The aim of this study was to isolate, cultivate and phenotypically characterize two types of human amnio-tic membrane (HAM)-derived cells, and to analyze their differentiation potential in vitro. Human amnion epithelial cells (hAEC) were derived from the embryonic ectoderm, while human amnion mesenchymal cells (hAMC) were derived from the embryonic mesoderm. The cells were characterized by flow cytometry and immunofluorescence, then immunofluorescence also was performed for the analysis of multipotentiality in differentiation. The results indicated that immunophenotypic characterization of both cell types demonstrated positive for HLA-A, B, C and mesenchymal stem cell markers (CD29, CD73, CD44, CD59, CD90, CD105, CD166), but did not express the hematopoietic markers (CD31, CD34, CD45, HLA-DR) and showed the weak expression of costimulatory molecules (CD40, CD40L, CD80, CD86). Phenotypes of both cell populations were maintained from passages 3 to 7. The immunofluorescence indicated that hAEC expressed cytokeratin 19, but did not express vimentin. On the contrary, hAMC expressed vimentin but did not express cytokeratin 19. The assessment of multilineage potential demonstrated that hAMC showed greater cardiomyocytes potential, while hAEC showed greater neural potential. It is concluded that hAEC and hAMC can be successfully isolated from the HAM. Both cell populations possess similar immunophenotype. However, they differ in cell yield and multipotential for differentiation into the major lineages, hAEC possess a much greater ectodermal differentiation capacity, while hAMC possess a much greater mesodermal differentiation capacity. This conclusion will be important for use of these cells in cell therapy.
Amnion ; cytology ; Cell Differentiation ; physiology ; Cell Lineage ; Epithelial Cells ; cytology ; Humans ; Immunophenotyping ; Stromal Cells ; cytology

Human leukocyte antigen G (HLA-G), a kind of non-classical major histocompatibility complex class I antigens, can inhibit inflammatory reaction, assist tumor cells to escape from immune surveillance and promote the immunologic tolerance of the graft. HLA-G, expressed and secreted by human amniotic mesenchymal cells (HAMC), suppresses the functions of NK cells, T cells and B cells and modulates the activity of dendritic cells (DC). These findings provide a theoretical basis for illustrating the mechanism of immunosuppression on HAMC. In this article, the recent advances on not only the gene and the molecular structure of HLA-G, but also the possible mechanisms of HLA-G in immunoregulatory function of HAMC, as well as the relation of HLA-G with HAMC, NK, DC, T and B cells are reviewed.
Amnion ; cytology ; HLA-G Antigens ; immunology ; Humans ; Immune Tolerance ; Mesenchymal Stromal Cells ; cytology ; immunology

The aim of this study was to investigate the combined effects of bortezomib (Bor) and daunorubicin (DNR) or each drug alone on proliferation of human multiple myeloma cell line KM3. KM3 cells were cultured with different concentrations of Bor and DNR, Bor or DNR alone for different times. The cell proliferation was analyzed by MTT assay, and the concentration of 50% growth inhibition (IC(50)) was calculated. The results indicated that both of Bor and DNR inhibited KM3 cell proliferation in dose dependent manner. The IC(50) of both drugs were 0.27 micromol/L and 0.16 micromol/L respectively. The inhibiting rate of Bor plus DNR on KM3 cells was much higher than that of Bor (p < 0.05). It is concluded that the Bor has synergistic inhibitory effect with DNR on the growth of KM3 cell in vitro.
Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Daunorubicin ; pharmacology ; Drug Synergism ; Humans ; Inhibitory Concentration 50 ; Multiple Myeloma ; Pyrazines ; pharmacology

This study was purposed to investigate the effect of bortezomib (Bor) and arsenic trioxide (As(2)O(3)) combination on multiple myeloma cell line KM3 and its mechanisms. KM3 cells were cultured with different concentration of Bor or As(2)O(3) as well as both for a certain time. The cell proliferation was analysed by MTT assay and the concentration of 50% proliferation inhibition (IC(50)) was calculated. Early apoptosis and late apoptosis of KM3 cells were detected by Annexin-V-FITC Kit, and the change of transmembrane potential was measured by flow cytometry. mRNA of Caspase-3, Bim and Bcl-xL were detected by RT-PCR. The results showed that the proliferation inhibitory rate of KM3 cells treated by Bor plus As(2)O(3) was much higher than that of KM3 cells treated by Bor only for 72 h [ (27.64 ± 0.81)% vs (21.67 ± 2.20)%, P < 0.05]. There were more KM3 cells treated by Bor plus As(2)O(3) in early apoptosis at 48 h and late apoptosis at 72 h than that of KM3 cells treated only by Bor [ (53.20 ± 3.70)% vs (35.40 ± 2.58)%, P < 0.01; (63.96 ± 2.97)% vs (54.08 ± 3.76)%, P < 0.01]. Transmembrane potential (Δψm) of KM3 cells treated by Bor plus As(2)O(3) decreased more at 48 h, as compared with Bor alone. The expression levels of caspase-3 mRNA and Bim mRNA in KM3 cells treated with Bor plus As(2)O(3) were higher than that in KM3 cells treated with Bor alone. But the expression level of Bcl-xL mRNA was lower than that in KM3 cells treated with Bor alone. It is concluded that As(2)O(3) can enhance the apoptosis-inducing effect of Bor on multiple myeloma cell line KM3, which is associated with decreasing the expression of Bcl-xl mRNA and increasing the expression of Caspase-3 and Bim mRNA.
Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Arsenicals ; administration & dosage ; pharmacology ; Bcl-2-Like Protein 11 ; Boronic Acids ; administration & dosage ; pharmacology ; Bortezomib ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Humans ; Membrane Proteins ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; Oxides ; administration & dosage ; pharmacology ; Proto-Oncogene Proteins ; metabolism ; Pyrazines ; administration & dosage ; pharmacology ; bcl-X Protein ; metabolism

OBJECTIVETo investigate the effect of bortezomib (Bor) alone or in combination with As(2)O(3) (ATO) and/or dexamethasone (DXM) on proliferation and apoptosis in KM3 human multiple myeloma cell line KM3.

METHODSKM3 cells were cultured with different concentrations of Bor and ATO and/or DXM in combination or Bor, ATO, DXM alone for different times. Cell proliferation was assayed by MTT assay, and IC(50) was calculated. Cell morphology was observed with light and electric microscopy. The agarose gel electrophoresis was used to evaluate DNA content, and the flow cytometry was used to exam Annexin V-FITC/PI stain.

RESULTSBor, ATO and DXM inhibited KM3 cell proliferation in a time-and dose-dependent manner with the IC(50) of 0.27, 3.10 and 8.01 micromol/L, respectively. The inhibition rate of KM3 cells by Bor plus ATO and DXM was significantly higher than Bor plus ATO or DXM \[(34.51 +/- 0.51)% vs (25.39 +/- 0.90)% and (34.51 +/- 0.51)% vs (23.80 +/- 0.78)% respectively\]. Typical morphology for apoptosis and DNA ladder were observed in KM3 cell treated with 0.25 micromol/L Bor for 48 h, by Annexin V positivity. The apoptosis rate induced by Bor plus both ATO and DXM was higher than that induced by Bor plus DXM.

CONCLUSIONBor can inhibit the proliferation and induce apoptosis of KM3 cells. Bor enhances the inhibitory effect of ATO and DXM on the growth of KM3 cell. ATO enhances the apoptosis effects of Bor and DXM on KM3 cells.

Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Humans ; Multiple Myeloma ; metabolism

This study was aimed to explore the effect of bortezomib on proliferation and apoptosis of acute monocytic leukemic cells SHI-1 and the function of Bcl-2 gene family including Bcl2l12, Bcl-2 and Bax in its apoptosis. SHI-1 cells were cultured and treated with bortezomib of different concentrations for different time. MTT assay was used to detect the proliferation and apoptosis, Annexin-V staining, mitochondrial transmembrane potential (DeltaPsim) and DNA aga-rose gel electrophoresis were used to investigate apoptosis of SHI-1 cells. RT-PCR was used to analyze the levels of Bcl2l12, Bcl-2 and bax mRNA in SHI-1 cells treated with bortezomib for 0, 6, 12 and 24 hours. The results showed that bortezomib inhibited the proliferation of SHI-1 cells in time-and doze-dependent manners, the IC(50) at 24 and 48 hours were 54.13 nmol/L and 5.45 nmol/L respectively. Bortezomib could induce apoptosis of SHI-1 cells in time-dependent manner, increase expression of Annexin-V positive cells, decrease DeltaPsim of SHI-1 cells and result in DNA fragmentation and morphologic changes of apoptosis. RT-PCR showed that Bcl2l12 mRNA expression was up-regulated, bcl-2 mRNA expression was down-regulated and bax mRNA expression was not changed obviously. It is concluded that bortezomib inhibits the proliferation of SHI-1 and induces apoptosis in which Bcl2l12 and Bcl-2 gene can be ones of the main genes taking part in.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Humans ; Leukemia, Monocytic, Acute ; genetics ; Muscle Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Pyrazines ; pharmacology ; RNA, Messenger ; bcl-2-Associated X Protein ; genetics

BACKGROUNDThe NASG gene has been confirmed as a tumor-suppressor gene candidate related to nasopharyngeal carcinoma (NPC) by previous studies. We further investigated the expression and the role of NASG in the homogeneous tissue cells by microdissecting the samples of tissue from human NPC, and introduced a new way to study the expression of specific genes in tumor tissue.

METHODSThe RNAlater reagent was used to preserve the samples of tissue from the nasopharynx of NPC patients. The samples were microdissected to harvest the homogeneous tissue cells and then total RNA was isolated from them. The antisense RNA (aRNA) was amplified from the total RNA by "in vitro transcription (IVT)". We investigated NASG expression in the homogeneous tumor cells of NPC (22 samples) and compared it with that in the pure epithelial pillar cells of normal nasopharyngeal (10 samples) by semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR).

RESULTSThe high quality total RNA could be harvested from the microdissected homogeneous tissue cells of the nasopharynx, then sufficient aRNA was derived from it. NASG gene expression was identified using aRNA by sqRT-PCR and showed that there was significant difference between the average value of case groups and that of control group (t = -5.275, df = 30, P < 0.001). The NASG gene in the subgroups WHOII tended to express lower levels than those in the subgroup WHOIII although this difference was not statistically significant (t = -1.584, df = 20, P = 0.129 > 0.05).

CONCLUSIONSMicrodissection was an effective method to obtain the homogeneous tissue cells of nasopharyngeal tissue (including the samples of NPC and non-NPC) in our study. Sufficient aRNA from amplifying total RNA could be used in sqRT-PCR to analyse the expression of NASG in the pure tissue cells. NASG should be a tumor-suppression gene candidate regarding to NPC.

Adult ; Aged ; Female ; Genes, Tumor Suppressor ; Humans ; Male ; Microdissection ; Middle Aged ; Nasopharyngeal Neoplasms ; genetics ; Nasopharynx ; metabolism ; RNA ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEThis study was aimed to investigate the effect of mesenchymal stem cells (MSCs) on subsets and cytokine secretion of T lymphocytes.

METHODSUmbilical cord blood-derived mesenchymal stem cells (UCBMSCs) were isolated by density gradient and were cultared by amplifying culture. The subsets and cytokine secretion of T lymphocytes were detected by flow cytomety after being co-cultured with UCBMSC.

RESULTSThe proliferation of lymphocytes was inhibited. CD4(+)T cell subsets were increased, CD8(+)T cell subsets decreased when co-cultured with UCBMSC; Th1 and Tc1 level significantly reduced, while Th2 and Tc2 level slightly increased.

CONCLUSIONThe UCBMSC can inhibit the proliferation of lymphocytes, especially CD8(+)T cell subsets. In addition, UCBMSCs can reduce Th1 and Tc1 cells, and increase Th2 and Tc2 cells. UCBMSC may have the clinical application potential for preventing and remedying GVHD.

Coculture Techniques ; Fetal Blood ; Humans ; Lymphocyte Count ; Mesenchymal Stromal Cells ; Stem Cells ; T-Lymphocyte Subsets

OBJECTIVE: To investigate the frequency, karyotype characteristics and prognosis significance of monosomal karyotype (MK) in newly diagnosed MDS patients. METHODS: The clinical, laboratorial and follow-up data of 202 MDS patients received the chromosome karyotype test in Department of Hematology, Ningbo Hospital of Zhejiang University from 2009 to 2018 were analyzed retrospectively, the monosomal karyotype features, clinical characteristics and their effects on the prognosis of MDS patients also were analyzed. RESULTS: Among 202 cases of MDS, 25 (12.38%) confirmed to be the MK. The abnormality of chromosome 5 (60.00%), 7 (56.00%), 17 (56.00%), 15 (56.00%), 13 (40.00%) and 20（40.00%）were common in monosomal karyotype. MK-MDS (MDS with monosomal karyotype) patients had higher bone marrow blast percentage than MK-MDS (MDS without monosomal karyotype) patients, the median are 6.25% and 3.00% (P＜0.01) respectively, but there were no difference in age, sex, hemoglobin level, white blood cell count, neutrophile granulocyte percentage, platelet count, blood blast percentage, serum ferritin, folic acid and vitaminB12 between MK-MDS and MK-MDS. The overall survival time of MK-MDS and MK-MDS patiens with chromosome 3, 5, 7, 13, 15, 17 abnormalities was significantly shorter than MK-MDS and AK+MK-MDS patients (MDS with abnormal karyotype but without monosomal karyotype) , the MK-MDS patients had a median survival time of 7.33 months, but the median survival time had not been reached in MK-MDS and AK+MK-MDS patients had not been reached by the end of the follow-up, and could not be assessed (P＜0.01). CONCLUSION The monosomal karyotype is a poor prognosis factor for newly-diagnosed MDS patients. The poor prognosis suggested by monosomal karyotype may be related with the abnormality of 3, 5, 7, 13, 15 and 17 chromosome.
Humans ; Karyotype ; Karyotyping ; Monosomy ; Myelodysplastic Syndromes ; Prognosis ; Retrospective Studies

OBJECTIVE: To investigate the genetic and prognostic characteristics of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) patients. METHODS: There were 230 non-M3 AML patients treated in Ningbo First Hospital enrolled, among which 58 patients were newly diagnosed AML-MRC, the patients were followed up and SPSS 25.0 was used to statistically analyze. RESULTS: There were 49 patients performed genetic testing, 29 patients (59.2%) showed chromosomal abnormalities, including 7q- 8 cases (16.3%), 5q- 6 cases (12.2%), 5 cases (10.2%) of 17p abnormalities, 13 cases (26.5%) of highly abnormal complex karyotypes (CK) (≥5 unrelated chromosomal abnormalities), CK contained chromosomal abnormalities such as +8, 5q-, and 12 cases (24.5%) of monosomal karyotypes (MK). Genetic testing was performed in 37 patients, and 24 (64.9%) patients showed genetic mutations, among which ASXL1 mutation was the most common (8 cases, 21.6%), followed by TET2 mutation in 6 cases (16.2%). Kaplan-Meier analysis showed that AML-MRC patients with high CK (P=0.012), 5q- abnormalities (P=0.038), and TP53 mutations (P=0.008) had poor overall survival. CONCLUSION AML-MRC has unique genetic characteristics, and high CK, 5q- and TP53 mutations are poor prognostic factors.
Humans ; Karyotype ; Karyotyping ; Leukemia, Myeloid, Acute/genetics* ; Myelodysplastic Syndromes ; Prognosis