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AIM:To investigate the direct effect of croton oil(CO)onhuman intestinal epithelial cells(HIEC)and guinea pigcolonic smooth muscle cells in vitro.METHODS:Growth curves of HIEC were drawn by MTTcolorimetry.The dynamics of cell proliferation was analyzedwith flow cytometry,and morphological changes wereobserved under light and electron microscopy after long-term(6 weeks)treatment with CO.Expression of cyclo-oxygenase-2(COX-2)mRNA was detected by dot blot inHIEC treated with CO.Genes related to CO were screenedby DD-PCR,and the direct effect of CO on the contractilityof isolated guinea pig colonic smooth muscle cells wasobservedRESULTS:High concentration(20-40 mg·L~(-1))COinhibited cell growth significantly(1,3,5,7d OD sequence:(20 mg·L~(-1))0.040±0.003,0.081±0.012,0.147±0.022,0.024±0.016;(40 mg·L~(-1))0.033±0.044,0,056±0.012,0.104±0.010,0.189±0.006;OD control 0.031±0.008,0.096±0.012,0.173±0.009,0.300±0.016,P<0.01),whichappeared to be related directly to the dosage.Comparedwith the control,the fraction number of cells in Gl phasedecreased from 0.60 to 0.58,while that in S phase increasedfrom 0.30 to 0.34,and DNA index also increased after 6weeks of treatment with CO(the dosage was increasedgradually from 4 to 40 mg·L~(-1)).Light microscopicobservation revealed that cells had karyomegaly,lessplasma and karyoplasm lopsidedness.Electron microscopyalso showed an increase in cell proliferation and in thequantity of abnormal nuclei with pathologic mitosis.Expression of COX-2 mRNA decreased significantly in HIECtreated with CO.Thirteen differential cDNA fragments werecloned from HIEC treated with CO,one of which was 100percent homologous with human mitochondrial cytochromeC oxidase subunit Ⅱ.The length of isolated guinea pigcolonic smooth muscle cells was significantly shortenedafter treatment with CO(P<0.05).CONCLUSION:At a high CO concentration(>20 mg·L~(-1)), cell growth and proliferation are inhibited in a dosage-dependent manner.Increase in cell proliferation and inmalignant conversion of the cellular phenotype is observedin cells cultured chronically with CO.COX-2 mRNAexpression decreases significantly,while humanmitochondrial cytochrome C oxidase subunit Ⅱ mRNAexpression increases markedly in HIEC treated with CO.COalso has a direct effect on the contractility of guinea pigcolonic smooth muscle cells.
AIM: To investigate the direct effect of croton oil (CO) onhuman intestinal epithelial cells (HIEC) and guinea pigcolonic smooth muscle cells in vitro. METHODS: Growth curves of HIEC were drawn by MTT colorimetry. The dynamics of cell proliferation was analyzed with flow cytometry, and morphological changes wereobserved under light and electron microscopy after long-term (6 weeks) treatment with CO.Expression of cyclo-oxygenase-2 (COX-2) mRNA was detected by dot blot inHIEC treated with CO.Genes related to CO were screenedby DD-PCR, and the direct effect of CO on the contractility of isolated guinea pig colonic smooth muscle cells wasobservedRESULTS: High concentration (20-40 mg · L -1) COinhibited cell growth significantly (1,3,5,7d OD (20 mg · L -1) 0.040 ± 0.003,0.081 ± 0.012,0.147 ± 0.022,0.024 ± 0.016 (40 mg · L -1) 0.033 ± 0.044,0,056 ± 0.012,0.104 ± 0.010 ± 0.089 ± 0.006; OD control 0.031 ± 0.008,0.096 ± 0.012,0.173 ± 0.009,0.300 ± 0.016, P <0.01), whichappeared to be related directly to the dosage.Compared with the control, the fraction number of cells in Gl phase created from 0.60 to 0.58, while that in S phase increased from 0.30 to 0.34, and DNA index also increased after 6 weeks of treatment with CO (the dosage was increasedgradually from 4 to 40 mg · L Light microscopicobservation revealed that cells had karyomegaly, lessplasma and karyoplasm lopsidedness. Electron microscopyalso showed an increase in cell proliferation and in thequantity of abnormal nuclei with pathologic mitosis. Expression of COX-2 mRNA decreased significantly in HIECtreated with CO .Thirteen differential cDNA fragments were cloned from HIEC treated with CO, one of which was 100percent homologous with human mitochondrial cytochrome C oxidase subunit II. The length of isolated guinea pigcolonic smooth muscle cells was significantly shortened after treatment with CO (P <0.05) .CONCLUSION: At a high CO concentration (> 20 mg · L -1), cell growth and proliferation are inhibited in a dosage-dependent manner.Increase in cell prolif eration and inmalignant conversion of the cellular phenotype was observed in cells cultured chronically with CO.COX-2 mRNA expression significantly, while humanmitochondrial cytochrome C oxidase subunit II mRNA expression significantly markedly in HIEC treated with CO. COalso has a direct effect on the contractility of guinea pigcolonic smooth muscle cells.