Animals ; Cell Differentiation ; End Stage Liver Disease ; therapy ; Hepatocytes ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology

Objective: To construct and identify a lentiviral vector harboring RNAi sequence targeting the human high mobility group A1(HMGA1) gene.Methods: The effective sequence of siRNA targeting the HMGA1 gene confirmed in our previous study,the complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pGCL-GFP vector diced by the restriction enzyme of HpaⅠ and XhoⅠ,which contained the U6 promoter and green fluorescent protein(GFP).The resulting lentiviral vector containing HMGA1 shRNA was named LV-sh HMGA1 and confirmed by PCR and DNA sequencing.A total of 293T cells were cotransfected with LV-sh HMGA1,pHelper 1.0 and pHelper 2.0.All the virus stocks were produced by Lipofectamine2000-mediated transfection.The titer of the virus was tested according to the expression level of GFP.Results: PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the human HMGA1 gene was successfully inserted into the lentiviral vector.The titer of the recombinant lentiviral vector was 5?107 TU/ml.Conclusion: The successful construction of the lentiviral vector of HMGA1 has prepared the ground for further studies on the functions of the HMGA1 gene with the RNAi technique.

Aim To explore the role of endoplasmic reticulum stress(ERS) in Astragaloside Ⅳ-induced cardioprotection in H9c2 cardiac cells, and to explore the potential mitochondrial mechanism.Methods Conventional culture was performed of rat heart tissue-derived H9c2 cells.Experiment was randomly divided into the control group, the ERS inducer 2-Deoxy-D-Glucose(2-DG) group, Astragaloside Ⅳ and 2-DG combination group, Astragaloside Ⅳ group.Confocal microscopy was used to observe the changes of TMRE fluorescence intensity so as to confirm the influence of ERS on the mitochondrial potential, and further speculate on the opening of the mitochondrial permeability transition pore(mPTP).Western blot analysis was applied to detect the expressions of ERS proteins GRP 78, GRP 94 and IRE1.Transmission electron microscopy was used to observe the ultrastructure of the cells.Results Different doses of 2-DG could mimic the mPTP opener atractyloside to induce the mPTP opening with the peak at 100 μmol·L-1;Astragaloside Ⅳ significantly reduced 2-DG-induced mPTP opening, the expression of GPR 78, GRP 94 and IRE1 and reduced injury of mitochondria and endoplasmic reticulum.Conclusions Endoplasmic reticulum stress could be induced by 2-DG.Astragaloside ⅳ-induced mitochondrial cardioprotection involves inhibition of the ERS through GRP 78, GRP 94 and IRE1 by prevention of the mPTP opening.

Objective To investigate the clinical significance of peripheral blood Th17 cells and their cytokines in the patients with tongue squamous cell carcinoma.Methods Sixty-six cases of tongue squamous cell carcinoma in our hospital from January 2013 to January 2016 served as the research subjects and contemporaneous 42 volunteers undergoing healthy physical examination were selected as the control group.Peripheral blood was collected in all subjects.The proportion of peripheral blood Th17 cells was detected by flow cytometry.The IL-17,TGF-β and IL-6 cytokine levels were detected by ELISA.Results The proportion of peripheral blood Th17 cells,IL-17,TGF-β and IL-6 in the observation group and the control group were (1.46±0.41)% vs.(0.31±0.12)%,(123.36±21.20)pg/mL vs.(20.76±8.95)pg/mL,(215.80±21.52)pg/mL vs.(26.90±10.41)pg/mL,(17.32±8.02)pg/mL vs.(5.85±1.49)pg/mL respectively,the differences were statistically significant(P<0.05).The proportion of peripheral blood Th17 cells,IL-17,TGF-β and IL-6 levels were increased with the increase of tongue squamous cell carcinoma clinical stage (P<0.01).The TGF-β level was positively correlated with the IL-17 level (r=0.626,P=0.021),the IL-6 level was positively correlated with the proportion of Th17 cells and IL-17 (r=0.626,0.597,P=0.021,0.034).Conclusion The increase of Th17 cells and IL-17 expression plays an important role in the development and progression of tongue squamous cell carcinoma,Th17 cells and IL-17 cytokine can become the important target spots of anti-tumor therapy.

OBJECTIVE: To investigate the protective effect of protein kinase C (PKC) inhibitor rottlerin on rat renal vascular endothelial injury induced by lipopolysaccharide (LPS). METHODS: Rat renal microvascular endothelial cells cultured for 3-6 generations were divided into three groups according to random number table: blank control group in which cells were not challenged, LPS group in which cells were only stimulated by LPS 10 mg/L for 24 hours, and PKC inhibitor group in which cells were treated with PKC inhibitor rottlerin 2 μmol/L 30 minutes before LPS stimulation. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-1β, IL-8) were determined by enzyme-linked immunosorbent assay (ELISA). Monolayer permeability was determined by Transwell assay. The expressions of PKC, RhoA and vascular endothelial-cadherin (VE-cadherin) were detected by Western Blot. The morphological characteristic and distribution of F-actin was measured by laser confocal fluorescence microscope. RESULTS: Compared with blank control group, the levels of inflammatory cytokines at 24 hours after 10 mg/L LPS stimulation were significantly increased in LPS group [TNF-α (ng/L): 397.3±25.4 vs. 46.8±8.9, IL-1β (ng/L): 76.7±11.2 vs. 12.6±3.2, IL-8 (ng/L): 574.5±31.4 vs. 73.2±9.6, all P < 0.05], the permeability of endothelial cells was significantly increased (A value: 1.32±0.03 vs. 0.36±0.02, P < 0.05), while the expressions of PKC and RhoA were significantly up-regulated (PKC/β-actin: 0.88±0.02 vs. 0.61±0.03, RhoA/β-actin: 0.96±0.01 vs. 0.49±0.03, both P < 0.05), VE-cadherin expression was significantly down-regulated (VE-cadherin/β-actin: 0.51±0.01 vs. 0.72±0.04, P < 0.05), and the F-actin distribution disorder had obvious stress fiber formation. Compared with LPS group, the levels of inflammatory cytokines were significantly lowered in PKC inhibitor group [TNF-α (ng/L): 127.4±14.6 vs. 397.3±25.4, IL-1β(ng/L): 43.2±7.8 vs. 76.7±11.2, IL-8 (ng/L): 212.7±18.2 vs. 574.5±31.4, all P < 0.05], the permeability of endothelial cells was significantly decreased (A value: 0.81±0.02 vs. 1.32±0.03, P < 0.05), the expressions of PKC and RhoA were significantly down-regulated (PKC/β-actin: 0.44±0.03 vs. 0.88±0.02, RhoA/β-actin: 0.63±0.05 vs. 0.96±0.01, both P < 0.05), the VE-cadherin expression was significantly up-regulated (VE-cadherin/β-actin: 0.69±0.03 vs. 0.51±0.01, P < 0.05), and the F-actin remodeling and stress fiber formation were significantly reduced. CONCLUSIONS PKC inhibitor could significantly attenuate the damage of vascular endothelial barrier induced by LPS, and plays an important role in endothelial cell barrier.
Acute Kidney Injury/prevention & control* ; Animals ; Endothelium, Vascular/drug effects* ; Interleukin-1beta ; Lipopolysaccharides/toxicity* ; Protein Kinase C/antagonists & inhibitors* ; Protein Kinase Inhibitors/pharmacology* ; Random Allocation ; Rats

This study was aimed to observe the effect ofIllicium verum water extract on excess-cold syndrome in rats. The intragastric administration of the decoction of gypsum, gentian, phellodendron andrhizoma anemarrhenae (2:1.2:1:1.5) was used to establish the excess-cold rat model. Effects ofI. verum water extract on general body signs of excess-cold syndrome rats were observed. The detection was made on the contents of LD, PA, TG, TSH, T4 in the serum and LD, PA, glycogen in liver tissues. The vitality of the LDH, SDH and ATPas was also detected. The results showed thatI. verum water extract can improve the general body signs of excess-cold syndrome rats. It reduced the content of TG; increased the content of PA, T4, TSH; reduced the content of LD, glycogen, and the vitality of LDH;increased the vitality of LDH, Ca2+-Mg2+-ATPase in liver tissues. It was concluded thatI. verum water extract can improve some general body signs of excess-cold syndrome rats, improve the substance and energy metabolism, and regulate the thyroid axis function, in order to achieve the warmingyang for dispelling cold effect.

Objective To investigate the differentiation of CD4+T helper cell in expressed prostatic secretions(EPS)in type Ⅲ prostatitis. Methods Seventy-six patients were studied,aged from 18 to 47(average 31.8).All patients presented with typical clinical symptoms for over 3 months.Cases were classified as type Ⅲ A(47 cases),type Ⅲ B(29 cases)and control group(16 cases)according to NIH classification system.Type Ⅲ A was also divided into group Ⅲ A1 with 26 cases(mild inflammation)and group Ⅲ A2 with 21 cases(severe inflammation).Th1 cytokine IFN-γ,Th2 cytokines IL-4 in EPS were detected by double antibody sandwich ELISA,and IFN-γ/IL-4 was determined.Results Compared with group control,IFN-γ in group Ⅲ A and Ⅲ B were significantly up regulated (134.78±43.67 pg/ml,109.82±30.09 pg/ml,P＜0.05),while IFN-γ level in group Ⅲ A was higher than in group Ⅲ B(P＜0.05).IL-4 in groupⅢ A was not changed(51.99±20.59 pg/ml,P＞0.05).IL-4 in group ⅢB was higher(76.40±17.99 pg/ml,P＜0.05).IFN-γ/IL-4 in group ⅢA was elevated significantly(2.94±1.12,P＜0.05),while IFN-γ/IL-4 in group ⅢB was not changed(1.49±0.48,P＞0.05).IFN-γ/IL-4 in group Ⅲ A2 was higher than in groupⅢA1 significantly(3.67±0.82vs 2.34±0.97,P＜0.05). Conclusions Th1 eell differentiation took the dominance in type Ⅲ A prostatitis.Th1/Th2 equilibrium was shifted to Th1.It is probably one of the pathogenies that Th1 dominant differentiation leads to local inflammation in prostate of type Ⅲ A prostatitis.

Objective To explore the effect of indoxyl sulfate (IS) on the differentiation, maturation and immunological function of human monocyte derived dendritic cells (mDCs), in order to provides evidence for mechanism of IS in atherosclerosis. Methods Human peripheral blood mononuclear cells isolated by double gradient centrifugation were cultured for immature mDCs by rhGM?CSF and rhIL?4 in vitro. All cases were randomly divided into PBS group, LPS group(1 μg/mL), IS.1 group(30 μmol/L), IS.2 group(300 μmol/L)and IS.3 group (600 μmol/L). The phenotypic maturation of mDCs was evaluated by flow cytometry (FCM) and functional maturation of mDCs was analyzed by measuring FITC?dextran uptake and ELISA. Results IS significantly upregulated the expression of CD80, CD83, CD86 and MHC II key membrane molecules on mDCs, while downregulating phagocytosis and increasing the secretion of IL?12p70 by mDCs (P<0.05). And the LPS and IS showed typical morphology with rough surface, long protrusions and fusiform. 300 μmol/L IS is the most appropriate stimulus concentration. Conclusion Stuctural, phenotypic and functional maturation of dendritic cells derived from human monocytes can be induced by indoxal sulphate at defined concentrations, which may be one of the mechanisms involved in the process of atherosclerosis.

Objective:To explore the changes of immune microenvironment and prognosis of bladder cancer patients with positive urinary nuclear matrix protein 22 (NMP22).Methods:Retrospective analysis was made on 86 patients who were diagnosed with bladder cancer in Xuzhou Central Hospital from January 2019 to September 2020. All patients were tested for urinary NMP22 by colloidal gold method. The patients with positive test results were NMP22 positive group, and the patients with negative test results were NMP22 negative group. The expression of CD8, programmed cell death-ligand 1 (PD-L1), programmed cell death protein-1 (PD-1) and PanCK were detected by multiple fluorescent immunohistochemical method on the pathological tissue sections of all enrolled patients with bladder cancer after surgery. Follow-up data of enrolled patients were collected after discharge, and univariate and multivariate Cox analysis was performed on the follow-up data.Results:There were 69 patients in the NMP22 positive group and 17 patients in the NMP22 negative group. The percentage of CD8 and PD-L1 positive cells in NMP22 positive group was significantly higher than that in NMP22 negative group, and the difference was statistically significant (all P<0.05). Univariate analysis showed that tumor stage was correlated with bladder cancer progression ( HR=2.67, P=0.017). Multivariate analysis showed that positive NMP22 was significantly correlated with bladder cancer recurrence and disease progression (all P<0.05). Conclusions:The density of CD8 + T cells and PD-L1 in tumor parenchyma of urinary NMP22 positive bladder cancer patients was higher than that of NMP22 negative patients. Urinary NMP22 positive can be one of the bad prognostic factors of bladder cancer, and the patients with NMP22 positive should strengthen reexamination.

Objective To study the differential diagnostic efficacy of α-enolase ( ENO1 ) autoantibodies in lung adenocarcinoma , benign pulmonary disease , and normal individuals , and to evaluate the improvement of the diagnostic efficiency of existing markers by establishing a binary logistic regression model.Methods This was a case-control study.Participants were from the public health welfare program led by the National Cancer Center/Chinese Academy of Medical Sciences Peking Union Medical College Cancer Hospital.Serum samples were collected June 2014 to June 2017 including 60 patients with lung adenocarcinoma , 50 patients with benign lung diseases , and 90 healthy controls.Luminex MAGPIX platform was applied to detect serum ENO1 autoantibodies, CEA and Cyfra21-1 proteins.The receiver operating characteristic curve (ROC)analysis and binary logistic regression were used to evaluate the performance and build diagnostic model.Results The median level of serum ENO1 autoantibody in patients with lung adenocarcinoma was 918.5 ( 665.5-2 043.3 ), which was significantly higher than that in the normal individuals (722.5, 585.5-921.8, Z＝-3.113, P＝0.002) and benign lung disease patients (693.0, 501.4-973.3, Z＝-3.395, P＝0.001).And no significant differences between benign disease groups and normal individuals (Z＝-1.155, P＝0.248).ROC was plotted, and the area under the curve (AUC) of ENO1 autoantibodies was 0.664 (95% confidence interval : 0.576-0.752), while the AUCs of existing diagnostic marker CEA and Cyfra21-1 were 0.680 (95% confidence interval : 0.594-0.767) and 0.617 (95% confidence interval: 0.532-0.703).A joint diagnostic model including ENO1 and CEA was built with an AUC of 0.757 (95%confidence interval : 0.675-0.838).The diagnostic efficacy of the model was significantly different from ENO1 autoantibodies (Z＝2.648, P＝0.008).When the specificity was 90%, the sensitivity of ENO1 autoantibodies was 38.3%, while the sensitivity of the combination with CEA was raised to 50%.Conclusion ENO1 autoantibodies could be a marker for the auxiliary diagnosis of lung adenocarcinoma, and can improve the efficacy of the existing diagnostic markers such as CEA .ENO1 has the potential use for the diagnosis and screening.