白原胶基因工程菌株 Xanthomonas campestris ΔrpfB ΔxanK发酵工艺优化

黄原胶是一种亲水杂多糖,广泛应用于食品、药品、化妆品等领域,但因其自身带有菌黄素而呈黄色,限制了其应用范围。野油菜黄单胞菌工程菌株Xanthomonas campestrisΔrpfB ΔxanK可以生产不含有菌黄素的黄原胶,命名为“白原胶”,具有极其广阔的市场应用前景,但其发酵培养条件亟待进一步优化。本研究通过单因素实验和正交实验对Xanthomonas campestrisΔrpfB ΔxanK菌株发酵培养基进行优化,以提升胶体产量,并用15 L机械搅拌发酵罐进行放大验证,对最终生产的胶体和市售黄原胶通过红外光谱进行比对验证。结果表明,最佳发酵培养基为玉米淀粉蔗糖(31,质量比)5%(质量分数,下同),蛋白胨0.2%,K_(2)HPO_(4)0.4%,CaCO_(3)0.5%,大豆0.3%,初始pH 8,接种量5%(体积分数),摇床转速220 r/min,发酵温度28℃,发酵时间72 h。摇瓶产量可以达到33.8 g/L。经15 L机械搅拌发酵罐扩大实验,前期通气量1 vvm,后期逐渐增大到2 vvm,前36 h转速250 r/min,后36 h转速400 r/min,发酵72 h最终产量为38.4 g/L。红外光谱分析显示,白原胶结构与市售黄原胶结构吻合,证明两者具有相同的结构。本研究为Xanthomonas campestrisΔrpfB ΔxanK菌株的工业化应用提供了科学合理的发酵策略,为白原胶进一步的工业应用提供参考。

Vascular Channel Formation by Osteosarcoma Cells in Vitro: Vasculogenic Mimicry

Objective: To observe whether there is evidence for vascular channel formation by osteosarcoma cellsin vitro and to illustrate mechanism of vasculogenic mimicry in osteosarcoma.Methods: Osteosarcoma cell lines (U-2OS) were tested for their ability to form tubular networks in three-dimensional culture containing type I collagen. The structures of the tubular networks were observed under a phase contrast microscope and an electron microscope.Results: Observation under light microscopy and electron microscopy showed that high aggressive osteosarcoma cells line (U-2OS) formed networks containing channels when grown in three-dimensional culture containing type I collagen in the absence of endothelial cells or fibroblasts.Conclusion: These observations strongly suggest that aggressive osteosarcoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis and have the ability of vasculogenic mimicry. Key words osteosarcoma cells line - vasculogenesis mimicry - angiogenesis - 3-dimensional cultures This study was supported in part by the National Natural Sciences Foundation of China (No. 30271314).

Analysis of Molecular Weight Distribution and Antioxidant Activities of Cirrhinus molitorella Skin Collagen Hydrolysates

[Objective] The aim of this work was to identify molecular weight （MW） distribution and antioxidant activity of fish skin col agen hydrolysates. [Method] The MW distribution of hydrolysates was determined using both size exclusion chromatography and matrix-assisted laser desorption ionization time-of-flight mass spec-trometry （MALDI-TOF-MS）. Fish skin were treated by the alkaline protease 2709. [Result] The optional conditions for hyerolysis were time 3 h, temperature 55 ℃, pH 10.0, substrate concentration 80 g/L and E/S 4%. The results of both methods indi-cated that the molecular weight of col agen hydrolysates was from 400 to 1 800 Da, and the peptides’ molecular weight was less than 1 400 Da mostly. The reducing power and antioxidant/radical scavenging activity [1, 1-diphenyl-2-picryl-hydrazyl （DPPH） free radical scavenging activity] were determined. [Conclusion] The results reveal that the fishskin hydrolysate is a potential source of antioxidants.

Morphological and serum hyaluronic acid, laminin and type Ⅳ collagen changes in dimethylnitrosamine-induced hepatic fibrosis of rats

AIM： To study the morphological and serum hyaluronic acid （HA）, laminin （LN）, and type IV collagen changes in hepatic fibrosis of rats induced by dimethylnitrosamine （DMN）.METHODS： The rat model of liver fibrosis was induced by DMN. Serum HA, type IV collagen, and LN were measured by ELISA. The liver/weight index and morphological changes were examined under electron microscope on d 7, 14, 21, and 28 by immunohistochemical alpha smooth muscle actin α-SMA staining as well as Sirius-red and HE staining.RESULTS： The levels of serum HA, type IV collagen and LN significantly increased from d 7 to d 28 （P = 0.043）. The liver/weight index increased on d 7 and decreased on d 28. In the model group, the rat liver stained with lie and Sirius-red showed evident hemorrhage and necrosis in the central vein of hepatic 10 Iobules on d 7. Thin fibrotic septa were formed joining central areas of the liver on d 14. The number of α-SMA positive cells was markedly increased in the model group. Transitional hepatic stellate cells were observed under electron microscope. All rats in the model group showed micronodular fibrosis in the hepatic parenchyma and a network of α-SMA positive cells. Typical myofibroblasts were embedded in the core of a fibrous septum. Compared to the control group, the area-density percentage of collagen fibrosis and pathologic grading were significantly different in the model group （P〈0.05） on different d （7, 14, and 28）. The area-density percentage of collagen fibrosis in hepatic tissue had a positive correlation with the levels of serum HA, LN, and type IV collagen.CONCLUSION： The morphological and serum HA, type IV collagen, and LN are changed in DMN-induced liver fibrosis in rats.

Effects of P2Y_1 receptor on glial fibrillary acidic protein and glial cell line-derived neurotrophic factor production of astrocytes under ischemic condition and the related signaling pathways

Objective The present study aimed to explore the role of P2Y1 receptor in glial fibrillary acidic protein （GFAP） production and glial cell line-derived neurotrophic factor （GDNF） secretion of astrocytes under ischemic insult and the related signaling pathways. Methods Using transient right middle cerebral artery occlusion （tMCAO） and oxygen-glucose-serum deprivation for 2 h as the model of ischemic injury in vivo and in vitro, immunofluorescence, quantitative real-time reverse transcription-polymerase chain reaction （RT-PCR）, Western blotting, enzyme linked immunosorbent assay （ELISA） were used to investigate location of P2Y1 receptor and GDNF, the expression of GFAP and GDNF, and the changes of signaling molecules. Results Blockage of P2Y1 receptor with the selective antagonist N^6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate diammonium （MRS2179） reduced GFAP production and increased GDNF production in the antagonist group as compared with simple ischemic group both in vivo and in vitro. Oxygen-glucose-serum deprivation and blockage of P2Y1 receptor caused elevation of phosphorylated Akt and cAMP response element binding protein （CREB）, and reduction of phosphorylated Janus kinase2 （JAK2） and signal transducer and activator of transcription3 （STAT3, Ser727）. After blockage of P2Y1 receptor and deprivation of oxygen-glucose-serum, AG490 （inhibitor of JAK2） reduced phosphorylation of STAT3 （Ser727） as well as expression of GFAP; LY294002, an inhibitor of phosphatidylinositol 3-kinase （PI3-K）, decreased phosphorylation of Akt and CREB; the inhibitor of mitogen-activated protein kinase kinase 1/2 （MEK 1/2） U0126, an important molecule of Ras/extracellular signal- regulated kinase （ERK） signaling pathway, decreased the phosphorylation of JAK2, STAT3 （Ser727）, Akt and CREB. Conclusion These results suggest that P2Y1 receptor plays a role in the production of GFAP and GDNF in astrocytes under transient ischemic condition and the related signaling pathways may be JAK2/STAT3 and PI3-K/Akt/CREB, respectively, and that crosstalk probably exists between them.

Expression and Identification of Mycoplasma penetrans P35 Lipoprotein

Objective: To study the biological activity of Myco-plasma penetrans 35kDa lipoprotein(P35) in vitro, prokaryotic expression vector pQE31//p35 was constructed and recombinant fusion protein P35 (rP35) was expressed in E.coli. Methods: The p35 gene was amplified by polymerase chain reaction(PCR), cloned to pQE31, and a positive clone was screened. PCR-mediated mutagenesis was used to change the two "TGA" triplets to "TGG" triplets within the p35 gene. Production of the recombinant protein was induced by the addition of IPTG to the E.coli culture. rP35 was purified with a Ni-NTA Spin Kit and rP35 purification was analyzed by Western blot. Results: About 1Kb PCR amplification was cloned into pQE31. The two "TGA" triplets within the p35 gene were successfully changed to "TGG" triplets. The pQE31/p35 vector expressed a protein with a calculated molecular mass of 37.4kDa in E.coli. Western blot indicated the 37.4kDa protein was rP35 . Conclusion: PQE31/p35, a prokaryotic expression vector containing p35 gene, was successfully constructed and expressed in E.coli.

Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepa1-6 cells and its relationship with collagen type Ⅳ

AIM： To investigate the regulation of activin receptor-interacting protein 2 （ARIP2） expression and its possible relationships with collagen type Ⅳ （collagen Ⅳ） in mouse hepatoma cell line Hepal-6 cells. METHODS： The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate （PMA）, forskolin and A23187. After pcDNA3- ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor （ActRⅡ） and collagen Ⅳ expression were evaluated. RESULTS： The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation （2 or 4 h）. The ARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 （25.3% ± 5.7% vs 48.1% ± 3.6%, P 〈 0.01）. ARIP2 overexpression could remarkably suppress the expression of ActRⅡA mRNA in dose-dependent manner, but has no effect on ActRⅡB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells. CONCLUSION： These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

Idiopathic fistula-in-ano

Fistula-in-ano is the most common form of perineal sepsis.Typically,a fistula includes an internal opening,a track,and an external opening.The external opening might acutely appear following infection and/or an abscess,or more insiduously in a chronic manner.Management includes control of infection,assessment of the fistulous track in relation to the anal sphincter muscle,and finally,definitive treatment of the fistula.Fistulotomy was the most commonly used mode of management,but concerns about post-fistulotomy incontinence prompted the use of sphincter preserving techniques such as advancement flaps,fibrin glue,collagen fistula plug,ligation of the intersphincteric fistula track,and stem cells.Many descriptive and comparative studies have evaluated these different techniques with variable outcomes.The lack of consistent results,level I evidence,or long-term follow-up,as well as the heterogeneity of fistula pathology has prevented a definitive treatment algorithm.This article will review the most commonly available modalities and techniques for managing idiopathic fistula-in-ano.

Effect of matrine and carvedilol on collagen and MMPs activity of hypertrophy myocardium induced by pressure overload

Objective： To explore the effect and mechanism of matrine （Mt.） on myocardial interstitial fibrosis induced by pressure overload. Methods： Pressure overloaded myocardial hypertrophy was produced by banding of aorta abdominalis in 67 male Sprague-Dawley rats weighing （200±15） g. The rats were assigned into one of the following groups： sham-operation control, operation control, operation group treated with matrine （15 mg/（kg·d）） and treated with carvedilol （Car.） （3.6 mg/（kg·d）） group. The rats were given drugs one day after operation. Five weeks after treatment, the left ventricular weight （LVW） was measured and the volume of myocardial cells was detected with Hematoxylin-Eosin （H-E） stain and Masson stain was used to assess the level of fibrosis of the myocardial matrix. Myocardial metalloproteinase activity was quantified with zymography, and survival rate was calculated. Results： Survival rate significantly decreased （P〈0.05）, LVW/BW （body weight）, MMP-2 （matrix metalloproteinase-2） activity （P〈0.05）, size of cardiomyocytes and interstitial fibrosis obviously increased in the operation group compared with sham control group. Mr. and Car. treatment can significantly increase survival rate （P〈0.05）, decrease LVW/BW （P〈0.05） and MMP-2 activity （P〈0.05）, decrease size of cardiomyocytes and interstitial fibrosis compared with operation group. But there was difference compared with sham group. Conclusion： Matrine was shown to be able to prevent cardiac remodelling of bypertrophy cardium induced by pressure overload including myocardial hypertrophy and fibrosis which may be associated with the decrease in MMP-2 activity of heart.

INFLUENCE OF QUERCETIN AND X-RAY ON COLLAGEN SYNTHESIS OF CULTURED HUMAN KELOID-DERIVED FIBROBLASTS

Objoctive To investigate the effects of quercetin and X-ray on collagen synthesis of cultured human keloid-derived fibroblast and the mechanism. Methods Collagen synthesis of cultured human keloid and normal fibroblasts were detected by hydroxyproline colorimetric analysis. Immunocytochemical staining was used to investigate collagen I and III expression, mRNA expression of collagen Ⅰ and Ⅲ, and transforming growth factor （ TGF）-β1 were assayed by reverse transcription-polymerase chain reaction （RT-PCR） and real-time PCR. Results Quercetin inhibited the collagen synthesis of both keloid and normal fibroblasts in a dose-dependent man- ner. Immunocytochemical staining indicated that collagenⅠ and Ⅲ were down-regulated by quercetin and X-ray （P 〈 0.05 ）, particularly collagen I （ P 〈 0. 05 ）. mRNA expression of both collagen I and III in quercetin groups significantly decreased compared with that in control group （ P 〈 0. 05 ）, especially in the group treated with both quercetin and X-ray （ P 〈 0. 01 ）. mRNA level of TGF-[31 gene was down-regulated by quercertin （ P 〈 0. 05 ）. Conclusions Quercetin will probably be one of the new medicines which could effectively treat keloid. Quercetin combined with X-ray could reduce the dose of radiation.

SERUM CONCENTRATIONS OF HYALURONIC ACID, PROCOLLAGEN TYPE III NH_2-TERMINAL PEPTIDE, AND LAMININ IN PATIENTS WITH CHRONIC CONGESTIVE HEART FAILURE

Objective To explore the role of serum fibrotic indices including hyaluronic acid （HA）, procollagen type Ⅲ NH2-terminal peptide （PCIIIP）, and laminin （LN） in assessing the severity of myocardial fibrosis in chronic congestive heart failure （CHF）. Methods Serum levels of HA, PCIIIP, and LN in 39 patients with CHF E [14 with New York Heart Association （NYHA） functional class II, 21 with class Ⅲ, 4 with class Ⅳ] and in 46 patients with NYHA functional class I were assessed by radioimmunoassay. Results The serum concentrations of HA, PCMP, and LN were 359.75 ± 84.59 μg/L, 77.88 ± 24. 67 μg/L, 86. 73 ± 23.90 μg/L in CHF group, and 211.60 ±54. 80 μg/L, 64.82 ±23.99 μg/L, 82. 26 ±23.98 μg/L in NYHA functional class Ⅰ group, respectively. The HA level was significantly higher in CHF patients as compared with NYHA functional class Ⅰ group （ P 〈 0.05 ）. However, no difference was found in the levels of PCIIIP and LN between CHF group and NYHA functional class Ⅰ group. The serum HA concentration was negatively correlated with left ventricular ejection fraction （ r = - 0.71, P 〈 0.05 ）. Conclusion Serum HA level may act as an indicator for myocardial fibrosis.

Collagen-based biological glue after Appleby operation for advanced gastric cancer

Pancreatic fistula is a common complication of distal pancreatectomy; although various surgical procedures have been proposed, no clear advantage is evident for a single technique. We herein report the case of a 38-year-old patient affected by an advanced gastric carcinoma infiltrating the pancreas body, with exten- sive nodal metastases involving the celiac trunk, who underwent total gastrectomy with lymphadenectomy, distal pancreatectomy and resection en bloc of the celiac trunk (Appleby operation). At the end of the demolitive phase, the pancreatic stump and the aorta at the level of the celiac ligature were covered with a layer of Tachosil, a horse collagen sponge made with human coagulation factors (fibrinogen and thrombin). Presenting this case, we wish to highlight the possible sealing effect of this product and hypothesize a role in preventing pancreatic fistula and postoperative lymphorrhagia from extensive nodal dissection.

Serum type Ⅳ collagen level is predictive for esophageal varices in patients with severe alcoholic disease

AIM： To determine factors predictive for esophagea varices in severe alcoholic disease （SAD）. METHODS： Abdominal ultrasonography （US） was performed on 444 patients suffering from alcoholism. Forty-four patients found to have splenomegaly and/ or withering of the right liver lobe were defined as those with SAD. SAD patients were examined by upper gastrointestinal （UGI） endoscopy for the presence of esophageal varices. The existence of esophageal varices was then related to clinical variables. RESULTS： Twenty-five patients （56.8%） had esophageal varices. A univariate analysis revealed a significant difference in age and type Ⅳ collagen levels between patients with and without esophageal varices. A logistic regression analysis identified type Ⅳ collagen as the only independent variable predictive for esophageal varices （P = 0.017）. The area under the curve （AUC） for type Ⅳ collagen as determined by the receiver operating characteristic （ROC） for predicting esophageal varices was 0.78. CONCLUSION： This study suggests that the level of type Ⅳ collagen has a high diagnostic accuracy for the detection of esophageal varices in SAD.

Kinetics of High Cell Density Fed-batch Culture of Recombinant Escherichia coli Producing Human-like Collagen

The kinetics of batch and fed-batch cultures of recombinant Escherichia coli producing human-like collagen was investigated. In the batch culture, a kinetic model of a simple growth-association system was concluded without consideration of cell endogeneous metabolism. The cell lag time, the maximum specific growth rate and Yx/s were determined as 1.75h, 0.65h^-1 and 0.51g·g^-1, respectively. In the fed-batch culture, different specific growth rates were set at （0.15, 0.2, 0.25h^-1） by the method of pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and product can reach 77.5g·L^-1 and 10.2g·L^-1 respectively. The modal predictions are in good agreement with the experimental data.

Effects of Collagen Peptide from Cyanea nozakii on Mouse Immune Function

[Objective ] The study aimed to provide a theoretical basis for the clinical application of collagen peptide from Cyanea nozakii. [ Method] After acute toxicity test on mice, collagen peptide from C. nozakii were given to mice by continuous intragastric administration for 30 d at the doses of 25, 50, 100 mg/kg, and then the phagocytosis of macrophage, delayed type hypersensitivity （DTH） and serum hemolysin level were determined. [ Result] Collagen peptide from C. nozakii was atoxic or low toxic, and the three immune indices of experimental groups were signifi- canUy higher than those of the control group （treated with same volume of normal saline） at 0.05 or 0.01 level. E Conclusion] Collagen peptide from C. nozakii has a certain immunopotentiation.

The protective effect of Dendrobium officinale polysaccharides on photoaging fibroblasts by scavenging reactive oxygen species and promoting the expression of TGF-β1

The purpose of this study was to explore the protective effect of Dendrobium officinale polysaccharides （DOP） onphotoaging human skin fibroblasts and its specific mechanism of action. The photoaging fibroblast model wasestablished by ultraviolet B （UVB） irradiation. The toxic effects of different concentrations of DOP were detected usingMTT. Senescent cells were detected using a β-galactosidase kit. Reactive oxygen species （ROS） in cells were detectedusing a flow cytometer. The expression of matrix metalloproteinase-1 （MMP-1）, type I collagen C-terminal peptide（CICP）, and transforming growth factor β-1 （TGF-β1） in spent culture medium was detected by ELISA. The resultsshowed that the low concentration of DOP （20, 40, 80 μg/mL） had no cytotoxicity on fibroblasts. After 60 mJ/cm2UVBirradiation, the number of aging β-gal-positive cells increased, the levels of CICP and TGF-β1 in spent culture mediumdecreased, while the levels of MMP-1 and ROS increased. After administration of DOP on photoaging fibroblasts, thenumber of aging β-gal-positive cells decreased, the levels of ROS and MMP-1 decreased, and the levels of TGF-β1 andCICP increased. This experiment suggests that DOP has the effect of removing ROS induced by UVB, regulating thebalance of collagen production and degradation, and protecting photoaging human skin fibroblasts.

Targeting collagen expression in alcoholic liver disease

Alcoholic liver disease （ALD） is a leading cause of liver disease and liver-related deaths globally, particularly in developed nations. Liver fibrosis is a consequence of ALD and other chronic liver insults, which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Liver fibrosis is characterized by accumulation of excess extracellular matrix components, including type I collagen, which disrupts liver microcirculation and leads to injury. To date, there is no therapy for the treatment of liver fibrosis; thus treatments that either prevent the accumulation of type I collagen or hasten its degradation are desirable. The focus of this review is to examine the regulation of type I collagen in fibrogenic cells of the liver and to discuss current advances in therapeutics to eliminate excessive collagen deposition.

A unique case of collagenous colitis presenting as protein-losing enteropathy successfully treated with prednisolone

A 76-year-old woman with a 5-mo history of recurrent diarrhea and generalized edema was admitted to our hospital. Colonoscopy revealed edematous mucosa,and histopathological examination was compatible with collagenous colitis. Protein leakage from the colon,particularly in the ascending portion,was identified on 99mTc-human serum albumin scintigraphy. Collagenous colitis associated with protein-losing enteropathy (PLE) without small bowel disease was diagnosed. Prednisolone treatment ameliorated diarrhea and hypoproteinemia. Collagenous colitis should be included in the differential diagnosis of chronic diarrhea with hypoproteinemia for appropriate management.

Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

Complex role of matrix metalloproteinases in angiogenesis

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP) play a significant role in regulating angiogenesis, the process of new blood vessel formation. Interstitial collagenase (MMP-1), 72 kDa gelatinase A/type IV collagenase (MMP-2), and 92 kDa gelatinase B/type IV collagenase (MMP-9) dissolve extracellular matrix (ECM) and may initiate and Promote angiogenesis. TIMP-1, TIMP-2, TIMP-3, and possibly,TIMP-4 inhibit neovascularisation. A new paradigm is emerging that matrilysin (MMP-7), MMP-9, and metalloelastase (MMP-12) may block angiogehesis by converting plasndnogen to angiostatin, which is one of the most potent angiogenesis antagonists. MMPs and TIMPs play a complex role in regulating angiogenesis. An understanding of the biochemical and cellular pathways and mechanisms of angiogenesis will provide importal information to allow the control of angiogenesis, e.g. the stimulation of angiogenesis for coronary collateral circulation formation; while the inhibition for treating arthritis and cancer.