Résumé
Objective:To investigate the effects of early enteral nutrition intervention on systemic inflammation and intestinal injury in rats with acute pancreatitis and its mechanism.Method:Rat acute pancreatitis model was established. The rats were divided into sham surgery groups, model group, 12 h nutrition support group, 24 h nutrition support group, 48 h nutrition support group, and 48 h nutrition support group +PMA group according to the random number chart method, with 10 rats in each group. After laparotomy, the rats in sham operation group were closed after gently turning the pancreas. The sham operation group and model group were injected with the same amount of physiological salt. Nutritional support group for 12 h, nutritional support group for 24 h and nutritional support group for 48 h were given enteral nutrition support for 12, 24 and 48 h, respectively. Nutritional support group for 48 h +PMA group, intraperitoneal injection of 5 mg/kg NF-κB signaling pathway activator PMA was given after modeling, and nutritional support was given for 48 h. The contents of lipase, amylase and creatinine in serum of each group were detected by automatic biochemical analyzer. The serum levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and D-lactic acid were detected by enzyme-linked immunosorbent assay (ELISA). The content of diamine oxidase (DAO) was detected by colorimetry. Hematoxylin-eosin (HE) staining was used to detect the pathological changes of intestinal mucosa. Western blot was used to detect the expression of NF-κB pathway-related proteins in pancreatic tissue of rats in each group.Results:(1) Lipase, amylase and creatinine in sham operation group, model group, 12 h nutrition support group, 24 h nutrition support group and 48 h nutrition support group were (4.37±0.61) vs (12.021±1.00) vs (8.77±0.62) vs (6.88±0.63) vs (5.20±0.41) U/ml, (1674.03±172.24) vs (4356.30±229.38) vs (3676.11±382.43) vs (2990.06±251.93) vs (1919.75±179.40) U/L, (32.12±3.37) vs (91.73±9.76) vs (72.38±6.83) vs (53.72±5.98) vs (41.82±4.00) U/L. Compared with sham operation group, the contents of serum lipase, amylase and creatinine in model group were significantly increased. Compared with model group, the contents of lipase, amylase and creatinine were significantly decreased after 12, 24 and 48 h of nutritional support, and were time-dependent ( P<0.05). (2) The levels of IL-6, IL-1β, TNF-α and IL-10 were (40.26±3.93) vs (123.34±13.19) pg/ml in sham operation group, model group, 12 h nutritional support group, 24 h nutritional support group and 48 h nutritional support group, respectively vs (108.97±12.70) vs (77.36±6.75) vs (49.18±4.97) pg/ml, (77.53±9.95) vs (316.36±23.76) vs (254.79±13.96) vs (177.92±17.20) vs (119.19±13.17) pg/ml, (62.94±5.39) vs (353.16±28.03) vs (275.87±22.11) vs (198.78±24.33) vs (94.60±9.41) pg/ml, (41.21±4.29) vs (6.92±1.01) vs (10.76±0.66) vs (21.24±1.64) vs (35.33±1.69) pg/ml. Compared with sham operation group, the contents of serum inflammatory cytokines IL-6, IL-1β and TNF-α in model group were significantly increased, while the content of IL-10 was significantly decreased. Compared with model group, the contents of IL-6, IL-1β and TNF-α were significantly decreased after 12, 24 and 48 h of nutritional support, while the contents of IL-10 were significantly increased in a time-dependent manner ( P<0.05). (3) The intestinal histopathological scores, DAO and D-lactic acid of sham operation group, model group, 12 h nutritional support group, 24 h nutritional support group and 48 h nutritional support group were (0.00±0.00) vs (4.20±0.60) vs (3.00±0.45) points, respectively vs (1.90±0.54) vs (1.30±0.64) points, (4.92±0.42) vs (14.95±1.20) vs (11.87±1.13) vs (9.02±0.53) vs (6.30±0.59) U/L, (2.39±0.22) vs (6.92±0.46) vs (5.21±0.28) vs (3.64±0.39) vs (2.95±0.15) nmol/ml. Compared with sham operation group, intestinal histopathological scores, DAO and D-lactic acid levels were significantly increased in model group. Compared with model group, intestinal histopathological scores, DAO and D-lactic acid levels were significantly decreased after 12, 24 and 48 h of nutritional support ( P<0.05). (4) The protein expressions of NF-κB p65 and p-IκBα were (0.23±0.03) vs (0.94±0.10) vs (0.75±0.06) vs (0.62±0.06) in sham operation group, model group, 12 h nutrition support group, 24 h nutrition support group and 48 h nutrition support group, respectively. vs (0.41±0.06), (1.06±0.12) vs (0.25±0.04) vs (0.47±0.03) vs (0.62±0.08) vs (0.85±0.08). Compared with sham operation group, NF-κB p65 protein level in model group was significantly increased, while p-IκBα protein level was significantly decreased. Compared with model group, the NF-κB p65 protein level was significantly decreased after 12, 24 and 48 h of nutritional support, while the P-iκBα protein was significantly increased ( P<0.05). (5) NF-κB p65, p-IκBα, IκBα, IL-6, IL-1β, TNF-α, IL-10, lipase, amylase and creatinine were (0.41±0.06) vs (0.82±0.06) in the 48 h group and the 48 h +PMA group, respectively. (0.85±0.08) vs (0.37±0.02), (1.05±0.11) vs (1.10±0.14), (49.18±4.97) vs (105.68±10.69) pg/ml, (119.19±13.17) vs (247.16±23.41) pg/ml, (94.60±9.41) vs (328.24±30.86) pg/ml, (5.20±0.41) vs (10.33±1.01) U/ml, (1919.75±179.40) vs (4023.40±334.56) U/L, (5.20±0.41) vs (10.33±1.01) U/ml, (41.82±4.00) U/L vs (81.33±7.96) U/L. Compared with the 48 h group, the expression level of NF-κB p65 protein, IL-6, IL-1β, TNF-α, lipase, amylase and creatinine in the 48 h +PMA group were significantly increased, while the expression level of P-iκBα protein and the content of IL-10 were significantly decreased ( P<0.05) . Conclusion:Early nutritional intervention can inhibit inflammatory response, reduce intestinal injury and control the development of acute pancreatitis by regulating NF-κB signaling pathway.
Résumé
Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.
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Objective Todetectthe level of plasma VEGF before and after treatment of arteriovenous mal-formationpatients,and the pathophysiological role of VEGF in arteriovenous malformationpatientswas also studied. Methods The blood samples of 17 arteriovenous malformation patients were collected according to the following three groups:group before operation(AVM),group 24 hours after operation(AVM24h)and group 30 days after operation(AVM30d).As a control(Con),22 blood samples were collected from lumbar laminectomypatients.The level of plasma VEGF was determined by ELISA assay. Results Compared with the control group,the plasma VEGF was significantly decreased in AVM group and AVM24h group(P < 0.05),while the plasma VEGF in AVM30d groupwas similarto that of the control group. Conclusions Abnormal blood vessel plays an important pathophysiological role in cerebral arteriovenous malformation,and the metabolism of VEGF is involved in the pro-cess of arteriovenous malformation.
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Objective To study the influence factors of infertility by analysis of semen sample and reference for clinical treat-ment.Methods 3 527 cases of semen sample were collected from Jan 2012 to Jun 2014.All samples were analysed by SQA-V analyzer and compared with 80 cases of normal semen.Results There were 358 normal samples (10.2%)and 3 169 ab-normal samples (89.8%).Among the abnormal samples low sperm motility had the highest ratio (2.7%)while abnormal pH had the lowest ratio (61.5%).All the indexes had significance difference to normal sample expect pH value (t=0.065, P =0.969).Among them,the comparison of rate had statistical significance(χ2 =3.214~24.712,P <0.05).The compari-son of mean also had statistical significance(t=2.523~15.324,P <0.05).Conclusion Infertility male almost has abnormal index of semen volume,liquefaction time,sperm motility,sperm density,sperm morphology,sperm viability.Accurately sperm analysis can provide objective basis to clinical diagnosis and treatment.