Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.

In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.     

Exogenous cytosine deaminase gene expression in Bifidobacterium breve I-53-8w for tumor-targeting enzyme/prodrug therapy

Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy.     

Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.     

Combined radiation and enzyme/prodrug treatment for head and neck cancer in an orthotopic animal model.

In an effort to improve the therapeutic outcome for squamous cell cancer of the head and neck, we have used the enzyme cytosine deaminase (CD) and the prodrug 5-fluorocytosine (5-FC) as a means to deliver the chemotherapeutic agent 5-fluorouracil (5-FU) in a tumor-specific manner and have evaluated the use of this treatment in combination with external-beam radiation. Infection of SCCVII cells in culture with a CD-expressing retrovirus and treatment with 5-FC was cytotoxic depending on the time of treatment and dose of 5-FC. An orthotopic model of squamous cell cancer of the head and neck was used in vivo to study the CD/5-FC system both alone and with concurrent radiation due to the radiosensitizing properties that 5-FU generates in situ. Treated mice were imaged using magnetic resonance imaging (MRI), and their survival was evaluated. Neither 5-FU nor radiation either alone or combined provided a survival advantage. In contrast, 5-FC treatment prolonged survival and decreased tumor burden compared to control animals, but the tumors recurred after the treatment ceased. Finally, combined treatment with concurrent administration of 5-FC and radiation resulted in a synergistic decrease in tumor growth and enhanced survival over treatment with 5-FC or radiation alone.     

Implication of functional activity for determining therapeutic efficacy of suicide genes in vitro

Gene-Directed Enzyme Prodrug Therapy (GDEPT), commonly known as suicide gene therapy, provides a selective approach to eradicate tumor cells that is currently considered as an alternate approach to conventional therapy for cancers due to its high efficacy. Herein, we have demonstrated functional activity of the cytosine deaminase (CD) and the hybrid cytosine deaminase-uracil phosphoribosyltransferase (CD-UPRT) suicide genes in transfected cell lines. We have monitored retention profiles of various metabolites that were formed during enzymatic conversion of the prodrug 5-flurocytosine (5-FC) using reverse phase HPLC method. Therapeutic effect of suicide genes was established by cell viability and toxicity assay, whereas apoptotic induction was confirmed by DNA fragmentation ELISA. Our results demonstrated that 5-FC/CD-UPRT-mediated apoptotic cell death was more than 5-FC/CD, which could be further potentiated with anticancer compound curcumin. Such results corroborated 5-FC/CD-UPRT in combination with curcumin as a better chemosensitization method.     

Molecular Mechanisms of Resistance to 5-Fluorocytosine in Laboratory Mutants of <Emphasis Type="Italic">Candida glabrata</Emphasis>

Resistance to 5-fluorocytosine (5-FC) has been poorly investigated in the yeast Candida glabrata. This study was conducted on laboratory mutants obtained by exposure of a wild-type isolate to 5-FC. Based on their susceptibility to 5-fluorouracil (5-FU), two of these mutants were selected for further analysis of the molecular mechanisms of 5-FC resistance. One mutant, resistant to both compounds, exhibited a missense mutation in the gene coding the cytosine deaminase and a decrease in the expression level of the gene coding the uridine monophosphate pyrophosphorylase. The other mutant that showed a reduced susceptibility to 5-FC and 5-FU exhibited an overexpression of the genes coding the thymidylate synthase and a cytosine permease, associated with a missense mutation in the last gene. Thus, beside mutations in the FUR1 gene which represent the most common cause of resistance to 5-FC, other mechanisms may also occur in C. glabrata.     

Anticancer effects of the engineered stem cells transduced with therapeutic genes via a selective tumor tropism caused by vascular endothelial growth factor toward HeLa cervical cancer cells

The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTECs) expressing bacterial cytosine deaminase (CD) and/or human interferon-beta (IFN-β) gene against HeLa cervical cancer and the migration factors of the GESTECs toward the cancer cells. Anticancer effect of GESTECs was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed so as to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells toward HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by CD gene and it caused the cell death in a co-culture system. When IFN-β was additionally expressed with CD gene by these GESTECs, the anticancer activity was significantly increased. In the migration assay, the GESTECs selectively migrated to HeLa cervical cancer cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTECs. These GESTECs transduced with CD gene and IFN-β may provide a potential of a novel gene therapy for anticervical cancer treatments via their selective tumor tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTECs.     

Augmenting podocyte injury promotes advanced diabetic kidney disease in Akita mice

To determine if augmenting podocyte injury promotes the development of advanced diabetic nephropathy (DN), we created mice that expressed the enzyme cytosine deaminase (CD) specifically in podocytes of diabetic Akita mice (Akita-CD mice). In these mice, treatment with the prodrug 5-flucytosine (5-FC) causes podocyte injury as a result of conversion to the toxic metabolite 5-fluorouracil (5-FU). We found that treatment of 4–5 week old Akita mice with 5-FC for 5 days caused robust albuminuria at 16 and 20 weeks of age compared to 5-FC treated Akita controls, which do not express CD (Akita CTLs). By 20 weeks of age, there was a significant increase in mesangial expansion in Akita-CD mice compared to Akita CTLs, which was associated with a variable increase in glomerular basement membrane (GBM) width and interstitial fibrosis. At 20 weeks of age, podocyte number was similarly reduced in both groups of Akita mice, and was inversely correlated with the albuminuria and mesangial expansion. Thus, enhancing podocyte injury early in the disease process promotes the development of prominent mesangial expansion, interstitial fibrosis, increased GBM thickness and robust albuminuria. These data suggest that podocytes play a key role in the development of advanced features of diabetic kidney disease.     

A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated ΔsupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the ΔsupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive.     

Purification and Characterization of Conjugated Bile Salt Hydrolase from Bifidobacterium longum BB536

Bifidobacterium species deconjugate taurocholic, taurodeoxycholic, taurochenodeoxycholic, glycocholic, glycodeoxycholic, and glycochenodeoxycholic acids. The enzyme level increases in the growth phase. No increase in activity is observed for the cytoplasmic enzyme after addition of conjugated bile acids to a stationary-phase culture. Conjugated bile salt hydrolase (BSH) was purified from Bifidobacterium longum BB536. Its apparent molecular mass in denaturing polyacrylamide gel electrophoresis was ca. 40,000 Da. The intact enzyme had a relative molecular weight of ca. 250,000 as determined by gel filtration chromatography, suggesting that the native BSH of B. longum is probably a hexamer. The purified enzyme is active towards both glycine and taurine conjugates of cholate, deoxycholate, and chenodeoxycholate. The pH optimum is in the range of 5.5 to 6.5. A loss of BSH activity is observed after incubation at temperatures higher than 42(deg)C; at 60(deg)C, 50% of the BSH activity is lost. The importance of free sulfhydryl groups at the enzyme active center is suggested. For B. longum BB536, no significant difference in the initial rate of deconjugation and enzymatic efficiency appears between bile salts. The enzymatic efficiency is higher for B. longum BB536 than for other genera. In this paper, a new method which permits a display of BSH activity directly on polyacrylamide gels is described; this method confirms the molecular weight obtained for B. longum BB536 BSH.     

Therapeutic effect of genetically modified human neural stem cells encoding cytosine deaminase on experimental glioma

The aim of this study was to determine the efficacy of neural stem cell-based suicidal gene therapy in rats bearing human glioma. F3 human neural stem cells (NSCs) were transduced to encode cytosine deaminase (CD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Intratumoral or intravenous transplantation of F3.CD human NSCs led to marked reduction in tumor burden and significantly prolonged the survival of brain tumor-bearing rats. The systemic administration of 5-FC with direct intratumoral/intravenous transplantation of F3.CD cells had remarkable therapeutic effect in rats with human glioma cells as compared with transplantation of parental F3 cells. There was 74% reduction in tumor volume in rats receiving direct transplantation of F3.CD cells into tumor site, and 67% reduction in tumor volume in rats receiving intravenous injection of F3.CD cells as compared to control animals transplanted with human glioma U373 cells alone. The combination of F3.CD and 5-FC was a highly effective in the glioma rat model. Our observations suggest that genetically engineered NSCs encoding suicide gene CD could provide clinical application of suicide gene therapy for patients with glioma.     

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.     

Antithyroid cancer effects of human neural stem cells expressing therapeutic genes on anaplastic thyroid cancer cells

Stem cells that express therapeutic proteins have been identified to have an anticancer effects on various types of cancer. In the present study study, human neural stem cells (hNSCs) that were genetically engineered to express cytosine deaminase (CD) and human interferon-β (IFN-β) were used for anaplastic thyroid cancer (ATC) treatment owing to their tumor-tropic properties and therapeutic effects. CD is an enzyme that converts 5-fluorocytosine (5-FC), a prodrug, to 5-fluorouracil (5-FU) which is a medication to suppress tumor growth through DNA synthesis inhibition. Also, IFN-β suppresses tumor growth by the induction of apoptotic process. In water soluble tetrazolium salt (WST) assay, SNU-80 cells which are human female ATC cells were cocultured with three cell types including engineered hNSCs such as HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-β cells on transwells and treated with 5-FC for 72 hours. Finally, the SNU-80 cell viability was reduced by the coculture with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In dichlorofluorescein diacetate (DCF-DA) and TdT-mediated dUTP nick-end labeling (TUNEL) assays, the production of reactive oxygen species (ROS) and the number of apoptotic cells were increased by HB1.F3.CD and HB1.F3.CD.IFN-β cells in the presence of 5-FC. In Western blot assay, ROS, and apoptosis-related genes were increased in SNU-80 cells when they were cocultured with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In transwell migration assay, hNSCs selectively migrated to SNU-80 cells because hNSCs interacted with chemoattractant factors like SDF-1α, uPAR, and CCR2 secreted by SNU-80 cells. Taken together, engineered hNSCs were revealed to selectively migrate to ATC cells and to inhibit growth as well as to induce apoptosis of ATC cells via ROS production through the actions of transgenes such as CD and IFN-β. Therefore, these engineered hNSCs can be promising candidates for the treatment of metastatic ATC.     

Oncosuppressive Suicide Gene Virotherapy “PVH1-yCD/5-FC” for Pancreatic Peritoneal Carcinomatosis Treatment: NFκB and Akt/PI3K Involvement

Peritoneal carcinomatosis is common in advanced pancreatic cancer. Despite current standard treatment, patients with this disease until recently were considered incurable. Cancer gene therapy using oncolytic viruses have generated much interest over the past few years. Here, we investigated a new gene directed enzyme prodrug therapy (GDEPT) approach for an oncosuppressive virotherapy strategy using parvovirus H1 (PV-H1) which preferentially replicates and kills malignant cells. Although, PV-H1 is not potent enough to destroy tumors, it represents an attractive vector for cancer gene therapy. We therefore sought to determine whether the suicide gene/prodrug system, yCD/5-FC could be rationally combined to PV-H1 augmenting its intrinsic oncolytic activity for pancreatic cancer prevention and treatment. We showed that the engineered recombinant parvovirus rPVH1-yCD with 5-FC treatment increased significantly the intrinsic cytotoxic effect and resulted in potent induction of apoptosis and tumor growth inhibition in chemosensitive and chemoresistant cells. Additionally, the suicide gene-expressing PV-H1 infection reduced significantly the constitutive activities of NFκB and Akt/PI3K. Combination of their pharmacological inhibitors (MG132 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) with rPVH1-yCD/5-FC resulted in substantial increase of antitumor activity. In vivo, high and sustained expression of NS1 and yCD was observed in the disseminated tumor nodules and absent in normal tissues. Treatment of mice bearing intraperitoneal pancreatic carcinomatosis with rPVH1-yCD/5-FC resulted in a drastic inhibition of tumor cell spreading and subsequent increase in long-term survival. Together, the presented data show the improved oncolytic activity of wPV-H1 by yCD/5-FC and thus provides valuable effective and promising virotherapy strategy for prevention of tumor recurrence and treatment. In the light of this study, the suicide gene parvovirotherapy approach represents a new weapon in the war against pancreatic cancer. Moreover, these preliminary accomplishments are opening new field for future development of new combined targeted therapies to have a meaningful impact on advanced cancer.     

Apoptotic Induction with Bifunctional <Emphasis Type="Italic">E.coli</Emphasis> Cytosine Deaminase-Uracil Phosphoribosyltransferase Mediated Suicide Gene Therapy is Synergized by Curcumin Treatment In vitro

Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloned cytosine deaminase gene expression of Bifidobacterium longum and application to enzyme/pro-drug therapy of hypoxic solid tumors

Bifidobacterium longum is a nonpathogenic anaerobic bacterium among normal bacterial flora. Recently, it was reported that B. longum accumulated in hypoxic solid tumors. The gene of interest was expressed in transfected B. longum by the shuttle vector pBLES100 in solid tumors. In this report, we constructed pBLES100-S-eCD, which included the cytosine deaminase gene. We confirmed by western blotting that transfected B. longum produced cytosine deaminase. In addition, transfected B. longum produced cytosine deaminase that converted 5-fluorocytosine into 5-fluorouracil. B. longum could be useful for enzyme/pro-drug therapy of hypoxic solid tumors.     

CD/5-FC对KSHV肿瘤转化基因K12诱导NIH3T3转化细胞的杀伤作用

采用分子克隆技术构建含卡波济肉瘤相关疱疹病毒(Kaposi’s sarcoma—associated herpesvirus,KSHV)转化基因K12和含胞嘧啶脱氨酶(CD)基因重组逆转录病毒表达质粒,分别命名为LZRS-K12和pLXSN—Fcy∷Fur。将LZRS-K12质粒转染NIH3T3细胞系,诱导细胞转化获得具有肿瘤细胞生物学特性的N／K12细胞,进一步将pLXSN—Fcy∷Fur质粒转染N／K12细胞,获得含CD基因的N／K12／FF细胞。加入5-氟胞嘧啶(5-FC)后,采用MTT法、流式细胞仪和免疫组化(IHC)等方法,分别检测细胞存活率、凋亡率及凋亡相关蛋白的表达。结果显示,N／K12／FF细胞在使用5-FC后存活率明显下降。流式细胞仪检测结果显示,5-FC作用后的N／K12／FF细胞的凋亡率与作用前相比增加了4．6％。与此同时,经5-FC作用后的N／K12和N／K12／FF细胞的生长周期发生了明显变化,均表现为G0-G1期细胞比率降低,S和G2-M期细胞比率增高。5-FC使得N／K12细胞的增殖主要阻滞在G2-M期,而N／K12／FF细胞的增殖主要阻滞在S期。IHC检测结果表明,N／K12细胞在5-FC使用前后Fas和Fas-L表达改变均不明显;而N／K12／FF细胞在5-FC使用后的Fas和Fas-L表达水平均明显高于5-FC使用前的水平。提示,CD／5-FC对KSHV肿瘤转化基因K12诱导NIH3T3转化细胞具有杀伤作用;凋亡有可能介导了部分杀伤过程;Fas和Fas-L有可能部分参与凋亡的诱导。     

Novel mutual prodrug of 5-fluorouracil and heme oxygenase-1 inhibitor (5-FU/HO-1 hybrid): design and preliminary in vitro evaluation

In this work, the first mutual prodrug of 5-fluorouracil and heme oxygenase1 inhibitor (5-FU/HO-1 hybrid) has been designed, synthesised, and evaluated for its in vitro chemical and enzymatic hydrolysis stability. Predicted in silico physicochemical properties of the newly synthesised hybrid (3) demonstrated a drug-like profile with suitable Absorption, Distribution, Metabolism, and Excretion (ADME) properties and low toxic liabilities. Preliminary cytotoxicity evaluation towards human prostate (DU145) and lung (A549) cancer cell lines demonstrated that 3 exerted a similar effect on cell viability to that produced by the reference drug 5-FU. Among the two tested cancer cell lines, the A549 cells were more susceptible for 3. Of note, hybrid 3 also had a significantly lower cytotoxic effect on healthy human lung epithelial cells (BEAS-2B) than 5-FU. Altogether our results served as an initial proof-of-concept to develop 5-FU/HO-1 mutual prodrugs as potential novel anticancer agents.     

富硒双歧杆菌缓解5-FU导致的化疗性肠粘膜炎的药效学研究

通过腹腔注射5-FU建立小鼠肠黏膜炎模型,探讨富硒长双歧杆菌(Selenium-enriched Bifidobacterium longum,Se-B.longum)能否改善5-FU所致的小鼠肠黏膜炎。将健康BALB/c小鼠随机分为对照组、5-FU组和Se-B.longum/5-FU组,分别灌胃生理盐水、生理盐水和Se-B.longum(Se0.3 mg/kg BW,1×106bacteria/只)6 d,然后5-FU组和Se-B.longum/5-FU组小鼠均腹腔注射5-FU(250 mg/kg),观察小鼠腹泻及死亡情况,5 d后处死小鼠,计算体重变化、脏器指数及考察肠道组织变化;将健康BALB/c小鼠随机分为对照组、5-FU组和Se-B.longum/5-FU组,分别灌胃生理盐水、生理盐水和Se-B.longum(Se 0.3 mg/kg BW,1×106bacteria/只)6 d,然后5-FU组和Se-B.longum/5-FU组小鼠均腹腔注射5-FU(300 mg/kg),观察小鼠死亡情况,绘制生存曲线。Se-B.longum能缓解5-FU导致的正常小鼠的肠粘膜炎、降低小鼠死亡率。