Objective To analyze the expression profile of long non-coding RNA (lncRNA) in the lipopolysaecharide (LPS)-induced inflammation of monocyte-derived macrophages.Methods Peripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. The macrophages were divided into blank control group and LPS (1 mg/L) stimulated 12 hours group. Culture supernatants and cell pellets were harvested in each group, enzyme linked immunosorbent assay (ELISA) was used to assay the production changes of interleukins (IL-1β and IL-6), and tumor necrosis factor-α (TNF-α) in the supernatant. The technique of lncRNA microarray was used to test the lncRNA expression profile in LPS-induced inflammation of macrophages and control macrophages. The raw data of lncRNA were pretreated for normalization. Five lncRNA expressions were validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Furthermore, qRT-PCR was used to detect the expression of NR_028034 in macrophages after LPS-induced inflammation.Results ① The contents of IL-1β (ng/L:562.93±61.17 vs. 59.74±15.68), IL-6 (ng/L: 702.46±92.31 vs. 71.66±18.25) and TNF-α (ng/L: 794.50±63.89 vs. 85.12±22.07) in the LPS group were significantly higher than those in the blank control group (allP < 0.01). These results indicated that the inflammatory model of human macrophages was constructed successfully. ② Compared with blank control group, and 1479 lncRNA which have more than 2 folds variation and significant difference (P < 0.05) by statistical analysis was defined as lncRNA with differential expression. Among these lncRNA, LPS group showed 953 up- regulated and 526 down- regulated genes by 2 folds and 49 up- regulated and 35 down- regulated genes by 5 folds. ③ qRT-PCR results were generally consistent with the microarray data. ④ The expression of NR_028034 was increased by (4.41±0.65), (11.56±2.04), (18.58±1.36) folds compared with blank control group at 3, 6, 12 hours after LPS stimulation (allP < 0.01).Conclusions These data show a significantly altered lncRNA expression profile in the LPS-induced inflammation of monocyte-derived macrophages, suggesting that lncRNA may be involved in regulation of macrophages inflammatory response.

Objective To establish and optimize the two-dimensional gel electrophoresis technical platform for the blood serum proteome research in patients with end stage renal disease(ESRD).Methods Immobiline pH gradients isoelectric focusing was used as the first dimensional gel electrophoresis and the vertical SDS-PAGE was used as the second dimensional gel electrophoresis(2-DE).The 4 differentially expressed protein spots were identified by mass spectrometry.Results Satisfactory 2-DE maps of ESRD patients serum protein were obtained and there were some differentially protein spots between the 2-DE maps of ESRD patients and normal controls.Conclusions The 2-DE technology for the serum proteome of ESRD patients is set up.

The different sera proteomic components between uremia patients and normal subjects were studied through two-dimensional gel electrophoresis technique. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE), silver staining, ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS) and IPI human database searching were used to separate and identify the proteome of the sera from the patients with uremia. The results showed that satisfactory 2DE patterns of the serum proteins were obtained. Twenty-six protein spots showed significant difference in quantity in uremia patients, and 20 protein spots were identified by MALDI-TOF-TOF-MS. It was concluded that good reproducibility could be obtained by applying immobilized pH gradient 2DE to separate and identify the proteome in serum, which provided the foundation for the further study on uremia toxins pertaining to protein.
Blood Protein Electrophoresis ; Blood Proteins/*analysis ; Case-Control Studies ; *Electrophoresis, Gel, Two-Dimensional/methods ; Proteome/*analysis ; Proteome/chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods ; Uremia/*blood

Objective To investigate the effects of lincRNA-cox2 on the polarization of murine RAW264. 7 macrophages by analyzing the expression of lincRNA-cox2 in RAW264. 7 macrophages of M1 and M2 phenotypes. Methods Murine RAW264. 7 cells were induced by IFN-γand LPS to polarize to M1 phenotype, and were induced by IL-4 to polarize to M2 phenotype. The expression of lincRNA-cox2 in M1 and M2 macrophages were analyzed by real-time quantitative PCR ( RT-PCR) . We designed and synthesized siRNA oligo for lincRNA-cox2 and unrelated sequences. Then the siRNA oligo and NC oligo were transfected into RAW264. 7 cells by LipofectmineTM 2000. The transfected RAW264. 7 cells were induced by IFN-γand LPS or by IL-4 to polarize to M1 or M2 macrophages. Enzyme linked immunosorbent assay ( ELISA) was performed to measure the secretion of IL-10 and IL-12 induced in different conditions. The expression of in-ducible nitric oxide synthase ( iNOS ) , TNF-α, arginase 1 ( Arg-1 ) and found in inflammatory zone 1 (Fizz1) at mRNA level were detected by RT-PCR. The M1 macrophages were transfected with siRNAs to knock down the expression of lincRNA-cox2 for analyzing the biological effects of lincRNA-cox2 on the polar-ization of macrophages. Results The relative expression of lincRNA-cox2 in M1 macrophages was signifi-cantly higher than that in RAW264. 7 cells and M2 macrophages. Compared with the control group, the RAW264. 7 cells transfected with lincRNA-cox2-siRNA showed decreased secretion of IL-12 and inhibited expression of iNOS and TNF-αat mRNA level after IFN-γand LPS induction, but increased secretion of IL-10 and enhanced expression of Arg1 and Fizz1 at mRNA level after IL-4 induction. Transfecting the M1 mac-rophages with lincRNA-cox2-siRNA inhibited the secretion of IL-12, but promoted the secretion of IL-10. Conclusion This study indicated that lincRNA-cox2 was involved in the regulation of macrophage pheno-types by promoting the polarization to M1 macrophages and inhibiting the polarization to M2 macrophages.

Objective To construct a lentiviral vector-based short hairpin RNA (shRNA) targeting the gene encoding tryptophan-aspartate containing coat protein ( TACO) and to evaluate its inhibitory effect on the expression of TACO , and to further elucidate its effects on the phagocytosing and intracellular killing of My-cobacterium tuberculosis (M.tb) by macrophages and the possible mechanisms.Methods Three shRNA frag-ments targeting TACO gene and a scrambling control shRNA fragments were designed and cloned into the lentivi -ral vector pSicoR .The recombinant lentiviral vectors were identified by sequencing analysis and then packed in 293T cells.Real-time RT-PCR and Western blot assay were performed to evaluate the gene-silencing efficiency of the recombinant lentiviral vectors among RAW 264.7 cells transfected with the concentrated lentivirus .The most effective lentivirus was screened out to transfect the RAW 264.7 cells for 48 hours, followed by infection those cells with M.tb strains.The entry and intracellular survival of M .tb strains in RAW264.7 cells were de-termined by bacterial culture at indicated time points .The colocalization of M .tb and lysosomes was detected by immunofluorescence staining .The cyto-ID autophagy kit was used to detect the cellular autophagy and the auto-phagy-associated protein LC 3 was determined by Western blot assay .Results The recombinant lentiviral vec-tors were successfully constructed and confirmed by sequencing analysis .Decreased expression of TACO in RAW264 .7 cells was detected after transfecting the cells with the lentiviral vector-based shRNA vectors targeting TACO gene for 48 hours.The most effective lentivirus , LV-pSRT1, decreased the expression of TACO by 85.24%and 69.00%at the mRNA and protein levels, respectively.The bacterial loads in LV-pSRT1 trans-fected RAW264.7 cells were significantly decreased at the time point of 0 h after M.tb infection as compared with those in the control lentivirus treated cells (5.50×104 vs 8.1 ×104, P<0.05).Compared with the RAW264 .7 cells transfected with control lentivirus , the survival rate of intracellular M .tb strains in LV-pSRT1 transfected cells was significantly decreased at the time point of 48 h (134.54% vs 213.58%, P<0.05) and 72 h (148.18%vs 262.96%, P<0.05) considering the bacterial load at the time point of 0 h as the standard. The immunofluorescence staining demonstrated that the colocalization of M .tb strains with lysosomes was signifi-cantly enhanced in LV-pSRT1 transfected cells as compared with that in control lentivirus treated cells (75.67%vs 10.66%, P<0.05).Moreover, significantly enhanced autophagy and relative expression of LC 3Ⅱ protein were observed in RAW264.7 cells with TACO gene knockdown (16.20%vs 8.50%, P<0.05;0.51 vs 0.34, P<0.05).Conclusion The lentiviral vector-based shRNA targeting TACO gene could effectively knockdown the expression of TACO protein , decrease the entry and increase the intracellular killing of M .tb strains in mac-rophages.The enhanced intracellular killing of M .tb strains by macrophages was associated with the increased fusion of M.tb-containing phagosome and lysosome .

Objective To observe the effects of fosinopril(FOS)on secretion of ColⅠ,expression of Smad2、Smad7 mRNA in TGF-β1-induced glomerulomesangial cells(GMC)in rat model. Methods Rat glomerular mesangial cells were cultured in vitro,passages 3 - 10 cells were used in the study after identification,and the cells were divided into 3 groups:control group(Ctrl group),TGF-β1 group,and fosinopril group. Expression of Col Ⅰ in cell culture supernatant was detected by the enzyme-linked immunosorbent assay(ELISA)at 6 h,24 h and 48 h. Changes of Smad2,Smad7 mRNA expression were evaluated by fluorescent quantitation PCR. Results Glomerular mesangial cells had Col Ⅰ protein expression. Secretion of Col Ⅰ was significantly higher in TGF-β1 group than those in Ctrl group at each time point(P ＜ 0.01),however the Col Ⅰ was significantly lower in fosinopril group at all time points than that in TGF-β1 groups(P ＜ 0.05). Glomerular mesangial cells also had Smad2,Smad7 mRNA expressions. The expressions of Smad2,Smad7 mRNA were significantly higher in TGF-β1 group than those in Ctrl group at each time point. Expression of Smad2 mRNA was significantly lower in fosinopril group than that in TGF-β1 group at all time points,while the difference in Smad7 mRNA expression between TGF-β1 group and fosinopril group showed no statistical significance(P ＞ 0.05). Conclusions Fosinopril could inhibit the secretion of Col Ⅰ and expression of Smad2 mRNA in glomerular mesangial cells induced by TGF-β1,suggesting that fosinopril might delay glomerular sclerosis through inhibiting the expression of Smad2 in TGF-β1/Smad signaling pathway.

Objective To investigate the relationship between low serum calcium concentration and hematoma volume in patients with intracerebral hemorrhage.Methods Between January 2012 and October 2014,870 consecutive patients with intracerebral hemorrhage admitted to the Department of Neurosurgery,West China Hospital,Sichuan University were enrolled prospectively.The patients completed laboratory serum calcium concentration and head CT examinations within 24 h after attack,and the baseline data and laboratory findings were collected.According to the normal reference value of laboratory serum calcium concentration,the patients were divided into a hypocalcemia calcium group (<2.1 mmol/L;n=193) and a normal calcium group (2.1-2.7 mmol/L;n=677).Spearman correlation analysis was used to analyze the correlation between the blood serum calcium concentration and the hematoma volume on admission.Results (1) The hypocalcemia group compared with normal calcium group,the proportion of male patients was high (73.6% [n=142] vs.66.0% [n=447]),the median score for Glasgow coma scale was lower (9 vs.11),and the median hematoma volume was larger (33.86 cm3 vs.21.69 cm3).The differences were statistically significant (all P<0.05).(2) Spearman correlation analysis showed that the lower serum calcium level on admission was weakly negatively correlated with the volume of hematoma in patients with intracerebral hemorrhage (r=-0.113,P<0.01).Conclusion The study suggested that the hypocalcemia on admission was mostly males in patients with intracerebral hemorrhage,the condition was serious,the volume of hematoma was larger,and the lower serum calcium concentration was negatively correlated with the hematoma volume.

In current study, the expressions of protein kinase C (PKC)-alpha, betaI and betaII as well as their correlation to the expression of transforming growth factor-betaI (TGF-betaI) and vascular endothelial growth factor (VEGF) were investigated in glomeruli of normal renal tissues taken from human kidney tumors and kidney tissues from patients with diabetic nephropathy (DN). The accumulation of glomerular extracelluar matrix (ECM) was determined by PAS staining, the expressions of PKC-a, PKC-betaI, PKC-betaII, TGF-betaI and VEGF were measured by semi-quantitative immunohistochemistry. Our results showed that in glomeruli of normal renal tissues, PKC-alpha and betaII had a strong expression whereas the expression of PKC-betaI was weak; in glomeruli of DN patients, the expressions of PKC-alpha, PKC-betaI, VEGF and TGF-betaI and the accumulation of ECM increased significantly, but the expression of PKC-betaII decreased markedly. Meanwhile, the expressions of PKC-alpha and betaI had a positive correlation to the expressions of VEGF and TGF-betaI respectively, whereas PKC-betaII showed no correlation to VEGF and TGF-betaI. It is concluded that the expressions of PKC-alpha, betaI and betaII in glomeruli of normal subjects and DN patients are different. PKC-alpha seems to play a critical role in human DN by up-regulating VEGF expression, whereas PKC-betaI is relatively important for the up-regulation of TGF-betaI and the accumulation of ECM under diabetic conditions.

The different sera proteomic components between uremia patients and normal subjects were studied through two-dimensional gel electrophoresis technique. Immobilized pH gradient twodimensional polyacrylamide gel electrophoresis (2DE), silver staining, ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDITOF-TOF-MS) and IPI human database searching were used to separate and identify the proteome of the sera from the patients with uremia. The results showed that satisfactory 2DE patterns of the serum proteins were obtained. Twenty-six protein spots showed significant difference in quantity in uremia patients, and 20 protein spots were identified by MALDI-TOF-TOF-MS. It was concluded that good reproducibility could be obtained by applying immobilized pH gradient 2DE to separate and identify the proteome in serum, which provided the foundation for the further study on uremia toxins pertaining to protein.

In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 (AQP2) was investigated in the renal medullary collecting duct of mice with diabetic nephropathy (DN). Mice were divided into three groups: normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group (2.39±0.11 vs 3.16±0.16, P<0.05). Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system (RAS) is implicated in the pathogenesis of DN by regulating the expression of AQP2.