[Etiological study of pelvic organ prolapse and stress urinary incontinence with collagen status and metabolism].

OBJECTIVE: To explore the alteration of collagen ultrastructure and content in uterine ligaments and paraurethral tissue and explore whether the alteration may contribute to stress urinary incontinence (SUI) and pelvic organ prolapse (POP). METHODS: The cardinal ligament, uterosacral ligament and paraurethral tissue samples were obtained from 90 subjects undergoing hysterectomy. Collagen ultrastructure was examined with transmission electron microscopy. And collagen content and expression of vasoactive intestinal peptide (VIP) were examined with immunohistochemistry. RESULTS: The smooth muscle fascicles were thinner in the patients of SUI and POP. Arrangement of smooth muscle fascicles was disorderly. Fibroblast was metabolically active. The mean collagen fibril diameters in the SUI and POP groups were larger than that in the control group (P < 0.01). The mean contents of collagen I and III in the SUI and POP groups were lower than that in the control group (P < 0.01). The expression of VIP was lower (P < 0.05). CONCLUSION: Predominance of collagen degradation during tissue repair may contribute to and promote POP and SUI. The decrease of VIP might be related with nerve damage or degeneration to cause or accelerate the progress of pelvic organ prolapse.

[Transfer and identification of multidrug resistance gene in mesenchymal stem cells derived from human placenta].

OBJECTIVE: To explore the feasibility and safety of mdr1 gene transferred into placenta derived mesenchymal stem cells (P-MSCs) by reconstructed retroviral vector. METHODS: Human P-MSCs were isolated and expanded by Percoll density gradient and then transduced repeatedly by reconstructed retroviral vector containing mdr1 gene. The transfection and expression of mdr1 gene was detected by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS). Meanwhile, the biological features of mdr1-MSCs were identified and analyzed. RESULTS: The expression of mdr1mRNA was found in transfected cells. The expression of P-glycoprotein (P-gp) encoded by mdr1 gene was (27.6 ± 5.1)% in the transfected P-MSCs cells versus (0.4 ± 0.1)% in the non-transfected P-MSCs cells (t = 14.291, P < 0.01). The percent of P-MSCs at quiescent phase (G0/G1 phase) was around 95.40% and it was in accord with the characterization of stem cells. The mdr1-MSCs exhibited typical ultrastructures of low-differentiated stem cells. Moreover, they still retained the potency of adipogenic and osteogenic differentiation in the presence of appropriate conditioned media. CONCLUSION: A stable expression of P-gp may be obtained by reconstructed retroviral-mediated transfection in vitro. And transfected MSCs retain the characteristics of stem cells.

[Retention of biological features in human placental mesenchymal stem cells transfected by multidrug resistance gene].

OBJECTIVE: To transfer multidrug resistance gene (mdr1) into human placental mesenchymal stem cells (P-MSCs) by retroviral vector and assess the effects of mdr1 gene transduction upon biological features of P-MSCs. METHODS: Human P-MSCs were isolated from trypsin-digested term placentas and then transduced by reconstructed retroviral vector containing mdr1 gene and green fluorescent protein (GFP) reporter gene. Flow cytometric analysis was employed to determine the immunophenotypes of transfected P-MSCs. And the proliferation and cell cycle were detected by methyl thiazolyl tetrazolium and propidium iodide staining. Ultrastructures of transfected P-MSCs were observed and different induction conditions used to direct the cells to differentiate into adipocytes and osteoblasts. RESULTS: The transfected P-MSCs still expressed stem cell markers such as CD29, CD44 and CD73. The mean cumulative time of population doubling was 23.9 hours. The cellular cycle retained the proliferative characterization of stem cells. Ultrastructural features of transfected P-MSCs included increased surface microvilli, abundant mitochondria and slightly swollen rough endoplasmic reticulum. Furthermore these transfected cells demonstrated osteogenic and adipogenic differentiation potentials under appropriate conditions. CONCLUSION: The mdr1 gene transduction by retroviral vector in vitro has no significant effect upon biological characteristics of P-MSCs. It might provide experimental references for the application of P-MSCs in high-dose tumor chemotherapy.

Retroviral-mediated transfer and functional expression of multidrug resistance gene in human placenta mesenchymal stem cells.

BACKGROUND: Most of gynecologic malignancies are sensitive to chemotherapy. Myelosuppression is the main dose-related toxicity of many chemotherapeutic drugs. The human multidrug resistance (mdr1) gene is well known for its ability to confer drug resistance. This study aimed to explore the feasibility of expression and resistance of mdr1 gene transduction into human placenta mesenchymal stem cells (P-MSCs) by retrovirus vector. METHODS: Human P-MSCs were isolated from trypsin-digested term placentas, and their immunophenotypes and differentiation potential were evaluated. Human P-MSCs were transduced by reconstructed retroviral vector containing the mdr1 gene and green fluorescent protein (GFP) reporter gene. The integration and expression of the mdr1 gene were observed indirectly by the expression of GFP, and fluorescence-activated cell sorter was used to evaluate the functional activity of permeability glycoprotein (P-gp) encoded by the mdr1 gene. The stimulating test was made in vitro to show pleiotropic drug resistance of transfected cells. RESULTS: The isolated, cultured and expanded P-MSCs expressed stem cell markers such as CD29, CD44 and CD73, and showed osteogenic and adipogenic differentiation potentials under appropriate conditions. The expression of P-gp in the non-transfected P-MSCs cells was (0.4 +/- 0.1)%, but increased to (28.1 +/- 4.7)% after gene transfection (P < 0.01). And positive staining of P-gp located mainly at cell membrane and cytoplasm. Accumulation and extrusion assays showed that P-gp expressed by the transfected cells had pump-functional activity and could efflux daunomycin out of cells. The analysis of cell survival confirmed that transfected P-MSCs had a characteristic of multidrug resistance with a significant increase in the resistance to anticancer agents. CONCLUSIONS: Transfer and expression of human mdr1 gene mediated by retrovirus vector conferred P-MSCs drug resistance. It might provide a new alternative to chemoprotection strategies.

[Cytotoxicity of dendritic cells pulsed by CpG ODN on ovarian carcinoma cells: in vitro experiment with ovarian carcinoma cells].

OBJECTIVE: To investigate the effect of dendritic cells (DCs) pulsed by oligonucleotide containing "un-methylated cytimidine-phosphodiester bond-guanylic acid" motif (CpG ODN) on ovarian carcinoma cells. METHODS: Dendritic cells were isolated form the peripheral monocytes and co-incubated with synthesized CpG2006, CAI125, important epithelial ovarian cancer-associated antigen, or CpG ODN + CA125 for 72 h. Flow cytometry (FC) was used to detect the expression of CD(1alpha), CD(63), CD(86), and human leucocyte antigen-DR (HLA-DR) on the cell surface. ELISA was used to detect the IL-12 level in the supernatant. T cells were obtained from the DCs. The suspensions of different groups of pulsed DCs were co-incubated with T cells. Mixed lymphocyte reaction (MLR) was used to detect the proliferating activity of the T cells. Ovarian carcinoma cells of the line OVCAR-3 were added into the culture fluid of T cells with different effector-target ratios, and then unpulsed DCs, CpG ODN-pulsed DCs, CpG ODN + CA125-pulsed DCs, and CA125-pulsed DCs were added respectively. MTT colorimetry was performed to measure the A values of different wells so as to calculate the killing rate. RESULTS: (1) The expression levels of CD(63), CD(86), and HLA-DR on the membranes of the CpG ODN-pulsed DCs and CpG ODN + C125-pulsed DCs were significantly higher than those of the un-pulsed DCs (all P < 0.01), however, there were no significant differences in the expression of CD(1alpha) among different groups (all P > 0.05). (2) The IL-12 levels in the supernatants of the CpG ODN-pulsed DCs and CpG ODN + C125-pulsed DCs were significantly higher than those of the unpulsed and CA125-pulsed groups (all P < 0.01). (3) MLR showed that the T cell proliferation rates of the T cells sensitized by the CpG ODN-pulsed DCs and CpG ODN + C125-pulsed DCs were both higher than those of the T cells stimulated by the unpulsed and CA125-pulsed groups when the effector: target ratio was 10:1 (all P < 0.01). (4) The killing rates on OVCAR-3 cells of the CTLs sensitized by CpG ODN + CA125-pulsed DCs at the same effector: target ratios were all higher than those of the CTLs sensitized by the CpG ODN-pulsed DCs, CA125-pulsed DCs, and unpulsed DCs (all P < 0.01), the killing activity of the cytotoxic T lymphocytes (CTLs) sensitized by CpG ODN+CA125-pulsed DCs on the OVCAR-3 cells was shown even when the effector: target ratio was as low as 10:1, and then increased along with the increase of the effector: target ratio to the height of 64.9%. CONCLUSION: Capable of inducing immune cytotoxicity on ovarian carcinoma, DCs sensitized by CpG ODN + CA125 may have a great implication on clinical application.

[Construction and expression of recombinant cecropin B-binding site of luteinizing hormone releasing hormone gene and its anticancer function].

OBJECTIVE: To construct and express recombinant cecropin B-binding site of luteinizing hormone releasing hormone (CB-LHRH') gene, and to evaluate the anticancer function of CB-LHRH' on human ovarian cancer cell line SKOV3 and human endometrial cancer cell line HEC-1B. METHODS: The sequence of the cDNA encoding CB-LHRH' was designed, artificially synthesized, verified by DNA sequence analysis and expressed by Bac-to-Bac baculovirus expression system. The expression of CB-LHRH' proteins were identified by western dot blot using rabbit polyclonal antibody against LHRH as the primary antibody. To determine the anticancer effects of the CB-LHRH' protein, ovarian cancer cell line SKOV3 and endometrial adenocarcinoma cell line HEC-1B were treated by different doses of the CB-LHRH' protein. Cell growth inhibition assay was performed using the 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl) 5 [(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) kit at different times, and cell morphologic changes were observed under the inverted microscope. RESULTS: The inhibitory rate of proliferation by CB-LHRH' increased with the increase of dose and time respectively: SKOV3 cell, from (5.03 +/- 0.08)% to (53.24 +/- 1.22)%; HEC-1B cell, from (5.13 +/- 0.37)% to (56.16 +/- 1.08)%. The inhibitory effect on HEC-1B cell was stronger than that on SKOV3 cell (P < 0.01). There were obvious cell morphologic changes. CONCLUSION: The CB-LHRH' protein has anticancer activity, probably through the LHRH receptor, and it may be expected to be a potential candidate as peptide drugs for targeted cancer therapy.

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.

[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.

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.

[Evaluation of construction and expression shiga toxin2 A-luteinizing hormone releasing hormone recombinant toxin].

OBJECTIVE: The purpose is to construct shiga toxin2A-luteinizing hormone releasing hormone (Stx2A-LHRH) recombinant toxin which can kill cancer cells specifically and try to get a new target-binding drug. METHODS: The fragment of Stx2A-LHRH DNA amplified by polymerase chain reaction (PCR) were cloned into plasmid pET-20b(+) vector. The recombinant plasmid pET-Stx2A-LHRH was constructed successfully and identified by endonucleases digestion and sequencing analysis then it was transformed to Escherichia coli BL21 (DE3) and expressed under the induction of isopropyl-beta-D-thiogalactopyranoside. RESULTS: The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel thin layer scanning proved a special band of a molecular weight of about 38,000, accounting for 10.32% of total amount of the supernatant protein. Stx2A-LHRH recombinant toxin can kill HeLa cells clearly. CONCLUSION: Stx2A-LHRH recombinant toxin may become a choice of target-binding drug in the future.

Expression of MiR200a, miR93, metastasis-related gene RECK and MMP2/MMP9 in human cervical carcinoma--relationship with prognosis.

AIM AND BACKGROUND: Cervical cancer remains the third most common cancer in women globally after breast and colorectal cancer. Well-characterized biomarkers are necessary for early diagnosis and to predict metastatic progression and effective therapy. MiRNAs can regulate gene expression, cell growth, differentiation and apoptosis by targeting mRNAs for translational repression or degradation in tumor cells. The present study was conducted to assess expression of miR93, miR200a, RECK, MMP2, MMP9 in invasive cervical carcinoma, and analyze their clinical significance. METHOD: A total of 116 patients with invasive cervical carcinoma and 100 patients undergoing hysterectomy for benign lesions were retrospectively examined. Quantitative real-time PCR was performed to determine expression of miR93 and miR200a while RECK, MMP2, MMP9 and MVD were assessed by immunohistochemical staining. RESULTS: Cervical carcinoma patients demonstrated up-regulation of miR-93, miR-200a, MMP2 and MMP9, with down-regulation of RECK as compared to benign lesion tissues. RECK was significantly inversely related to invasion and lymphatic metastasis. The 5-year survival rate for patients with strong RECK expression was significantly higher than that with weakly expressing tumors. CONCLUSION: MiR-93 and miR-200a are associated with metastasis and invasion of cervical carcinoma. Thus together with RECK they are potential prognostic markers for cervical carcinoma. RECK cooperating with MMP2, MMP9 expression is a significant prognostic factor correlated with long-term survival for patients with invasive cervical carcinoma.

Transduction of mesenchymal stem cells with multidrug resistance gene provides protection for bone marrow toxicity after being transplanted into a nude mice model.

BACKGROUND: Myelosuppression is the main dose-related toxicity of many chemotherapeutic drugs. The human multidrug resistance (mdr1) gene is well-known for its ability to confering drug resistance. In this study, we meant to transplant the placenta mesenchymal stem cells (P-MSCs) moderated by mdr1 gene into a nude mice model radiated by γ-Co(60) and to explore the chemoprotection for bone marrow (BM) toxicity. METHODS: Human P-MSCs were isolated from trypsin-digested term placentas and then transduced by with reconstructed retroviral vector containing mdr1 gene and green fluorescent protein (GFP) reporter gene. The integration and expression of mdr1 gene was observed indirectedly by the expression of GFP. A nude mice model was constructed after irradiation with a sublethal dosage of γ-Co(60). These irradiated mice were transplanted with mdr1-MSCs through the caudal vein and then received paclitaxel (PAC) intraperitoneal chemotherapy. The Peripheral peripheral blood (PB) of the nude mice was collected, and the PB cells counts and values were determined using an automatic analyzer. RESULTS: After PAC treatment, mdr1-MSCs transplanted mice showed markedly improved survival upon compared to MSCs transplanted mice (85.7% vs. 57.1%). White blood cell (WBC) and red blood cell (RBC) counts as well as the hemoglobin (Hb) values were significantly increased in PAC treated mdr1-MSCs mice compared to PAC treated control mice when PAC chemotherapy had been finished (all P < 0.05), but the difference was not found in the plateltes (PLT) count (P > 0.05). CONCLUSION: Human P-MSCs moderated by mdr1 gene when transplanted into nude mice may provide chemoprotection for hematopoietic toxicity.

[Distribution patterns of PAHs in soils from coking plant and the particle-size cut points of soil washing].

Soil particle size distribution and contaminants distribution patterns in different soil size fractions are the basis of soil treatability using soil washing method. Soil particle-size cut points are important parameters of soil washing process. According to ex situ soil washing technology, soil samples were collected in a former coking plant. The soil particle size distribution and the concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in USEPA priority list were analyzed. Tween 80 and Triton X-100 solutions were used to clean the polluted soil with different particle size. Results showed that the total concentrations of 16 PAHs ranged from 6.27 to 40.18 mg/kg dry weight in the six soil size fractions and present a bimodal distribution. The maximum individual PAH concentration mostly occurred in the 250-500 microm size fraction. The lowest individual PAH concentration was in the 50-75 microm size fraction. The removal efficiencies of PAHs in different soil size fractions depended on their initial concentrations and the characteristics of soil. The PAHs removal efficiencies in coarser size fractions were lower than that in the finer size fractions owing to their higher organic carbon content. Based on the removal efficiency of PAHs in each soil size fractions by surfactant solution and the requirements of waste volume reduction, 50 microm was determined as the particle-size cut point. Then, 82.95% volume reduction can be achieved.

[Effect of human bone marrow mesenchymal stem cell on cord blood T lymphocyte transformation].

To study the effect of mesenchymal stem cell (MSC) on immune function, MSCs were isolated and cultured from human bone marrow cells. The purity of MSCs were identified with the spindle-fibroblastic morphology characterization by microphotograph and the phenotypes were tested by flow cytometry. MSCs were plated in 96-well plates (2,000/well and 1,000/well), and cocultured for 3 days with T cells isolated from cord blood. Cord blood T cells non-cocultured with MSC acted as control group. After cord blood T cells stimulated by PHA for 60 hours, [(3)H]-thymidine was added to each well and T cell proliferation was assessed by [(3)H] thymidine incorporation. The results showed that cord blood T cell proliferation was suppressed when 2,000 MSCs were plated each well and cord blood T cell proliferation was activated when 1,000 MSCs were plated. Our results suggested that the immunomodulatory function of MSC seemed dependent on cell dose. High concentration of MSC most often resulted in inhibition, while low concentration resulted in stimulation.

[Isolation and characterization of human placenta derived adherent cells and their hematopoietic growth factor expression].

OBJECTIVE: To isolate and culture human placenta derived adherent cells (hPDAC) and assay their hematopoietic growth factor expression. METHODS: By enzyme digestion, hPDAC were isolated from human placenta tissue and cultured, and their biological characteristics were studied. The hematopoietic growth factor (HGF) mRNA expression of hPDAC was assayed by RT-PCR. RESULTS: hPDAC was successfully isolated from human placenta tissue, which was further confirmed as mesenchymal stem cell-like cells. HGF including SCF, FL, G-CSF, GM-CSF, M-CSF and IL-6 were expressed in hPDAC. CONCLUSION: hPDAC could be used as feeder layer for umbilical cord blood CD(34)(+) cells ex vivo expansion.