OBJECTIVE: Uterine leiomyomas are the most common tumor of the uterus. But the molecular causes of uterine leiomyoma remain unclear. We conducted the current investigation in order to elucidate the molecular mechanisms in the development of uterine leiomyoma. METHODS: We employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) method that involved annealing control primers (ACPs) to identify the genes that are differently expressed in uterine leiomyoma. RESULTS: Using 120 ACPs, we identified and sequenced 14 differently expressed genes (DEGs) in uterine leiomyoma compared with normal myometrium. Basic Local Alignment Search Tool (BLAST) searches were performed to examine the known functions of these genes associated with uterine leiomyoma. We confirmed differently expressed patterns in more cases using the RT-PCR method. We also detected two novel genes, Tenascin-X and Leukemia Inhibitory Factor Receptor (LIFR), which had not yet been reported to have any functions associated with uterine leiomyoma. RT-PCR confirmation shows that both of these two genes are down-regulated in uterine leiomyoma. CONCLUSION: Our results suggest that Tenascin-X and LIFR may play a role in the development of uterine leiomyoma. Although further studies are required to establish the precise mechanisms with which these genes are involved in the genesis of uterine leiomyoma, the present research is significant in that it is the first study which detects down-regulated novel genes in uterine leiomyoma using the ACP system.
Animals ; Female ; Leiomyoma ; Leukemia ; Leukemia Inhibitory Factor ; Mice ; Myometrium ; Receptors, OSM-LIF ; Tenascin ; Uterus

OBJECTIVETo investigate the expression and clinical significances of HGFA, Matriptase, HAI-1 and HAI-2 in patients with acute myeloid leukemia (AML).

METHODSThe bone marrow samples from 91 AML patients, 41 AML patients in complete remission, and 32 normal controls were collected. Real time fluorescence quantitative RT-PCR (qRT-PCR) was used to detect the mRNA expressions levels of HGFA, Matriptase, HAI-1, HAI-2 . The expressions of these genes were compared among AML untreated group, the complete remission group and the healthy control group. The correlation of their expression with clinical characteristics was analyzed.

RESULTSThe level of HGFA in the AML untreated group was higher than that in the healthy control group（P<0.05）, while the HAI-2 mRNA level was lower than that in the healthy control group（P<0.05）. The mRNA levels of HAI-1 and Matriptase were not changed significantly in all groups. The HAI-2 mRNA expression level was significantly lower in the high white blood cell group (P<0.05).

CONCLUSIONThe abnormal activation of HGF/c-Met signaling system in AML may result from the increase of HGFA expression and the decrease of HAI-2 expression of the upstream regulatory factors.

OBJECTIVE: To determine the expression of endometrial leukemia inhibitory factor (LIF) in infertile women and to compare it with that of fertile women during the window of implantation METHODS: Endometrial biopsies were performed during the window of implantation from 10 normal fertile and 15 infertile women (5 unexplained infertility, 5 endometriosis and 5 tubal disease with hydrosalpinx). We performed immunohistochemistry using paraffin-embedded endometrial tissues. RESULTS: In fertile and infertile women, the staining intensities of LIF in the glandular epithelium were significantly higher (p<0.01) than those of stromal cells. There was no significantly difference in the expression of LIF between the glandular epithelium of infertile women with endometriosis or hydrosalpinx and those of fertile women. However, the staining intensities of LIF in the glandular epithelium of unexplained infertility patients were significantly lower (p<0.01) than those in fertile women. The staining intensities of LIF in stromal cell of women with unexplained infertility, endometriosis and hydrosalpinx were similar to those in fertile women. CONCLUSIONS: This study demonstrated that the glandular epithelial cells are more important sites of LIF secretion than stromal cells. The expression of endometrial LIF may be decreased in women with unexplained infertility during the window of implantation. Therefore, this result suggest that abnormalities of LIF expression in infertile women may underlie endometrial dysfunction in the adhesive phase of implantation.
Adhesives ; Biopsy ; Endometriosis ; Endometrium* ; Epithelial Cells ; Epithelium ; Female ; Humans ; Immunohistochemistry ; Infertility ; Leukemia Inhibitory Factor* ; Leukemia* ; Stromal Cells

OBJECTIVE: To assess the effect of recombinant human leukemia inhibitory factor on in vitro development of 1-cell ICR mouse embryo. MATERIALS AND METHOD: ICR mice were superovulated with PMSG/hCG and 1-cell stage mouse embryos were recruited. 1-cell mouse embryo were cocultured on human oviductal cells in a CO2 incubator (coculture group) and were cultured on 0.4% BSA+HTF media (control group). And anti-hLIF Ab was added the cocultured group in a different concentration (1pg, 10pg, 100pg, 1ng) and developmental rate was compaired to the control group, and rhLIF was added to the preincubated 0.4% BSA+HTF media in a different concentration (2000U, 1000U, 100U, 10U) and its developmental rate was compaired to group which was cultured on 0.4% BSA+HTF media only. RESULT: 1. The cleavage rate of 2-cell mouse embryo co-cultured with human tubal epithelial cell was significantly higher than that of cultured with media alone (HTF with 0.4% BSA) (p<0.05). 2. When LIF antibody was added to the medium with human tubal epitherlial cell, the mouse embryo could not cleave more than 2-cell in 1 ng of LIF antibody, and less than 1 ng, the cleavage rate was lower than cultured without LIF antibody group(p<0.05). 3. Two cell blocked ICR mouse embryos were developed into four cells under LIF(p<0.05), but no further development was observed. CONCLUSIONS: These results shows that LIF enhances the development of preimplantation embryo, and when rhLIF is applicated in vitro, it has positive effects on the development of early mouse embryo and can help overcoming the two-cell block.
Animals ; Blastocyst* ; Coculture Techniques* ; Embryonic Structures ; Epithelial Cells ; Humans ; Incubators ; Leukemia Inhibitory Factor* ; Leukemia* ; Mice ; Mice, Inbred ICR ; Oviducts

Bovine embryos (day 5) were cultured to day 10 with or without 100 ng/mL PGF2α in medium supplemented with control; 100 nM Dex; 1,000 U/mL recombinant human leukemia inhibitory factor (rhLIF); or Dex+rhLIF. Although the rates to development to the blastocyst were not significantly different among groups, the hatching rate after additional culture with Dex +/or rhLIF was significantly higher in all supplemented groups than the control (p < 0.05). In the presence of PGF2α, the hatching rate was significantly restored in all supplemented groups relative to the group treated with only PGF2α and the control (p < 0.05). Embryo transfer (ET) was performed with blastocysts (day 7). PGF2α levels of control recipient cows were significantly higher in the circulatory blood samples collected 60 min after ET than in samples collected 60 min before ET (p < 0.005), and were decreased in cows injected with loading medium supplemented with Dex+rhLIF (p < 0.005). Pregnancy rate was significantly higher in the ET group that received supplemented embryo-loading medium than in the non-supplemented control (p < 0.05). The intrauterine administration of Dex and rhLIF at ET prevented increased PGF2α in circulatory blood and resulted in enhanced pregnancy rate.
Animals ; Blastocyst ; Cattle* ; Dexamethasone* ; Embryo Transfer* ; Embryonic Structures* ; Fertilization in Vitro ; Humans* ; Leukemia Inhibitory Factor* ; Leukemia* ; Pregnancy Rate* ; Pregnancy* ; Prostaglandins F

In order to observe the effect of Bushenantai recipe on the expression of endometrial leukemia-inhibitory factor (LIF) in mice with embryonic implantation dysfunction (EID), 120 Kunming mice post coition were randomized into three groups: normal control group, model group and traditional Chinese medicine group (TCM group) (n=40 in each group). Uterus was collected on the pregnancy day (Pd) 4, 5, 6 after an intravenous injection of Evan's blue. The endometrium was dyed by Evan's blue and the mean points of response were observed on Pd 5. The expression of LIF mRNA and protein was detected by RT-PCR and immunohistochemistry respectively and analyzed statistically by image system. The results showed that the number of implantation sites in model group was remarkably less than in normal control group and TCM group. There was no significant difference between normal control group and TCM group. The expression of LIF mRNA and protein in model group was delayed. Bushenantai recipe could increase the expression of LIF mRNA and protein in endometria of mice with EID. It was suggested that Bushenantai recipe could improve embryo implantation of mice with EID by promoting the endometrial LIF expression and endometrial decidualization.
Blastocyst/cytology ; Embryo Implantation ; Endometrium/*metabolism ; Gene Expression ; Gene Expression Regulation, Developmental ; Leukemia Inhibitory Factor/*biosynthesis ; Leukemia Inhibitory Factor/*genetics ; Medicine, Chinese Traditional ; Models, Biological ; Plant Extracts/pharmacology ; RNA, Messenger/metabolism ; Time Factors

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.
Animals ; Cell Differentiation ; Cell Line ; Cell Separation ; Embryonic Stem Cells ; Feeder Cells ; Green Fluorescent Proteins ; Leukemia Inhibitory Factor ; Mice

Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest-related precursor cells that can be isolated from dermis. It is known that the properties of porcine SKPs can be enhanced by leukemia inhibitory factor (LIF) which is an essential factor for the generation of embryonic stem cells in mice. In our present study, to enhance or maintain the properties of murine SKPs, LIF was added to the culture medium. SKPs were treated with 1,000 IU LIF for 72 hours after passage 3. Quantitative real time RT-PCR was then performed to quantify the expression of the pluripotent stem cell specific genes Oct4, Nanog, Klf4 and c-Myc, and the neural crest specific genes Snai2 and Ngfr. The results show that the expression of Oct4 is increased in murine SKPs by LIF treatment whereas the level of Ngfr is decreased under these conditions. Interestingly, LIF treatment reduced Nanog expression which is also important for cell proliferation in adult stem cells and for osteogenic induction in mesenchymal stem cells. These findings implicate LIF in the maintenance of stemness in SKPs through the suppression of lineage differentiation and in part through the control of cell proliferation.
Adult Stem Cells ; Animals ; Cell Proliferation ; Dermis ; Durapatite ; Embryonic Stem Cells ; Leukemia ; Leukemia Inhibitory Factor ; Mesenchymal Stromal Cells ; Mice ; Neural Crest ; Pluripotent Stem Cells

OBJECTIVE: Successful implantation depends on a complex interaction between the developing blastocyst and the endometrium. Among the steroid hormones, growth factors, and cytokines which participate in preparing the uterus for implantation, leukemia inhibitory factor (LIF) plays an essential role in implantation. We compared the expression of LIF in normal pregnancies to that of recurrent abortions in placenta to elucidate whether spontaneous abortion and expression of LIF has correlation. METHODS: Placental tissues from normal pregnancies and recurrent abortions were fixed and embedded in paraffin. Standard immunohistochemical staining was used to identify LIF. RESULTS: LIF expressions on cytotrophoblast of recurrent abortion were lower than those of normal pregnancy. There were no expressions on syncytiotrophoblast and stroma in the both groups. In the decidua and gland, LIF was expressed in mild degree and there were no differences in LIF expression between normal pregnancy and recurrent abortion. CONCLUSION: LIF expression on cytotrophoblast of recurrent abortion was lower than that of normal pregnancy. LIF may provide paracrine and autocrine signals to both embryonic tissues and uterine epithelium during implantation. The dysfunction of LIF production may be a cause of the unexplained recurrent abortions.
Abortion, Habitual* ; Abortion, Spontaneous ; Blastocyst ; Cytokines ; Decidua ; Endometrium ; Epithelium ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; Leukemia Inhibitory Factor* ; Leukemia* ; Paraffin ; Placenta ; Pregnancy* ; Trophoblasts ; Uterus

The aim of this study was to investigate the feasibility of the human cord blood adult stem cells (ASCs) to differentiate into hepatocytes in vitro induced by combined stimulation with hepatocyte growth factor (HGF), stem cell factor (SCF) and leukemia inhibitory factor (LIF). The adult stem cells were obtained through density gradient centrifugation and magnetic activated cell sorting (MACS). The adult stem cells were cultured in DMEM with HGF (10 ng/ml)+SCF (10 ng/ml)+LIF (10 ng/ml) in induced group I. In induced group II the enriched cells were cultured in DMEM with SCF (10 ng/ml)+LIF (10 ng/ml) and the undifferentiated cells acted as the control group without the factors. The morphology of cells was observed by the inverted phase contrast microscopy; the expression of albumin (Alb), human hepatocyte cytokeratin (CK18) and alpha-fetoprotein (AFP) were detected by immunofluorescence, immunohistochemistry and RT-PCR assay in the 21-day culture. Alb secreted by hepatocytes in the medium was determined by radioimmunoassay (RIA) at day 7, 14, 21, 23 and 25. The results showed that the shapes of ASCs changed and their sizes and number increased in the course of culture in group I. After being induced for three weeks, the cells turned round and resembled hepatocyte-like cells. The mRNA for Alb could be detected by RT-PCR in the differentiated adult stem cells in group I, and the mRNA for AFP was poorly detected by RT-PCR at day 21. Alb and CK18 were positive through immunofluorescence and immunohistochemistry at day 21, compared with group II and the control group. In group I, Alb in the medium significantly increased, compared with control group, and reached the highest level at day 21, then decreased at day 23. It is concluded that under some definite inducing conditions, human cord blood adult stem cells can differentiate into hepatocyte-like cells and HGF plays a critical role during the course.
Adult Stem Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; Humans ; Leukemia Inhibitory Factor ; pharmacology