| Objective：Establish the model of vascular endothelial cell in vitroMethods: Human umbilical vein endothelial cells was isolated fromhealthy fetus umbilical cord and cultured in vitro by enzymatic digestion,the cell morphology of was observed under inverted Phase-contrastmicrographs. The expression of coagulation factor Ⅷ was detected byimmunofluorescence assay. The expression of cadherin was detected byWestern blotting.Results：（1） the morphology of primary HUVEC was round, shortspindle-shape or flat. After being subcultured the morphology of cells aresimilar to primary HUVEC, and cells grew vigorously in cobblestoneappearance.（2） primary culture and subcultured cells all expresscoagulation factor Ⅷ， However, The positive expression rate ofcoagulation factor Ⅷ of HUVEC in subcultured were significantly higherthan that of primary culture（with F value all equal to7.481，P values allblow0.05）.(3) Primary culture and the passaged cells all express cadherin Conclusion: Confirm that the cells which separated and cultured invitro is HUVEC Objective: To explore a new pathogenesis for fibrosis disease, weobserved the role of tumor necrosis factor-alpha (TNF-α) inendothelial-mesenchymal transition (EnMT).Methods: The third to fifth generations of cultured HUVEC inlogarithmic phase were harvested and seed in12-well plates、6-well platesand6cm plates,12-well plates and6-well plates were divided into controlgroup (ordinary culture without any stimulation),5ng,10ng,25ng,50ng,100ng/ml TNF-α group(with contain5ng,10ng,25ng,50ng,100ng/ml of TNF-α was respectively added into the nutrient solution)according to the random number table. However,6cm plates was dividedintocontrol group (ordinary culture without any stimulation),5ng,25ng,50ng/ml TNF-α group (with contain5ng,25ng,50ng/ml of TNF-α wasrespectively added into the nutrient solution) according to randomly number table. Three sample in each group. After being cultured for72hours, the cellmorphology was observed under inverted Phase-contrast microscope. Theexpression level of coagulation factor Ⅷ and α-smooth muscle actin(α-SMA) was detected by immunofluorescence assay. The mRNAexpression level of cadherin, α-SMA, and typeⅠcollagen was detected byRT-PCR. The protein expression of cadherin and α-SMA was detected byWestern blotting. Data were processed with one-way analysis of varianceand LSD test.Results:(1) As compared with control group, the appearance of cells in5ng,10ng,25ng,50ng,100ng/ml TNF-α group was gradually transformedfrom round, short spindle-shape or flat to long-spindle with reducedintercellular junction and larger intercellular gap along with the increase inthe concentration of TNF-α.（2）The ratios of coagulation factor anda-SMA double-positive cells in control group were significantly lower thanthat in5ng,10ng,25ng,50ng,100ng/ml TNF-α group (with F valuerespectively45.009，values all below0.01).（3）the expression level ofVE-cadherin mRNA in5ng,10ng,25ng,50ng,100ng/ml TNF-α group weregradually reduced, and it was significantly lower in latter four group thanthat in control group (with F value11.593，P <0.05or P <0.01). theexpression level of α-SMA and Ⅰcollagen mRNA in5ng,10ng,25ng,50ng,100ng/ml TNF-α group were gradually increased, and it wassignificantly higher in latter three group than that in control group (with F value respectively7.839、2.898，P <0.05or P <0.01).（4）The proteinexpression of cadherin5ng,10ng,25ng/ml TNF-α group were graduallyreduced, and it was significantly lower in latter two group than that incontrol group (with F value66.551, P <0.05or P <0.01), However, Theprotein expression quality of α-SMA in5ng,10ng,25ng/ml TNF-α groupwas significantly higher than that in control group.Conclusions: TNF-α can promote endothelial-mesenchymal transition(EnMT) in a dose-dependent manner. These data show that endothelial cellsthrough EnMT may be a significant source for myofibroblasts within fibrotictissue in chronic inflammation disease and scar formation，Proinflammatorycytokines is a key incentive in this transformation. Objective: To observed the role of interleukin6(IL-6) inendothelial-mesenchymal transition (EnMT), and to explore a newmechanism for fibrosis disease,Methods: The third to fifth generations of cultured HUVEC in logarithmic phase were harvested and seed in12-well plates、6-well platesand6cm plates,12-well plates and6-well plates were divided into controlgroup (ordinary culture without any stimulation),5ng,10ng,25ng,50ng,100ng/ml IL-6group(with contain5ng,10ng,25ng,50ng,100ng/mlof IL-6was respectively added into the nutrient solution) according to therandom number table, however,6cm plates was divided into control group(ordinary culture without any stimulation),5ng,25ng,50ng/ml IL-6group(with contain5ng,25ng,50ng/ml of IL-6was respectively addedinto the nutrient solution) according to randomly number table. Threesample in each group. After being cultured for72hours, the cell morphologywas observed under inverted Phase-contrast microscope. The expressionlevel of coagulation factor Ⅷ and α-smooth muscle actin (α-SMA) wasdetected by immunofluorescence assay. The mRNA expression level ofcadherin, α-SMA, and typeⅠcollagen was detected byRT-PCR. Theproteinexpressionqualityofcadherinand α-SMAwas detectedbyWesternblotting.Data were processed with one-way analysis of variance and LSD test.Results:(1) As compared with control group, the whirlpool appearanceof cells in5ng,10ng,25ng,50ng,100ng/ml IL-6group was graduallytransformed from round, short spindle-shape or flat to long-spindle withreduced intercellular junction and cell amount，larger intercellular gap andcell volume along with the increase in the concentration of IL-6.（2）Theratios of coagulation factor Ⅷ and a-SMA double-positive cells in control group were significantly lower than that in IL-6group (with F valuerespectively79.72，P values all below0.01).（3）the expression level ofcadherin mRNA in5ng,10ng,25ng,50ng,100ng/ml IL-6group weregradually reduced, and it was significantly lower in latter four group thanthat in control group (with F value24.637，P <0.05or P <0.01). theexpression level of α-SMA and Ⅰcollagen mRNA in5ng,10ng,25ng,50ng,100ng/ml IL-6group were gradually increased，the expression levelof α-SMA mRNA was significantly higher in latter four group than that incontrol group (with F value23.955，P <0.05or P <0.01),and the expressionlevel of Ⅰcollagen mRNA was significantly higher in100ng/ml IL-6groupthan that in control group(with F value2.250，P <0.05).（4）The proteinexpression level of cadherin in IL-6group were gradually reduced， and itwas significantly lower than that in control group (with F value69.822, P<0.01). However, the protein expression level of α-SMA in IL-6weregradually increased， and it was significantly higher in latter two group thanthat in control group (with F value5.766, P <0.01).Conclusions: IL-6could promote endothelial-mesenchymal transition(EnMT) in a dose-dependent manner. EnMT may be a significant source formyofibroblasts within fibrotic tissue in chronic inflammation disease andscar formation. Indicate proinflammatory cytokine possess promote fibrosispotency。... |
PDF Full Text Request