Effect Of Thyroid Stimulating Antibody On Thyroid Cell Proliferation And Its Mechanism

Objective To observe the effect of thyroid stimulating antibody(TSAb) on the growth and proliferation in thyrocytes and MAPK signal transduction pathway; to probe into the mechanism of TSAb stimulating proliferation of thyrocytes; and elucidate the preliminary molecular mechanism of TSAb leading to the goitor and Graves' disease(GD).Methods 1. A human primary thyrocyte culture was established. To observe the effect of TSAb on proliferation of thyrocytes by these measurements such as cell counting, MTT assay and 3H-thymidine uptake into the DNA synthesis. 2. MAPK protein expression in thyrocytes was determined by flow cytometry. 3. In the same time, to evaluate the signal transduction pathways through which TSAb regulates the thyroid cells proliferation with inhibitors of signal pathways.Results 1. TSAb could enhance proliferation of thyrocytes. When 2mg/ml TSAb was added, the cell numbers increased by 19.4%, 24.0%, 28.7%, 27.7% for 24> 48, 72, 96h respectively; With MTT assay ,we could observe the same results; The degree of TSAb on the 3H-thymidine uptake was increased when the dose of TSAb was increased, and the cpm reached maximum by 2mg/ml TSAb, which was 2.47-fold higher than the control(P<0.05). 2. TSAb upregulated the expression of p42/44 MAPK and p38 MAPK protein in thyrocytes. The protein expression of p42/44 MAPK and p38 MAPK reached maximum at 20 min with 2mg/ml TSAb, and increasing by 2.08, 1.70 -fold respectively than the control(P<0.05). After addition of PKA inhibitor, BAPTA-AM and PD98059, the protein expression of p42/44 MAPK was decreased by 43.1 %, 29.3 % , 59.6%. 3, After addition of inhibitors involved above for blocking of c-AMP/PKA pathway, scavenging intracellutar Ca2+ and MAPK pathway, PKA inhibitor could mostly block the effect of TSAb on proliferation , BAPTA-AM couldpartly block the effect and PD98059 could block the effect completely.Conclusion 1. TSAb can stimulate thyroid cell proliferation, thus TSAb leads to the goitor and may be the main pathogenesis of GD. 2. Through upregulating the protein expression of p42/44MAPK and p38MAPK, TSAb can activite MAPK signal pathway and result in proliferation of thyrocytes. 3. The effect of TSAb stimulating cell proliferation can be blocked by PKA mostly, BAPTA-AM partly and PD98059 completely, which indicates that TSAb regulate firstly thyrocyte proliferation by c-AMP/PKA mainly and PIP2/Ca2+ pathway secondarily, at last TSAb regulates thyrocyte proliferation by MAPK pathway. And it may be the molecular mechanism that TSAb induces thyroid cell proliferation.