Comparative study on the stem cell phenotypes of C6 cells under different culture conditions / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Glioma stem cell (GSC) hypothesis posits that a subpopulation of cells within gliomas have true clonogenic and tumorigenic potential. Significantly, a more controversial correlate to GSC is that cells in different culture conditions might display distinct stem cell properties. Considering these possibilities, we applied an approach comparing stem cell characteristics of C6 glioma cells under different culture conditions.</p><p><b>METHODS</b>C6 cells were cultured under three different growth conditions, i.e., adherent growth in conventional 10% serum medium, non-adherent spheres growth in serum-free medium, as well as adherent growth on laminin-coated flask in serum-free medium. Growth characteristics were detected contrastively through neurosphere formation assay and cell cycle analysis. Markers were determined by immunofluorescence, relative-quantitative reverse transcription (RT)-PCR, Western blotting and flow cytometry. Side population cells were analyzed via flow cytometry. Tumor models were detected by magnetic resonance imaging and hematoxylin & eosin staining. Data analyses were performed with SPSS software (17.0).</p><p><b>RESULTS</b>C6 cells (C6-Adh, C6-SC-Sph and C6-SC-Adh) showed distinctive growth patterns and proliferation capacity. Compared to suspending C6-SC-Sph, adherent C6-Adh and C6-SC-Adh displayed higher growth ratio. C6-SC-Sph and C6-SC-Adh showed enhanced capability of neurosphere formation and self-renewal. High side population ratio was detected in C6-SC-Sph and C6-SC-Adh. CD133 was not detected in all three kinds of cells. Conversely, Nestin and β-III-tubulin were demonstrated positive, nonetheless with no statistical significance (P > 0.05). Interestingly, lower expression of glial fibrillary acidic protein was demonstrated in C6-SC-Sph and C6-SC-Adh. C6-Adh, C6-SC-Sph and C6-SC-Adh were all displayed in situ oncogenicity, while statistical difference of survival time was not confirmed.</p><p><b>CONCLUSIONS</b>C6 glioma cell line is endowed with some GSC phenotypes that can be moderately enriched in vitro when transferred into stem cell culture condition. The resultant tumor-spheres may be not a prerequisite or sound source of GSCs and adherent culture in stem cell medium is not a growth condition in favor of GSCs expanding in vivo.</p>

The relative analysis of clinical endocrine features and pathological types of pituitary microadenomas / 中华外科杂志

<p><b>OBJECTIVES</b>To study the relationship between the clinical presentation, endocrinal findings and pathological types in patients with pituitary microadenomas, so as to improve the accuracy of clinical diagnosis and choose the best therapy strategy before the operation.</p><p><b>METHODS</b>From January 2007 to June 2009, the clinical data of 94 patients who were surgically removed pituitary microadenomas were obtained, including the clinical presentation, endocrinal findings and pathological diagnosis. The analysis was accomplished with Chi-square test.</p><p><b>RESULTS</b>Hormonal symptoms were found in 86 patients (91.5%), it occurred more frequently in immunopositive patients (85/92, 92.4%) than in immunonegative patients (1/2, 50.0%) (P < 0.05). The coincidence of hormonal symptoms and immunohistochemistry diagnosis was 71.7%; 88.9% patients had the symptoms of amenorrhea, galactorrhea and sexual function diseases in prolactin (PRL) positive group and 28.1% patients had the symptoms of gigantism or acromegaly in growth hormone (GH) positive group. The coincidence of endocrinal findings and immunohistochemistry diagnosis was 69.0%; 87.7% patients had high level of blood PRL in PRL positive group and 21.9% patients had high level of blood GH in GH positive group.</p><p><b>CONCLUSIONS</b>There is an obvious relationship between the clinical presentation, endocrinal findings and pathological diagnosis in patients with pituitary microadenomas, which may contribute to the clinical diagnosis and treatment of pituitary secreting microadenomas.</p>

Effects of human HSF1 on transcriptional regulation of prohibitin promoter / 中国应用生理学杂志

<p><b>AIM</b>To assay the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.</p><p><b>METHODS</b>The total length of hHSF1 exon was amplified by PCR method and cloned into pcDNA3. 1(+) vector. pcDNA3. 1(+)-hHSF1 and pGI3 prohibitin were co-transfected into HEK293 cells. The luciferase activity was detected by Dual-Luciferase Reporter Assay System to evaluate the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.</p><p><b>RESULTS</b>The pcDNA3.1(+)-hHSF1 vector was constructed successfully. The assay of luciferase activity showed that the transcription of pGL3-prohibitin was dramatically upregulated by hHSF1.</p><p><b>CONCLUSION</b>hHSF1 can transcriptionally regulate prohibitin expression.</p>

Construction and effect identification of MiR RNAi eukaryotic expression vectors of prohibitin / 中国应用生理学杂志

<p><b>AIM</b>To construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection.</p><p><b>METHODS</b>The specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting.</p><p><b>RESULTS</b>The DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1.</p><p><b>CONCLUSION</b>A RNAi eukaryotic vector containing prohibitin gene was successfully constructed.</p>

LRIG3 gene regulates biological activity of GL15 cell line / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the biological activity of glioblastoma cell line GL15.</p><p><b>METHODS</b>Glioblastoma GL15 cells were cultured and transfected with LRIG3-EGFP plasmid. The location of LRIG3 in GL15 cells was observed with confocal microscopy. The proliferation and invasiveness of GL15 cells were detected with methyl thiazolyl tetrazolium (MTT) and Transwell methods respectively; the expression of epidermal growth factor receptor (EGFR) and LRIG3 mRNA and protein were detected with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot respectively.</p><p><b>RESULT</b>After transfection with the plasmid LRIG-EGFP, LRIG3 fusion protein was found in cytoplasm of GL15 cells and cell proliferative and invasiveness were reduced. The expression of EGFR and LRIG3 varied with the duration of EGF treatment (100 ng/ml): the expression of EGFR decreased while the expression of LRIG3 increased as time prolonged.</p><p><b>CONCLUSION</b>LRIG3 can inhibit the proliferation and invasiveness of glioblastoma cells and may be used as a target gene in gene therapy of glioblastoma.</p>

The assay of transcriptional activation effect of TR3 and deletion mutation in the yeast two hybrid system / 中国应用生理学杂志

<p><b>AIM</b>To assay the transcriptional activation effect of TR3 and it's deletion mutation in yeast two hybrid system.</p><p><b>METHODS</b>The total length of TR3 and TR3/delta1-690 gene was amplified by PCR method and cloned into pGBKT7 vector. Bait vector of pGBKT7-TR3 and pGBKT7-TR3/delta1-690 was transformed into AH109 competence yeast. Then self activation of the recombination vector was tested by assay the activity of beta-galactosidae.</p><p><b>RESULTS</b>The pGBKT7-TR3 and pGBKT7-TR3/AM 690 vector was successfully constructed. The filter paper containing beta-galactosidae didn't changed to blue showed that the reporter gene wasn't activationed.</p><p><b>CONCLUSION</b>TR3 and TR3/delta1-690 hadn't the activity of transcriptional activation.</p>

Effects of LRIG1 gene on the invasion of bladder cancer in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the change of invasion ability of the bladder cancer cell line BIU87 after transfected with LRIG1 gene.</p><p><b>METHODS</b>Plasmid pLRIG1-GFP was transfected into bladder cancer cells (BIU87) by Lipofectamine 2000, and cells that could express LRIG1 stably was screened out by G418. The alteration of cellular invasion property of BIU87, BIU87-neo and BIU87-LRIG1 cells was evaluated by Boyden chamber. The expression of mRNA and protein of LRIG1 and EGFR was detected by RT-PCR and Western-blot.</p><p><b>RESULTS</b>Compared with BIU87 and BIU87-neo cells, after BIU87 cell line was transfected with plasmid pLRIG1-GFP, the invasion ability decreased significantly, the expression of mRNA and protein of LRIG1 was obviously up-regulated and that of EGFR was down-regulated (P < 0.01).</p><p><b>CONCLUSION</b>LRIG1 gene can significantly inhibit the invasion ability of BIU87 cell line.</p>