OBJECTIVE: Trefoil factor 3 plays a pivot role in oncogenic transformation, growth and metastatic extension of solid tumours besides mucosal protection. We screened the best siRNA sequence targeting human TFF3 by the transient-transfection of the lentiviral mediated shRNA into thyroid carcinoma K1 cells which secrete TFF3 themselves. METHOD: Four siRNA transcription template hairpin structure target potential sites in human TFF3 mRNA sequence(132,170,258 and 537 bp,seperately) were selected and synthesized,as well as one negative shRNA(shRNAC). After annealing in vitro, insert pLVX-shRNA-puro construct recombinant plasmid, then enzyme digestion and sequencing analysis. The lentiviral-shRNAs were transient-transfected into K1 cells. TFF3 mRNA and protein levels were test by real-time PCR and western blot respectively in K1 cells at 48h post transient-transfected. RESULT: Genetic mutations in two sequences of shRNA1~2, so the follow-up study terminated. The TFF3 expression were obviously inhibited in K1 cells at 48 hours post transient-transfected of shRNA3 and shRNA4. TFF3 (258-276) showed the highest silencing efficiency (TFF3 mRNA reduced 60.67% and TFF3 protein reduced 56.44%, P < 0.01) when the transfection efficiency was 76.83%. CONCLUSION pLVX-shRNA-puro-TFF3 expression plamid were successfully constructed and the highest efficiency sequences were screened. All these laid a foundation for further study about the function of TFF3 gene.
Cell Line, Tumor ; Genetic Vectors ; Humans ; Lentivirus ; Peptides ; genetics ; Plasmids ; RNA, Small Interfering ; genetics ; Transfection ; Trefoil Factor-2

OBJECTIVETo clone human intestinal trefoil factor (hITF/hTFF3) gene into an eukaryotic expression vector for its expression in eukaryotic cells.

METHODSThe total RNA was extracted from normal human colon mucosa, and transcribed into cDNAs using RT-PCR. hTFF3 gene was amplified by PCR and ligated into pGEMT vector by TA cloning method. After sequencing, the hTFF3 gene was transfered into the eukaryotic expression vector pCMV5-myc. The recombinant vector was transfected into 293-T cells, and the expression of the recombinant protein was detected by Western blotting.

RESULTS AND CONCLUSIONhTFF3 gene was successfully cloned from normal human colon mucosa. The vector pCMV5-myc-hTFF3 was reconstructed, and in 293-T cells transfected with the vector, hTFF3 expression was detected by Western blotting.

Blotting, Western ; Cell Line ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; Humans ; Intestinal Mucosa ; metabolism ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; methods ; Trefoil Factor-2 ; Trefoil Factor-3

OBJECTIVEGrowth, regeneration and reparation of gastric mucosal epithelium may relate to the expression of peptides. This study aimed to investigate the effect of pS2, TGF-alpha and PCNA in endotoxin-induced acute gastric mucosal injury in young rats.

METHODSEighteen-day-old Wistar rats were randomly injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg) or normal saline (control). The gastric mucosal specimens were harvested 1.5, 3, 6, 24, 48, and 72 hrs after LPS or normal saline injection (n=8 each). The pathological changes of the gastric mucosa were observed by hematoxylin-eosin staining. The expression of pS2,TGF-alpha and PCNA was measured by immunohistochemistry SP method.

RESULTSGastric mucosal injuries were the most serious 6 hrs after LPS injection, characterized by massive erosion, bleeding and cord necrosis of the gastric mucosa paralleling with gastric longitudinal axis. PCNA expression in the gastric mucosa in the LPS group 3, 6, 24 and 48 hrs after LPS injection was significantly lower than that in the control group (P<0.01). pS2 expression in the gastric mucosa weakened 1.5 hrs after LPS injection, recovered to the control level at 3 hrs and was significantly higher than the control at 6, 24, 48 and 72 hrs of LPS injection (P<0.01). TGF-alpha expression in the gastric mucosa in the LPS group increased significantly 6, 24 and 48 hrs after LPS injection when compared with the control group (P<0.01).

CONCLUSIONSPCNA expression may be associated with the proliferation activity of the gastric mucosa in the process of gastric mucosal injury/reparation. pS2 and TGF-alpha might participate in the defense and reparation of gastric mucosal cells through mediating cell proliferation following acute gastric mucosal injury.

Animals ; Endotoxemia ; metabolism ; Female ; Gastric Mucosa ; chemistry ; pathology ; Immunohistochemistry ; Male ; Peptides ; analysis ; Proliferating Cell Nuclear Antigen ; analysis ; Rats ; Rats, Wistar ; Transforming Growth Factor alpha ; analysis ; Trefoil Factor-2

In order to produce relatively large amounts of recombinant human intestinal trefoil factor and assess its biological activity. The expression plasmid pPIC9-hITF containing AOX1 promotor and the sequences of secreting signal peptides was transformed into the yeast cells. Then through selection, positive transformants were cultivated in fermentation basal salts medium in a 5L fermenter to obtain large amount product with low cost. The secreted peptides were then purified by a combination of ionic exchange chromatography and molecular sieve. To verify the product, electrospray mass spectrometry analyses was used to determine the structure of rhITF and Western Blotting was performed to test the immunological activity. Furthermore, the biological activity of the peptide was examined by experiments from cell to tissue. The nucleotide sequence of rhITF was the same as expected. With a 5-L fermenter, 253mg of hITF was isolated at the purity of 96% from 3.5 L of yeast fermentation broth. The expression level for recombinant human ITF in this yeast system was 73.33mg/L. In our study, we provided a way to gain a production among milligram to gram of recombinant human ITF by the use of a yeast expression system. As human ITF are difficult to purify in any significant amount from tissue extraction, the way described may become a valuable tool in obtaining pure peptide for further studies of trefoil peptide function.
Cell Movement ; drug effects ; Epithelial Cells ; cytology ; drug effects ; Fermentation ; Humans ; Intestine, Small ; cytology ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Trefoil Factor-2

OBJECTIVETo observe the effect of intestinal trefoil factor (ITF) combined with mucin on immune function of intestinal epithelial cells (IEC) after being treated with burn rat serum.

METHODSThe rat IEC-6 cell lines were divided into control group (C, cultured in DEME medium containing 10% calf serum), burn control group (BC, cultured in DEME medium containing 10% burn rat serum), burn serum + ITF group (B + I, cultured in DEME medium containing 10% burn rat serum and 25 microg/mL ITF), burn serum + mucin group (B + M, cultured in DEME medium containing 10% burn rat serum and 250 microg/mL mucin), and burn serum + ITF + mucin group (B + I + M, cultured in DEME medium containing 10% burn rat serum, 25 microg/mL ITF, and 250 microg/mL mucin) according to the random number table. Meanwhile, 200 microL suspension of E. coli with density of 1 x 10(8) CFU/mL was added to each culture. At post culture minute (PCM) 15, 30 and post culture hour (PCH) 1, 2, 3, the number of bacteria adherent to IEC-6 was counted after Wright-Giemsa staining, and cell survival rate was calculated after trypan blue staining, with 20 samples in each group at each time point. (2) Other samples of IEC-6 cells without addition of E. coli were divided into BC, B + I, B + M, and B + I + M groups with the same treatment as above. The supernatant contents of IL-6, IL-8, and TNF-alpha were determined by radioimmunoassay at PCH 3, 6, 12, 24, 48, with 6 samples in each group at each time point. Data were processed with t test.

RESULTS(1) Compared with that in C group, count of adherent bacteria to IEC-6 in BC group at each time point was significantly increased (with t values from 2.947 to 8.149, P values all below 0.01). Compared with those in BC group, the counts in B + I, B + M, B + I + M groups at the major time points were significantly decreased (with t values from -4.733 to -2.180, P < 0.05 or P < 0.01). (2) Compared with that in C group, cell survival rate in BC group at each time point was obviously lowered (with t values from -4.126 to -2.363, P values all below 0.05). Cell survival rates in B + I and B + M groups at some time points were significantly elevated as compared with those in BC group (with t values from 2.120 to 3.423, P < 0.05 or P < 0.01). Cell survival rate in B + I + M group at PCM 15 and PCH 3 was respectively (96.7 +/- 2.4)% and (84.0 +/- 6.7)%, which was respectively higher than that in B + I and B + M groups [(94.5 +/- 3.1)%, t = 2.507, P < 0.05; (77.1 +/- 8.2)%, t = 2.934, P < 0.01]. (3) The contents of TNF-alpha in supernatant of B + I + M group at PCH 6, 12, 24, 48 were significantly lower than those in the other 3 groups (with t values from -6. 914 to -2.889, P < 0.05 or P < 0.01). The contents of IL-6 in supernatant of B + I + M group at some time points were significantly lower than those in the other 3 groups (with t values from -7. 657 to -2.580, P < 0.05 or P < 0.01). The contents of IL-8 in supernatant of B + I + M group at PCH 6, 12, 24, 48 were significantly lower than those in BC and B + M groups (with t values from - 8.802 to - 3.640, P values all below 0.01), and those in B + I + M group at PCH 12, 24 were lower than those in B + I group (with t value respectively -2.786, -2.740, P value all below 0.05).

CONCLUSIONSITF can maintain immune function and homeostasis of IEC, prevent bacterial adherence, decrease cell death rate, and reduce release of inflammatory mediators. The effect can be strengthened with addition of mucin.

Animals ; Bacterial Adhesion ; Burns ; blood ; Cell Line ; Epithelial Cells ; drug effects ; immunology ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Intestines ; cytology ; immunology ; metabolism ; Mucins ; pharmacology ; Peptides ; pharmacology ; Rats ; Serum ; immunology ; Trefoil Factor-2 ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVEThis study aimed to investigate the protective effects of recombinant intestinal trefoil factor (rITF) against intestinal injuries and the possible mechanism by examining the changes of diamine oxidase (DAO) and TNF-alpha and the intestinal ultrastructural changes in lipopolysaccharide (LPS) induced intestinal injuries.

METHODSNinety-six ten-day-old Wistar rats were randomly injected with either normal saline (1 mL/kg, Control group), LPS (1 mL/kg) or LPS (1 mL/kg) + rITF (0.1 mL) intraperioneally. At 2, 6, 24 and 72 hrs after administration plasma DAO activity was determined using absorption spectrometry; and the intestinal protein and mRNA expression of TNF-alpha were measured using immunohistochemistry and RT-PCR methods. The intestinal ultrastructural changes were observed by electron microscopy.

RESULTSThe plasma DAO activity in the LPS group began to increase at 2 hrs, peaked at 6 hrs and remained at significantly higher levels until 72 hrs after administration compared with the Control group (P < 0.01). The plasma DAO activity in the LPS + rITF group decreased noticeably compared with the LPS group at all time points (P < 0.01 or 0.05). A significant difference in the plasma DAO activity was only observed at 6 hrs after administration between the LPS + rITF and the Control group. The expression of TNF-alpha protein in the LPS group significantly increased at each time point, peaking at 6 hrs after LPS administration, with the IODT of TNF-alpha of 37,247.64 +/- 3,387.59 vs 6,191.02 +/- 482.32 (P < 0.01) compared with the Control group. rITF treatment decreased the expression of TNF-alpha protein although it remained significantly higher than in the Control group (P < 0.01). The TNF-alpha mRNA was weakly expressed in the Control group but strikingly increased after LPS injection (P < 0.01). Compared with the LPS group, the TNF-alpha mRNA expression in the LPS + rITF group decreased at all time points (P < 0.01 or 0.05). Vacuole changes of mitochodrium, cell nucleus condense, break and depletion of part of microvilli, and widen and disrupted tight junction were observed in the LPS group. The ultrastructural changes of intestinal tissues were improved in the LPS + rITF group.

CONCLUSIONSrITF can decrease the plasma DAO activity and inhibit the expression of TNF-alpha, resulting in a protective effect against intestinal injuries induced by LPS in young rats.

Amine Oxidase (Copper-Containing) ; blood ; Animals ; Intestines ; drug effects ; metabolism ; pathology ; Lipopolysaccharides ; toxicity ; Peptides ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Recombinant Proteins ; pharmacology ; Trefoil Factor-2 ; Tumor Necrosis Factor-alpha ; analysis ; genetics

OBJECTIVETo explore the effects of intestinal trefoil factor (ITF) on intestinal epithelial proliferation and its possible signal transduction mechanism.

METHODS1). The intestinal epithelial cytoplasmic membrane was isolated and harvested from Wistar rats, and it was treated with various doses of ITF in the concentration of 0.01, 0.10, 1.00 and 10.00 micro g/ml. The tyrosine protein kinase (TPK) activity from cytoplasmic membrane ITF receptor was determined by membrane-bound method. 2). The intestinal epithelial cells 6 (IEC-6) cultured in vitro were employed in the study. Some of the cells were used as normal control, while a group of cells were stimulated by 1 micro g/ml ITF, and others were treated by PD098059, SB202190 and SB202474, respectively. The last three agents were inhibitors of three members of mitogen-activated protein kinase family (MAPKs), i.e. extracellular signal regulated protein kinases (ERKs), protein kinase p38 (p38), and stress-activated protein kinase (SAPK). They were used before the addition of 1 micro g/ml of ITF. The changes in DNA synthetic rate and the MAPK activity of IEC-6 after being treated by above agents were assessed by (3)H-TdR incorporation method.

RESULTSITF receptor possessed TPK activity. TPK activity of ITF receptor, MAPKs activity and DNA synthetic rate of IEC-6 were increased obviously under the stimulation of ITF (P < 0.01). The above ITF effects could be evidently blocked by PD098059 and partially attenuated by SB202474. But SB202190 showed no effect in this respect.

CONCLUSIONITF could promote intestinal epithelial proliferation and transmit extracellular signals mainly by means of ERKs signalling pathway.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Epithelial Cells ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Intestines ; cytology ; Peptides ; pharmacology ; Rats ; Rats, Wistar ; Signal Transduction ; Trefoil Factor-2

OBJECTIVETo investigate the effect of Rhizoma Atractylodis extract (ERA) in protecting gastric mucosa and modulating gastrointestinal immune function of a rat model of spleen deficiency syndrome and elucidate the mechanism by which ERA improves spleen deficiency syndrome.

METHODSMale rats were fed with Xiaochengqi decoction and subjected to irregular feeding to induce spleen deficiency syndrome. The established models were randomized into model group, high-, moderate- and low-dose ERA groups, and domperidone group. After corresponding treatment for 30 days, the content of IgA in the intestinal lavage fluid, serum IgG, and the indices of the spleen and thymus were determined. The pathological changes in the gastric mucosa was observed with HE staining, gastric mucosal blood flow was evaluated with laser Doppler rheometry, and the expression of TFF1 in the gastric mucosa and TLR4 expression in the colon tissue were detected with immunohistochemistry.

RESULTSThe rat models of spleen deficiency syndrome showed obvious abnormalities in gastric mucosal morphology, blood flow and immunological indexes. Compared with the model rats, the rats receiving ERA treatment as different doses all showed significant improvements in gastric mucosal morphology, blood flow volume, gastric mucosa trefoil factor 1 (TFF1) expression, intestinal lavage fluid IgA content, serum IgG content, indices of the spleen and thymus, and TLR4 expression in the colon TLR4 (P<0.05 or P<0.01).

CONCLUSIONERA can inhibit gastric mucosal damage, protect and repair the damaged mucosal tissues, and improve the immune function of in rats with spleen deficiency.

Animals ; Atractylodes ; chemistry ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Gastric Mucosa ; drug effects ; pathology ; Immunoglobulin A ; metabolism ; Immunoglobulin G ; blood ; Immunohistochemistry ; Male ; Peptides ; metabolism ; Rats ; Rhizome ; chemistry ; Spleen ; physiopathology ; Trefoil Factor-2

Biomarkers ; blood ; Child ; Child, Preschool ; Critical Illness ; Early Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Female ; Gastrointestinal Diseases ; blood ; diagnosis ; etiology ; Humans ; Infant ; Male ; Peptides ; blood ; Severity of Illness Index ; Trefoil Factor-2

OBJECTIVETo observe the effect of intestinal trefoil factor (ITF) combined with mucin on the ability of proliferation and migration of intestinal epithelial cells (IEC) after being treated by burn rat serum.

METHODSThe rat IEC-6 cell lines were subcultured and divided into control group (C, cultured with DMEM medium containing 10% calf serum), burn serum group (BS, cultured with DMEM medium containing 10% burn rat serum), burn serum + ITF group (B + I, cultured with DMEM medium containing 10% burn rat serum and 25 microg/mL ITF), burn serum + mucin group (B + M, cultured with DMEM medium containing 10% burn rat serum and 250 microg/mL mucin), and burn serum + ITF + mucin group (B + I + M, cultured with DMEM medium containing 10% burn rat serum, 25 microg/mL ITF, and 250 microg/mL mucin) according to the random number table. Cells were counted on post culture day (PCD) 0, 1, 2, 3, 4, reflecting cell proliferation ability. Cell migration distance was measured at post scratch hour (PSH) 12, 24, 36, 48, 72. Then, cells of each group were placed in upper compartment of Transwell chamber while the corresponding medium was respectively added into lower compartment of Transwell chamber. Cells in lower compartment of Transwell chamber were counted at post culture hour (PCH) 4, 6, 8, 10, 12, reflecting cytomorphosis ability. Data were processed with t test.

RESULTS(1) Cell proliferation ability. The cell numbers in BS group on PCD 0, 1, 2, 3, 4 were significantly less than those in C group (with t values from -16.569 to -2.613, P < 0.05 or P < 0.01). The cell number showed no statistical difference between B + I and BS groups, and between B + M and BS groups at each time point (with t values respectively from 0.037 to 0.740 and 0.116 to 0.429, P values all above 0.05). The cell number in B + I + M group on PCD 2 was respectively larger than that in BS group (t = 6.484, P < 0.01) and B + I group ( t = 3.838, P < 0.01). (2) Cell migration distance in BS group at PSH 12, 24, 36, 48, 72 was significantly shorter than that in C group (with t values from -37.594 to -6.727, P values all below 0.01). There was no obvious difference in cell migration distance between BS and B + M groups at each time point (with t values from 0.055 to 0.589, P values all above 0.05). Cell migration distance in B + I group at PSH 12, 24, 36 was respectively (47 +/- 6), (126 +/- 13), (170 +/- 11) microm, all longer than those in BS group [(42 +/- 7), (98 +/- 14), (154 +/- 22) microm, with t values from 2.230 to 4.817, P < 0.05 or P < 0.01]. Cell migration distance in BS group at PSH 12, 24, 36, 48, 72 and B + I group at PSH 12, 24, 36, 48 was respectively shorter than that in B + I + M group (with t values respectively from 2.982 to 7.390 and 2.707 to 2.918, P < 0.05 or P < 0.01). (3) Cytomorphosis ability. Compared with those of C group, cell counts in lower compartment of BS group at PCH 4, 6, 8, 10, 12 were significantly decreased (with t values from -23.965 to -6.436, P values all below 0.01). Cell count in lower compartment of BS group at PCH 4, 6, 8, 10, 12 was respectively less than that of B + I group (with t values from 3.650 to 10.028, P values all below 0.01) and similar to that of B + M group (with t values from 0.199 to 0.797, P values all above 0.05). Cell counts in lower compartment of B + I + M group at PCH 4, 6, 8, 10, 12 were significantly larger than those of BS group (with t values from 4.313 to 15.100, P values all below 0.01). Cell count in lower compartment of B + I + M group at PCH 10 (328 +/- 47) and PCH 12 (465 +/- 37) was respectively larger than that in B + I group (277 +/- 25, 353 +/- 34, with t value respectively 3.051, 6.945, P values all below 0.01).

CONCLUSIONSITF can improve cytomorphosis ability for promoting cell migration with limited effect on cell proliferation, which can be enhanced with addition of mucin. The main mechanism of ITF in maintaining intestinal mucosal barrier may be attributed to acceleration of cell migration.

Animals ; Burns ; blood ; Cell Line ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Intestinal Mucosa ; Intestines ; cytology ; metabolism ; Mucins ; pharmacology ; Peptides ; pharmacology ; Rats ; Serum ; immunology ; Trefoil Factor-2