<i>LINC00609</i> inhibits A549 cells progression through the regulation of <i>miR-128-3p/RND3</i> axis

Background: Long-chain non-coding RNA (lncRNA) LINC00609 is a potential tumor suppressor, but the mechanism of action in non-small cell lung cancer (NSCLC) is yet to be understood.Objectives: The effects of LINC00609 on A549 cell proliferation, apoptosis, and cell cycle arrest were investigated. Methods: The LINC00609 levels in NSCLC and normal tissues were analyzed by bioinformatics. Expressions of LINC00609, miR-128-3p, and Rho family GTPase 3 (RND3) in NSCLC cells (A549) were determined by qRT-PCR. Bioinformatics analysis predicted target genes and dual-luciferase reporter assays to ensure that LINC00609 targeted miR-128-3p and miR-128-3p targeted RND3. The proliferation of cells was determined using EDU and CCK-8. Flow cytometry was used to evaluate cell apoptosis rate and cell cycle. The western blotting assay identified proteins related to proliferation and apoptosis. Results: In NSCLC tissues, LINC00609 was expressed in low levels, while its high expression was associated with a higher survival rate. LINC00609 affected cell proliferation, apoptosis, cell cycle arrest, and expression of related proteins. Dual-luciferase reporter assay showed that LINC00609 binds specifically to miR-128-3p, and miR-128-3p binds to RND3. MiR-128-3p overexpression could neutralize the effects of LINC00609. A siRNA targeting RND3 could reverse the effect of the miR-128-3p inhibitor. Silencing RND3 resulted in a decrease in apoptosis rate and the number of cells in the S-phase and an increase in the number of cells in the G1-phase. Furthermore, phosphorylation levels of the AKT protein and mTOR protein, and Bcl2 expression, increased; however, the expression of RND3, Bax, and caspase3 decreased. Conclusions: LINC00609 regulated miR-128-3p/RND3 axis to modulate A549 cell proliferation, apoptosis, and cell cycle arrest. In the case of NSCLC, LINC00609 could be a potential target for therapy.     

A549/DDP derived exosomes can affect cisplatin chemosensitivity via transporting CXCR4 to A549 cells

The resistance of cancer cells to the anti-cancer drugs is the most important reason that affecting the efficacy of the non-small cell lung cancer (NSCLC) chemotherapy; thus, to explore the underlying mechanism of drug resistance of NSCLC medications is urgently needed for improving the therapeutic efficacy of current anti-NSCLC chemotherapies. The aim of the present study is to explore the roles of exosomes in the chemosensitivity of A549 cells and the related mechanism. A549 cells and cisplatin resistant cell line A549/DDP derived exosomes were isolated, and the expressions of CXCR4 were compared. Then, after cisplatin treatment, A549 cells were treated with exosomes, and the proliferation, apoptosis, migration, and invasion of the cells were examined. Finally, the tumorigenic effect of A549/DDP derived exosomes were also evaluated by cisplatin treated xenograft tumor mice models in vivo. We found that A549/DDP derived exosomes increased the proliferation, migration, and invasion, and inhibited the apoptosis and cisplatin sensitivity of A549 cells. CXCR4 was also significantly increased in cells treated with A549/DDP derived exosomes. Furthermore, A549/DDP derived exosomes may also decrease the chemosensitivity of NSCLC cells to cisplatin in vivo. Our data suggested that A549/DDP derived exosomes can affect the chemosensitivity of A549 cells to cisplatin, possibly by transporting CXCR4 to A549 cells. Our data may provide novel evidence for the investigation of drug resistance of NSCLC.     

Association between preterm birth risk and polymorphism and expression of the DNA repair genes <i>OGG1</i> and <i>APE1</i> in Saudi women

Genomic instability and mutations caused by increases in oxidative stress during pregnancy can damage the fetoplacental unit and can upshot preterm birth. Oxidative damage to DNA may possibly be involved in etiology of preterm birth (PTB) which can be repaired by DNA repair gene. In the present study, we assessed the association of base excision repair gene family by analyzing the association of single nucleotide polymorphisms and genes expression in 8-oxoguanine glycosylase-1 (OGG1) and apurinic-apyrimidinic endonuclease 1 (APE1) genes with risk of preterm birth in Saudi women. We analyzed genotypes of four single nucleotide polymorphisms (SNPs) (rs1052133, rs293795, rs2072668 and rs2075747) in OGG1 gene and three SNPs (rs1130409, rs3136814, and rs3136817) in APE1 gene using TaqMan Genotyping assay kits in 50 pairs of preterm cases and individually matched controls. Also, gene expression level was explored by RT-PCR in 10 pairs of preterm placental tissues and individually matched normal placental tissues. Two OGG1 SNP, rs1052133 (OR=0.497; c2=1.11; p=0.292) and rs2072668 (OR=0.408; c2=1.90; p=0.167) and one APE1 SNP rs3136817 (OR=0.458; c2=0.40; p=0.527) showed nonsignificant protective effect against PTB development. The expression of both genes under study was found lower in the PTB patients. Genotype and allele frequencies of both gene SNPs did not show any association with the risk of preterm delivery in Saudi women (P˃0.05). However, synthesis and release of OGG1 and APE1 proteins decreased in preterm placental tissues compared to term delivery reflects the probability of being one of the mechanisms leading to preterm birth.     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

Agmatine pretreatment protects retinal ganglion cells (RGC-5 cell line) from oxidative stress <i>in vitro</i>

Agmatine, 2-(4-aminobutyl)guanidine, has been reported to have neuroprotective effects against various neuronal damages. In this study it was investigated whether agmatine pretreatment rescues the retinal ganglion cells from oxidative injury in vitro. After differentiation of transformed rat retinal ganglion cells (RGC-5 cell line) with staurosporine, agmatine (0.0 to 100.0 μM) pretreatment was performed for 2 hours. Subsequently, they were exposed to hydrogen peroxide (0.0 to 2.5 mM) as an oxidative stress. Cell viability was monitored for up to 48 hours with the lactate dehydrogenase (LDH) assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. As a result, differentiated RGC-5 cells were found to have decreased viability after addition of hydrogen peroxide in a dose-dependent manner. This hydrogen peroxide induced cytotoxicity caused apoptosis characterized by DNA fragmentation. Agmatine pretreatment not only increased cell viability but also attenuated DNA fragmentation. In conclusion, agmatine pretreatment demonstrated neuroprotective effects against oxidative stress induced by hydrogen peroxide in differentiated RGC-5 cells in vitro. This suggests a novel therapeutic strategy rescuing retinal ganglion cells from death caused by oxidative injury     

Fractions of a chloroform extract of ajenjo leaves (<i>Artemisia mendozana</i> DC. var. <i>mendozana</i>) inhibit the proliferation,viability and clonogenicity of B16-F0 melanoma cells

The ajenjo, Artemisia mendozana DC. var. mendozana (Asteraceae), grows in the Andean foothills of Mendoza and San Juan, Argentina, and is used as a medicinal plant for its antispasmodic and antifungal properties. The aim of this work was to obtain fractions of a chloroform extract of ajenjo leaves and to evaluate the in vitro effects on proliferation, viability and clonogenicity of B16-F0 melanoma cells. Using a silica gel chromatography column, 120 fractions were collected and grouped according to the chromatographic profile in 9 main fractions (F1–F9). Their major compounds identified were: terpenes (F1), terpenes and sesquiterpene lactones (F2–F3), sesquiterpenes (F4–F6) and phenols and sesquiterpenes (F7-9). B16-F0 cells were incubated for 72 h with DMSO (vehicle) or 0.1 mg/ml F1–F9. At 72 h of culture, F1 decreased both the growing index (GI) and cell viability. F2 and F3 both decreased GI and only F3 decreased clonogenic activity. F4 and F5 both decreased GI. Only F5 decreased cell viability and F4 decreased clonogenicity. Consequently, fractions F6–F8 did not affect any of the cell parameters assayed, while F9 decreased cell viability and inhibited clonogenicity.     

LncRNA <i>ZFAS1</i> regulates cardiomyocyte differentiation of human embryonic stem cells

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.     

Neural stem cell-conditioned medium upregulated the PCMT1 expression and inhibited the phosphorylation of MST1 in SH-SY5Y cells induced by Aβ_25-35

A progressive neurodegenerative disease, Alzheimer’s disease (AD). Studies suggest that highly expressed protein isoaspartate methyltransferase 1 (PCMT1) in brain tissue. In the current study, we explored the effects of neural stem cell-conditioned medium (NSC-CDM) on the PCMT1/MST1 pathway to alleviate Aβ_25-35-induced damage in SH-SY5Y cells. Our data suggested that Aβ25-35 markedly inhibited cell viability. NSC-CDM or Neural stem cell-complete medium (NSC-CPM) had a suppression effect on toxicity when treatment with Aβ_25-35, with a greater effect observed with NSC-CDM. Aβ_25-35 + NSC-CDM group exhibited an increase in PCMT1 expression. sh-PCMT1 markedly decreased cell proliferation and suppressed the protective role of NSC-CDM through the induction of apoptosis and improved p-MST1 expression. Overexpression of PCMT1 reversed the Aβ_25-35-induced decrease in cell proliferation and apoptosis. In summary, our findings suggest that NSC-CDM corrects the Aβ_25-35- induced damage to cells by improving PCMT1 expressions, which in turn reduces phosphorylation of MST1.     

Expression analysis of <i>OsSERK</i>, <i>OsLEC1</i> and <i>OsWOX4</i> genes in rice (<i>Oryza</i> sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.     

Nicotinic acid induces apoptosis of glioma cells <i>via</i> the calcium-dependent endoplasmic reticulum stress pathway

Malignant glioma is one of the most common and deadly tumors in the central nervous system while developing effective treatments for this devastating disease remains a challenge. Previously, we demonstrated that the vitamin nicotinic acid (NA) inhibits glioma invasion. Here, we show that high-dose NA induces apoptosis of malignant glioma cells in vitro and in vivo. In cultured U251 glioma cells treated with NA, we detected ER stress that was likely caused by elevated intracellular calcium levels. The elevated calcium can be attributed to the activation of TRPV1, a cation channel that has been implicated in cutaneous flushing caused by NA administration. Our data further suggested that NA-induced apoptosis is mediated by the calcium-dependent proteases called calpains, whose activities are drastically upregulated by NA. NA-induced apoptosis of U251 cells can be attenuated by blocking calpain activity or knocking down TRPV1. These results reveal a novel function of NA in regulating glioma cell apoptosis via the calcium-dependent ER stress pathway and imply a potential application of NA for the treatment of malignant glioma.     

<i>In vivo</i> protective effect of late embryogenesis abundant protein (ApSK₃ dehydrin) on <i>Agapanthus praecox</i> to promote post-cryopreservation survival

Dehydrins (DHNs), as members of the late embryogenesis abundant protein family, play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses. Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage. In this study, ApSK₃ type DHN was genetically transformed into embryogenic calluses (EC) of Agapanthus praecox by overexpression (OE) and RNA interference (RNAi) techniques to evaluate the in vivo protective effect of DHNs during cryopreservation. The cell viability showed a completely opposite trend in OE and RNAi cell lines, the cell relative death ratio was decreased by 20.0% in ApSK₃-OE EC and significantly increased by 66.15% in ApSK₃-RNAi cells after cryopreservation. Overexpression of ApSK₃ increased the content of non-enzymatic antioxidants (AsA and GSH) and up-regulated the expression of CAT, SOD, POD, and GPX genes, while ApSK₃-RNAi cells decreased antioxidant enzyme activities and FeSOD, POD, and APX genes expression during cryopreservation. These findings suggest that ApSK₃ affects ROS metabolism through chelating metal ions (Cu²⁺ and Fe³⁺), alleviates H₂O₂ and OH· excessive generation, activates the antioxidant system, and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation. DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.     

Effect of microRNA-143-3p-mediated CTNND1 on the biological function of lung cancer cells

Lung cancer poses a serious threat to human life with high incidence and miRNA is an important biomarker in tumors. This study aimed to explore the effect of miR-143-3p on the biological function of lung cancer cells and the underlying mechanism. Eighty-seven samples of lung cancer tissues and 81 samples of tumor-adjacent tissues from patients undergoing radical lung cancer surgery in our hospital were collected. The lung cancer cells and lung fibroblast cells (HFL-1) were purchased, and then miR-143-3p-mimics, miR-NC, si-CTNND1, and NC were transfected into A549 and PC-9 cells to establish cell models. MiR-143-3p and CTNND1 expression levels were measured by the qRTPCR, Bax, Bcl-2, and CTNND1 expression levels by the Western Blot (WB), and cell proliferation, invasion, and apoptosis by the MTT assay, Transwell assay, and flow cytometry. Dual luciferase report assay was used to determine the relationship between miR-143-3p and CTNND1. In this study, miR-143-3p was lowly expressed in lung cancer and CTNND1 was highly expressed in lung cancer. The overexpression of miR-143-3p inhibited cell proliferation and invasion, promoted cell apoptosis, significantly increased Bax protein expression, and decreased Bcl-2 protein expression. The inhibition of CTNND1 led to opposite biological characteristic in cells. The dual luciferase reporter assay demonstrated that miR-143-3p was a target region of CTNND1. Such results suggest that miR-143-3p can inhibit the proliferation and invasion of lung cancer cells by regulating the expression of CTNND1 and promote the apoptosis of lung cancer cells, sott is expected to be a potential target for lung cancer.     

Immune strategies of phagoctytic cells stimulated <i>in vitro</i> with live and heat-inactivated <i>Streptococcus pyogenes</i>

Streptococcus pyogenes (group A Streptococcus) is frequently involved in a wide range of human diseases. Here we evaluated polymorphonuclear neutrophils and mononuclear cells from healthy subjects for their bactericidal function after stimulation with live and inactivated Streptococcus pyogenes (Streptococcus Group A). Mononuclear cells and Neutrophils were isolated from heparinized blood samples (n=18) using a Ficoll-Hypaque gradient and cultured in RPMI 1640 for 18 hours with a suspension of either live or inactivated Streptococcus pyogenes. Both the respiratory burst (flow cytometry) and nitrite, TNF and IL17 production (ELISA) were measured in the cell culture supernatants. An increased respiratory burst (expressed as R index) was induced by both live and inactivated bacteria. Also, increased nitrite, TNF and IL17 concentrations were found in cell culture supernatants in both cases. These findings may provide some explanation as to the roles played by neutrophils and mononuclear cells in Streptococcus pyogenes immunopathogenicity.     

MicroRNA-382 inhibits the proliferation of mouse spermatogonia by targeting <i>Kmt5a</i>

Spermatogenesis is a highly efficient and intricate process in the testis by which mature spermatozoa are produced daily to maintain lifelong male fertility. Essential to this process are spermatogonia capable of both proliferation and differentiation. Nevertheless, the underlying mechanisms for spermatogonial proliferation and differentiation remain poorly understood. MicroRNAs (miRNAs) are a category of non-coding small RNAs with regulatory functions by binding to the 3’ untranslated region (UTR) of the target mRNA. Previous studies have demonstrated that miRNAs are capable of modulating cell proliferation, differentiation and apoptosis, but the roles of individual miRNAs in spermatogonial fate determination remain largely elusive. Here, by using a mouse spermatogonial cell line (GC-1), we investigated the role for miRNA-382 in spermatogonial proliferation. We found that pre-miRNA-382 was expressed in spermatogonia. The luciferase reporter assay demonstrated Kmt5a but not Top1 as a target gene of miRNA-382. Overexpression of miRNA-382 by transfecting a miRNA mimic downregulated Kmt5a at both RNA and protein levels, and further reduced the proliferation and viability of spermatogonia. Knockdown of Kmt5a by RNA interference (RNAi) resulted in a uniform phenotype in spermatogonia. We therefore conclude that miRNA-382 inhibits the proliferation of mouse spermatogonia by targeting Kmt5a. Our finding extends the knowledge about the regulatory roles of miRNAs in spermatogonia and lays the groundwork for diagnosis and treatment of male infertility.     

3-<i>epi</i>-bufotalin suppresses the proliferation in colorectal cancer cells through the inhibition of the JAK1/STAT3 signaling pathway

Traditional Chinese medicine (TCM) has been increasingly employed in the last decades in China for both preventing and treating a variety of cancers. 3-epi-bufotalin is an active ingredient of TCM “Chanpi” with anti-tumor potential. However, the effect and mechanism of 3-epi-bufotalin on colorectal cancers were not well disclosed. The present study demonstrated that 3-epi-bufotalin could reduce viability, trigger apoptosis, and block the cell cycle at the G₂/M stage in colorectal cancer cell lines HT29, RKO, and COLO205 in vitro. Moreover, 3-epi-bufotalin inhibited the JAK1/STAT3 signaling pathway. These results indicated the anti-proliferation ability of 3-epi-bufotalin in colorectal cancer cells.     

The extracellular secretion of <i>miR-1825</i> wrapped by exosomes increases <i>CLEC5A</i> expression: A potential oncogenic mechanism in ovarian cancer

Background: Ovarian cancer (OC) is a leading cause of gynecological cancer-linked deaths worldwide. Exosomal miR-1825 and its target gene C-type lectin domain family 5 member A (CLEC5A) are associated with tumorigenesis in cancers that was further probed. Methods: Exosomal miR-1825 expression in exosomes and its impact on overall survival (OS) prediction were determined using Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) data. Target genes of miR-1825 were searched in five prediction databases and prognostically significant differentially expressed genes were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out. The ability of CLEC5A to predict OS was evaluated using univariate and multivariate Cox regression analyses and Kaplan-Meier curves. The CLEC5A expression pattern in OC was validated using immunohistochemistry. The CIBERSORT algorithm was used to compare the immune cell landscape, and the results were validated in a GEO cohort. Finally, the predicted half maximal inhibitory concentration (IC50) values for five commonly used chemotherapy agents were also compared. Results: MiR-1825 level was higher in exosomes derived from OC cells and served as a tumor suppressor. The CLEC5A gene was found to be a target of miR-1825, the upregulation of which was correlated with a poor prognosis. M2 macrophage infiltration was significantly enhanced in the CLEC5A high expression group, while T follicular helper cell infiltration was reduced in it. While the predicted IC50 for cisplatin and doxorubicin was higher in the CLEC5A high expression group, that of docetaxel, gemcitabine, and paclitaxel was lower. Conclusion: MiR-1825, a promising OC biomarker, may promote OC progression by increasing CLEC5A expression via exosome-mediated efflux from tumor cells.     

Isolation of epithelial cells,villi and crypts from small intestine of pigeons (<i>Columba livia</i>)

The isolation of viable enterocytes, villi and crypts from the small intestine of a feral bird (Columba livia) is important for performing physiological experiments in ecologically relevant processes of membrane transport. The effectiveness of mechanical disruption, enzymatic digestion and chelating agents were compared. The objectives were to isolate enterocytes, villi and crypts from the small intestine of young pigeons; to evaluate the viability of the isolated intestinal epithelial cells isolated; and to verify the integrity of enterocytes by biochemical features. Enzymatic and mechanical methods yielded both elongated columnar and spherical cells. With the chelating method villi and crypts were obtained. All methods produced a high yield of intestinal epithelial cells with about 50 % viability. Brush border enzymes (sucrase-isomaltase and alkaline phosphatase) activities were high and, as reported in chickens, they did not differ along the intestinal villus-crypt axis. Although the three methods have good viabilities, the enzymatic technique gives the best yield in cell number, while the chelating method provides the highest populations of morphologically distinctive villi and crypts.