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Objective To study the changes of immune function of endotoxin tolerant dendritic cell (ETDC) and to observe its effect on sepsis in mouse model.Methods ETDC were prepared by pretreated bone marrow dendritic cells derived from BALB/c mice with lipopolysaccharide stimulation.The cells were collected and the expressions of surface markers including major histocompatibility complex ( MHC)Ⅱ, CD86 and CD11c were detected by flow cytometry.The proliferation rate of T lymphocytes was evaluated by cell counting kit-8 and the concentrations of cytokines in the supernatant were detected by enzyme linked immuno sorbent assay.Afterwards, 36 mice were randomly assigned into 4 groups.The blank control group did not receive any treatment, the sham-operated group underwent simple incision suture, the sepsis group and ETDC reinfusion group underwent cecal ligation and puncture to establish sepsis.Before sepsis model establishment, 0.9% sodium chloride solution or suspension of ETDC and 0.9%sodium chloride were reinfused by tail vein.The serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), tumor necrosis factor (TNF)-α, interleukin(IL)-6 and IL-10, and the proportion of help T cell ( Th) 17/regulatory T cell ( Treg) in spleen of each group were detected.The pathological manifestations of liver, kidney and ileum in each group were observed.T test and χ2 test were used for comparisons between groups.Results The results of flow cytometry showed that MHCⅡ, CD86 and CD11c of ETDC were 70.4%, 43.1%, and 73.1%, respectively, which were significantly lower than those of mature dendritic cell (mDC) (96.1%, 89.5%, and 84.6%, respectively) (χ2 ＝56.47, 83.78, and 23.29, respectively, all P＜0.01).The concentrations of IL-10, TNF-αand IL-6 in the supernatant of ETDC were (978.04 ±56.70), (980.34 ±111.96) and (12 743.03 ±865.81) ng/L, respectively, and those of mDC were (741.35 ±99.23), (1 703.11 ±117.00) and (19 052.28 ±1 145.84) ng/L, respectively.The differences were all statistically significant (t＝5.073, 10.93, and 10.76, respectively, all P＜0.01).The proliferation rates of T lymphocytes co-cultured with ETDC in 1∶5 and 1∶10 ratio group were (676.95 ±85.99)%and (514.00 ±106.39)%, respectively, which were lower than those of the mDC group (956.25 ±127.12)%and (772.07 ±214.08)%, respectively.The pathological injuries of liver, kidney and ileum in ETDC treatment group were significantly lighter than those in sepsis group.Serum ALT and AST levels in the ETDC reinfusion group were (299.71 ±36.91) and (690.39 ±154.92) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.067 ±0.005), (0.428 ±0.051) and (0.058 ±0.005) ng/L, respectively.Serum ALT and AST levels in the sepsis group were (620.67 ±69.27) and (1 430.17 ±134.05) U/L, respectively, and TNF-α, IL-6 and IL-10 were (0.085 ±0.007), (0.774 ±0.088) and (0.036 ±0.005) ng/L, respectively.The differences were all statistically significant (t＝11.60, 10.96, 5.991, 8.657, and 8.04, respectively, all P ＜0.01).The proportion of Treg/Th17 in the ETDC reinfusion group was 23.4%, and that in the sepsis group was 60.8%(χ2 ＝28.69, P＜0.01).Conclusion The reinfusion of ETDC has a protective effect on sepsis in mouse model, which may play a negative immune regulatory role by regulating the differentiation of T cells.
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Objective To investigate the expression levels of serum visfatin ,leptin and other indexes and their correlation and clinical significance in the patients with type 2 diabetes mellitus(T2DM ) .Methods Eighty-two age-matched and gender-matched cases of T2DM (T2DM group)and 71 cases of healthy subjects(health control group)were tested serum visfatin ,leptin ,insulin ,lipid and glycemia levels .Results The serum visfatin ,leptin ,triglycerides(TG) ,total cholesterol(TC) ,high-density lipoprotein choles-terol(HDL-C) and low density lipoprotein cholesterol(LDC-C) ,fasting plasma glucose ,fasting insulin(FINS) ,homeostasis model assessment of insulin resistance(OMA-IR) ,2 h FPG ,2 h FINS had statistically significant differences between the T 2DM group and the health control group(P< 0 .05);the stepwise regression analysis showed that visfatin were positively correlated with BMI , FINS ,HOMA-IR ,TC ,TG ,LDL-C (P<0 .05)and negatively correlated with HDL-C(P<0 .05);leptin were positively correlated with BMI ,FINS ,HOMA-IR(P<0 .05) .Conclusion Serum visfatin and leptin in the T2DM patients are hither than those in the healthy subjects and closely related with blood lipid ,blood glucose and insulin ,which may become the new diagnostic indexes of T2DM .
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ObjectiveTo study the expression of microRNA-21 ( miR-21 )in serum of patient with diffuse large B cell lymphoma (DLBCL) and DLBCL cell lines and validate the significance of miR-21 in early diagnosis,genotyping and prognosis estimates of DLBCL.MethodsmiR-21 expression were detected by fluorescent quantity polymerase chain reaction (FQ-PCR)in 9 lymphoma cell lines (OCI-Ly1,OCI-Ly3,OCI-Ly4,OCI-Ly7,OCI-Ly8,OCI-Ly10,OCI-Ly18,OCI-Ly19 and HBL),the serum from DLBCL patients (n =62) and health controls (n =50 ).Kaplan-Meier survival analysis was carried out during the relapsefree survival period of DLBCL patients to explore the relationship between the prognosis and microRNA expression level.ResultsReal time FQ-PCR result indicated that miR-21 expression was higher in DLBCL cell lines than that in normal B cells (BC).miR-21 expression in normal B cell and 9 DLBCL cell lines separately were 1.04 ± 0.02,2.30 ± 0.35,237.97 ± 56.19,5.27 ± 0.83,3.40 ± 0.30,11.22 ± 2.70,133.55 ± 16.78,6.63 ±0.24,4.91 ±0.37 and 81.59 ±6.64.Compared with BC,the expression of miR-21 were higher in all 9 DLBCL cell lines ( t =7.3,13.7,21.0,6.2,8.8,13.6,6.5,39.5,18.1 ;P ＜ 0.01 ).miR-21 expression segregates with specific molecular subgroups of DLBCL The expression was higher in the ABC type cell lines (OCI-Ly3,OCI-Ly10,HBL) than GCB type cell lines (OCI-Ly1,OCI-Ly4,OCI-Ly7,OCI-Ly8,OCI-Ly18,OCI-Ly19;t =11.18,P ＜ 0.01 ).Consistent with the cell line models,miR-21 expression levels were higher in serum from DLBCL patients [21.38 (10.26-45.21 )] than from controls [1.87 ( 1.05-3.97 ),U =168,P =0.000],and the levels were higher in DLBCL cases with an ABC-type [28.68 ( 14.92-98.44 )] than those in GCB-type [18.30 ( 7.32-33.46 ),U =336,P =0.043].MiR-21 expression levels were different in sera from different clinical stage DLBCL patients.The miR-21 level in serum of patients with subgroup ABC and subgroup GCB in stage Ⅰ and Ⅱ were 47.49( 25.65-295.41 ) and 24.74( 16.08-50.38) respectively and in stage Ⅲ and Ⅳ were 16.66 ( 5.35-44.30 ) and 11.96 ( 4.10-21.05) respectively.The levels were higher in DLBCL cases withⅠ -Ⅱ stage than those with Ⅲ-Ⅳ stage (U =62,P =0.013 in GCB type; U =53,P =0.014 in ABC type).Moreover,compare with relapse-free survival in DLBCL patients,high miR-21 expression was associated with well prognosis ( U =259,P =0.035).ConclusionsMiR-21 is high expression in DLBCL cell lines and DLBCL patients serum.miR-21 level in sera from DLBCL patients is associated with clinical stage,molecular subgroup and prognosis estimates.MiR-21 may serve as a new biomarker to early detection,genotyping and prognosis estimates of DLBCL.
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Objective To study the expression of microRNA-301 in pancreatic carcinoma andvalidate the significance of miR-301 in invasion and metastasis of pancreatic carcinoma.Methods miR-301 expression were detected by FQ-PCR in 5 pancreatic cancer eell lines(PANC-1,PaCa-2,AsPC-1,Hs766T.BxPC-3).Further immunohistochemistry in pancreatic cancer tissue microarrays was detected miR-301 expression,which contained 60 pancreatic cancer specimens along with 10 normal adjacent tissues and 10 normal pancreas tissues.After high expression of miR-301 in pancreatic carcinoma being confirmed.the clinical significance of high expression of miR-301 in invasion and metastasis of pancreatic carcinoma were studed.Pancreatic cancer cell lines(PANC-1.PaCa-2)were transfected by 100 nmoml/L miR-301 inhibitor(anti-miR-301)or negative eontrol(Anti-miR~(TM) Negative Control#1).COX-2 and MMP-2 protein expression in pancreatic cancer cell lines were detected by WB.and cell migration assays were performed using transwell technology.Results FQ-PCR resuhs indicated that miR-301 expression was higher in pancreatic cancer cell lines than normal pancreatic cells.The relative level of miR-301 in 5 pancreatic cancer cell lines(PANC-1,PaCa-2,AsPC-1,Hs-766T,BxPC-3)and normal pancreatic cell were 33.09± 4.21,30.76±3.18,47.57±3.56,20.20 ±1.21,76.75±13.51 and 1.00±0.08 respectively.The miR-301 level in all 5 pancreatic cancer cells were significantly higher than those of normal pancreatic cell(t=8.86,9.53,6.39,6.77,11.18,P<0.01).Immunohistochemistry results also showed miR-301 expression was higher in pancreatic carcinoma tissues than those in the cancer adjacent tissues and normal pancreatic tissues.The relative levels of miR-301 in pancreatic carcinoma tissues.normal adjacent tissues and normal pancreas tissues were 0.88±0.09,0.22±0.04 and 0.14±0.05 respectively.The miR-301 levels in pancreatic carcinoma tissues were significantly higher than those of normal adjacent tissues and normal pancreatic tissues(t=15.1,10.6,P<0.01).There was no significant difference between normal adjacent tissues and normal pancreas tissues(t=1.32,P=0.22).After miR-301 inhibitor was introduced into pancreatic cancer cells PANC-1 and PaCa-2.miR-301 levels were reduced while the protein levels of COX-2 and MMP-2.which were invasion and metastasis related factors,were down-regulated.The cell migration assay indicated the numbers of PANC-1 and PaCa-2 cells,which migrated to lower chamber.were 587±27 and 363±13 respectively after miR-301 inhibitor was applied.The numbers of migrated cells were 1091 4-15.737±44 when the netative control was applied.The cell invasion ability was decreased significantly in the inhibitor group compared with the negative group(t=7.89,7.56,P<0.01).Conclusions miR-301 is highly expressed in pancreatic cancer cell lines and pancreatic cancer tissues.Inhibition of miR-301 expression can effectively supress the invasion of pancreatic cancer cells.miR-301 may serve as a new biomarker for early detection of pancreatic cancer and molecular target for early treatment of pancreatic cancer.