BACKGROUND: Protooncogene CD117 is a cytokine receptor important for hematopoietic element development. Currently, we are not able to routinely separate a sufficient quantity of bone marrow CD117+ cells for experimental purposes. AIM: The aim of this study was to establish an immunomagnetic separation method for CD117+ hematopoietic stem cell isolation and to estimate the expression of chosen BCl-2 family protein members in these elements. MATERIAL AND METHODS: 120 samples of human and murine bone marrow were acquired using the magnetic separation system. The cells were stained for CD117, BCl-2, BAX, and CD33 by an indirect fluorescent immunocytochemistry. RESULTS: The flow cytometry analysis showed only 2.6% CD117+ cells from human as well as mouse bone marrow which is insufficient for further experiments. Cytospin was not good for morphologic characterization and immunophenotyping due to the fragility and destruction of the studied cells. Therefore, cell suspension staining was selected and by this method we found CD117 positivity in 70% of the mononuclerar (CD33 positive) elements in the case of chronic myeloid leukaemia. Labelling of the BCl-2 family in this case showed antiapoptotic BCl-2 expression in 80 %, proapoptotic BAX expression in approximately 5%. CONCLUSION: Our results show that CD117 immunomagnetic separation from bone marrow material is not acceptable for experimental purposes. They demonstrate that the only practical useful for the bone marrow cell examination (morphology and immunophenotype) is cell suspension staining which uncovers the distribution of both cytoplasmic proteins and surface antigens of immature blood elements.
The quaternary benzo[c]phenanthridine alkaloid sanguinarine (SG) is the main component of Sangrovit, a natural livestock feed additive. Dihydrosanguinarine (DHSG) has recently been identified as a SG metabolite in rat. The conversion of SG to DHSG is a likely elimination pathway of SG in mammals. This study was conducted to evaluate the toxicity of DHSG in male Wistar rats at concentrations of 100 and 500 ppm DHSG in feed for 90 days (average doses of 14 and 58 mg DHSG/kg body weight/day). No significant alterations in body or organ weights, macroscopic details of organs, histopathology of liver, ileum, kidneys, tongue, heart or gingiva, clinical chemistry or hematology markers in blood in the DHSG-treated animals were found compared to controls. No lymphocyte DNA damage by Comet assay, formation of DNA adducts in liver by 32P-postlabeling, modulation of cytochrome P450 1A1/2 or changes in oxidative stress parameters were found. Thus, repeated dosing of DHSG for 90 days at up to 500 ppm in the diet (i.e. approximately 58 mg/kg/day) showed no evidence of toxicity in contrast to results published in the literature. In parallel, DHSG pharmacokinetics was studied in rat after oral doses 9.1 or 91 mg/kg body weight. The results showed that DHSG undergoes enterohepatic cycling with maximum concentration in plasma at the first or second hour following application. DHSG is cleared from the body relatively quickly (its plasma levels drop to zero after 12 or 18 h, respectively).
- MeSH
- adukty DNA MeSH
- benzofenantridiny farmakokinetika krev toxicita MeSH
- biochemická analýza krve MeSH
- časové faktory MeSH
- financování organizované MeSH
- ileum patologie účinky léků MeSH
- isochinoliny farmakokinetika krev toxicita MeSH
- játra patologie účinky léků MeSH
- kometový test MeSH
- krmivo pro zvířata MeSH
- krysa rodu rattus MeSH
- náhodné rozdělení MeSH
- orgánová specificita MeSH
- oxidační stres účinky léků MeSH
- pilotní projekty MeSH
- plocha pod křivkou MeSH
- poškození DNA účinky léků MeSH
- potkani Wistar MeSH
- systém (enzymů) cytochromů P-450 účinky léků MeSH
- testy genotoxicity MeSH
- tkáňová distribuce MeSH
- velikost orgánu účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
Cíl studie: Porovnat změny exprese bcl-2 jako regulačního mechanismu apoptózy v sekrečním endometriu v přirozeném cyklu a po orální estrogen-progesteronové substituci u stejné skupiny pacientek. Typ studie: Klinicko-laboratorní prospektivní studie. Název a sídlo pracoviště: LF UP a FN Olomouc – Gynekologicko-porodnická klinika, Ústav histologie a embryologie, Gynekologicko-porodnické odd. Baťovy nemocnice Zlín. Metodika: Vzorky sekrečního endometria (n=56) byly získány od 14 žen 5. a 7. den po ovulaci v přirozených cyklech a ve stejném období v dalších cyklech po hormonální přípravě endometria orální substitucí estrogenem a progesteronem. Pacientky zařazené do studie měly více než roční anamnézu neplodnosti, pravidelný menstruační cyklus, věk nižší než 40 let, BMI v rozpětí 20–32 a bazální hladiny FSH mezi 5 až 9 IU/l. Biopsie endometria byly provedeny 5. a 7. den po ovulaci nebo po zahájení substituce progesteronem. Materiál byl zpracován rutinním způsobem a ke stanovení bcl-2 byla použita nepřímá třístupňová imunohistochemická metoda. Výsledky: Prokázali jsme výskyt proteinu bcl-2 ve všech sledovaných strukturách děložní sliznice u obou sledovaných cyklů. V přirozených cyklech nedošlo k výraznějším diferencím v expresi bcl-2 mezi 5. a 7. dnem sekreční fáze. V cyklech s hormonální substitucí byl pozorován signifikantní nárůst bcl-2 jen v povrchovém epitelu (p<0,05). Při porovnání obou typů cyklů byla vyšší exprese bcl-2 v 7. dnu substituce progesteronem oproti spontánnímu cyklu v povrchovém epitelu (p<0,001), v buňkách spongiózy (p<0,01) i kompakty (p<0,05). Závěr: Exprese proteinu bcl-2 v endometriu ve střední sekreční fázi cyklu je výrazně vyšší při podávání estrogen-progesteronové substituce než v cyklu přirozeném. Hormonální substitucí jsou výrazněji ovlivněny struktury epiteliální (povrchový epitel, žlázky v pars compacta i spongióza), méně stromální.
Objective: To compare the changes of the protein bcl-2 expression, regulatory mechanism in the process of apoptosis, in the secretory endometrium throughout natural and artificial (estrogen-progesterone substitution) cycles in the same group of patients. Design: A prospective study. Setting: Dept. of Obstetrics and Gynecology, University Hospital Olomouc, Dept. of Histology and Embryology, Faculty of Medicine, Palack˘ University in Olomouc, Dept. of Obstetrics and Gynecology, Bata Hospital, Zlín Methods: Endometrial samples (n=56) were obtained on days 5 and 7 after ovulation, progesterone addition, resp. Patients (n=14) included in the study had regular menstrual cycle, age under 40, BMI range 20–32 and basal FSH level range 5–9 IU/l. The collected samples were processed routinely and bcl-2 was estimated by indirect three level imunohistochemic method. Results: We demonstrated the bcl-2 expression in all evaluated layers (epithelial surface, stroma, glands) of the mid-secretory endometrium in both cycles. No difference was found in bcl-2 expression between days 5 and 7 in the secretory phase of the natural cycle. In artificial cycles higher bcl-2 expression was found only in epithelial surface (p<0.05) between days 5 and 7. On day 7 higher bcl-2 expression was found in the artificial cycle in the endometrial epithelial surface (p<0.001) as well as in spongy layer (p<0.01) and compact layer (p<0.05) comparing to day 7 of the natural cycle. Conclusion: The bcl-2 expression in the mid-secretory endometrium is significantly higher in the cycle with estrogen-progesterone substitution comparing to the natural cycle. These changes were more significant in endometrial and glandular epithelium than in stromal cells.
Apoptosis plays a significant role in differentiation of many organs and helps to maintain homeostasis. The occurrence of apoptosis (using the apoptotic index) and expression of regulation protein Bcl-2 in the human endometrium was evaluated within the secretory phase of both the natural cycle, and an artificial one. Oral hormonal substitution used in this design induced similar, but more marked dynamic changes in Bcl-2 expression in the mid-secretory endometrium as were observed in the natural cycle, primarily in the surface and glandular epithelium of the endometrium. The apoptosis revealed similar a trend, but not significantly different.
- MeSH
- apoptóza účinky léků MeSH
- endometrium cytologie metabolismus MeSH
- estradiol analogy a deriváty farmakologie MeSH
- konjugované estrogeny (USP) farmakologie MeSH
- lidé MeSH
- luteální fáze MeSH
- menstruační cyklus účinky léků MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The objective of the study was to ascertain if the estradiol-progesterone substitution used for the preparation of endometrium which has been shown to elicit supraphysiological levels of circulating progesterone may also induce demonstrable changes in the ultrastructural morphology of human secretory endometrium which is characterised by three progesteron- dependent structures, the so called postovulatory triad: nuclear or nucleolar channel system, giant mitochondria and subnuclear glycogen. The study was approved by the institutional Ethic Committee and included 24 patients of one IVF centre who signed informed consent. Two sequentional endometrial biopsy specimens were taken on the 5th and 7th day after ovulation in one natural cycle and on the 5th and 7th day after the initiation of progesterone addition in another substituted cycles in the same patient. The surface and glandular epithelial cells were examined under transmission electron microscope (TEM). The appearance of nuclear channel system (NCS), giant mitochondria and glycogen was the same in both natural and substituted cycles, without abnormalities and deformations. Cells with NCS were found, mostly in clusters on discrete parts, in 55 to 70 % of ultrathin sections. The relative number of NCS positive specimens was not statistically different between the cycles or between the day +5 and the day +7 within different type of cycle. Some NCS positivity was detected more often (in 79 % and 66 %, resp.) in substituted cycles than in spontaneous ones. The percentage of NCS positive cells was also higher (median 17 % and 12 %, resp.) in substituted cycles but the differences were not statistically significant. With some caution regarding the inherent limited reproducibility of the TEM findings we can conclude that the oral hormonal substitution used in our setting does not markedly alter endometrial ultrastructural morphology.
Objective: Exogenous hormonal manipulation may alter physiological cyclic pattern of endometrium. This article evaluates the effect of oral estrogen-progesterone substitution on the expression of estrogen and progesterone receptors and the level of apoptosis in the secretory endometrium of substituted cycles by comparison with natural cycles in the same patient. Methods: Serum levels of estradiol (E2), sex hormone-binding globulin (SHBG) and progesterone (P) were determined and bioptic samples of endometrium were taken on the 5th and 7th day of luteal phase in 26 infertile women who entered the assisted reproduction program first in a spontaneous and later in an estrogen-progestagen substituted cycle. E and P receptors (ER and PR) were estimated by immunostaining and image analysis in stroma and glandular epithelium. The level of apoptosis was evaluated by the TUNEL assay. Wilcoxon matched-pairs test was used for the statistical analysis of data. Results: The mean serum levels of E2, SHBG and P were higher in substituted cycles (p<0.05, p<0.001, p<0.01, resp.). The expression of ER and PR in stroma was lower than in glandular epithelium in both types of cycle and did not change between the 5th and the 7th day after ovulation or P addition. The expression of ER and PR in glandular epithelium in substituted cycles was higher than in spontaneous cycles on the day 5th (p=0.01, p=0.02, resp.). A decline between the 5th and the 7th day was seen in both types of cycle, but was much more prominent in substituted cycles (p<0.01, p<0.001, resp.). The mean level of apoptosis was always higher in glands than in stroma. Higher levels of apoptosis were more often found in compact layer than in spongy layer of glands, in substituted cycles than in spontaneous cycles and in the second biopsy samples than in the first ones. Conclusions: Our findings are in accordance with the supposed effect of oral ovarian steroid cycle substitution which may often elicit supraphysiological peripheral P levels. The higher expression of ER and PR observed in substituted cycles seemed to have no negative effect on endometrial receptivity.
- MeSH
- apoptóza genetika imunologie MeSH
- dospělí MeSH
- endometrium metabolismus MeSH
- estradiol aplikace a dávkování terapeutické užití MeSH
- exprese genu MeSH
- lidé MeSH
- progesteron aplikace a dávkování terapeutické užití MeSH
- receptory estradiolu analýza MeSH
- receptory progesteronu analýza MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- ženské pohlaví MeSH
