中华按蚊幼虫肠细胞膜级分的制备及其鼠抗血清对幼虫的效应 (英文)

已知芽孢杆卤杀虫蛋白的毒效依赖于它们与幼虫肠细胞膜的亲合力。对于其机理的研究和应用蛋白质工程手段改造杀蚊蛋白来说,蚊幼虫肠细胞膜的制备是必不可少的。本研究收集了万余只中华按蚊幼虫肠道,用于分离其细胞膜级分,进行蛋白组分分析,鼠抗血清的制备以及该抗血清对幼虫的作用的观察。仅在一龄幼虫观察到致死效应。

用基因融合技术进行杀蚊幼毒蛋白基因的研究Ⅱ.蚊幼虫肠壁细胞膜抗体基因的克隆

为了提高 Ｂ．ｓ．对蚊幼虫肠壁细胞膜的识别与结合力从而提高其杀蚊活性，本实验解剖了万余只中华按蚊幼虫肠道，制备了按蚊幼虫肠壁膜泡抗原及其鼠抗血清。抗原蛋白组分分析表明膜泡的纯度较高，而抗血清实验则显示出抗体组分与肠壁的结合作用。利用 Ｐ Ｃ Ｒ 技术成功扩增了小鼠抗体重链区基因，借助 Ｔ４ Ｄ Ｎ Ａ 聚合酶的修饰，使抗体基因得以成功克隆，为研制抗体基因与毒蛋白基因融合编码的“生物导弹”打下了基础。

铜绿假单胞菌体外粘附肠上皮细胞对其细胞膜生物特性的影响

目的 探索铜绿假单胞菌粘附肠上皮细胞后细胞膜生物特性的变化规律及可能的机制。方法 采用体外肠上皮细胞培养模型 ,研究绿脓杆菌粘附后对肠上皮细胞活力、细胞膜磷脂酶A2 、细胞内钙、细胞膜磷脂成分、膜流动性的影响。结果 细菌粘附后 3h肠上皮细胞活力明显下降 ,PLA2 活性增高 ,细胞内钙离子浓度增加 ,肠上皮细胞膜荧光偏振度、微粘度和分子排列有序性系数即出现增加 ,膜流动性降低 ,细胞膜磷脂含量降低 ,磷脂酰肌醇 (PI)、磷脂酰胆碱 (PC)含量逐渐降低。结论 绿脓杆菌粘附后肠上皮细胞胞内钙超载所导致的膜PLA2

Effects of basic fibroblast growth factor on ischemic gut and liver injuries

AIMS To explore the possible effects of basic fibrob- last growth factor (bFGF) on ischemic gut and liver in- juries after trauma. METHODS Animal model of super mesenteric artery (SMA) occlusion was used in this study. Seventy-two Wistar rats were divided into three groups of 24 rats each. Each animal in group 1 (bFGF treated) was in- jected with 4 μg of bFGF in 0.15 ml of normal saline solution containing 0.1%(w/v) heparin through the jugular vein at the onset of reperfusion. Animals in group 2 (saline treated) received the same vehicle, but without bFGF. Group 3 (sham-operated) ani- mals were treated with the same operations,but without SMA occlusion. Liver function parameters, serum TNFα,bacterial examination and pathological study were used to evaluate the results. RESULTS In group 1,the amounts of ALT and AST and serum TNFα were reduced significantly at 6,24 and 48 hours as compared with group 2. Bacterial ex- amination showed that the bacterial translocation from gut to liver,spleen and MLN in group 1 was much lower than that in group 2. The pathological results support the concept of significant protecting effects of bFGF. CONCLUSIONS Venous administration of bFGF may help reduce gut and liver injuries after ischemia and reperfusion. Its mechanism of action may involve the mitogenic and non-mitogenic effects of bFGF.

病理生理学

0020064 健康身躯在于健康精神之中,健康精神亦在健康身躯之中"不可控制"应激的损害力/Chrousos G P∥J Clin EndocMetab.-1998,83(6).-1842～1845四军大0020065 清醒兔急性应激器的血液动力学效应/Schadt J C∥Am J Physiol.-1998,274(3 Pt 2).-R814～821 医科情0020066

Decreased expression of serotonin in the jejunum and increased numbers of mast cells in the terminal ileum in patients with irritable bowel syndrome

AIM: To investigate if there are changes in serotonin (5-HT) levels, enterochromaffin (EC) cells and mast cells in small intestinal mucosa of patients with irritable bowel syndrome (IBS). METHODS: Diarrhea-predominant (IBS-D, n = 20), or constipation-predominant (IBS-C, n = 18) IBS patients and healthy controls (n = 20) underwent colonoscopy and peroral small intestinal endoscopy, and mucosal samples were obtained at the descending part of the duodenum, proximal end of jejunum and terminal ileum. High-performance liquid chromatography- electrochemistry and immunohistochemical methods were used to detect 5-HT content, EC cells and mast cells. RESULTS: (1) There were no differences in the number and distribution of EC cells between IBS patients and the normal group. (2) The mucosal 5-HT contents at the duodenum, jejunum and ileum in IBS-C patients were 182 ± 90, 122 ± 54, 61 ± 35 ng/mg protein, respectively, which were all lower than those in the normal group (256 ± 84, 188 ± 91, and 93 ± 45 ng/ mg protein, respectively), with a significant difference at the jejunum (P < 0.05). There were no differences in the small intestinal mucosal 5-HT contents between IBS-D patients and the normal group. The mucosal 5-HT contents at the duodenum were significantly higher than those at the ileum in the three groups (P < 0.001). (3) The numbers of mast cells in patients with IBS-C and IBS-D at the ileum were 38.7 ± 9.4 and 35.8 ± 5.5/highpower field (hpf), respectively, which were significantly more than that in the normal group (29.8 ± 4.4/hpf) (P < 0.001). There was no significant difference in the numbers of mast cells at the other two parts between IBS patients and the normal group. The numbers of mast cells in IBS-C, IBS-D, and normal groups were all significantly higher at the ileum (38.7 ± 9.4, 35.8 ± 5.5, 29.8 ± 4.4/hpf, respectively) than at the duodenum (19.6 ± 4.7, 18.5 ± 6.3, 19.2 ± 3.3/hpf, respectively, P < 0.001). CONCLUSION: The changes in the 5-HT signaling pathway at the jejunum of IBS-C patients and the increase in mast cells in patients with IBS at the terminal ileum may offer evidence to explain the pathogenesis of IBS.

Involvement of aquaporins in a mouse model of rotavirus diarrhea

Rotavirus diarrhea is a major worldwide cause of infantile gastroenteritis; however, the mechanism responsible for intestinal fluid loss remains unclear. Water transfer across the intestinal epithelial membrane seems to occur because of aquaporins(AQPs). Accumulating evidence indicates that alterations in AQPs may play an important role in pathogenesis. Here, we focus on changes in AQPs in a mouse model of rotavirus diarrhea. In the present study, 32 of 35 mice developed diarrhea and mild dehydration within 24 hours after infection with rotavirus strain SA11. Intestinal epithelial cells demonstrated cytoplasmic vacuolation, malaligned villi, and atrophy. AQP1 expression was significantly attenuated in the ileum and colon in comparison with controls; likewise, AQP4 and-8 protein expression were significantly decreased in the colon of rotavirus diarrhea-infected mice. In contrast, AQP3 protein expression was significantly increased in the colon of rotavirus-infected mice in comparison with controls. These results indicate that rotavirus diarrhea is associated with the downregulation of AQP1,-4, and-8 expression. Therefore, AQPs play an important role in rotavirus diarrhea.

Caffeic acid phenethyl ester modifies the Thl/Th2 balance in ileal mucosa afterγ-irradiation in the rat by modulating the cytokine pattern

AIM： To pharmacologically modulate Th polarization in the ileum exposed to ionizing radiation by using the immuno-modulatory/apoptotic properties of Caffeic Acid Phenethyl Ester （CAPE）. METHODS： Rats received CAPE （30 mg/kg） treatment ip 15 min prior to intestinal 10 Gy γ-irradiation and once a day for a 6 d period alter irradiation. Expression of genes implicated in Th differentiation in ileal mucosa （IL-23/IL-12Rβ2）, Th cytokine responses （IFN-γ, IL-2, IL-4, IL-13）, Th migratory behaviour （CXCR3, CCR5, CCR4）, Th signalling suppressors （SOCS1, SOCS3）, transcription factor （T-Bet, GATA-3） and apoptosis （FasL/Fas, TNF/TNFR, XIAP, Bax, caspase-3） was analyzed by RT-PCR 6 h and 7 d post-irradiation. CD4^＋ and TUNEL positive cells were visualized by immunostaining. RESULTS： The expression of Thl-related cytokine/ chemokine receptors （IFN-γ, IL-2, CXCR3, CCR5） was repressed at 7 d post-irradiation while Th2 cell cytokine/ chemokines （IL-4, IL-13, CCR4） were not repressed or even upregulated. The irradiation-induced Th2 profile was confirmed by the upregulation of both Th2-specific transcription factor GATA-3 and SOCS3. Although an apoptosis event occurred 6 h alter 10 Gy of intestinal γ-irradiation, apoptotic mediator analysis showed a tendency to apoptotic resistance 7 d post-irradiation. CAPE amplified apoptotic events at 6h and normalized Bax/FasL expressions at 7 d. CONCLUSION： CAPE prevented the ileal Th2 immune response by modulating the irradiation-influenced cytokine environment and apoptosis.

Role of mucosal dendritic cells in inflammatory bowel disease

The gastrointestinal innate and adaptive immune system continuously faces the challenge of potent stimuli from the commensal microflora and food constituents. These local immune responses require a tight control, the outcome of which is in most cases the induction of tolerance. Local T cell immunity is an important compartment of the specif ic intestinal immune system. T cell reactivity is programmed during the initial stage of its activation by professional presenting cells. Mucosal dendritic cells (DCs) are assumed to play key roles in regulating immune responses in the antigen-rich gastrointestinal environment. Mucosal DCs are a heterogeneous population that can either initiate (innate and adaptive) immune responses, or control intestinal inflammation and maintain tolerance. Defects in this regulation are supposed to lead to the two major forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC). This review will discuss the emerging role of mucosal DCs in regulating intestinal inflammation and immune responses.

B1a lymphocytes in the rectal mucosa of ulcerative colitis patients

AIM:To assess B1a cell expression in the rectal mucosa of ulcerative colitis (UC) patients in comparison with healthy controls.METHODS:Rectal mucosa biopsies were collected from 15 UC patients and 17 healthy controls.CD5 + B cells were analysed by three colour flow cytometry from rectal mucosal samples after mechanical disaggregation by Medimachine.Immunohistochemical analysis of B and T lymphocytes was also performed.Correlations between,on the one hand,rectal B1a cell concentrations and,on the other,erythrocyte sedimentation rate and C-reactive protein levels and clinical,endoscopic and histological disease activity indices were evaluated.RESULTS:Rectal B-lymphocyte (CD19 + /CD45 +) rate and concentration were higher in UC patients compared with those in healthy controls (47.85% ± 3.12% vs 26.10% ± 3.40%,P=0.001 and 501 ± 91 cells/mm 2 vs 117 ± 18 cells/mm 2,P < 0.001);Rectal B1a cell density (CD5 + CD19 +) was higher in UC patients than in healthy controls (85 ± 15 cells/mm 2 vs 31 ± 6.7 cells/mm 2,P=0.009).Rectal B1a cell (CD5/CD19 +) rate correlated inversely with endoscopic classification (Rs=-0.637,P < 0.05).CONCLUSION:B1a lymphocytes seem to be involved in the pathogenesis of UC,however,the role they play in its early phases and in disease activity,have yet to be defined.

Influence of dexamethasone on mesenteric lymph node of rats with severe acute pancreatitis

AIM: To study the influence and mechanisms of dexamethasone on mesenteric lymph node of rats with severe acute pancreatitis (SAP). METHODS: The SAP rats were assigned to model, treated or sham-operated groups. The mortality, pathological changes of mesenteric lymph nodes, expression levels of NF-kB, P-selectin, Bax, Bcl-2 and caspase-3 protein and changes in apoptotic indexes in lymph nodes were observed at 3, 6 and 12 h after operation. The blood levels of endotoxin, superoxide dismutase (SOD), malondialdehyde (MDA), and endothelin-1 (ET-1) in blood were determined. RESULTS: SOD content, expression of Bax protein and apoptotic index were significantly higher in the treated group than in the model group at different time points (P < 0.05 or P < 0.01). Other blood-detecting indexes and histopathological scores of mesenteric lymphnodes were lower in the treated than in the model group (P < 0.05, P < 0.01 or P < 0.01). NF-kB protein expression was negative in all groups. Comparing P-selectin and caspase-3 expression levels among all three groups, there was no marked difference between the model and treated group. CONCLUSION: Dexamethasone can protect mesen-teric lymph nodes. The mechanism may be by reducing the content of inflammatory mediators in the blood and inducing lymphocyte apoptosis.

Advances in small animal mesentery models for in vivo flow cytometry,dynamic microscopy,and drug screening

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.

Small intestinal submucosa improves islet survival and function during in vitro culture

AIM： To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa （SIS）. METHODS： Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. Purified islets were cultured in plates coated with multilayer SIS （SIS-treated group） or without multilayer SIS （standard cultured group） for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge best with low （2.7 mmol/L） and high glucose （16.7 mmol/L） solution supplemented with 50 mmol/L 3-isobutyl-1- methylxanthine （IBMX） solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA. RESULTS： After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture （95.8±1.0% vs 90.8±1.5%, P〉0.05）. When incubated with high glucose （16.7 mmol/L） solution, insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture （20.7±1.1 mU/L vs 11.8±1.1 mU/L, P〈0.05）. When islets were placed in high glucose solution containing IBMX, stimulated insulin secretion was higher in SlS-treated group than in control group. Calculated stimulation index of SlS-treated group was about 23 times of control group. In addition, the stimulation index of SlS-treated group remained constant regardless of short- and long- term periods of culture （9.5±0.2 vs 10.2±1.2, P〉0.05）. Much less apoptosis of islet cells occurred in SlS-treated group than in control group after the culture. CONCLUSION： Co-culture of isolated rat islets with native sheet-like SIS might build an extracellular matrix for islets and provide possible biotrophic and growth factors that promote the recovery and subsequent function of islets.

Ghrelin attenuates gastrointestinal epithelial damage induced by doxorubicin

AIM:To examine the influence of ghrelin on the regenerative potential of gastrointestinal(GI)epithelium.METHODS:Damage to GI epithelium was induced in mice by two intravenous injections of doxorubicin(10 and 6 mg/kg).Some of the doxorubicin-treated mice received a continuous subcutaneous infusion of ghrelin(1.25μg/h)for 10 d via implanted mini-osmotic pumps.To label dividing stem cells in the S-phase of the cell cycle,all mice received a single intraperitoneal injection of 5'-bromo-2'-deoxyuridine(BrdU)one hour before sacrifice.The stomach along with the duodenum were then removed and processed for histological examination and immunohistochemistry using anti-BrdU antibody.RESULTS:The results showed dramatic damage to the GI epithelium 3 d after administration of chemotherapy which began to recover by day 10.In ghrelintreated mice,attenuation of GI mucosal damage was evident in the tissues examined postchemotherapy.Immunohistochemical analysis showed an increase in the number of BrdUlabeled cells and an alteration in their distribution along the epithelial lining in response to damage by doxorubicin.In mice treated with both doxorubicin and ghrelin,the number of BrdUlabeled cells was reduced when compared with mice treated with doxorubicin alone.CONCLUSION:The present study suggests that ghrelin enhances the regenerative potential of the GI epithelium in doxorubicintreated mice,at least in part,by modulating cell proliferation.

Inhibition of invasiveness and expression of epidermal growth factor receptor in human colorectal carcinoma cells induced by retinoic acid

Human amniotic basement membrane (HABM) model and agarose drop explant method were used to in-vestigate the effects of retinoic acid(RA) on the invasive-ness alld adhesiveness to the basement membrane, and the migration of a highly invassive human colorectal cancer cell line CCL229. Results showed that 5 ×106 MRA markedly reduced the in vitro invasiveness and adhesiveness to the HABM, and the migration of the CCL229 cells. In addi-tion, to elucidate the relation between expression of epider-mal growth factor receptor (EGFR) and the invasiveness of the colorectal carcinoma cells, two well-differentiated, but with different invasiveness colorectal cancer cell lines were compared at mRNA level for expressioll of EGFR by using EGFR cDNA probe labeled with digoxigenin (DIG). Expression of EGFR was showll to be markedly higher in the highly invassive CCL229 cells than that in the low in- vasive CX-1 cells. Furthermore, expression of EGFR in RA treated CCL229 cells gradually decreased with time,the level being the lowest on day 6 of the RA treatment.

Muscularis mucosae in desmoplastic stroma formation of early invasive rectal adenocarcinoma

The origin of myofibroblasts or myofibroblastic cells in the desmoplastic stroma associated with carcinoma invasion has been controversial. In the early invasive area of a rectal adenocarcinoma reported here, an obvious transition between the muscularis mucosa and the bundles of eosinophilic stromal cells observed in the carcinomatous stroma was demonstrated both in morphology and in their cytoskeletal phenotype, which conceivably suggests that the smooth muscle cells of the muscularis mucosa could convert to the eosinophilic stromal cells, namely myofibroblasts. Moreover, type I procollagen was demonstrated in both protein and mRNA levels in the areas of eosinophilic stromal cells with a lesser degree of differentiated smooth muscle phenotype that showed a transition from the muscularis mucosa, implying that the myofibroblastic cells converted from smooth muscle cells of the muscularis mucosa could be responsible for type I collagen production. These findings suggest that the muscularis mucosae may not be a passive barrier through which colorectal carcinomas infiltrate into the submucosa, but may play an active role in the formation and remodeling of tumor stroma.

Relationship between p-catenin expression and epithelial cell proliferation in gastric mucosa with intestinal metaplasia

AIM： To investigate β-catenin expression in patients with intestinal metaplasia, and to look for a possible relationship between β-catenin expression and either epithelial proliferation values or Helicobacter pylori （ H pylori） infection.METHODS： Twenty patients with complete type intestinal metaplasia were studied. β-Catenin expression and epithelial cell proliferation in antral mucosa were assessed using an immunohistochemical analysis. H pylori infection was detected by histology and a rapid urease test.RESULTS： Reduced β-catenin expression on the surface of metaplastic cells was detected in 13 （65%） out of 20 patients. Moreover, in eight （40%） patients intranuclear expression of β-catenin was found. When patients were analyzed according to H pylori infection, the prevalence of both β-catenin reduction at the cell surface and its intranuclear localization did not significantly differ between infected and uninfected patients. Cell proliferation was higher in patients with intranuclear β-catenin expression as compared to the remaining patients, although the difference failed to reach the statistical significance （36±8.9 vs 27.2±11.4, P= 0.06）. On the contrary, a similar cell proliferation value was observed between patients with reduced expression of β-catenin on cell surface and those with a normal expression （28.1±11.8 vs 26.1±8.8, P= 0.7）.H pylori infection significantly increased cell proliferation （33.3±10.2% vs 24.6±7.4% , respectively, P= 0.04）.CONCLUSION： Both cell surface reduction and intranuclear accumulation of β-catenin were detected in intestinal metaplasia. The intranuclear localization of β-catenin increases cell proliferation. H pylori infection does not seem to play a direct role in β-catenin alterations, whilst it significantly increases cell proliferation.

Construction of cDNA Library from Intestine, Mesentery and Coelomocyte of Apostichopus japonicus Selenka Infected with Vibrio sp. and a Preliminary Analysis of Immunity-Related Genes

The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.

Primary rectal signet ring cell carcinoma with peritoneal dissemination and gastric secondaries

Disseminated signet ring cell carcinomas frequently arise from the stomach. However, primaries in the colon and rectum have also been reported. We present a 68 year old lady who presented with a change in her bowel habit. Colonoscopy showed a stenosing rectal tumour at 7 cm to 8 cm from the anal verge. Multiple scattered ulcers were also noted along the entire length of the colon. Biopsy of the lesions revealed signet ring cell adenocarcinoma. Gastroscopy showed multiple nodules with ulceration over several areas of the stomach which were similar in appearance to the colonic lesions. However, no primary tumour of the stomach was seen. Biopsy of the gastric lesions also showed signet ring cell adenocarcinoma. Computed tomography scan of the abdomen and pelvis revealed circumferential tumour at the rectosigmoid junction with possible invasion into the left ischiorectal fossa. The overall picture was that of a primary rectal signet ring cell carcinoma with peritoneal dissemination. The patient was referred for palliative chemotherapy in view of the disseminated disease. In the present report, we discuss this interesting pathological entity and review the role of various histolological techniques in helping to identify the primary tumor.