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ObjectiveTo obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. MethodsThe COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsSequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of ＞1×108. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P＜0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P＜0.05), suggesting that COX-2-sgRNA was active. ConclusionA CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2-/- cells with stable COX-2 gene knockout are obtained.
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ObjectiveTo investigate the effect and significance of cyclooxygenase-2 (COX-2) inhibitors on the expression of the Acsl gene family in the ileum of rats with nonalcoholic fatty liver disease (NAFLD). MethodsA total of 45 Sprague-Dawley rats were randomly divided into normal control group (15 rats given normal diet), NAFLD model group (15 rats given high-fat diet), and nimesulide group (15 rats given high-fat diet and nimesulide). All rats were sacrificed after 12 weeks of feeding, and then blood samples were collected from the inferior vena cava to measure total cholesterol (TC) and triglyceride (TG). HE staining and oil red O staining were performed for the liver to evaluate the degree of hepatic steatosis in each group, and quantitative real-time PCR was used to measure the mRNA expression of the Acsl family genes in the ileum. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the normal control group, the NAFLD model group had significant increases in serum TC and TG and marked hepatic steatosis (all P＜0.05); compared with the NAFLD model group, the nimesulide group had significant reductions in serum TC and TG and degree of hepatic steatosis (all P＜0.05). Compared with the normal control group, the NAFLD model group had a significant increase in the expression of COX-2 in the ileum (P＜0.05), and compared with the NAFLD model group, the nimesulide group had a significant reduction in the expression of COX-2 in the ileum (P＜005). Compared with the normal control group, the NAFLD model group had significant increases in the mRNA expression of Acsl3 and Acsl5 in the ileum (both P＜0.05), and compared with the NAFLD model group, the nimesulide group had significant reductions in the mRNA expression of Acsl3 and Acsl5 (both P＜0.05). ConclusionThe COX-2 inhibitor nimesulide can regulate the expression of the Acsl gene family in the ileum of rats with NAFLD, suggesting that COX-2 inhibitors may inhibit the progression of NAFLD through the Acsl gene.
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Long-chain acyl coenzyme A synthetase (ACSL) is a member of the synthetase family encoded by a multigene family; it plays an important role in the absorption and transport of fatty acid. Here we review the roles of ACSL in the regulating absorption and transport of fatty acid, as well as the connection between ACSL and some metabolic diseases.
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In recent years, increasingly evidences show that autophagy plays an important role in the pathogenesis and development of liver diseases, and the relationship between them has increasingly become a focus of concern. Autophagy refers to the process through which the impaired organelles, misfolded protein, and intruding microorganisms is degraded by lysosomes to maintain stability inside cells. This article states the effect of autophagy on liver diseases (hepatic fibrosis, fatty liver, viral hepatitis, and liver cancer), which aims to provide a new direction for the treatment of liver diseases.
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Cellular senescence refers to a process that cellular proliferation and differentiation modulated by the multiple stimulating factors gradually decline. Aging cells present the irreversible stop of proliferation and differentiation and change in secretory function because the cell cycle of aging cells is steadily blocked at some point. It has have been shown that cellular senescence plays an important role in the occurrence and development of liver diseases. In this paper, we review the advances in relations between cellular senescence and liver diseases.
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Objective To investigate the therapeutic effects of early enteral nutrition by gastroscopeguided naso-jejunal feeding tube placement on the intestinal endotoxemia of patient with severe acute pancreatitis (SAP).Methods Fourty-three patients were randomized into two groups:patients receiving early enteral nutrition (EN) by gastroscope-guided naso-jejunal feeding tube placement (24 cases) and those receiving total parenteral nutrition (TPN) ( 19 cases).The serum endotoxin(ET),albumin (ALB) and amylase (AMY) levels were measured.Abdominal distension and other complications were observed in the two groups.Results The abdominal pain and distension relief time,intestinal bleeding,infectious complications of EN group were significantly improved compared with that of TPN group ( P ＜ 0.01 或 P ＜ 0.05 或 P ＜ 0.001 ).The serum ET levels of EN group was much lower ( [ 0.19 ± 0.11 ] EU/ml) than that of TPN group ( [ 0.85 ± 0.28 ] EU/ml)on day 14 post-treatment (t =10.456,P ＜ 0.001 ).The serum AMY levels were decreased significantly in two groups after treatment,and the difference between two groups was not significant (t =3.l17,t =1.889,P ＞0.05 ).The serum ALB recovery of two groups was not significantly different ( P ＞ 0.05 ).Conclusion Gastroscope-guided Naso-jejunal feeding tube placement for early enteral nutrition can protect intestinal mucosa,reduce complications,alleviate symptoms of patients with SAP,which are benefitial factors for the treatment of intestinal endotoxemia in patients with SAP.
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Objective To investigate the feasibility, safety and short-term efficacy of endoscopic implantation of 5-FU Slow-release Particles for advanced gastroenteric tumor. Methods During the endoscopy procedure,slow-releasing 5-FU agents were implanted densely into the tumors and infiltrated area. Forty-five to sixty pieces of agents ( each piece equivalent to 1.67 mg 5-FU)were injected, which containing an average dose of 100 mg 5-FU. Results A total of 13 advanced gastric cancer patients were enrolled into this study. Significant effects were observed in 3 patients and good effects in 8 patients, but 2 cases with no effects. The total effective rate was 84.62%. The endoscopy examination showed that the size of tumors reduced in various degree at 1 - 2months after the implantation. No hemorrhage or perforation was observed. Parameters of hepatorenal function and routine blood test were stable after implantation. Conclusion Endoscopic implantation of 5-FU Slow-release Particles can relieve the symptoms of patients and limit tumor growth in advanced gastroenteric tumor with no marrow and hepatorenal functional impair.
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Objective To observe and analyze the effect of B. Adolescentis and L. Acidophilus on the proportion of Th cell Th1/Th2 in peripheral blood of UC-mice in acute stage and recovery stage. Method 40 BABL/c mice were induced by 3% DSS water for 7 clays free drinking and then with distilled water for 10 days. They were randomly allocated in 4 groups: NS group, SASP group, BF0624 group and LT0637 group, also the fifth group-10 normal animals. The blood of mice were collected by removing their eyes at day 8 and day 18, and then the mononuclear cells were iso]atecl. The proportion of Th1/Th2 was analyze through flow eytometry,hy labeling the specific antibody ot Th cellular membrane with the CD4 antibody, and the cytoplastie antigen of Thl or Th2 with IIA antibody or IF'N-γ antibody. Result The proportion of Th1/Th2 in normal mouse was 0. 84 -0. 94,and it raised up in DSS-mice at both acute stage and recovery stage. It decreased unequally after 7 or 17 days'B, adoleseentis, L. Acidophilus and SASP treatment, but that of all three groups were lower than NS group (2.21±0. 83). Even the proportion got close to the normal animals after 17 days'L. Acidophilus -treated. Conclusion The proportion of Thl/Th2 increased at the acute stage and recovery stage of DSS-mice. Both B. Adoleseentis and L. Acidophilus had more effective than SASP on decreasing the proportion of Th1/Th2 at two stages,ospecially L. Acidophilus.