Objective To observe pathological process of intestinal epithelial cells subjected to ischemia,ischemia/reperfusion injury and inflammation simulated hypoxia/reoxygenation (H/R) and lipopolysaccharide (LPS) challenged human fetal normal colonic cell (FHC) line in vivo,and to observe the changes when the assaulted intestinal epithelial cells were treated with emodin,in order to explore the possible intervention targets of emodin.Methods Normoxia group:the FHC cells were cultured in 95％ air and 5％ CO2 at 37 ℃.Hypoxia (H) group:the cells were cultured with a mixed anaerobic gas of 1％ O2,5％ CO2 and 94％ N2 at 37 ℃ for 1,2,3,4 hours.H + LPS group:the cells were cultured in hypoxic condition as H group with simultaneous challenge of LPS (1 mg/L).H/R group:the cells were cultured in hypoxia for 3 hours followed by reoxygenation for 1,2,3 and 4 hours,respectively.H/R + LPS group:the cells were cultured in H/R as H/R group and LPS (1 mg/L) simultaneously.Emodin intervention group:the cells were cultured in H3 h/R2 h + LPS and emodin (20,40,60,80 μmol/L) simultaneously.The variation trends of phosphorylation nuclear factor-κB profilin-o (pIκB-α),phosphorylation NF-κBp65 (pNF-κBp65) and their downstream target gene cyclooxygenase-2 (COX-2),and hypoxia-inducible factor-1α (HIF-1 α) were determined by Western Blot.The morphological changes in intestinal epithelium in different groups were observed using light microscope.The effect of emodin on the proliferation of intestinal epithelial cell was measured by methyl thiazolyl tetrazolium (MTT) assay.Results ① H group:the expressions of pIκB-α,pNF-κBp65 and COX-2 were upregulated,peaking at H1 h (0.350 ± 0.018,1.083 ± 0.054,0.903 ± 0.045),and then they gradually lowered (F value was 3.011,7.247,5.754,P value was 0.013,0.000,0.005,respectively).The expression of HIF-1 α peaked at H3 h (1.511 ± 0.076),but there was no significant difference among different groups (F=1.881,P=0.062).H + LPS group:the expressions of pIκB-α,pNF-κBp65,COX-2,HIF-1α were increased with elongation of duration of hypoxia,and a maximal induction was observed at H3 h (0.504 ± 0.025,1.255 ± 0.063,0.812 ± 0.041,1.209 ± 0.075,F value was 2.683,8.774,9.765,2.432,and P value was 0.011,0.000,0.000,0.026,respectively).H/R group:with the prolonged duration of reoxygenation,the expressions of NF-κB signaling pathway proteins (pIκB-α,pNF-κBp65,COX-2) were decreased and dropped to nadir at H3 h/R4 h (0.712 ± 0.034,1.202 ± 0.048,0.691 ± 0.042,F value was 1.923,6.765,2.719,and P value was 0.063,0.000,0.016,respectively).Compared with H group,HIF-1α was decreased with a prolonged duration of reoxygenation in H/R group,but there was no significant difference in value among different time points (F=1.280,P=0.081).H/R + LPS group:pIκB-o,pNF-κBp65,COX-2,HIF-1α showed no sign of degradation with the prolonged duration of reoxygenation,and their expression increased to maximum analogously at R2-3 h (3.302 ± 0.061,2.315 ± 0.055,2.017 ± 0.043,2.413 ± 0.098,Fvalue was 4.614,1.652,5.970,2.076,and Pvalue was 0.001,0.067,0.000,0.037,respectively).Emodin group:emodin when co-treated with H/R + LPS inhibited the expression of HIF-1o and NF-κB pathways with a dose-effect relationship (P＜0.05 or P＜0.01).Emodin at the dose of 80 μmol/L showed most marked inhibition (2.599 ± 0.130,1.772 ± 0.089,2.590 ± 0.129,2.518 ± 0.125).However,after treatment of emodin did not show such effect.② After treatment with H/R + LPS,there were morphological changes in cells:vacuoles,deformation and fusion.The speed of cell growth became much slower compared with H group.③ Emodin (20-80 μmol/L) had no significant effect on cell proliferation.Although emodin produced biological effect in this concentration range,it had no cellular toxicity.Conclusions Both hypoxia and inflammation can activate the hypoxia pathway of HIF-1α and the pro-inflammatory pathway of NF-κB,but different stimuli cause varying degrees of activation in these two pathways.In H/R group,both pathways were weakened during reoxygenation.However,in H/R + LPS group,the proteins remained to show a relatively high expression during the process of reoxygenation.This may be related to the pathophysiological mechanism of intestinal ischemia/reperfusion injury:hypoxia/reperfusion injury and LPS act together to destroy the intestinal epithelial cells and induce gut-derived sepsis.Emodin may inhibit inflammation by blocking HIF-1α/NF-κB-COX-2 signaling pathways.

Objective To observe the level of hypoxia-inducible factor-1 alpha (HIF-1α) and its downstream target gene cyclooxygenase-2 (COX-2) in LPS-treated intestinal epithelial cells,and to explore the possible intervention targets of Rheum emodin.Methods Human intestinal epithelial cells were cultured in vitro treated with LPS to establish the experimental model.The protein level trends of HIF-1α and COX-2 were measured by Western blot in LPS dose-dependent and time-dependent manners.The protein level trends of HIF-1α,COX-2,Phospho-IκB-α and Phospho-NF-κB p65 were measured in LPS plus various concentrations of Rheum emodin treated groups.The expression of HIF-1α mRNA were detected by PCR after cells treated with LPS or LPS plus Rheum emodin,respectively.The effect of Rheum emodin on the proliferation of intestinal epithelial cells was measured by MTT assay in each group.Data were analyzed with ANOVA,and P ＜0.05 was considered significant.Results LPS induced the protein level of HIF-1α in a dose-dependent and a time-dependent manners.With increasing concentrations of LPS,the protein level of HIF-1α increased to the peak when cells were treated with LPS at 10-30mg/mL,and then gradually decreased (P ＜0.05).Firstly the protein level of HIF-1α reached the peak at 0.5 h after treatment,and then decreased to the lowest level at 4 h,and finally returned to a high level (P＜0.05).The protein level trend of COX-2 went a similar way to that of HIF-1α (P ＜0.05).Rheum emodin inhibited the protein levels of LPS-induced HIF-1α,COX-2,Phospho-IκB-α and Phospho-NF-κB p65 with a significant dose-effect relationship (P ＜ 0.05).The PCR showed Rheum emodin inhibited LPS-induced increasing expression of HIF-1α mRNA.MTT assay showed different concentrations of Rheum emodin (0 μmol/L,20 μmol/L,40 μmol/L,60 μmol/L,80 μmol/L) had no significant effect on cell proliferation (0.95 ± 0.02,0.89 ± 0.03,0.88 ± 0.04,0.91 ± 0.03,0.83 ± 0.03,P ＞ 0.05).Although Rheum emodin produced biological effect at this concentration range,and it had no toxicity to intestinal cells.Conclusions LPS induces HIF-1α/COX-2 signaling pathway in a time-dependent and a dose-dependent manners in intestinal epithelial cells.Rheum emodin blocks the hypoxia pathway of LPS/HIF-1α/COX-2 and the inflammatory pathway of LPS/IκB-α/NF-κB/COX-2,which may play a protective effect on intestinal epithelial cells.

Objective:To express and purify recombinant human collagen type Ⅲ and evaluate its properties.Methods:The recombinant genetic engineering strain pET30a(+)-1880/pACYCDuet-hy726/bL21(DE3) was constructed to stably co-express recombinant human type Ⅲ collagen (rhCol) and prolyl hydroxylase. rhCol was prepared and purified by E. coli high-density fermentation, salting out and column chromatography protein purification technology. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to determine the purity of rhCol. The N-terminal amino acid sequences of rhCol were determined by automatic protein polypeptide sequencing instrument. The hydroxyproline content of rhCol was determined by ultraviolet spectrophotometry. The cellular compatibility of rhCol was evaluated by MTT assay. Results:The final wet weight of high-density fermentation was about 200 g/L. The expression level was about 3 g/L. The purity of rhCol by affinity chromatography was over 95%. The results showed that the hydroxyproline content of rhCol was 11.44%, and the rhCol products have good water solubility and cell compatibility.Conclusions:RhCol can be widely applied to the field of skin care and biomedicine as an excellent biological material.

BACKGROUND:A large amount of studies have confirmed that synovial mesenchymal stem cells have the similarity in cellmorphology, immune phenotype, colony forming ability and differentiation potential with bone marrow mesenchymal stem cells. But bone marrow mesenchymal stem cells are better than synovial mesenchymal stem cells in the ability to differentiate into cartilages. OBJECTIVE:To discuss the possibility of using synovial mesenchymal stem cells as seed cells for meniscal tissue engineering. METHODS:The synovial mesenchymal stem cells were isolated from rabbit synovial tissues with limiting dilution monoclonal culture method, and then the cells were purified. The morphology, ultrastructure, molecular phenotype, proliferation kinetics, karyotype and tumorigenicity of the in vitro cultured cells were analyzed. RESULTS AND CONCLUSION:The synovial mesenchymal stem cells isolated from the rabbit synovial cells had high proliferation capacity during in vitro monolayer culture. The synovial mesenchymal stem cells grew to peak at 6 days, and the doubling time was (30.2±2.4) hours. Flow cytometry results showed the synovial mesenchymal stem cells could express some molecular makers of mesenchymal stem cells, such as CD44 and CD90. DNA contents check, karyotype test and oncogenicity test confirmed isolated and purified synovial mesenchymal stem cells were the normal diploid cells without tumorigenicity, so the cells can be used as seed cells for meniscal tissue engineering.

BACKGROUND:Smal intestinal submucosa is characterized as antimicrobial activity, good biocompatibility, bio-mechanical properties, and rapid degradation in vivo, similar to the extracellular matrix of meniscal
fibrochondrocytes.
OBJECTIVE:To observe whether there exists a good histocompatibility between smal intestinal submucosa and synovial mesenchymal stem cells.
METHODS:Smal intestinal submucosa was treated with physical and chemical treatment. And hematoxylin-eosin staining and scanning electron microscopy observation were performed. Then, smal intestinal submucosa extracts were prepared for the fol owing experiments. (1) Pyrogenic test:smal intestinal submucosa extracts were injected into the ear vein of New Zealand white rabbits. (2) Skin sensitization test:smal intestinal submucosa extracts, paraformaldehyde solution and normal saline were respectively injected intradermal y into New Zealand white rabbits. (3) General toxicity test:smal intestinal submucosa extracts and normal saline were respectively injected into the ear vein of New Zealand white rabbits. Smal intestinal submucosa was co-cultured with osteogenic rabbit synovial mesenchymal stem cells, and smal intestinal submucosa cultured alone served as control. RESULTS AND CONCLUSION:There were some intestinal mucosal epithelial cells, fat cells and other cells adhered onto the surface of smal intestinal submucosa after physical treatment. While, the amount of residual cells decreased sharply after chemical treatment. But the main structure and the component had not been changed. The surface of smal intestinal submucosa was smooth and no cells remained, and there was a three-dimension network spatial structure. The porosity was 80%. Smal intestinal submucosa is a non-toxic, nonirritating, non-immunogenic biomaterial with very good biocompatibility, which has a good histocompatibility with rabbit synovial mesenchymal stem cells.

Purpose To investigate the expression of the protein of CD151 and MT1-MMP in adenocarcinoma of esophagogastric junc-tion tissues and to explore their relationship with the invasiveness and metastasis of the tumor. Methods CD151 and MT1-MMP pro-tein were detected by immunohistochemical staining respectively in 15 cases of paraneoplastic normal gastric mucosa tissue, 40 cases dysplasia and 172 cases of adenocarcinoma of esophagogastric junction tissues. Results All of the expression level of CD151 and MT1-MMP protein were increasing according to the order of normal-dysplsia-carcinoma. The expression rate of CD151 was 6. 67%, 20. 0% and 78. 3%, while the rate of MT1-MMP with 13. 3%, 22. 5% and 79. 1% in the normal, dysplasia and carcinoma subgroup. The expression rate of each protein were significant difference among the three goroups (P<0. 05). In the adenocarcinoma of esopha-gogastric junction tissues, the expression of them in the subgroup of poorly-differentiated with serosa invasion and lymph nodes metasta-sis was significantly higher than the other subgroup of well-differentiated with non serosa invasion or lymph nodes metastasis ( P <0. 05 ) . There was a positive correlation between the expression of CD151 and MT1-MMP protein in adenocarcinoma of esophagogastric junction tissues ( P<0. 05 ) . Conclusions CD151 and MT1-MMP assuredly and highly express in the adenocarcinoma of esopha-gogastric junction tissues and they have close relationships, which could involved in the invasiveness and metastasis synergistically in this tumor.

BACKGROUND:Anterior cruciate ligament (ACL) injury is a commonly sport-induced knee joint injury that does serious harm to the knee stability. ACL reconstruction is a commonly used treatment method, but researches on 1/2 peroneus longus tendon (PLT) graft are rarely reported. OBJECTIVE: To investigate the clinical outcomes of removing the autologous ipsilateral 1/2 PLT under arthroscopy for ACL reconstruction. METHODS:106 patients with complete ACL rupture in the Affiliated Hospital of Traditional Chinese Medicine, Southwest Medical University from December 2010 to December 2014 were enrolled, and autologous ipsilateral 1/2 PLT was removed under arthroscopy for ACL reconstruction. At baseline, 3, 6 and 12 months postoperatively, the knee stability was evaluated manually through the anterior drawer test, Lachman test, and pivot-shift test, and the knee function was evaluated by Tegner activity scale, Lysholm and International Knee Documentation Committee scores. RESULTS AND CONCLUSION: Postoperative anterior drawer test, Lachman test, and pivot-shift test tests were negative in all patients. In terms of Tegner activity scale, Lysholm and International Knee Documentation Committee scores, there were significant differences at baseline and postoperative 3 months as compared with postoperative 6 months (P < 0.05); the scores at baseline and postoperative 3 months showed significant differences compared with 12 months postoperatively (P < 0.05); the scores showed no significant difference between 6 and 12 months postoperatively (P > 0.05). These results indicate that autologous ipsilateral 1/2 PLT is a good choice for ACL reconstruction under arthroscopy, achieving rapid and satisfactory functional recovery of the knee joint, which is not only minimally invasive and easy to operate, but also exhibits good therapeutic efficacy.

Objective To establish the fingerprint analysis method of Schisandrae Chinensis Fructus by HPLC; To analyze the similarity on the fingerprints of Schisandrae Chinensis Fructus from different producing areas. Methods The chromatographic separation was performed by HPLC on a Diamonsil C18 column (250 mm×4.6 mm, 5 μm). Acetonitrile-water was used as gradient mobile phase. The flow rate was 1.0 mL/min. The column temperature was maintained at 30℃. The detection wavelength was set at 254 nm.Results The HPLC fingerprint analysis method of Schisandrae Chinensis Fructus was established. Twenty-nine common fingerprint peaks were identified. The similarities of the fingerprints of ten samples from different producing areas were above 0.95.Conclusion The method is simple and reliable, which can provide a scientific basis for quality evaluation of Schisandrae Chinensis Fructus.

Obgective To observe the effect of Milrinone combined with Esmolol in the treatment of severe hand-foot-and-mouth disease (HFMD) so as to improve the prognosis.Methods Eighty-two cases of children with critically severe HFMD,who were hospitalized in the Intensive Care Unit of Xuzhou Children's Hospital,were enrolled in the study from may of 2013 to June of 2014,and were randomly divided into a control group and an observation group.The control group was given intravenous Milrinone,and the observation group was given Milrinone combined with Esmolol.The heart rate (HR),systolic blood pressure (SBP),cardiac output (CO),left ventricular ejection fraction (LVEF) and brain natriuretic peptide (BNP),norepinephrine (NE) were detected on admission and checked again 1 hour and 48 hours again after treatment.The changes in the above indicators were compared before and after therapy to evaluate the clinical curative effect.Results (1) There was no significant difference in the HR,SBP,CO,LVEF,BNP and NE between the 2 groups before treatment(all P ＞ 0.05).(2) The HR,SBP,CO and LVEF of 2 groups were significantly improved after 1 hour treatment compared with those before treatment (all P ＜ 0.05),and the BNP and NE of the control group were not obviously improved compared with those before therapy (all P ＞ 0.05),but the significant changes were seen in the observation group (all P ＜ 0.05).Forty-eight hours after the treatment,all the observed indicators in 2 groups were significantly improved compared with those before treatment(all P ＜ 0.01).(3)Compared with control group,the HR,SBP,CO,LVEF and BNP of the observation group were significantly improved after 1 hour treatment (t =2.08,2.12,-2.11,-2.37,2.07,all P ＜ 0.05),but the NE of the observation group had no obvious improvement (t =0.83,P ＞ 0.05).All the observed indicators of the observation group were significantly improved compared with the control group after 48 hours treatment(t =3.76,2.48,-2.70,-2.27,5.37,2.74,all P ＜ 0.05).Conclusions Milrinone combined with Esmolol can significantly improve the cardiac function and the vital signs of the children with critically severe HFMD,which can be recommended clinically.

OBJECTIVETo observe the effect of octyl-a-cyanoacrylate upon bone healing and its degradation in vitro after middle tibial transverse fracture in rabbitsì and to establish treatment of higher efficacy with the application of octyl-a-cyanoacrylate.

METHODSMiddle tibial transverse fracture model of New Zealand rabbits was established. In the experimental group, internal fixation with 2 mm Kirschner wires was performed and the broken ends were fixed with octyl-a-cyanoacrylate. In the control group, only internal fixation with 2 mm Kirschner wires was conducted. Animals were killed at preset time intervals of 2, 4, 6, 8, 10 and 12 weeks postoperatively and samples were harvested.

RESULTSTwo weeks after operation, clear fracture lines were observed in both the experimental and the control groups. Fibrous soft tissue connection was noted between the broken ends and there was soft tissue adhesion around the fracture site. There was no callus formation and the broken ends were surrounded by adhesive soft tissues. Obvious external callus formation was confirmed at 8 weeks after operation in both groups with partial disappearance of fracture lines. Ten and twelve weeks after the operation, fracture lines disappeared completely and there was obvious external callus formation and bone union. In the fourth week, fibrous cells and chondrocytes were found to grow into the colloid and surround it at the 6th week. The adhesive material was degraded and gradually absorbed at the 8th week. Chondrification was observed.

CONCLUSIONSTwo weeks after fixation for tibial fracture in rabbits, octyl-a-cyanoacrylate begins in vivo degradation. Chondrocytes and fibrocytes gradually grow into the degradation area and surround the adhesive material, which broke into pieces at 8 weeks. Complete degradation and disappearance of the adhesive material is present between 10 and 12 weeks. No barrier effect hampering fracture healing is noted.

Adhesives ; therapeutic use ; Animals ; Cyanoacrylates ; therapeutic use ; Rabbits ; Radiography ; Tibial Fractures ; diagnostic imaging ; pathology ; therapy