The gene expressions of Interleukin-6 (IL-6) from human peripheral blood lymphocytes (HPBL) stimulated by C. albicans were investigated by using ELISA (Enzyme linked immunosorbent assay), reverse transcription polymerase chain reaction (RT-PCR) and northern blotting. HPBL (1 X 10'/ml) obtained from normal human peripheral blood lymphocytes were cultured with live C. albicans (LCA) or heat killed C. albicans type A 311 (KCA, 3 X 10/ml) for various times (0.5, 1, 4, 8, 18, 24, 48 and 72 hours). On the purpose of this experiment, we also used lipopolysacchalide (LPS, 10 ug/ml), zymosan (1, 10, 100 ug/ml) as a polysacchaide component of the wall of yeast cells or TNFa (50, 100 ng/ml) as a IL-6 inducers. For observation of the level of IL-6 gene expression, actinomycin D (AD, 5 pg/ml) or cyclohexamide (CHX, 25 ug/ml) was added to HPBL stimulated with LCA for 0.5, 2, 4 hours and the HPBL were assessed for IL-6 mRNA. The highest value for IL-6 activity by LCA were observed at 48 hours reaction, but in the case of KCA, highest value of IL-6 activity was observed at 72 hours reaction and the value was also higher (500 pg/ml) than that of LCA (188 pg/ml). 1L-6 mRNA induced by LCA were detected up to 48 hours but in the case of KCA, the band for IL-6 mRNA were far stronger and appeared until lately than that of LCA. Therefore, the results of IL-6 gene expression agreed with that of ELISA.
Blotting, Northern ; Dactinomycin ; Enzyme-Linked Immunosorbent Assay ; Gene Expression* ; Hot Temperature ; Humans* ; Interleukin-6 ; Lymphocytes* ; Polymerase Chain Reaction ; Reverse Transcription ; RNA, Messenger ; Yeasts ; Zymosan

FSH is the pivotal hormone in the regulation of ovarian function and acts by binding to specific receptor, FSH receptor (FSHR), which is belong to the family of G-protein coupled receptor. It have been considered that ovary is the only target organ of FSH because FSHR mRNA was first detected in ovarian follicles. However expression of FSHR mRNA was also detected on fallopian tube in experimental animal study and it is related wih tumorigenesis in postmenopausal women.In this study, in order to understand the FSH function in female genital organs, the ontogeny of the production profile of FSHR and the pattern of its localization in female genital organs were studied. We obtained the fresh tissues of ovary, fallopian tube, uterine body and uterine cervix with blood samples during proliferative phase in women with regular menstrual cycle. To establish FSHR mRNA expression of human internal genital organ, we studied by using in situ hybridization and quantitative competitive reverse transcription polymerase chain reaction (QC RT-PCR). To localize FSHR transcripts by in situ hybridization, we synthesized digoxigenin-labelled ssRNA probe (about 800 bp) from the cloned FSHR cDNA. For QC RT-PCR, we designed oligonucleotide primers (antisense: 5'-GGCCCTGCTCCTGGTCTCTTTG-3', sense: 3'-AACAGCGGGAGTACCTTCGG-5') which produced 799 bp sized PCR products. Simultaneously we synthesized 149 bp deleted DNA competitor by site-directed mutagenesis to quantify target FSHR mRNA expression comparing as internal control.In situ hybridization with digoxigenin-labelled ssRNA probe showed no signal above the background in primordial follicles. FSHR mRNA was first detected in the single layer of cuboidal granulosa cells surrounding primary follicles. As follicular growth progressed, FSHR mRNA expression increased gradually in antral and graafian follicles. Similary, in fallopian tube, the epithelium stained intensly. But FSHR mRNA expression was absent in uterine body including endometrium and myometrium and uterine cervix. Total RNA was extracted and quantitated by QC RT-PCR. The amounts of FSHR transcript measured were 840.00+/-516.29 in the ovarian tissue, 240.00+/-154.91 in the fallopian tube, 6.06+/-4.13 in the uterine body, 5.48+/-5.00 fg in the uterine cervix. These experiments demonstrated that FSHR mRNA is expressed in the ovary and fallopian tube, albeit only small amount was expressed in uterine body and cervix.In conclusion, the presence of FSHR mRNA in female internal genital organ with site specific pattern suggested that FSH may have some role in female genital organs during the adult reproductive cycle and may act as an factor in the tumorigenesis. Further study about the functional role and tumorigenesis of FSH should be performed in human internal genital organ.
Adult ; Animals ; Carcinogenesis ; Cervix Uteri ; Clone Cells ; DNA ; DNA Primers ; DNA, Complementary ; Endometrium ; Epithelium ; Fallopian Tubes ; Female ; Female* ; Genitalia ; Genitalia, Female* ; Granulosa Cells ; GTP-Binding Proteins ; Humans ; In Situ Hybridization ; Menstrual Cycle ; Mice ; Mutagenesis, Site-Directed ; Myometrium ; Ovarian Follicle ; Ovary ; Polymerase Chain Reaction ; Receptors, FSH ; Reverse Transcription ; RNA ; RNA, Messenger*