[Effects of activating and inhibiting Wnt/ß-catenin signaling pathway on murine model of eutopic endometrium and endometriosis].

OBJECTIVE: To employ the classical Wnt/ß-catenin signaling pathway interference to explore the effects on the functional changes of eutopic endometrium stromal cells and the differences between endometriosis in a murine model. METHODS: Two out of three mouse groups received an injection of either Wnt/ß-catenin signaling pathway activator or blocker. Later the endometrial tissue samples were obtained to develop endometrial stromal cell cultures for the detection of cell invasion ability via Boyden chamber invasion assay and Western blot (WB). Then the methods of WB and Immunohistochemical staining (IHC) were used to examine the factors of eutopic endometrium. And an endometriosis model was established to investigate the factors of signaling pathway via quantitative polymerase chain reaction (QPCR) and IHC. RESULTS: According to WB test, the level of ß-catenin, GSK-3ß and APC in the activation group were significantly higher than in the inhibition group (P < 0.01). In Boyden chamber invasion assay, the number of cells on membranes in the trial group was significantly higher than the control group [(113 ± 12) vs (64 ± 13)]. The expressions of VEGF and MMP-9 in the endometrial stromal cells culture from Boyden chamber assay analyzed via WB were ranked from highest to lowest respectively as activation group (vs control group was 35.6% and 27.4% higher), control group and inhibition group (vs control group was 12.3% and 30.4% lower). Furthermore, the endometrial E-cadherin and VEGF examined via IHC respectively showed a positive expression in inhibitor group and strong positive expression in activation group. QPCR showed the level of Wnt3, Wnt7, GSK3ß, Lef and E-cadherin in the activation group was higher than those in the inhibition group (P < 0.05). CONCLUSION: The intervention of WNT signaling pathway in vivo cause the changes of eutopic endometrial invasion and adhesion function, and further affect the development of endometriosis. Wnt/ß-catenin signaling pathway may promote the eutopic endometrial cell proliferation and improve the ability of eutopic endometrial implantation, invasion, metastasis and angiogenesis.

[Analysis of high risk factors associated with cervical intraepithelial neoplasia in married women aged 25 - 54 years in Beijing between 2007 - 2008].

OBJECTIVE: to investigate high risk factors associated with cervical intraepithelial neoplasia (CIN) in married women aged 25 to 54 years in Beijing. METHODS: from Mar. 2007 to Sep. 2008, 6339 married women at age of 25 to 54 years were selected randomly by cross sectional survey in a total of 137 communities of 12 districts or counties in Beijing. The interview was carried out with unified questionnaires, gynecological examination. The cervical smear cytology and high-risk human papillomavirus (HR-HPV) infection of cervical secretion were detected. Women with abnormal cervical cytology underwent colposcopy and cervical biopsy. Odd ratio (OR) and 95% confidence interval (CI) of related high risk factors with CIN were studied by logistic regression analysis. RESULTS: among 6339 women, the prevalence rate of CIN including 4 squamous cell carcinoma (SCC) was 5.90% (374/6339). By multinomial regression analysis, HR-HPV infection (95%CI: 9.953 - 15.811), History of trichomonas vaginitis (95%CI: 1.046 - 2.104), oral contraceptives (95%CI: 1.087 - 1.806), age less than 45 years old (95%CI: 1.069 - 1.828) were related with CIN. CONCLUSION: infection rate of HR-HPV is an independent risk factor of CIN, however, the history of trichomonas vaginitis, oral contraceptives and age less than 45 years old are related risk factors of CIN.

[Epidemiological study on the prevalence of cervical intraepithelial neoplasia among married women aged 25-54 years in Beijing between 2007-2008].

OBJECTIVE: To investigate prevalence of cervical intraepithelial neoplasia (CIN) among the married women aged 25-54 years in Beijing. METHODS: With method of cross sectional survey, 6339 married women at age of 25 to 54 years were selected randomly in 137 communities of 12 districts or counties in Beijing from March 2007 to September 2008. The cervical smear cytology and high-risk human papillomavirus (HPV) infection of cervical secretion were detected. Women with abnormal cervical cytology were underwent colposcopy and cervical biopsy. RESULTS: Among 6339 women, 9.58% (607/6339) cases had abnormal cytological results, the colposcopy and cervical biopsy showed the rate of CIN was 5.84% (370/6339) in total selected women and 60.96% (370/607) in women with abnormal cervical cytology, including 4.65%(295/6339) in CIN I, 0.80% (51/6339) in CIN II, 0.38% (24/6339) in CIN III; 0.06% (4/6339) in early invasive carcinoma (SCC). Based on geographical distribution, the rate of cervical lesions was 4.46% (42/942) in urban areas, 6.27% (188/3000) in suburbs and 6.01% (144/2397) in outer suburbs (P > 0.05). CONCLUSION: It was found that the incidence of CIN was 5.84% in married women aged 25-54 years in Beijing, which did not show significant prevalence in urban, suburb and outer suburbs region.

[Analysis of correlation factors for cervical intraepithelial neoplasia in married women in Beijing, China].

OBJECTIVE: To investigate the characteristic of precancerous conditions of cervical intraepithelial neoplasia (CIN) and its related high-risk factors among the married women in Beijing, China. METHODS: Based upon the method of cross-sectional survey, 6339 married women at reproductive age from 25 to 54 years old were selected randomly in a total of 137 communities of 12 districts or counties in Beijing from March 2007 to September 2008. The interview was carried out with unified questionnaires and gynecological examinations. And the cervical Pap smear was used for liquid-based cytological examination and the cervical secretion for detection of high-risk human papillomavirus (HPV) DNA. Furthermore, the women with abnormal cervical cytology received colposcopy and cervical biopsy. RESULTS: (1) Among 6, 339 study subjects, 374 cases had CIN and the prevalence rate was 5.9%; (2) For the cases with cervical cytology positive results, cervical biopsy showed an elevated level of abnormal cytology and an increased incidence of cervical lesions; (3) The peak age of CIN prevalence was 30 to 34 years old and there was a high grade of cervical neoplasia; (4) Among the population, the infection rate of high-risk HPV was 9.9% and the infection rate of high-risk HPV in positive cytological group (41.2%) was significantly higher than that in negative cytological group (6.6%); (5) The infection rate and DNA load of high-risk HPV increased following the severity grade of CIN. CONCLUSION: In Beijing, married women at 30 to 34 years old are the high-risk group in CIN incidence and the infection of high-risk HPV is an independent risk factor. Liquid-base cytology combined with high-risk HPV DNA test is a viable method to discover CIN in time and prevent the incidence of cervical cancer.

[Effects of ovarian carcinoma cells on the maturation and function of human dendritic cells].

OBJECTIVE: To investigate the effects of ovarian carcinoma cells on the differentiation, maturation, and function of the dendritic cells (DC). METHODS: Human epithelial ovarian carcinoma cells were isolated from specimens of ovarian carcinoma from 12 patients obtained during operation, and cultured. Peripheral blood samples were collected from these patients. DC were isolated and co-cultured with recombinant human tumor necrosis factor (TNF)-alpha to promote their maturation. Immunostaining and flow cytometry (FC) were used to detect the DC specific marker CD1a, maturation marker CD83, co-stimulating factors CD86 and human leucocyte antigen (HLA)-DR. FITC-labeled glucan was added and FC was used to detect the phagocytic function. T cells were co-cultured with immature and mature DC of different concentrations respectively for 96 h, 18 h before the end of culture 3H-TdR was added, liquid scintillation counter was used to measure the level of count per minute (cpm) Ovarian carcinoma cells and peripheral mononuclear cells were put into Transwell to contact each other directly or indirectly, TNF-alpha was add to promote the maturation, and then FC was used to detect the CD1a, CD83, CD86, and HLA-DR levels, and the glucan uptake level. ELISA was used to detect the levels of IL-12, IL-10, and interferon (IFN)-gamma, and Western blotting was used to detect the p-38 protein and phosphorylated p-38 protein in the supernatant. RESULTS: After stimulation of TNF-alpha the expression; levels of CD1a, CD86, CD83, and HLA-DR of the DCs were remarkably increased. Immature DCs showed a strong ability at phagocytosis of glucan, and this ability was decreased by the stimulation of TNF-alpha. The proliferative function of mature DCs on allogenic T lymphocytes was significantly increased dose-dependently (P<0.05). Co-culture with ovarian carcinoma cells decreased the expression levels of CD1a, CD86, and HLA-DR, increased the expression level of CD83, and decreased the ability in up taking glucan by 43.05%. The proliferative function of the DC cultured in direct contact with ovarian carcinoma cells was decreased by 56.35%, and such inhibitory function of the DCs cultured not in direct contact with ovarian carcinoma cells was weaker, and the levels of CD1a, CD86, CD83, and HLA-DR expressed by the DC cultured not in direct contact with ovarian carcinoma cells was between those of the DC cultured in direct contact with ovarian carcinoma cells and those of the DC undergoing routine culture. The levels of IL-12 and phosphorylated p-38 protein were decreased in the supernatant of the DC co-cultured with ovarian carcinoma cells. CONCLUSION: Ovarian carcinoma cells suppress the differentiation of monocyte into DC, negatively regulates the phagocytotic and antaean managing functions of DC, thus the DC can not effectively stimulate the proliferation of T lymphocytes and inhibit the activity of cytotoxic T lymphocytes. That may be one of the underlying mechanisms of immune escape of tumor and immune tolerance of T lymphocytes.

[Simulated uterus microenvironment induced human placental mesenchymal stem cells differentiation to uterus smooth muscle cells in vitro].

OBJECTIVE: To develop a new method to promote the differentiation of mesenchymal stem cells derived from human placenta (pMSC) to uterus smooth muscle cells (uSMC) in simulated uterus microenvironment. METHODS: MSCs were isolated from human placenta, cultivated, and analyzed for their phenotype by flow cytometry. The multipotential differentiation of the pMSC was examined by chondrogenic, adipogenic, and osteogenetic induction. uSMC were isolated from uteri resected during operation and co-cultivated with the pMSC in a Transwell chamber simulating Two, 4, and 8 days later RT-PCR and Western blotting were used to detect the mRNA and protein expression of alpha-actin, calmodulin, and myosin heavy chains (MHC), the markers of smooth muscle differentiation at the early, middle, and late stages. On day 8 RT-PCR was used to detect the expression of estrogen receptor in these 2 groups of cells, then estrogen was used to stimulate these cells and the protein kinase C (PKC) activity was examined. RESULTS: The pMSC could be induced into adipocytes, osteocytes, and chondrocytes respectively. After co-culture with uSMC, the morphology of the pMSC changed closely into that of the uSMC, and MHC was expressed in the pMSC. Estrogen receptor was positive in both groups of cells. The PKC activity increased, especially in the cell membrane, after stimulation of estrogen. CONCLUSION: The postpartum human placenta can be used as an important and novel source of multipotent stem cells for tissue engineering and genetic engineering. Placental MSC have the potential to differentiate into smooth muscle cells under the simulated uterus microenvironment in vitro.

[Effect of human placenta derived mesenchymal stem cells on cord blood lymphocyte transformation].

OBJECTIVE: To study the effect of human placenta derived mesenchymal stem cells (MSCs) on the immune function of lymphocytes derived from human umbilical cord blood. METHODS: Mononucleated cells (MNC) were isolated from human placenta tissue perfusate by density gradient fractionation. Individual colonies were selected and cultured. The culture-expanded cells were characterized by immune phenotyping so as to identify the MSCs. The MSCs were cultured under conditions promoting differentiation to osteoblasts or adipocytes. MNCs were isolated from adult peripheral blood and human umbilical cord blood and cultured, then the adherent cells were excluded and the suspended cells, lymphocytes, were inoculated in the culture fluids of MSCs of different concentrations and phytohemagglutinin (PHA), a nonspecific mitogenic stimulant, was added for 84 hours (MSC + PHA groups), then (3)H-thymidine deoxyribose ((3)H-TdR) was added for 12 hours. The cells were collected and scintillation counter was used to calculate the counts per minute (cpm). Pure lymphocytes without MSC and stimulated by PHA were used as control group (non-MSC Group) and pure lymphocytes and pure MSCs without PHA were used as blank control groups (non-PHA Group). RESULTS: From human placenta MSCs were successfully isolated and exhibited fibroblast-like morphology. Flow cytometric analysis showed that the placental MSCs were a homogeneous cell population devoid of hematopoietic cells positive for CD29, CD44, CD73, CD105, CD166, and HLA-ABC positive and negative for CD34, CD45, and HLA-DR. They could be induced into adipocytes or osteocytes. The cpm value of the non-MSC Group was 171 855 +/- 31 454, significantly higher than that of non-PHA Group (26 453 +/- 5268). The cpm values of the different concentrations MSC + PHA groups were all significantly lower than that of non-MSC Group in a dose-dependent manner; when the dose of MSCs was 2 x 10(5) the suppression rate was 79.97% in PB and 64.06% in UCB. CONCLUSION: MSCs derived from postpartum human placenta, an important and novel source of multipotent stem cells, suppress blood lymphocyte proliferation, thus may be used to reduce graft -versus-host disease (GVHD) in recipients.

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation.

Human placenta-derived mononuclear cells (MNC) were isolated by a Percoll density gradient and cultured in mesenchymal stem cell (MSC) maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblast-like morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and alpha-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class I major histocompatibility complex (MHC-I), but they did not express MHC-II molecules. Additionally these cells could suppress umbilical cord blood (UCB) lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells (HSC) from UCB to reduce the potential graft-versus-host disease (GVHD) in recipients.

Comparison of mesenchymal stem cells from human placenta and bone marrow.

BACKGROUND: Nowadays bone marrow represents the main source of mesenchymal stem cells (MSCs). We identified a new population of MSCs derived from human placenta and compared its biological characterization with bone marrow derived MSCs. METHODS: Mononucleated cells (MNC) were isolated from the human placenta tissue perfusate by density gradient fractionation. Individual colonies were selected and cultured in tissue dishes. At the same time, human bone marrow derived MSCs were identified. Culture-expanded cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. The hematopoietic cytokines were assayed using reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Human placental MSCs exhibited fibroblastoid morphology. Flow cytometric analyses showed that the placental MSC were CD29, CD44, CD73, CD105, CD166, HLA-ABC positive; but were negative for CD34, CD45, and HLA-DR. Functionally, they could be induced into adipocytes or osteocytes. Moreover, several hematopoietic cytokine mRNA was found in placenta-derived MSCs by RT-PCR analysis, including IL-6, M-CSF, Flt3-ligand and SCF. These results were consistent with the properties of bone marrow derived MSCs. CONCLUSION: These observations implicate the postpartum human placenta as an important and novel source of multipotent stem cells that could potentially be used for investigating mesenchymal differentiation and regulation of hematopoiesis.