Construction and screening effective sequence of shRNA targeting human trefoil factor 3 / 临床耳鼻咽喉头颈外科杂志

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.

Effect of Rhizoma Atractylodis extract in protecting gastric mucosa and modulating gastrointestinal immune function in a rat model of spleen deficiency / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Male 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>ERA can inhibit gastric mucosal damage, protect and repair the damaged mucosal tissues, and improve the immune function of in rats with spleen deficiency.</p>

The expression and significance of trefoil factor 3 and SDF-1/CXCR4 biological axis in papillary thyroid carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To study the expression of trefoil factor 3 (TFF3)with stromal cell-derived factor (SDF-1) and its receptor (CXCR4) in papillary thyroid carcinoma(PTC), and investigate the function of TFF3, SDF-1/ CXCR4 and the relationship among them during the tumor genesis,development and outcome of PTC.@*METHOD@#Detecting the expression of TFF3 and SDF-1/CXCR4 by immunohistochemical method (SP) in 92 cases of PTC and para-carcinoma tissue. Semiquantitative analysis of the results of immunohistochemistry was conducted by image analysis software.@*RESULT@#(1) TFF3 protein was expressed in the cytoplasm of cancer cells,while TFF3 was negative or weakly positive in follicular cells of para-carcinoma tissue. The positive expression rate of TFF3 was 92.39%, of which the strong positive rate of clinical stage III-IV accounted for 71.79% (42/59) and that of clinical stage I-II was 33.33% (11/33) (P < 0.01). The positive rate of TFF3 was significantly higher in the cases with lymph node metastasis than those without lymph node metastasis (100.00% vs 86.27%, P < 0.05). The AOD value of TFF3 was higher in PTC than in para-carcinoma tissue, that in cases with lymph node metastasis was higher than those without lymph node metastasis, and that in stage III-IV was higher than those in I-II (P < 0.05 or P < 0.05). (2) There was high expression of SDF-1 in the cytoplasm of malignant tissues. The para-carcinoma tissue was weakly positive or negative to SDF 1 and metastatic lymph nodes was weakly positive to SDF 1. The positive rates and AOD values of SDF-1 protein were similar to those of TFF3 in PTC,that is to say the positive rate and AOD values were higher in PTC than in para-carcinoma tissue, those in cases with lymph node metastasis were higher than those without lymph node metastasis, those in stage III-IV were higher than those in I-II, and those in patients older than 45 years old was obviously higher than those in patients under 45 years old (P < 0.05 or P < 0.01); CXCR4 was also mainly expressed in cytoplasm with few expression in nuclei, while negative or weakly positive in para-carcinoma. The positive rate and AOD values of CXCR4 in PTC were similar to SDF-1, meaning that they were higher in PTC than in para-carcinoma tissue and associated with the clinical stage, lymph node metastasis and age (P < 0.05 or P < 0.01). (3) There was positive relationship between TFF3 and SDF-1 as well as between SDF-1 and CXCR4 in PTC (r = 0.971, P < 0.01).@*CONCLUSION@#The high expression of TFF3, SDF-1 and CXCR4 in PTC are correlated with carcinogenesis and progression, and may play a significant role in evaluating the malignancy degree and progression of PTC.

Effects of intestinal trefoil factor combined with mucin on ability of proliferation and migration of intestinal epithelial cells after being treated by rat burn serum / 中华烧伤杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSIONS</b>ITF 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.</p>

Effects of intestinal trefoil factor combined with mucin on immune function of burn serum treated intestinal epithelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>(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).</p><p><b>CONCLUSIONS</b>ITF 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.</p>

Effects of intestinal trefoil factor on Toll-like receptors 2 and 4 expression in intestinal tissue in young rats with endotoxemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effects of intestinal trefoil factor (ITF) on Toll-like receptors (TLR) 2 and 4 expression in intestinal tissue and on intestinal injury in young rats with endotoxemia.</p><p><b>METHODS</b>A total of 24 10-day-old Wistar rat pups were equally randomly divided into three groups: a control group, intraperitoneally injected with normal saline 1 mL/kg; an endotoxemia group, intraperitoneally injected with lipopolysaccharide (LPS) 5 mg/kg and an ITF group,intraperitoneally injected with rITF 0.1 mL/each plus LPS 5 mg/kg. Rats were sacrificed 3 h after injection. A segment of distal ileum was dissected for pathologic examinations under an optical microscope (hematoxylin-eosin staning). The mRNA expressions of TLR2 and TLR4 were detected by reverse transcriptase polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The structure of the small intestine remained normal in the control group. Edema of interstitial substance and epithelium were observed in both LPS and ITF groups, whereas such changes were significantly lower in the ITF group than in the LPS group. The ITF group had significantly higher TLR2 mRNA expression than the NS and LPS groups (P<0.01), whereas the mRNA expression of TLR4 in the ITF group was significantly lower than in the NS and LPS groups (both P<0.01).</p><p><b>CONCLUSIONS</b>ITF can alleviate intestinal injury in young rats with endotoxemia, which may be related to the down-regulation of TLR4 mRNA expression.</p>

Expression of pS2, TGF-alpha and PCNA in the gastric mucosa of young rats with endotoxemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Growth, 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.</p><p><b>METHODS</b>Eighteen-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.</p><p><b>RESULTS</b>Gastric 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).</p><p><b>CONCLUSIONS</b>PCNA 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.</p>

Construction of a recombinant eukaryotic vector of human intestinal trefoil factor and its expression in 293-T cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone human intestinal trefoil factor (hITF/hTFF3) gene into an eukaryotic expression vector for its expression in eukaryotic cells.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS AND CONCLUSION</b>hTFF3 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.</p>

Expression of human intestinal trefoil factor in Pichia pastoris and its biological activity on intestinal epithelium in vitro / 生物工程学报

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.

Construction of Lactococcus lactis expression vector of recombinant human trefoil factor family 2 / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a Lactococcus lactis expression vector of c-myc-tagged human trefoil factor family 2 (hTFF2) fusion gene to prepare for genetic modification of Lactococcus lactis that can secrete bioactive c-myc-hTFF2 protein.</p><p><b>METHODS</b>Based on the amino sequence of hTFF2 and optimal Lactococcus lactis codon usage, the cDNA of hTFF2 was designed and extended at their 5' ends with a sequence encoding c-myc as the molecular tag. According to the restriction sites of pBluescript II sk (+), the SalI and BamHI sites were arranged at the 5' and 3' ends of the fusion gene respectively. The sequence of the fusion gene c-myc-hTFF2 was designed as 14 oligonucleotides that overlapped with each other, and by means of PCR, all the oligonucleotides were spliced to complete the construction of c-myc-hTFF2 fusion gene. The target gene of c-myc-hTFF2 was inserted into pBluescript II sk (+) to construct the cloning vector pBS-hTFF2 of c-myc-hTFF2 followed by verification by enzyme digestion and DNA sequencing. By digestion of pBS-TFF2 with BamHI/SalI and of pNBC1000 with BamHI/XhoI, we connected c-myc-hTFF2 with pNBC1000 to construct the expression vector c-myc-hTFF2 in E. coli named as pNTFF2. After digestion of pNTFF2 and pTRKH2 with XbaI, the target gene was subcloned into pTRKH2 and the construction of the expression vector pTRTFF2 in Lactococcus lactis was completed. The constructed vector was identified by restriction enzyme digestion.</p><p><b>RESULTS AND CONCLUSION</b>The expression vector pTRTFF2 of c-myc-hTFF2 fusion gene has been successfully constructed. Assembly of oligonucleotides in vitro is an effective means to synthesize the target fusion gene and this prepares the ground for constructing engineered bacterium of Lactococcus lactis.</p>

Protective effects of recombinant intestinal trefoil factor against intestinal injuries induced by endotoxin in young rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>This 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.</p><p><b>METHODS</b>Ninety-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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>rITF 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.</p>

Experimental study on the effects of intestinal trefoil factor on intestinal epithelial proliferation and its signal transduction mechanism / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effects of intestinal trefoil factor (ITF) on intestinal epithelial proliferation and its possible signal transduction mechanism.</p><p><b>METHODS</b>1). 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.</p><p><b>RESULTS</b>ITF 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.</p><p><b>CONCLUSION</b>ITF could promote intestinal epithelial proliferation and transmit extracellular signals mainly by means of ERKs signalling pathway.</p>