The Phenotypic Change Of Colonic Epithelial Cells Induced By TGFβ1 And The Intervention Effect Of Traditional Chinese Medicine

Objective:1、To preliminary establish the organic culture system of colon tissues from neonatal rat in vitro, to analyze the effect of transforming growth factor-β1(TGF-β1) on polarity characteristics of epithelial cells and to probe the role of TGFβ1 in mesenchymal microenvironment.2、To find the effective drug targets of evodiamine and berberine which are monomer of Chinese traditional herbs, and to state the potential mechanism by intervening biological activity of TGFβ1, and then analysis their latent function in mesenchymal microenvironment that were immediately activated when tumor started early.Method:1、The colonic tissue from new born rats was removed under aseptic conditions, cut into pieces at approximately 1 mm3 in size, The pieces were placed, luminal-side up, on a 100μm cell strainer which was inserted into a 6-well dish.2-3 mL culture medium was added. During cultivation process, We use microscope to observe the growing status, DAPI was applied to identify the activity of cells which climbed out of the tissues. Colon tissues at 0 h、24h、48h were respectively fixed by Neutral formalin, and then we used HE to compare culured tissues to uncultured tissues. The expression of Epithelial markers-CK18、Crumbs3、E-cadherin in tissues were detected by IHC.2、Forty-eight hours after 10 ng/mL TGF-β1 was added during the process of culture of colon tissues from neonatal rats, the expression changes of Crumbs3, E-cadherin and alpha smooth muscle act in (α-SMA) in the control group (without TGF-β1) and intervention group (with TGF-β1) were detected by IHC and RT-PCR.3、After the colon tissues were induced by TGF-β1 for 48 h, we added berberine treatment group and the evodiamine treatment group. We utilize PCR to respectively test the Expression changes of Crumbs3、E-cadherin、 α-SMA and relevant MiRNA in culture group、intervention group (with TGF-β1)、berberine treatment group and evodiamine treatment group.Result:1、Neonatal rats colon tissue organic culture in vitro can maintain the organization activity more than a week, however, the organic culture model with cell strainer had more advantages in maintaining the colon tissue structure than direct adherent cultivation model. HE staining showed that Neonatal rats colon mucosal layer had a large number of columnar epithelial cells, and they closely arranged, they were identified to be colonic epithelial cells and they own the characteristics of apical-base polarity.2、after 48 h of the organic culture of Neonatal rats colon tissue, Crumbs3 was mainly located at the apical membrane of epithelial cells (P<0.01); a single layer of α-SMA-positive myofibroblasts along the crypt axis was visible, and α-SMA was lowly expressed in the smooth muscle. However, cells in the intervention group showed different morphology and size; E-cadherin was mainly present in the cytoplasm, and E-cadherin expression was lower than that in the control group (P<0.05); α-SMA was abundantly expressed in activated f ibroblasts (P<0.01). The mRNA expression of E-cadherin and Crumbs3 in the control group was significantly lower than that in the intervention group (P< 0.05), while the mRNA expression of α-SMA in the intervention group was significantly higher than that in the control group (P<0.01).3、The expression of Crumbs3、E-cadherin in culture group was always higher than intervention group (with TGF-β1),on the contrary, the expression of α-SMA was always lower than intervention group (with TGF-β1). however, afer intervention treatment with berberine and evodiamine for 24 h, The expression of Crumbs3、E-cadherin in berberine treatment group and evodiamine treatment group was significantly higher than intervention group (with TGF-β1).The expression of α-SMA was visibly lower than intervention group (with TGF-β1),and close to control group (without TGF-β1). After Traditional Chinese medicine treatment for 6 h, the expression of rno-miR-1193-3p in Traditional Chinese medicine treatment group was up-regulated than intervention group (with TGF-β1) and control group. The expression of rno-miR-9a-5p was significantly lower than control group.Conclusion:1、Neonatal rats colon tissue could keep activity for a long time in vitro afer we optimized the composition of culture medium and applied the organic culture model with cell strainer.2、Berberine and evodiamine can inhibit the biological effect of TGF-β1 by restoring the expression of crumbs3、E-cadherin and suppressing the expression of α-SMA.3、Our date could proof that berberine and evodiamine had a control role for rno-miR-1193-3p、rno-miR-9a-5p, cause there was no negative control relationship between them and their molecular target protein-crumbs3、E-cadherin.