Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells / 中国中药杂志

To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 μmol·L~(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 μmol·L~(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.

Angiopoietin-1 Modified Human Umbilical Cord Mesenchymal Stem Cell Therapy for Endotoxin-Induced Acute Lung Injury in Rats

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.

Effect of Human Umbilical Cord Mesenchymal Stem Cells on Etoposide-induced Nalm-6 Cell Apoptosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on VP16-induced apoptosis of Nalm-6 cells.</p><p><b>METHODS</b>hUC-MSC were isolated and identified using morphological observation and flow cytometry, then Nalm-6 cells were treated with hUC-MSC with or without VP16. Apoptosis and cell cycle were assayed by FACS. The mRNA levels of apoptosis-related genes BCL-2, BAX and caspase-3 were detected by quantitative RT-PCR, and the protein levels of BCL-2, BAX and caspase-3 were examined by Western blot.</p><p><b>RESULTS</b>FACS showed that hUC-MSC inhibited the proliferation and decreased apoptosis of Nalm-6 cells resulted from VP16. The quantitative RT-PCR showed that hUC-MSC increased the mRNA expression level of BCL-2 and decreased the expression level of BAX and caspase-3 (P < 0.05). Western blot showed that the protein expression level of BCL-2 increased, and expression level of BAX and caspase-3 decreased in Nalm-6 cells after co-culture with hUC-MSC (P < 0.05).</p><p><b>CONCLUSION</b>hUC-MSC may protect Nalm-6 cells from apoptosis induced by VP16 through regulation of BCL-2, BAX and caspase-3.</p>

The expression and regulatory mechanism of microRNA-708 in pediatric common B-cell acute lymphoblastic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the expression of microRNAs and reveal the regulatory mechanism of miRNA-708 in pediatric common acute lymphoblastic leukemia (ALL) (common-ALL).</p><p><b>METHODS</b>The expressions of microRNAs in common-ALL patients were detected by microarrays in 3 pediatric common-ALL samples, and then verified by stem-loop quantitative RT-PCR in 34 common-ALL samples. The target genes of miR-708 were found by bioinformatics software, and verified by dual-luciferases reporter assay, RT-PCR and Western blot.</p><p><b>RESULTS</b>Compared to normal bone marrow samples, of all the 2006 detected miRNAs, the expression of miR-708, miR-181b and miR-210 were 16.886 ± 16.854, 5.710 ± 4.652, and 9.789 ± 1.178, retrospectively, being significantly up-regulated expressed than those in normal control (1.872 ± 0.339, 1.276 ± 0.531 and 1.005 ± 0.080, retrospectively) (P < 0.05), while miR-27b and miR-345 were the two most down-regulated ones (0.524 ± 0.085 and 0.675 ± 0.086, retrospectively) (normal control: 1.123 ± 0.066 and 1.204 ± 0.140, retrospectively) (P < 0.05). And the expression of miR-708 and miR-181b were significantly correlated with the clinical types in common-ALL. In high risk common-ALL, miR-708 and miR-181b were much higher than in standard and middle risk common-ALL (P < 0.05). The further verification research in 293 cell line showed that miR-708 decreased the expression level of its target genes CNTFR, NNAT and GNG12 by combining with 3'-UTR of the 3 genes, moreover, miR-708 combined with CNTFR 3'-UTR in 394 ∼ 400 bp sequence region.</p><p><b>CONCLUSION</b>MicroRNAs plays an important regulatory role during the occurrence and development of the pediatric common-ALL and miR-708 is an important factor for high risk common-ALL.</p>

Immunomodulatory ability of senile mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the immunomodulatory ability of human umbilical cord mesenchymal stem cells (UB-MSC) along with prolonging of culture time and increasing of passages in vitro. Mesenchymal stem cells (MSC) were isolated from human umbilical cord and cultured in vitro. The morphological changes and nucleocytoplasmic ratio of MSC were observed using Giemsa staining. MSC of the 5th passage were selected as control group, and MSC of the 13th passage were taken as senile group. The degree of cell senescence was detected by aging cells in situ test kit. Cell Counting Kit-WST-8 was used to determine the proliferation of lymphocytes in mixed lymphocytes coculture system with different passages of MSC. The expression of immunomodulation-related genes was detected by RT-PCR. The results showed that the length-breadth ratio of MSC increased and nucleocytoplasmic ratio decreased along with the increasing of passages. The senium degree of cells of the 13th passage was higher than that of the 5th passage cells. The capacity of suppressing lymphocyte proliferation of the 13th passage MSC was enhanced, compared with the 5th passage. Moreover, the expression of immunosuppression-related genes of senile MSC increased and the expression of most anti-inflammation associated genes declined as compared with young MSC by RT-PCR. It is concluded that the degree of MSC senescence gradually develops with increasing of culture passage, but the immunosuppressive ability of MSC strengthens with continuous culture.

Changes of biological characteristics and gene expression profile of umbilical cord mesenchymal stem cells during senescence in culture / 中国实验血液学杂志

This study was purposed to investigate the changes of biological properties and expression patterns of the aging related genes in umbilical cord mesenchymal stem cells (UC-MSC) during in vitro culture. UC-MSC at passage 3 were served as the control cells and those at passage 15 were considered as the aged cells. The biological features of those two kinds of cells including morphology, proliferation activity and phenotypic profile were observed, and the differences of gene expression were analysed by the whole human genome oligo microarray. Several differential genes were selected for further confirmation by quantitative reverse transcription-polymerase chain reaction. The results showed that UC-MSC at passage 15 were larger in size and their proliferation rate was slower compared with those of cells at passage 3, while the positivity of CD44 and CD105 remained unchanged. Compared with UC-MSC at passage 3, relatively aged cells expressed higher levels of genes that are associated with small subunit of ribosome. Further analysis with Gene Ontology functional categories showed that the up-regulated genes were concentrated in those related to steroid biosynthesis, galactose metabolism and the development of autoimmune diseases and degenerative diseases and the down-regulated genes in UC-MSC at passage 15 were concentrated in cytoskeleton molecules, DNA structure binding, mRNA binding and protein function. Functional analysis with Kyoto Encyclopedia of Genes and Genomes functional pathway revealed that the expression of some genes responsible for ribosome composition was elevated while those of associated with extracellular matrix, focal adhesion and cell cycle progression were down-regulated. It is concluded that UC-MSC become senescent due to the declines in metabolism and proliferation activities.

Influence of vascular endothelial growth factor on endothelial components in human bone marrow and umbilical cord mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to compare the proportion of endothelial cells (EC) in human bone marrow mesenchymal stem cell (BM-MSC) and human umbilical cord mesenchymal stem cells (UC-MSC), and to investigate the influence of vascular endothelial growth factor (VEGF) on proportion of EC in MSC. Flow cytometry was used to detect the proportion of CD34(+)CD133(+) and vWF(+)CD31(+) double positive cells in MSC. Wright's staining was employed to observe the influence of VEGF on morphology of MSC. The expressions of CD34, CD133, CD31, vWF were detected by immunofluorescence. qRT-PCR was performed to detect the influence of VEGF on EC marker genes' expression of MSC. The results showed that there were a small amount of EC and endothelial progenitor cells (EPC) in obtained BM-MSC and UC-MSC. After exposed to VEGF 10 ng/ml for 24 h, aspect ratio of MSC and the proportion of EC increased, while proportion of EPC decreased. Expression of EC related marker genes such as Tie-2 and ecNOS up-regulated, especially in UC-MSC. It is concluded that small amount of EC and EPC exists in cultured BM-MSC and UC-MSC, VEGF can enhance the proportion and function of EC in MSC.

Effect of TGF-β1 on proliferation and extracellular matrix and gene expressions of hUC-MSC / 中国实验血液学杂志

This study was purposed to investigate the effects of transforming growth factor-β1 (TGF-β1) on proliferation and extracellular matrix (ECM) and gene expressions of human umbilical cord derived mesenchymal stem cells (hUC-MSC). The hUC-MSCs were isolated and cultured by transplantation technique from umbilical cords. Surface antigens of the fifth passage cultured hUC-MSCs were detected by flow cytometry. The effects of TGF-β1 0.1 - 20.0 ng/ml on hUC-MSC proliferation were determined by Cell Counting Kit-8. The mRNA expression of ECM components and the components involved in the mechanism of ECM production regulation were detected by qRT-PCR. Expressions of collagen I (Col-I), collagen IV (Col-IV), fibronectin (FN) and laminin (LN) were observed by immunocytochemistry. The gene expression profiles of hUC-MSC in response to TGF-β1 were examined by microarray. The results showed that no significant changes of the OD values were observed with the increasing doses of TGF-β1, while all OD values of 0.1 ng/ml group showed relative peaks at 24, 48 and 72 hour time points. The mRNA expression level of Col-I, MMP-2, MMP-9 and TIMP-2 increased to maximum in 0.5 ng/ml group, and the mRNA expression of Col-IV, FN, LN, Integrin, Tenascin-C, MMP-1 and TIMP-1 reached the peak level in 0.1 ng/ml group. Immunocytochemical analysis for Col-I and Col-IV revealed that their production increased slightly in 0.5 and 0.1 ng/ml groups, respectively, while immunostaining for FN showed diffuse cell membrane and cytoplasmic staining randomly distributed, but LN staining was absent in both groups. Microarray analysis showed that 9 genes closely related to cell movement and migration were up-regulated. Analysis by Pathway and Go indicated that differentially expressed genes were involved in transcription, translation, biosynthesis, metabolism, signal transduction, migration and adhering junction etc. It is concluded that TGF-β1 0.1 ng/ml promotes the proliferation of hUC-MSC. TGF-β1 of different concentrations upregulates the expression of different ECM components. TGF-β1 may play important roles in various aspects of hUC-MSC including transcription, translation, biosynthesis, metabolism, signal transduction, migration and adherens junction and so on.

In vitro expansion and function of cord blood megakaryocyte / 中华儿科杂志

<p><b>OBJECTIVE</b>Cord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.</p><p><b>METHODS</b>CB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.</p><p><b>RESULTS</b>Different results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.</p><p><b>CONCLUSION</b>Ex vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.</p>

Study of single cell PCR for HLA typing / 中华血液学杂志

<p><b>OBJECTIVE</b>To apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.</p><p><b>METHODS</b>Single cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.</p><p><b>RESULTS</b>Enzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.</p><p><b>CONCLUSION</b>The modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.</p>

Differentiating ability of non-hematopoietic adult stem cells from rat fetal blood and bone marrow in vitro / 中国实验血液学杂志

To compare the growth characteristics of non-hematopoietic adult stem cells (NASC) derived from rat fetal blood and rat bone marrow in vitro, and to study the differentiation of these stem cells into neuron-like cells in vitro, the fetal blood of pregnant rats and bone marrow of adult rats were sterilely collected; mononuclear cells (MNC) were isolated by using standard Ficoll-hypague techniques and then cultured in DMEM/LG containing 10% fetal bovine serum (FBS). The acquired NASCs were subcultured for passage. The immunophenotype of NASCs was detected by flow cytometry. The expanded NASCs were induced to differentiate into neurons-like cells by beta-mercaptoethanol (beta-ME), dimethylsulfoxide (DMSO), butylated hydroxyanisole (BHA). The specific markers of these neuron-like cells were detected by immunocytochemistry. The results showed that two kinds of subcultured NASCs showed homogeneous spindle-shaped and expressed antigens CD44 and CD54, but did not expressed CD11b and CD45. The both induced cells were similar to neuron in morphology and were positive for nestin and neuron-specific enolase (NSE), but negative for glial fibrillary acidic protein (GFAP). It is concluded that no significant difference of NASCs derived from pregnant rat fetal blood and adult rat bone marrow found in cell morphology and biological characteristics. NASCs of both origins can be induced to differentiate into neuron-like cells, so fetal blood can be regarded as another resource of NASC.

Biological characteristics and induced differentiation ability of in vitro expanded umbilical cord blood mesenchymal stem cells / 中华儿科杂志

<p><b>OBJECTIVE</b>Mesenchymal stem cells (MSCs) from bone marrow are capable of differentiating into cells of different tissue lineages such as bone, cartilage, and adipose tissue and are the best candidates for tissue engineering. It is well accepted that umbilical cord blood (UCB) is a source for hematopoietic stem cells. However, controversy exists as to whether UCB contains MSCs and can serve as a source of MSCs. Therefore, the aim of this study was to explore the biological characteristics and inducing differentiation ability of in vitro expanded UCB MSCs.</p><p><b>METHODS</b>UCB was collected on normal full term delivery of infants with informed consent (n = 35) obtained from the mothers. Mononuclear cells (MNCs) were isolated from UCB by gravity centrifugation and cultured with DMEM including 10% fetal bovine serum. The morphology was observed under microscope per day. Cytochemical staining was carried out and flow cytometry was used to examine the surface antigen phenotype. Fifth passage cells were transferred into a different medium and osteogenic differentiation, adipogenic differentiation, and neurogenic differentiation were assessed.</p><p><b>RESULTS</b>MSCs could be isolated and cultured from MNCs of a few UCB sources. These cells displayed fibroblast-like morphology. They withstood over 20 passages without significant structural changes. These MSCs were negative for alkaline phosphatase (ALP) staining and positive for alpha-naphthol butyric acid esterase (NBE) staining. Expression of CD(29), CD(44)and CD(105), especially the human MSCs-specific markers SH-2 and SH-3 were observed, but CD(3), CD(14), CD(19), CD(34) and CD(45) could not be found, indicating that these cells were not of hematopoietic origin. Exposure of these MSCs to serum-free osteogenic condition, they could differentiate into bone cells and form mineralized matrix as evidenced by Alizarin red staining 2 weeks later. When these UCB-derived MSCs were cultured in adipogenic medium, morphologic changes in cells as well as the formation of neutral lipid vacuoles were noticeable as early as 1 week after induction and visualized by staining with oil-red O. Surprisingly, these MSCs were also able to differentiate into neuroglial-like cells. Morphology of these induced cells resembled that of neurons. Immunocytochemistry showed that they expressed Nestin and neuron-specific enolase (NSE), but not glial fibrillary acidic protein (GFAP).</p><p><b>CONCLUSION</b>UCB does contain MSCs. These MSCs, which are multipotent, could be isolated and cultured from a few UCB sources. UCB might serve as an alternative source of MSCs to bone marrow.</p>

Double chimerism in recipient by transplantation of two allogeneic MHC-mismatched mouse fetal blood units / 中国实验血液学杂志

We have constituted a mouse model for fetal blood transplantation (FBT) to cross over major histocompatibility complex (MHC) without causing serious GVHD. It seems that full matching at the MHC appears not necessary for FBT, while the nucleated cell dose is critical. Two fetal blood units were combined from different donors to increase the stem/progenitor cell dose so as to explore the possibility of MHC-mismatched allogeneic transplantation. 26 out of 40 mice in mixed FBT group survived in the observation period of 60 days after transplantation without obvious GVHD. Double chimerism was demonstrated by PCR and flow cytometric analysis; and skin transplantation test proved the induction of donor specific immune tolerance. Our data suggest that two MHC-mismatched allogeneic donor fetal blood units could simultaneously engraft and reconstitute immune and hematopoietic system in a mouse model. The result may be beneficial for the expansion of cord blood application and enables more patients to share the advantages of cord blood transplantation.