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Objective To investigate the expression of girders of actin filaments (Girdin) in pituitary adenomas, and its role in promoting cell proliferation and the related molecular mechanism. Methods Two prolactinoma, growth hormone adenoma and non-functioning pituitary adenoma tissues, and one normal pituitary gland tissue were collected. The protein expression of Girdin in the different tissues was detected by Western blotting, and then the expression of Girdin was further confirmed by immunofluorescence. Rat pituitary tumor cell lines GH3 cell model with Girdin knockdown and overexpression was established by RNA interference and overexpression of Girdin, respectively. The protein expression of Girdin and Akt and phosphorylation level of Akt in the GH3 cell models were detected by Western blotting. The function and biological behavior of Girdin in pituitary adenomas tissues were studied by cell proliferation assay and cell apoptosis assay. Results The expression of Girdin in the non-functioning pituitary adenomas was the highest, followed by growth hormone pituitary adenomas. The high expression of Girdin in the non-functioning pituitary adenomas was also verified by immunofluorescence assay. RNA interference and overexpression of Girdin effectively knocked down and increased the expression of Girdin, respectively, accompanied by the simultaneous changes of Akt phosphorylation. In addition, overexpression of Girdin promoted the proliferation of GH3 cells. Conclusion Girdin is highly expressed in non-functioning pituitary adenomas and can promote the proliferation of pituitary adenoma cell by regulating the Akt phosphorylation.
ABSTRACT
<p><b>BACKGROUND</b>Thyrotropin-secreting pituitary adenomas (TSHomas) are a rare cause of hyperthyroidism. Somatostatin (SST) analogs work by interacting with somatostatin receptors (SSTRs). This study aimed to evaluate short-term preoperative octreotide (OCT) use in TSHoma patients and to investigate SSTR2 and SSTR5 expression and observe structural changes in tumor tissue.</p><p><b>METHODS</b>We reviewed records and samples from eight TSHoma patients treated between July 2012 and July 2015. We tested immunohistochemically for SSTR2/5 expression and examined TSHoma cells for morphological changes. Signed rank sum test was used to compare the efficacy of short-term preoperative OCT treatment.</p><p><b>RESULTS</b>OCT treatment (median time: 7.9 days, range: 3-16 days; median total dose: 1.8 mg, range: 0.9-4.2 mg) led to significant decrease in all patients' thyroid hormone levels (FT3 [nmol/L]: 8.33 [7.02, 12.29] to 4.67 [3.52, 5.37] [P = 0.008]; FT4 [pmol/L]: 25.36 [21.34, 28.99] to 16.66 [14.88, 21.49] [P = 0.016]; and TSH [μU/ml]: 5.80 [4.37, 6.78] to 0.57 [0.19, 1.24] [P = 0.008]). All the eight tumor specimens expressed high SSTR2 protein levels; 5/8 expressed high SSTR5, but 3/8 that expressed low SSTR5 presented a significantly higher TSH suppression rate (P = 0.036). Electron microscopy showed subcellular level impairments, including clumped nuclear chromatin and reduced cytoplasmic volume. Golgi complexes were observed in the OCT-treated TSHoma specimens.</p><p><b>CONCLUSIONS</b>OCT can control hormone levels and damage the ultrastructure of tumor cells and organelles. Short-term response to OCT may be related to SSTR5 expression. Preoperative SST analog treatment for TSHoma could be considered as a combination therapy.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Immunohistochemistry , Microscopy, Electron , Octreotide , Therapeutic Uses , Pituitary Neoplasms , Drug Therapy , Metabolism , Receptors, Somatostatin , Metabolism , Thyrotropin , Bodily SecretionsABSTRACT
Objective: To compare the efficiencies of two methods (serum and non-serum) in inducing mouse embryonic stem cell differentiation into definitive endoderm cells. Methods: The serum and non-serum methods were used to induce differentiation of mouse embryonic stem cells into definitive endoderm cells. Fluorescence activated cell sorter (FACS) was used to analyze the inducing time and efficiency of definitive endoderm using their surface protein marks (Cxcr4, c-Kit and E-cadherin). Meanwhile, RT-PCR was used to analyze the gene profile of definitive endoderm induced by the two methods. Realtime PCR was used to analyze the gene expression in definitive endoderm during the induction course in the non-serum group. The Cxcr4 and c-Kit double positive definitive endoderm cells were sorted by flow cytometry and gene profile was characterized by RT-PCR. Results: Definitive endoderm cells were induced from mouse embryonic stem cells by both serum and non-serum methods. However, the efficiency of non-serum group (74. 19%) was higher than that in the serum group, and the induction outcome reached a climax at the 4th day of induction. Conclusion: We have established a highly efficient method to induce differentiation of mouse embryonic stem cells into definitive endoderm, which lays a foundation for further differentiation into liver and pancreatic cells.
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<p><b>OBJECTIVE</b>To establish a gene-modified embryonic stem (ES; E14.1-2) cell line with hepatoblast differentiation reporter genes, albumin (ALB) and cytokeratin 19 (CK19), labeled to facilitate study of their potential applicability as differentiated hepatoblasts.</p><p><b>METHODS</b>Two expression vectors were constructed, one with the ALB promotor driving the enhanced green fluorescent protein (EGFP) and anti-neomycin genes (pAlb-EGFP), and the other with the CK19 promotor driving the red fluorescence protein and anti-hygromycin genes (pCK19-hCD25-IRES-tdTOMATO). The linearized vectors were electroporated into the E14.1 line, and double reporter genes-modified ES cells (E14.1-2) were selected by neomycin and hygromycin. E14.1-2 hepatoblast differentiation was induced by exposure to growth factors (BMP4 and bFGF) and evidenced by embryoid body formation. Fluorescence-activated cell sorting (FACS) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm whether differentiated cells were hepatoblast-like and to quantify the differentiation efficiency.</p><p><b>RESULTS</b>The pAlb-EGFP and pCK19-hCD25-IRES-tdTOMATO vectors were shown to specifically activate ALB and CK19 expression. The E14.1-2 cell line with labeled ALB and CK19 was established, and shown to have pluripotency by RT-PCR detection of pluripotent markers' expression, namely Oct4 and SSEA-1. After 22 days of induction, 21.27% of the differentiated hepatoblasts were detected by FACS as positive for ALB and CK19 expression.</p><p><b>CONCLUSIONS</b>A gene-modified ES cell line was generated with hepatocyte differentiation reporter genes ALB and CK19 labeled. The differentiation of the resultant E14.1-2 line was technically simple to qualify and quantify, and will likely aid future studies of hepatoblast characteristics.</p>