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| Untersuchte Arbeit: Seite: 41, Zeilen: 1-5, 10-26 |
Quelle: Gu et al 2009 Seite(n): 2, 4, Zeilen: 2: re.Sp. 9-24, 28-36; 4: re.Sp. 7-12 |
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| 3.2 Methods
3.2.1. Cell culture The Caco2 human colon cancer cell lines and IEC06 non transformed intestinal cells were obtained from the American Type Culture Collection (Manassas, VA) and were studied between passages 60 and 70. CACO2 at 20,000/ml were cultured in DEME medium supplemented with 20% fetal bovine serum in culture flasks in a CO2 incubator at 37°C. Based on previous titration experiments [Gu et al., 2009] we have used throughout this study a 10-7 M testosterone-HAS concentration for mAR stimulation. 3.2.2. Preparation of steroid solution Before each experiment testosterone-3- (O-carboxymethyl) oxime-Human Serum Albumin, referred to as testosterone-HSA (or Testo-HSA), DHT and estradiol, were dissolved in serum-free culture medium at a final concentration of 10-5 M. This stock solution was incubated for 30 min at room temperature with 0.3% charcoal and 0.03% dextran, centrifuged at 3000 x g and passed through a 0.45 μm filter to remove any potential contamination with free steroid. Testosterone-HSA, estradiol and DHT solutions were used at a final concentration of 10-7 M throughout all studies. If not otherwise stated all treatments and incubations with steroids including apoptosis assays were performed in serum-containing medium. Testosterone-HSA-FITC or control HSA-FITC constructs were generated by conjugating Testosterone-HSA or HSA with FITC using standard techniques. 3.2.3. In vivo animal experiment Colon carcinoma was generated as described previously (Wang et al., 2004). In a first series of experiments, 7-week old Balb/c mice (both male and female) were divided into two groups, A (n=5) and B (n=7). Both groups [underwent carcinogenic treatment.] |
[Seite 2]
Materials and methods Cell cultures The Caco2 and HCT116 human colon cancer cell lines and the non transformed intestinal IEC06 cells were obtained from the American Type Culture Collection (Manassas, VA) and were studied between passages 55 and 70. Preparation of steroid solution Before each experiment testosterone-3-(O-carboxymethyl) oxime human serum albumin, (testosterone-HSA or Testo-HSA; Sigma) was dissolved in serum-free culture medium at a final concentration of 10-5 M. This stock solution was incubated for 30 min at room temperature with 0.3% charcoal and 0.03% dextran, centrifuged at 3,000 x g and passed through a 0.45 μm filter to remove any potential contamination with free steroid. This is highly important for the interpretation of the results to disconnect any possible intracellular testosterone- and/or iAR-interference with the effects mainly induced by the mAR activation. Testosterone HSA, estradiol and dihydrotestosterone (DHT) (Sigma) solutions were used at a final concentration of 10-7 M throughout the study unless otherwise mentioned. All treatments and incubations with steroids including apoptosis assays were performed in serum-containing medium. Testosterone-HSA-FITC or control HSA-FITC constructs were generated by conjugating Testosterone-HSA or HSA with FITC (Sigma) using standard techniques. [Seite 4] Induction of colon carcinoma Colon carcinoma was generated as described previously [26]. In a first series of experiments, 7-week old Balb/c mice (both male and female) were divided into two groups, A (n = 5) and B (n = 7). Both groups underwent carcinogenic treatment. 26. Wang JG, Wang DF, Lv BJ, Si JM: A novel mouse model for colitis-associated colon carcinogenesis induced by 1,2-dimethylhydrazine and dextran sulfate sodium. World J Gastroenterol 2004, 10:2958-2962. |
Obwohl Shg als Coautor von Gu et al (2009) genannt wird, stammt keine der Formulierungen dieses Artikels von Shg (vgl. die Anmerkungen zu Quelle:Shg/Gu_et_al_2009). Ergo: Übernahme eines Fremdtextes ohne jede Kennzeichnung. Ferner: aus dem "10-7" der Vorlage wird bei Shg in der Einleitung "10-7". Eine Referenz für "(Wang et al., 2004)" fehlt im Literaturverzeichnis von Shg. |
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