Study Of The Expression Of Id1Gene To Invasion Of The Pituitary Adenoma

[Objective]To preliminarily study the expression and significance of Idl protein in the tissue of invasive pituitary adenoma, establish primary invasive pituitary adenoma cell strain coded NQ-04, study the alteration of invasiveness of NQ-04cells after siRNA transfection, and thereby provide new theories and experimental basis for the clinical diagnosis and treatment of invasive pituitary adenoma.[Methods]1. Western blot separately detects the expression of Idl protein in invasive and non-invasive pituitary adenoma.We selected randomly15invasive pituitary tumors and non-invasive pituitary tumors from45case of intraoperative frozen fresh pituitary adenoma tissues our hospital collected to extract holoprotein. Then,2xSDS-PAGE loading buffer (proportion1:1) was added to holoprotein and the mixture was boiled to100℃for5min. After testing by Westernblot method, we observed gel image and scanned Western blot gel image by AdobePhotoshop, and obtained gray value. Analyze the difference of expressions of Idl protein in invasive and non-invasive pituitary adenoma.2. Establish primary invasive pituitary adenoma cell strain coded NQ-04After pituitary adenoma tissues were removed by surgery, we placed them into PBS solution in aseptic conditions, sealed the opening with film, and put them in ice bucket, and then moved them to laboratory. The cells were separated by mechanical and enzymic digestion. The research group utilized the primary generation of fresh tissue specimens of invasive pituitary adenoma to cultivate NQ-04cells.3.siRNA-id1transfects NQ-04cells.SiRNA was diluted to20umol/L with RNA-free enzyme H2O and then stored in-20℃. During transfection, we diluted the solution with cell culture medium and then mixed it with lipotaminelTM2000by the proportion2.5:1. The mixture was kept still in room temperature for20min and then added to the serum-free culture solution according to different proportions. After6hours, the culture solution was changed to the one containing10%serum and continued to be cultured for24-72h.4. Western blot detects the idl protein expression in NQ-04cells after siRNA-idl transfection to detect the efficiency of silence of Idl genes.5. Detect the influence of siRNA-transfected NQ-04cells on invasive ability by Transwell invasion experiment. Select5views under200x light microscope to count the numbers of transmembrane cells and take the average value. Repeat the experiment for3times.[Results]1.The expression of Idl gene is significantly higher in invasive pituitary tumor than non-invasive pituitary tumor and the difference has statistical significance (n=15, t=2.725, p=0.013, p<0.05),and further indicates that Idl gene has adjusting effect in invasive pituitary tumor.2.Expression of Idlin NQ-04cells interfered by small interfering RNA (siRNA) reduce Significantly.3.Select5views under200x light microscope to count the numbers of cells in control group and interference group. The result finds that the siRNA-idl-transfected NQ-04cell group is (40±16.54) and non-siRNA-idl-transfected NQ-04cell group is (67±17.45). The difference has statistical significance (t=2.972, p=0.016, p<0.05).This indicates that after transfected siRNA disturbed id1, the transfer ability of NQ-04cells is significantly reduced. After NQ-04cells are transfected by siRNA-idl, expression of idl protein is significantly reduced.[Conclusions]1. The expression of Idl protein is higher in invasive pituitary adenoma, which has significant difference from non-invasive pituitary adenoma and statistical significance.2. Idl protein can promote the invasion and transfer of pituitary adenoma cells. Targeted therapy of Id1gene may become one of the new strategies of treatment.