Transduction of mesenchymal stem cells with multidrug resistance gene provides protection for bone marrow toxicity after being transplanted into a nude mice model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>Human P-MSCs moderated by mdr1 gene when transplanted into nude mice may provide chemoprotection for hematopoietic toxicity.</p>

Retroviral-mediated transfer and functional expression of multidrug resistance gene in human placenta mesenchymal stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Comparison of mesenchymal stem cells from human placenta and bone marrow / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effect of human bone marrow mesenchymal stem cell on cord blood T lymphocyte transformation / 中国实验血液学杂志

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 / 中华血液学杂志

<p><b>OBJECTIVE</b>To isolate and culture human placenta derived adherent cells (hPDAC) and assay their hematopoietic growth factor expression.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>hPDAC could be used as feeder layer for umbilical cord blood CD(34)(+) cells ex vivo expansion.</p>

Construction and expression of recombinant cecropin B-binding site of luteinizing hormone releasing hormone gene and its anticancer function / 中华妇产科杂志

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