69 on T lymphocytes under resting states. After the stimulation with PDB or Con A, the expressional rates of CD69 on T lymphocytes in the present of quercetin (10 μmol/L) showed significant decrease compared with those of control groups at different time points (P＜0.01). The inhibitory rate of quercetin on CD69 expression stimulated by PDB dropped sharply from 2 h to 24 h, whereas the inhibitory rate of quercetin on Con A action were relatively stable.CONCLUSION:Quercetin has inhibitory effects on the activation of T lymphocytes by Con A or PDB, suggesting that the action site of quercetin may be on PKCθ or its downstream. Furthermore,these inhibitory effect seems to be reversible.     

中国园艺学会第七届青年学术讨论会

中国园艺学会第九届第8次常务理事扩大会决定,“中国园艺学会第七届青年学术讨论会”由山东农业大学园艺科学与工程学院和山东省园艺学会承办,将于2006年7月或8月在山东泰安举行。会议交流主题:(1)园艺作物种质资源、遗传育种与生物技术;(2)园艺作物有机、无公害及标准化安全生     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

Role of humoral immune response in the protection induced by H. pylori vaccine with chitosa as adjuvant

AIM:To study the immunological protection of H. pylori vaccine with chitosa as adjuvant. METHODS:One-grade female BALB/c mice were randomly divided into nine groups and immunized by ①PBS alone; ②chitosan solution alone; ③chitosan particles alone; ④H. pylori antigen alone; ⑤H. pylori antigen plus chitosan solution; ⑥H. pylori antigen plus chitosan particles; ⑦H. pylori antigen plus CT; ⑧H. pylori antigen plus chitosan solution and CT; ⑨H. pylori antigen plus chitosan particles and CT. At 4 weeks after the last immunization, these mice were challenged by alive H. pylori（1×1012CFU/L） twice at two-day intervals. At 4 weeks after the last challenge, these mice were all killed and gastric mucosa were embedded in paraffin, sectioned and assayed with Giemsa staining. The other gastric mucosa were used to quantitatively culture with H. pylori. ELISA was used to detect H.pylori IgA in saliva and gastric mucosa and anti-H.pylori IgG, IgG1, IgG2a in serum, and immunohistochemical method was used to examine sIgA in gastric mucosa. RESULTS:①In the groups with chitosan as adjuvant, 60% mice achieved immunological protection, which was according to that with CT as adjuvant (58.33%), and was significantly higher than H. pylori antigen alone and other groups without H. pylori antigen（P<0.01 or P<0.05）. While the rates of protection in the groups with chitosan plus CT as adjuvant were 84.62%,85.71% and the H. pylori colonization score in it was significantly lower than that in the groups with CT as an adjuvant and without adjuvants（P<0.01 or P<0.05）. ②The each isotype IgG levels induced by H. pylori vaccine with adjuvants were significantly higher than those in control group and non-adjuvant groups（P<0.01 or P<0.05），while the anti-H. pylori IgG levels in the groups with CT plus chitosan as adjuvant were significantly higher than those in the groups with CT or chitosan as an adjuvant alone（P<0.05）. ③ The labeling index for sIgA-positive lumen of glands and special anti-H. pylori IgA levels in gastric mucosa in the groups with chitosan as an adjuvant had no difference with those in the group with CT as an adjuvant（P>0.05）and were significantly higher than those in non-adjuvant groups, while those in the groups with chitosan plus CT were significantly higher than those in the group with CT as an adjuvant（P<0.01 or P<0.05）. CONCLUSION:H. pylori vaccine with chitosan as adjuvant could protect against H. pylori infection, suggested that chitosan is a mucosa adjuvant of H. pylori vaccine.     

Effect of TMB-8 on the activation,proliferation and cell-cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of ［8-(diethylamino) octyl-3, 4, 5 -trimethoxybenzoate］ (TMB-8), an intracellular Ca2+ antagonist, on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (Con A) in vitro. METHODS: After stimulated with Con A, T cells were treated with different concentrations of TMB-8 alone and its combination with cyclosporine A (CsA). The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation-related index was determined by carboxyl fluorescin diacetate succinmidyl ester (CFDA-SE) flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining.RESULTS: After 6 h culture, the activation rate of CD69+ T cell in Con A group was (74.88±1.88)%. 10, 20 and 40 μmol/L of TMB-8 inhibited the expression of CD69 (P<0.01), especially in 40 μmol/L (52.55%±1.54%). After 48 h and 72 h culture, the PI of Con A group was 1.24±0.01, 2.05±0.07, respectively. TMB-8 with the concentration up to 5 μmol/L exerted a definite inhibitory effect on the proliferation with a maximal inhibition in 40 μmol/L(P<0.01). In the combination of 10 μmol/L of TMB-8 with 25 μg/L of CsA, an evident synergistic effect was observed (P<0.01). Moreover, the cell-cycle distribution analysis showed that after 48 h culture, the concentration of TMB-8 over 10 μmol/L showed an evident suppression in S phase.CONCLUSION: TMB-8 significantly inhibites the early steps of the Con A-induced T cell activation and proliferation, as well as the progression of T lymphocytes in S phase.     

Expression of proteins in mouse thymocytes induced by low dose X-rays and their biological activity

AIM:To observe the expression of proteins induced by whole-body irradiation in mice with low dose X-rays and their biological activity.METHODS:To analyse the expression of protein induced by whole-body irradiation with 75mGy X-rays and sham-irradiation, the method of gel filtration with Sephadex G-100 and HPLC were used.The biological activity of protein expressed in thymocytes was observed by mouse splenocyte proliferation and chromosome aberration of human peripheral blood lymphocytes.RESULTS:HPLC analysis showed that there was a marked decrease in expression of 24.5 kD protein molecule in the fractions of thymocyte extract in comparison with the corresponding fractions from the sham-irradiated control mice. These protein fractions from the thymocytes of the irradiated mice at the concentration of 6.25 mg/L showed both inhibitory effect on normal T cell proliferation and protective effect on the chromosome damage induced by high dose radiation. CONCLUSION:Down regulation of 24.5 kD protein molecule expression may have implications in the mechanism of immunoenhancement and cytogenetic adaptive response induced by low dose radiation.     

桦褐孔菌发酵液提取物小鼠体内抗H_(22)肝癌的作用

为确定桦褐孔菌发酵液提取物的毒性级别,研究对H22肝癌小鼠的抗肿瘤及免疫调节作用。依据2004年实施的《急性毒性试验国家标准》进行急性毒性研究和级别鉴定,同时建立H22肝癌的荷瘤小鼠模型进行小鼠体内抗肿瘤的研究,并采用MTT法检测荷瘤小鼠自然杀伤细胞活性(NK)及T淋巴细胞增殖情况。结果表明,桦褐孔菌发酵液提取物(IOFE)的LD50为15.02 g·kg-115 g·kg-1,属无毒级别。与阴性对照和阳性对照比,体重、采食量均稳定增长,抑瘤率为44.25%,可显著提高NK细胞活性67.13%(p0.01),但对T淋巴细胞增殖率效果不明显。桦褐孔菌发酵液提取物具有较好的抗肿瘤作用,且毒性极低,抗肿瘤作用可能是通过提高机体免疫活性尤其是NK细胞的杀伤活性实现的。     

Inhibitory effect of berberine on the activation and proliferation of T lymphocytes

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 μmol/L and 50 μmol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 μmol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G₀/G₁ phase to S and G₂/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.     

WT1 peptide-loaded DC triggers cytotoxic T lymphocytes and killing effects on K562 cells in vitro

AIM: To study the effects of WT1 peptide-loaded dendritic cells (DC) stimulating the cytotoxic T lymphocytes (CTL) on K562 cells in vitro. METHODS: DC were generated from normal human peripheral blood mononuclear cells (PBMC) in the presence of granulocyte-macrophage colony stimulating factor(GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α) , DC were cultured with WT1 peptides , and then triggered T cells into specific CTL. RESULTS: Most suspended cells exhibited distinctive morphological features of DCs and they stimulated proliferation of allogenic lymphocytes. Under the effector : target ratio of 20∶1, CTLs derived from cultures with DC and WT1 peptides were showed 86.1%±26.8% cytotoxicity against K562 cells, cytotoxicity by CTLs derived from cultures with unloaded DC against K562 cells were 47.1%±20.8% and cytotoxicity by lymphocytes were 27.7%±15.3%. Cytotoxicity by CTLs derived from culture with WT1 peptides-loaded DC were the strongest among three groups （P＜0.05）. CONCLUSION: CTLs derived from cultures containing DC pulsed with WT1 peptides show the strongest cytolytic activities on K562 cells.     

Hypoxia upregulates CD73 expression in mouse microvascular endothelial cells

AIM: To study the effect of hypoxia on CD73 expression in mouse microvascular endothelial cell line bEnd.3. METHODS: ① bEnd.3 cells were exposed to different periods of hypoxia. ② Concentration of LDH released by bEnd.3 cells into the culture medium was detected. ③ Surface CD73 activity in bEnd.3 cells was measured by HPLC according to the conversion of E-AMP to E-ADO. ④ CD73 mRNA expression were analyzed by semiquantitative RT-PCR. ⑤ Cell surface proteins were biotinylated and CD73 was detected by avidin blots of immunoprecipitation with mAb TY23. RESULTS: ① bEnd.3 cells exposed to hypoxia for 24 h demonstrated a significant increase in LDH release (P<0.01). ②Hypoxia induced CD73 activity in bEnd.3 cells in a time-dependent manner. ③ CD73 mRNA expression increased markedly in hypoxia for 4 h and 8 h (P<0.05). ④ bEnd.3 cells exposured to hypoxia induced a time-dependent increase in expression of CD73 (P<0.05). CONCLUSION: Hypoxia induces CD73 mRNA, protein expression and increases CD73 activity in mouse microvascular endothelial cells.     

Effect of 7-hydroxyisoflavone on HCT116 cells proliferation by Id1

AIM:To investigated the effect of 7-hydroxyisoflavone (7-HIF) on the proliferation, apoptosis and stem-like cell feature of colorectal cancer cells. METHODS:The effect of 7-HIF on the proliferation of HCT116 cells was detected by WST-1 assay and colony formation assay. The effects of 7-HIF on the cell cycle distribution and apoptosis in the HCT116 cells were analyzed by flow cytometry. The expression of cell-cycle related proteins and the stemness related proteins was determined by Western blot. RESULTS:After treated with 7-HIF (200 μmol/L), the viability of HCT116 cells was inhibited, and the size and number of the colony were decreased as compared with control group (P<0.05). The G₀/G₁ phase of the cell cycle was increased. The proportion of S phase was decreased and the cells were mainly arrested in G₀/G₁ phase. The apoptotic rate of HCT116 cells was 21.4%, which was significantly higher than that in the control group (1.1%). The results of Western blot revealed that the expression of inhibitor of differentiation 1(Id1) was significantly decreased (P<0.05). The expression of cell cycle markers cyclin D1 and cyclin E, the proliferative markers survivin and PCNA, and stem cell markers CD133, ALCAM and EpCAM were all down-regulated by 7-HIF treatment (P<0.05). CONCLUSION:7-HIF inhibits the proliferation and induces the apoptosis of colorectal cancer cells, and inhibits the stem-like cell feature, which may be related to Id1 inhibition.     

Influence of complement bypass-activation on IL-8 expression in endothelial cells

AIM:To study the induction of IL-8 expression by bypass-activated complement in human umbilical vein endothelial cells (HUVECs) and regulatory effect of nuclear factor-kappa B on the expression of IL-8. METHODS:In vitro, zymosan-activated human serum(ZAHS) directly challenged the HUVECs monolayers. Following techniques were used in the experiment: ① RIA for measurement of IL-8,ISH for measurement of their mRNA.② EMSA for measurement of nuclear factor-kappa B(NF-κB). RESULTS:①After HUVECs monolayers were stimulated with ZAHS, the level of IL-8 increased significantly at 4 h. ②The NF-κB activity began upregulated within 30 min after ZAHS stimulation, maximal NF-κB activity was observed at 120 min. Pretreatment of endothelial monolayers with PDTC (20 μmol/L) significantly inhibited the secretion of IL-8 (P＜0.05). CONCLUSION:Bypass-activated complement directly challenged HUVECs to secret IL-8. Cytoplasma to nuclear translocation of NF-κB was necessary for this response.     

Growth inhibition of colorectal cancer HCT116 cells by lincRNA-p21 through STAT3 signaling pathway

AIM: To study the effect of long intergenic non-coding RNA-p21 (lincRNA-p21) on the growth inhibition of colorectal cancer HCT116 cells via STAT3 signaling pathway. METHODS: The human colorectal cancer cell line HCT116 was used to construct the cells with over-expression of lincRNA-p21 by transfection of pcDNA-lincRNA-p21, and negative control cells were also set up. After transfection, the expression level of lincRNA-p21 was detected by RT-qPCR. The cell viability and proliferation were examined by MTT assay and plate colony formation assay, respectively. The protein levels of STAT3 and phosphorylated STAT3 (p-STAT3) were determined by Western blot. After STAT3 signaling pathway activator SD19 was used to treat the colorectal cancer HCT116 cells with over-expression of lincRNA-p21, Western blot was used to detect the protein levels of STAT3 and p-STAT3, MTT assay was used to measure the viability of the cells, and flow cytometry analysis was used to determine the cell apoptosis. RESULTS: Compared with control group and pcDNA group, the expression of lincRNA-p21 in pcDNA-lincRNA-p21 group was significantly up-regulated, the cell proliferation was inhibited, and the protein levels of STAT3 and p-STAT3 were significantly decreased (P<0.05). After treatment with STAT3 activator SD19, the protein levels of STAT3 and p-STAT3 in pcDNA-lincRNA-p21+SD19 group were higher than those in pcDNA-lincRNA-p21 group, the cell viability was increased, and the apoptotic rate was decreased significantly (P<0.05). CONCLUSION: Over-expression of lincRNA-p21 inhibits the growth of colorectal cancer HCT116 cells. STAT3 signaling pathway activator abolishes the growth inhibitory effect of lincRNA-p21 over-expression. lincRNA-p21 inhibits the growth of colorectal cancer cells by inhibiting the activation of STAT3 signaling.     

Immunomodulatory effect of human umbilical cord mesenchymal stem cells with interleukin-17 receptor-like molecule overexpression on spleen lymphocytes from mice with trinitrobenzene sulfonic acid-induced colitis

AIM: To investigate the immunomodulatory effect of human umbilical cord mesenchymal stem cells with interleukin-17 receptor-like molecule overexpression (IL-17RLM-hUCMSCs) on the spleen lymphocytes from the mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis for providing optimal seed cells to treat inflammatory bowel disease. METHODS: Mesenchymal stem cells were isolated from human umbilical cord. The IL-17RLM gene was transferred into mesenchymal stem cells by lentivirus vector to establish IL-17RLM-hUCMSCs. The experimental colitis mice were induced by TNBS enema, and the spleen lymphocyte suspension was isolated. The lymphocytes were co-cultured with different concentrations of IL-17RLM-hUCMSCs and hUCMSCs under the stimulation of concanavalin A (ConA) for 72 h. The proliferation of lymphocytes was detected by the methods of CCK8 assay and CFSE labeling with lymphocytes+ConA as positive control. The changes of lymphocyte subsets (Th1, Th2, Th17 and Treg) were examined by flow cytometry.RESULTS: Both hUCMSCs and IL-17RLM-hUCMSCs inhibited T-cell proliferation in vitro co-culture system (P < 0.05). When the ratios of MSCs to lymphocytes ranged from 1:1 to 1:10, the inhibitory rates were in a dose-dependent manner. IL-17RLM-hUCMSCs showed higher inhibitory rate than hUCMSCs within the effective concentration range (P < 0.05). Both hUCMSCs and IL-17RLM-hUCMSCs reduced the proportions of Th1 and Th17 cell subsets and increased Treg cell population of spleen lymphocytes from TNBS-induced colitis mice, and IL-17RLM-hUCMSCs showed a stronger inhibitory effect on Th17 cell subset (P < 0.05). CONCLUSION: IL-17RLM-hUCMSCs remarkably inhibit the proliferation of spleen lymphocytes from TNBS-induced colitis mice in a dose-dependent manner. Meanwhile, they regulate immune balance of T cells and have stronger inhibitory effect on Th17 subset.     

CCK-8 upregulates B7.1 and B7.2 expressions and enhances the costimulatory activity of murine peritoneal macrophages

AIM: To investigate in vitro effects of cholecystokinin octapeptide(CCK-8) on the expressions of B7.1 and B7.2 and the costimulatory activity of T lymphocytes in unstimulated macrophages.METHODS: Mouse peritoneal macrophages were isolated and incubated with CCK-8（10-12-10-6 mol/L） for indicated time. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry. CD4+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages which were pretreated with CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, CCK1R antagonist CR1409, CCK2R antagonist CR2945 for 24 h. ConA was added into the culture medium to stimulate CD4+T cell proliferation. The proliferation was determined by measuring ［3H］-TdR incorporation in a β-scintillation counter. RESULTS: B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were enhanced by CCK-8 in a dose-dependent manner, and the maximal effects occurred at the concentrations of 10-9 mol/L to 10-7 mol/L. Anti-B7.2 antibody, but not anti-B7.1 antibody, reduced the modulatory role of CCK-8 on costimulatory activity. Both CR1409 and CR2945 reversed the effect of CCK-8 on costimulation, and the role of CR1409 was more significant. CONCLUSION: CCK-8 enhances macrophage costimulatory activity by upregulating B7.2 expression, which is mediated by CCK1R and CCK2R. CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.     

辣椒胞质雄性不育（CMS）基因型的鉴定方法

利用材料与不育系测交,F1代不育则材料的基因型为N（msms）;F1代全恢复则材料的基因型为N（MsMs）或S（MsMs）,为进一步判断,分别以其为母本与基因型为N（msms）的材料杂交,F2代分离出不育株的则其基因型为S（MsMs）;F1代一半不育,自交后分离,后代出现1/4不育株则材料的基因型为S（Msms）,否则...     

Inhibitory effect of HET0016 on LPS-induced proliferation and migration of microglia

AIM To study the effects of HET0016 on the proliferation and migration of microglia stimulated by lipopolysaccharide (LPS). METHODS Primary microglia from neonatal SD rats were isolated, purified and cultured. CCK-8 assay was performed to detect the effect of HET0016 on the viability of microglia after treatment with LPS. The levels of 20-hydroxyeicosatetraenoic acid （20-HETE）, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured by ELISA. The proportion of S phase was evaluated by flow cytometry. The cell migration ability was detected by Transwell assay and scratch wound healing assay. The protein expression of NF-κB p50 and p65 was determined by Western blot. RESULTS LPS induced the increases in the proliferation and migration of microglia and the release of inflammatory cytokines (P<0.05). Compared with LPS group, HET0016 inhibited the cell proliferation and migration (P<0.05), decreased the levels of TNF-α and IL-1β (P<0.05), and reduced the expression of NF-κB p50 and p65 (P<0.05). CONCLUSION HET0016 has inhibitory effects on the proliferation and migration of microglia induced by LPS, and reduces the release of inflammatory cytokines. The mechanism may be related to NF-κB signaling pathway.