幼鼠和老龄鼠尾胶原体外纤维化的形态学比较

目的:通过观察幼鼠和老龄鼠尾胶原体外的纤维化过程以及幼鼠、老龄鼠腰椎松质骨胶原纤维形态学的变化,探讨年龄对胶原纤维化的影响。方法:取2周、20月的鼠尾肌腱各5g,分别利用稀醋酸进行胶原的提取,提取出的胶原低温冻干。取2周龄和20月龄的鼠尾胶原各15mg,分成3组,每组5mg,在4℃下分别熔解于10mmol.L-1醋酸溶液中;将钛金属螺钉浸润到醋酸溶液中,加入缓冲液,37℃下进行胶原的纤维化,分别在2、4和8h3个时间点取出螺钉,扫描电镜下观测螺钉表面的胶原形态。取2周、20月鼠各10只,处死后取出L3椎体,制成规则试件,反复生理盐水冲洗后,脱水,进行喷金后,行扫描电镜观察。结果:纤维化2h,2周龄及20月龄组鼠尾胶原纤维均未在螺钉表面微纤维化。纤维化4h,20月龄鼠尾胶原原纤维在螺钉的表面聚集成形态不规则的团块状胶原聚合物,未见胶原纤维形成;2周龄鼠尾胶原在螺钉的表面微纤维化成宽为0.07～0.20μm,纵横交错的胶原纤维。纤维化8h,20月龄鼠尾胶原在螺钉的表面聚集成原形或其他形态大小不等的胶原聚合物,未见胶原纤维形成;2周龄鼠尾胶原原纤维进一步纤维化,形成宽0.65～1.35μm的胶原纤维。骨小梁扫描电镜下显示:2周龄鼠骨胶原交联成粗细均匀的胶原纤维,纤维排列紧密靠拢,取向一致;20月龄鼠骨小梁胶原纤丝粗细不等,排列松散、稀疏、紊乱,其间联接相临的较粗纤丝的更细的胶原纤丝的取向杂乱无章。结论:随着年龄的增长,Ⅰ型胶原纤维化能力逐渐降低,导致骨胶原纤维形态的改变,矿物质沉积的异常,这可能是老年骨质疏松、骨折危险性增加的非常重要原因之一。

Single Polar Compound Bioelectret Material and Its Influence on the Cell Growth

To explore the influence of compound bioelectret material′s dielectric property on the cell growth,several kinds of compound bioelectret materials of collagen/chitosan were developed Their TSDC(thermally stimulated depolarization current)spectra were analyzed,and the compound bioelectret collagen/chitosan whose t α and I α were 37℃ and 2×10 -9 A respectively at polarized state was selected The cell culture study showed that the compound bioelectret material could promote normal cell growth when singly negatively polarized,and could inhibit cancer cell growth when singly positively polarized It proves that the rational designation of compound bioelectret has a broad application for clinical medicine

Establishment of a Collection and Detection Technology of Mycoplasma hyopneumoniae in Aerosol

ObjectiveThis study was to establish a simple method for collecting and detecting Mycoplasma hyopneumoniae （Mhp） in aerosol. MethodBased on the mechanisms of liquid impinger and filtration sampler, a double concentration aerosol sampler was designed for collecting Mhp aerosol. Firstly, the collection was performed in a closed environment full of artificial aerosol of Mhp. Secondly, collection efficiency was detected by real-time PCR. Thereafter, the clinical feasibility of the designed equipment was tested by collecting aerosol samples in different pig herds. In one assay, the samples were collected at different times from one pig house challenged with Mhp. In another assay, the samples was collected from the delivery room, nursery and fattenning house of a MPS outbreak farm as well as a Mhp infection positive pig farm without obvious clinical symptoms. All the aerosol samples were then detected by real-time PCR or nested PCR. ResultThe collection efficiency of the designed bioaerosol sampler was （37.04±6.43） %, Mhp could be detected 7 d after intratracheal challenge with pneumonic lung homogenate suspension. Aerosol samples of 11 pig houses from the two Mhp positive pig farms with or without clinical symptoms all showed a positive result of PCR, the positivity rate was 100%. ConclusionA high sensitive collecting and detecting technology of aerosol was successfully established, which can be applied to clinical detection of Mhp in aerosol.

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.

Effect of Cyclic Deformation on Magnetorheological Elastomers

Fatigue properties of magnetorheological elastomer （MRE） samples were investigated based on cis-polybutadiene rubber by using a fatigue test machine. Three MRE samples with iron particles mass fraction of 60%, 70%, and 80% were fabricated, and their properties dependence of three strain amplitudes （50%, 75%, and 100%） were measured. The absolute magnetorheological （MR） effect, storage modulus, and loss modulus of MRE samples after fatigue were evaluated by a modified dynamic mechanical analyzer. The results revealed that MR effect, storage modulus, and loss modulus of MREs containing 80% iron particles depended strongly on the strain amplitudes and the number of cycles, while storage mod-ulus and loss modulus of MREs containing 70% iron particles also depended on the strain amplitudes and the number of cycles but not as strongly as sample which contains 80% iron particles, but the properties of MREs containing 60% iron particles after cyclic deforma-tion were almost independent of the fatigued conditions. In order to investigate the fatigue mechanism of MREs, the sample was carried out with a quasi-static tensile testing and its surface morphology during testing was observed in situ by scanning electron microscopy.

Antifibrotic Effects of Genistein and Quercetin In Vitro

Objective: To study the antifibrotic effects of genistein(GE) and quercetin(QU) on rat hepatic stellate HSC-T6 cell proliferation stimulated with platelet-derived growth factor (PDGF), collagen synthesis and type I procollagen messenger RNA (mRNA) expression stimulated with transforming growth factor b1 (TGFb1). Methods: Cell proliferation was measured by crystal violet staining assay. Collagen synthesis was determined by 3H-proline incorporation assay. Type I procollagen mRNA level was determined by reverse transcription polymerase chain reaction (RT-PCR). Results: GE (25~70 mmolL-1) and QU (6.25~50 mmolL-1) concentration-dependently attenuated PDGF-driven HSC-T6 cell proliferative activity. TGFb1-stimulated collagen synthesis was also reduced. This was associated with a decrease in type I procollagen mRNA expression, indicating an effect at a pretranslational level. Conclusion: GE and QU may have therapeutic potential against liver fibrosis by regulating PDGF and TGFb1 actions.

Adult islets cultured in collagen gel transdifferentiate into duct-like cells

AIM: To establish a model of islet-ductal cell transdifferen-tiation to identify the transdifferentiated cells. METHODS: Collagen was extracted from rat tail at first. Purified rat islets were divided into three groups, embedded in collagen gel and incubated respectively in DMEM/F12 alone (control group), DMEM/F12 plus epidermal growth factor (EGF), DMEM/F12 plus EGF and cholera toxin (CT). Transdifferentiation was proved by microscopy, RT-PCR, immunohistochemistry and RIA. RESULTS: Islets embedded in collagen gel plus EGF and CT were cystically transformed and could express new gene cytokeratin 19 while still maintaining the expression of insulin and Pdx-1 genes. Immunohistochemistry demonstrated that the protein of cytokeratin 19 was only expressed in the third group. The insulin content secreted by islets in the third group decreased significantly during the transdiffe-rentiation. CONCLUSION: CT is a crucial factor for the islet-ductal cell transdifferentiation.

Process Control for Production of Human-like Collagen in Fed-batch Culture of Escherichia coli BL 21

Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)] and 19.8% (g·g-1 DCW) respectively.

Expression of Peptidylarginine Deiminase 4 and Protein Tyrosine Phosphatase Nonreceptor Type 22 in the Synovium of Collagen-Induced Arthritis Rats

Objective To study the expression level of peptidylarginine deiminase 4(PADI4) and protein tyrosine phosphatase nonreceptor type 22(PTPN22) in the synovium of rat model of collagen-induced arthritis, and to explore their possible therapeutic role in rheumatoid arthritis. Methods Thirty-two female Wistar rats weighing 100±20 g were randomly assigned into 3-week collagen-induced arthritis(CIA) model group(n=8), 4-week CIA model group(n=8), 6-week CIA model group(n=8), and the control group(n=8). The body weight changes of each group were recorded. The expression levels of PADI4 and PTPN22 were detected and compared by the methods of immunohistochemical staining and Western blot. Results Arthritis of rat began to form 14 days after sensitization and the joint swelling reached peak at 28 days. The weights of the rats slowly grew both in CIA model groups and the control group. Immunohistochemical staining results showed that the positive expression of PADI4 and PTPN22 was mainly located in cartilage peripheral mononuclear cells, the cytoplasm of infiltrated cells, and bone marrow cavity. There were significant differences in the optical density of PADI4 and PTPN22 among CIA model groups and the control group(PADI4, 0.2898±0.012, 0.2982±0.022, 0.2974±0.031, 0.2530±0.013 in 3-week CIA model, 4-week CIA model, 6-week CIA model and control groups; PTPN22, 0.2723±0.004, 0.2781±0.010, 0.2767±0.008, 0.2422±0.019; all P <0.05). The expression bands of PADI4 were observed in Western blot 3 weeks after initial immunization, the thickest in the 4th week, and decreased in the 6th week. The expression bands of PTPN2 were observed at all the time points, with no obvious time-dependent trend. Conclusions PADI4 and PTPN22 are obviously correlated with CIA in rat model. PADI4 is expressed at early stage of the disease, while the expression of PTPN22 sustains throughout the course.

Influence of Bioelectret Materials on cell Culture

The aim of this work is to study the effects of the electret property of natural biopolymers (such as collagen) on tissue repair. Type I collagen was prepared from pigskin,and polarized by using the TSDC (thermally stimulated depolarization current) technique. The polarized materials are used for cell culture. and then the cell growth curve is drawn. It was found that the polarized biomaterials accelerated cell differentiation. which indicates that they may be applied in the biomedical field.

Antitumor activity of anti-type IV collagenase monoclonal antibody and its lidamycin conjugate against colon carcinoma

AIM： Type IV collagenase including MMP-2 and -9 plays an important role in cancer cell invasion and metastasis and is an attractive target for rnAb-directed therapy. The irnrnunoreactivity of rnAb 3G11, a rnAb directed against type Ⅳ collagenase in human colorectal carcinomas, was studied by irnrnuno-histochernical （IHC） staining, rnAb 3G11 was conjugated to an antiturnor antibiotic lidarnycin （LDM）. The antiturnor activity of 3G11-LDM conjugate against colon carcinoma was investigated in mice. METHODS： ELISA, gelatin zyrnography, and Western blot assay were used for the biological characterization of rnAb 3G11. The irnrnunoreactivity of rnAb 3G11 with human colorectal carcinomas was detected by IHC staining. The cytotoxicity of LDM and 3G11-LDM conjugate to human colon carcinoma HT-29 cells was examined by clonogenic assay and MTT assay. The therapeutic effect of conjugate 3G11-LDM was evaluated with colon carcinoma 26 in mice. RESULTS： As shown in ELISA, mAb 3Gll reacted specifically with type IV collagenase, while 3G11-LDM conjugate also recognized specifically its respective antigen. In IHC assay, mAb 3G11 showed positive irnrnunoreactivity in most cases of colorectal carcinoma, and negative irnrnunoreactivity in the adjacent non-malignant tissues. By gelatin zyrnography, the inhibition effect of rnAb 3G11 on the secretion activity of type IV collagenase was proved. In terms of IC50 values in MTT assay, the cytotoxicity of LDM to human colon carcinoma HT-29 cells was 10 000-fold more potent than that of rnitornycin C （MMC） and adriarnycin （ADM）. 3G11- LDM conjugate also displayed extremely potent cytotoxicity to human colon carcinoma HT-29 cells with an IC50 value of 5.6× 10^-19 mol/L. 3G11-LDM conjugate at the doses of 0.05 and 0.1 mg/kg inhibited the growth of colon carcinoma 26 in mice by 70.3 and 81.2%, respectively. CONCLUSION： mAb 3G11 is immunoreactive with human colorectal carcinoma and its conjugate with LDM is highly effective against colon carcinoma in mice.

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.

Single center experience of capsule endoscopy in patients with obscure gastrointestinal bleeding

AIM:To identify optimum timing to maximize diagnostic yield by capsule endoscopy (CE) in patients with obscure gastrointestinal bleeding (OGIB).METHODS:We identified patients who underwent CE at our institution from August 2003 to December 2009.Patient medical records were reviewed to determine type of OGIB (occult,overt),CE results and complications,and timing of CE with respect to onset of bleeding.RESULTS:Out of 385 patients investigated for OGIB,284 (74%) had some lesion detected by CE.In 222 patients (58%),definite lesions were detected that could unequivocally explain OGIB.Small bowel ulcer/erosions secondary to Crohn's disease,tuberculosis or non-steroidal anti-inflammatory agent use were the commonest lesions detected.Patients with overt GI bleeding for < 48 h before CE had the highest diagnostic yield (87%).This was significantly greater (P < 0.05) compared to that in patients with overt bleeding prior to 48 h (68%),as well as those with occult OGIB (59%).CONCLUSION:We established the importance of early CE in management of OGIB.CE within 48 h of overt bleeding has the greatest potential for lesion detection.

Rejection of Permacol~ mesh used in abdominal wall repair:A case report

Permacol mesh has shown promise when used in abdominal wall repair,especially in the presence of a contaminated surgical field.This biomaterial,derived from porcine dermis collagen,has proposed advantages over synthetic materials due to increased biocompatibility and reduced foreign body reaction within human tissues.However,we present a case report describing a patient who displayed rejection to a Permacol mesh when used in the repair of abdominal wound dehiscence following an emergency laparotomy.Review of the English language literature using PubMed and Medline, showed only two previously published cases of explantation of Permacoldue to sepsis or wound breakdown. The authors believe this is the first case of severe foreign body reaction leading to rejection of Permacol.Both animal and human studies show conflicting evidence of biocompatibility.There are several reports of successful use of Permacolto repair complex incisional herniae or abdominal walls in the presence of significant contamination.It appears from the literature that Permacolis a promising material,but as we have demonstrated,it has the potential to evoke a foreign body reaction and rejection in certain subjects.

Effect of Traditional Chinese Medicine Antiviral Capsules On Animal Model Genital Herpes and HSV-2 in Cell Culture

Objective: To study the effect of traditional Chinese medicine antiviral capsules in the treatment of genital herpes. Methods: Using female guinea pig genital herpes as the animal model, this study used oral administration of two formulations of antiviral capsules (AC) and observed the effect on vaginal HSV-2 titers and vulvar symptoms. Cell cultures were also used to examine the direct inactivation of HSV-2 by the antiviral capsules and the suppression of HSV-2 via three drug administration methods. Results: There was no significant difference of mean vaginal virus titers between the antiviral capsule groups and that of the positive acyclovir (ACV) control (P>0.05). Mean vulvarsymptom scores of the two antiviral capsule groups were also significantly lower than that of the saline negative control group on days 2, 3, 5, 7 and 8 (P<0.05) and similar to that of the ACV control (P>0.05). Cell culture showed the minimum inhibitory concentrations of antiviral capsules No. 1 and No. 2 were 0.390625 mg/ml and 1.5625 mg/ml, respectively. Conclusion: The traditional Chinese medicine antiviral capsules had suppressive effects on HSV-2 in both animal model GH and in vitro cell culture.

Involvement of 90-kuD ribosomal S6 kinase in collagen type Ⅰ expression in rat hepatic fibrosis

AIM： To investigate the relationship between 90-kuD ribosomal $6 kinase （pg0RSK） and collagen type I expression during the development of hepatic fibrosis in vivo and in vitro.METHODS： Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type I were determined by co-immunofluoresence and confocal microscopy.Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell （HSC） line. The expression of collagen type I in HSC-T6 cells was assessed by Western blotting and real-time polymerase chainreaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, thencollagen type I promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated withor without platelet-derived growth factor （PDGF）-BB at a final concentration of 20μg/L and the cell growthwas determined by MTS conversion.RESULTS： In fibrotic liver tissues, p90RSK was over-expressed in activated HSCs and had a significantpositive correlation with collagen type I levels.In HSC-T6 cells transfected with RNAi targeted top90RSK, the expression of collagen type I was down-regulated （61.8% in mRNA, P 〈 0.01, 89.1% inprotein, P 〈 0.01）. However, collagen type ] promoteractivity was not increased with over-expression of p90RSK and not decreased with low expression either,compared with controls in the same cell line （P = 0.076）.Furthermore, p90RSK siRNA exerted the inhibitionof HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation.CONCLUSION： p90RSK is over-expressed in activatedHSCs and involved in regulating the abnormalexpression of collagen type I through initiating theproliferation of HSCs.

Preparation and surface characterization of nanodisk/nanoflower-structured gallium-doped zinc oxide as a catalyst for sensor applications

Nanostructured gallium‐doped zinc oxide （GZO） thin films were fabricated on piezoelectric sub‐strates. The GZO thin films with nanodisk/nanoflower morphologies were prepared by a simple spin‐coating process followed by one‐step hydrothermal treatment. Addition of polymer during hydrothermal treatment resulted in nanodisk and nanoflower morphologies. The morphology, microstructure and chemical composition of thin films prepared under different conditions were examined by field‐emission scanning electron microscopy （FE‐SEM）, X‐ray diffraction （XRD） and Raman spectroscopy. The XRD and FE‐SEM investigations confirmed that the GZO nanodisks, na‐norods and nanoflowers formed on the AlN/Si substrates were all wurtzite phase. Green fluorescent protein （GFP） was immobilized on the as‐synthesized GZO nanostructured materials by a dipping process. Atomic force microscopy （AFM） and fluorescence spectroscopy measurements were con‐ducted to confirm the surface binding nature of GFP on the GZO nanostructures to determine their suitability for use in sensor applications and bioimaging techniques. Trace‐level addition of GFP to the GZO nanostructures resulted in a fluorescence response, revealing good activity for ultraviolet light sensor applications.