OBJECTIVETo study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.

METHODSExperiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.

RESULTSThe cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.

CONCLUSIONLIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.

Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Genes, Homeobox ; Humans ; Leukemia Inhibitory Factor ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Octamer Transcription Factor-3 ; metabolism ; Organic Chemicals ; Proliferating Cell Nuclear Antigen ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Umbilical Cord ; cytology

OBJECTIVETo investigate effects of retinol on the expressions of epidermal growth factor (EGF), stem cell factor (SCF), colony-stimulating factor 1 (CSF1) and leukemia inhibitory factor (LIF) in cultured human umbilical-derived mesenchymal stem cells (UCMSCs).

METHODSHuman UCMSCs were isolated from human umbilical cord and identified for immunophenotypes. The cells were then cultured in DMEM/F12 media supplemented with 12% fetal bovine serum (FBS), 12% FBS+1 µmol/L retinol, 15% knockout serum replacement (KSR) and 15% KSR+ 1 µmol/L retinol. The expressions of the cytokines EGF, SCF, CSF1 and LIF in the cells were detected using RT-PCR and ELISA.

RESULTSThe isolated cells exhibited characteristic immunophenotypes of human UCMSCs and expressed EGF, CSF1 and SCF at both mRNA and protein levels but not LIF protein. Retinol (1 µmol/L) significantly promoted the expressions of SCF and CSF1 at both mRNA and protein levels but did not result in changes of EGF and LIF expressions in human UCMSCs.

CONCLUSIONRetinol at the concentration of 1 µmol/L can promote expression of SCF and CSF1 in human UCMSCs in vitro.

Cell Differentiation ; Cells, Cultured ; EGF Family of Proteins ; metabolism ; Humans ; Immunophenotyping ; Leukemia Inhibitory Factor ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Stem Cell Factor ; metabolism ; Umbilical Cord ; cytology ; Vitamin A ; pharmacology

PURPOSE: To investigate the effect of macrophage migration inhibitory factor (MIF) on corneal sensitivity after laser in situ keratomileusis (LASIK) surgery. METHODS: New Zealand white rabbits were used in this study. A hinged corneal flap (160-microm thick) was created with a microkeratome, and -3.0 diopter excimer laser ablation was performed. Expressions of MIF mRNA in the corneal epithelial cells and surrounding inflammatory cells were analyzed using reverse transcription polymerase chain reaction at 48 hours after LASIK. After LASIK surgery, the rabbits were topically given either 1) a balanced salt solution (BSS), 2) MIF (100 ng/mL) alone, or 3) a combination of nerve growth factor (NGF, 100 ug/mL), neurotrophine-3 (NT-3, 100 ng/mL), interleukin-6 (IL-6, 5 ng/mL), and leukemia inhibitory factor (LIF, 5 ng/mL) four times a day for three days. Preoperative and postoperative corneal sensitivity at two weeks and at 10 weeks were assessed using the Cochet-Bonnet esthesiometer. RESULTS: Expression of MIF mRNA was 2.5-fold upregulated in the corneal epithelium and 1.5-fold upregulated in the surrounding inflammatory cells as compared with the control eyes. Preoperative baseline corneal sensitivity was 40.56 +/- 2.36 mm. At two weeks after LASIK, corneal sensitivity was 9.17 +/- 5.57 mm in the BSS treated group, 21.92 +/- 2.44 mm in the MIF treated group, and 22.42 +/- 1.59 mm in the neuronal growth factors-treated group (MIF vs. BSS, p < 0.0001; neuronal growth factors vs. BSS, p < 0.0001; MIF vs. neuronal growth factors, p = 0.815). At 10 weeks after LASIK, corneal sensitivity was 15.00 +/- 9.65, 35.00 +/- 5.48, and 29.58 +/- 4.31 mm respectively (MIF vs. BSS, p = 0.0001; neuronal growth factors vs. BSS, p = 0.002; MIF vs. neuronal growth factors, p = 0.192). Treatment with MIF alone could achieve as much of an effect on recovery of corneal sensation as treatment with combination of NGF, NT-3, IL-6, and LIF. CONCLUSIONS: Topically administered MIF plays a significant role in the early recovery of corneal sensitivity after LASIK in the experimental animal model.
Animals ; Epithelium, Corneal/*drug effects/innervation/physiology ; Female ; Humans ; Interleukin-6/pharmacology ; Keratomileusis, Laser In Situ/*methods ; Leukemia Inhibitory Factor/pharmacology ; Macrophage Migration-Inhibitory Factors/genetics/*pharmacology ; Models, Animal ; Nerve Growth Factor/pharmacology ; Nerve Regeneration/*drug effects/physiology ; Neurotrophin 3/pharmacology ; RNA, Messenger/metabolism ; Rabbits ; Recovery of Function/*drug effects/physiology ; Sensation/*drug effects/physiology

OBJECTIVETo study the effects and underlying mechanisms of Zhuyun Recipe (ZR) on the endometrial receptivity in ovarian stimulation (OS) and blastocyst implantation dysfunction (BID) mice.

METHODSTotally 200 normal female Kunming mice were randomly divided into 6 groups, i. e., the control group (Group A), the OS group (Group B), the OS + ZR group (Group C), the BID group (Group D), the BID + ZR group (Group E), and the ZR group (Group F). The pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) were intraperitoneally injected to mice in Group B. Mifepristone was subcutaneously injected to mice in Group D at 9:00 am on the 4th gestation day. Corresponding medications were given to mice in Group C, E, and F at 1.5 mL/100 g by gastrogavage at 8:00 am from the first to the 4th gestation day. Eight uterus samples were collected at 9:00 pm on the 4th gestation day and fixed. The expression levels of leukemia inhibitory factor (LIF) and integrin beta3 were detected using immunohistochemical assay. The pregnant mice were sacrificed at 9:30 pm on the 8th gestation day, and their uterus were taken out. The number of blastocysts was counted.

RESULTSCompared with Group A, the pregnant rate was 6.67% (1/15 cases) in Group B and 18.75% (3/16 cases) in Group D, the mean OD value of LIF was 0. 18 +/- 0.02 in Group B and 0.23 +/- 0.02 in Group D, and the mean OD value of integrin beta3 was 0.20 +/- 0.05 in Group B and 0.19 +/- 0. 02 in Group D, showing statistical difference (P < 0.01). The pregnant rate was 54.55% (12/22 cases) in Group C and 65. 22% (15/23 cases) in Group E, the mean OD value of LIF was 0.37 +/- 0. 09 in Group C and 0.39 +/- 0.02 in Group E, and the mean OD value of integrin beta3 was 0.34 +/- 0.04 in Group C and 0.38 +/- 0.08 in Group E, showing statistical difference when compared with those of Group B and Group D respectively (P < 0.05).

CONCLUSIONSOS and BID had negative effects on the endometrial receptivity and hindered the blastocyst implantation. ZR could improve the uterine receptivity and elevate the pregnant rate by up-regulating the expressions of endometrial LIF and integrin beta3.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Embryo Implantation ; drug effects ; Endometrium ; drug effects ; physiology ; Female ; Integrin beta3 ; metabolism ; Leukemia Inhibitory Factor ; metabolism ; Mice ; Mice, Inbred Strains ; Ovulation Induction ; Pregnancy

OBJECTIVETo investigate whether signaling through Toll-like receptor-2 (TLR-2) can affect the expression of some cytokines in human gingival fibroblasts.

METHODSThe gingival fibroblasts were isolated and cultured in vivo, divided into blank control group, lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) group and Escherichia coli (Ec) group. mRNA expression levels were measured by real-time polymerase chain reaction (PCR). The protein expression levels were detected by the enzyme linked immunosorbent assay (ELISA). The data was statistically analyzed by SPSS16.0 software package.

RESULTSLPS from Pg could stimulate the expression of interleukin (IL)-6 and leukemia inhibitory factor (LIF) mRNA and protein, which reached the peak (5.87 ± 0.83) at 10 h, and the expression level increased with the increase of the Pg concentration. IL-11 or oncostatin-M (OSM) mRNA expression was not affected by LPS. After treated with Pg for 48 h, the protein expression of IL-6 and LIF was up-regulated, (962 ± 57) ng/L and (47 ± 18) ng/L respectively.

CONCLUSIONSSignaling through TLR-2 controls the expression of cytokines of IL-6 family in human gingival fibroblasts.

Adolescent ; Adult ; Cells, Cultured ; Child ; Dose-Response Relationship, Drug ; Escherichia coli ; chemistry ; Fibroblasts ; cytology ; metabolism ; Gingiva ; cytology ; Humans ; Interleukin-11 ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia Inhibitory Factor ; genetics ; metabolism ; Lipopeptides ; pharmacology ; Lipopolysaccharides ; administration & dosage ; isolation & purification ; pharmacology ; Oncostatin M ; genetics ; metabolism ; Porphyromonas gingivalis ; chemistry ; RNA, Messenger ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; agonists ; Young Adult

OBJECTIVETo search for the optimal approach for hepatocyte-directed differentiation of hepatic progenitor cells and investigate the molecular mechanism of the hepatic differentiation.

METHODSHepatic progenitor cells were infected with recombinant adenovirus which containing human LIF, BMP2 or BMP9 gene. The maturation and differentiation of progenitor cells were examined by PAS staining and ICG uptake methods at 4, 7 and 10 days post infection. The production of Albumin (Alb) was measured by luciferase activity at day 4, 7, 10 and 14.

RESULTSPAS staining assay revealed that BMP2 and BMP9 enhanced glycogen storage in hepatic progenitor cells most obviously at day 7. The percentages of positive cells were 30% and 45% respectively at 7 days post-infection. Meanwhile, 40% and 30% cells were positive by ICG uptake assay after BMP2 and BMP9 induction. Luciferase activity indicated that BMP9 induced ALB-Luc activity most significantly at day 7. However, less inductive activity was found in LIF-treated group.

CONCLUSIONThese results indicated tuat hepatic progenitor cells were differentiated into hepatocyte-like cells by BMPs and LIF induction.

Adenoviridae ; Bone Morphogenetic Proteins ; pharmacology ; Cell Differentiation ; Cells, Cultured ; Hepatocytes ; cytology ; metabolism ; virology ; Humans ; Leukemia Inhibitory Factor ; pharmacology ; Stem Cells ; cytology ; metabolism ; virology

OBJECTIVETo evaluate the influence of superovulation by GnRHa protocol and pregnant mare's serum gonadotropin (PMSG) alone on the expression of estrogen receptor (ER), progesterone receptor (PR) and leukemia inhibitory factor (LIF) mRNA on endometrium.

METHODSForty-five female ICR mice were randomly allocated into 3 groups:(1) GnRHa+PMSG group: alarelin was give first for desensitizing the pituitary, then superovulation with PMSG; (2) PMSG group: mice were injected with PMSG only; (3) Natural cycle group: mice were given with same volume of saline. Endometrium samples were taken at 48 hours after given hCG or ovulation (control group). ER and PR in glandular cells were detected with SP immunohistochemistry semiquantitatively. Expression of LIF mRNA on endometrium was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

RESULTThe positive rate(%) and expression intense (AU) of ER and PR on glandular epithelium cells were significantly lower in GnRHa+PMSG group and PMSG group than those in natural cycle group (all P <0.01). The expression of LIF mRNA was significantly lower in GnRHa+PMSG group and PMSG group than that in natural cycle group (all P <0.01); but the expressions of ER, PR and LIF in GnRHa+PMSG group were higher than those in PMSG group.

CONCLUSIONThe protocol with GnRHa down regulates the expressions of ER, PR and the LIF mRNA on the mice of secretive phase endometrium, suggesting it may have an adverse effect on the endometrial receptivity in mice, but it may still be better than PMSG alone.

Animals ; Clinical Protocols ; Endometrium ; metabolism ; Female ; Gonadotropin-Releasing Hormone ; analogs & derivatives ; pharmacology ; Gonadotropins ; pharmacology ; Leukemia Inhibitory Factor ; metabolism ; Mice ; Mice, Inbred ICR ; Ovulation Induction ; methods ; RNA, Messenger ; metabolism ; Random Allocation ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism ; Superovulation ; metabolism

Undifferentiated embryonic stem (ES) cells can be maintained in vitro if cultured in the presence of the cytokine leukaemia inhibitory factor (LIF). ES cells can also differentiate in vitro. A particularly efficient method for inducing ES cell differentiation is to culture ES cells as aggregates in the absence of LIF. Under these conditions they form structures known as embryoid bodies (EBs). However the current protocols for EB formation are still diverse. In order to facilitate further study, we carefully controlled the culture conditions for EB formation, and here we report an efficient protocol by which uniformly differentiated EBs were obtained, monitored by measuring the differentiation of beating cardiomyocytes. Furthermore, by using this protocol we observed in long-term cultured plating EBs (> 60 days) there still exist cell colony with pluripotency. This observation raised a potential possibility that ES cells may keep pluripotent in a niche provided by differentiated cells.
Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Culture Media ; Embryonic Development ; physiology ; Embryonic Stem Cells ; cytology ; Leukemia Inhibitory Factor ; pharmacology ; Mice ; Stem Cell Niche ; physiology ; Time Factors

OBJECTIVETo study the effects of growth factors on the survival and proliferation of human spermatogonial stem cells (SSCs) in vitro.

METHODSSSCs were treated with the growth factors SCF, LIF and bFGF added to the culture, each at the concentrations of 0, 5, 10 and 20 microg/L and repeated three times. The survival time and proliferation rate of the cells were determined every 8-12 hours and their morphological features observed with the light microscope and electron microscope.

RESULTSThe survival time and proliferation rate of the SSCs were significantly increased in the treated groups as compared with the control (P < 0.05).

CONCLUSIONThe growth factors SCF, LIF and bFGF can promote the survival and proliferation of SSCs in vitro.

Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Leukemia Inhibitory Factor ; pharmacology ; Male ; Spermatogonia ; cytology ; drug effects ; Stem Cell Factor ; pharmacology ; Stem Cells ; cytology ; drug effects

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