Study On The Mechanism Of Lin28B And CyclinD1 In The Proliferation Of Rat Tenon's Capsule Fibroblasts Induced By TNF-?

Objective: Through the primary cultured rat Tenon's capsule fibroblasts,using tumao necrosis factor alpha(TNF-?)to stimulate proliferation of Tenon 's' capsule fibroblasts,to evaluate the changes of expression of RNA binding protein LIN28 B and cyclin Cycin D1,to study the mechanism of Lin28 B and Cyclin D1 in the proliferation of rat Tenon 's capsule fibroblasts,and explore its effect on bleb fibrosis.Methods: Rat Tenon's capsule fibroblasts were cultured using tissue adherent method,HE staining and immunohistochemistry were used to identify.Plant the cell suspension which were digested to the 2×104/ml in 96 well plate,and the cells were respectively treated with 10ng/ml,50ng/ml and 100ng/ml TNF-? for 3 hours,6 hours,12 hours,18 hours and 24 hours,and then the Cell Counting Kit-8(CCK-8)method was used to detect the growth status of fibroblasts,to detect optimal concentration and best time of TNF-?.Plant the cell suspension which were digested to the 2×104/ml in 12 well plate,after one night of serum starvation,the cells were divided into three groups: group A: the cells were cultured with 50ng/ml TNF-?,cultrue 12 hours;group B: the cells were cultured with 50ng/ml TNF-? after the intervention of 100?mol/L PDTC for 0.5 hour.culture 12 hours.group C: the cells were cultured with 50ng/ml TNF-? after the intervention of 100?mol/L PDTC for 0.5 hour,culture 24 hours.detect the growth status of fibroblasts by CCK-8,then detect and compare the expression of Lin28 B and Cyclin D1 among 3groups by immunocytochemistry and Real-time PCR.Results:1.Rat Tenon's capsule fibroblasts cultured by explant culture Line HE staining results show:The cells showed typical long spindle shape,the nuclei are clearly blue and purple,oval,forsingle or multiple,the cytoplasm was purplish red.Immunocytochemical analysis revealed that these cells were positive for keratin and negative for vimentin.2.The expression of Lin28 B showed significant difference among the three groups(F=580.903,P=0.000).The expression of Lin28 B m RNA in group B and group C was significantly lower than that in group A(all P=0.000);group B showed a small amount of Lin28 Bm RNA,but the expression of in group B Lin28 B m RNA was higher than that in group C(P=0.003);A cells in the cytoplasm of Lin28 B showed strong positive expression,the average optical density value of group B,C was significantly less than that in the group A;In group B,the expression of Lin28 B was positive in cytoplasm.In group C,the expression of Lin28 B in the cytoplasm of a small number of cells was weakly positive,the average optical density was significantly lower than that of group B.3.The expression of Cyclin D1 showed significant difference among the three groups(F=558.667,P=0.000).The expression of Cyclin D1 m RNA in group B and group C was significantly lower than that in group A(all P=0.000);and the expression level of Cyclin D1 m RNA in group C was significantly lower than that in group B(P=0.000);In group A,the expression of Cyclin D1 in cell nucleus was strongly positive,and the average optical density of group B and group C was significantly less than that of group A;In group B,the positive expression of Cyclin D1 was found in the cell nucleus;In group C,the expression of Cyclin D1 in the nucleus of a small number of cells was weakly positive,and the average optical density was significantly lower than that of group B.4.The correlation between Lin28 B and Cyclin D1 m RNA expression was analyzed.,the results showed that r=0.953,P < 0.001.Lin28 B was positively correlated with Cyclin D1.Conclusion:1.The expression of protein and mRNA of Lin28 B ? CyclinD1 in TNF-?induced proliferation of Tenon 's capsule fibroblasts was significantly increased,which indicated that the expression of Lin28 B and Cyclin D1 were closely related to the proliferation of Tenon's capusle fibroblasts.2.The expression of Lin28 B m RNA was positively correlated with the Cyclin D1 m RNA,it is suggested that the activation of Lin28 B can directly regulate the expression of Cyclin D1,accelerate cells from G1 phase into S phase,shorten the process of cell cycle,promote the Tenon 's capsule fibroblast proliferation.3.Regulation of Lin28B-Cyclin D1 pathway to inhibit the proliferation of Tenon 's capsule fibroblasts may be a new target for inhibiting postoperative bleb scarring after glaucoma surgery.