Comparison of the Biological Functions between Human Bone Marrow Derived CD106 Mesenchymal Stem Cells and CD106 Subgroup / 中国医学科学院学报

Objective To analyze the differences in biological functions between bone marrow(BM)-derived CD106 mesenchymal stem cells(MSCs)and the CD106 subgroup. Methods The MSCs from normal BM were isolated and expanded.The subgroups of CD106 and CD106 MSCs were sorted.The cell proliferation and adhesion functions,chemotactic activities,adipogenic and osteogenic potentials,senescence,and senescence protein 21(p21)were detected.The capacity of translocation into nucleus of nuclear factor-kappa B(NF-κB)when stimulated by tumor necrosis factor(TNF-α)was measured. Results The proliferative ability was higher in CD106 MSCs than that in CD106 MSCs.In 48 hours,the value of optical density(OD)was significantly higher in CD106 MSCs than that in CD106 subgroup(1.004±0.028 0.659±0.023,=3.946,=0.0225).In 72 hours,this phenomenon was even more pronounced(2.574±0.089 1.590±0.074,=11.240,=0.0000).The adhesive capacity of CD106 MSCs was significantly stronger than that of CD106 subgroup(0.648±0.018 0.418±0.023,=7.869,=0.0002).Besides,the metastasis ability of CD106 MSCs were significantly stronger than that of CD106 subgroup(114.500±4.481 71.000±4.435,=6.900,=0.0005).The CD106 MSCs had signifcnatly lower proportions of senescent cells.The expression of aging protein p21 in CD106 MSCs was significantly lower than that in CD106 MSCs [(17.560±1.421)% (45.800±2.569)%,=9.618,=0.0000].Furthermore,there were no visible pigmenting cells after β-galactosidase staining in CD106 MSCs subgroup.However,in CD106 MSCs,some colored green cells were detected.The rate of NF-κB translocation into nucleus after stimulated by TNF-α was significantly higher in CD106 MSCs than CD106 MSCs [(37.780±3.268)% (7.30±1.25)%,=8.713,=0.0001]. Conclusion Bone marrow-derived CD106 MSCs possess more powerful biological functions than CD106 MSCs.

Ultrastructure and Raman Spectral Characteristics of Two Kinds of Acute Myeloid Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the Raman spectral characteristics of leukemia cells from 4 patients with acute promyelocytic leukemia (M) and 3 patients with acute monoblastic leukemia (M), establish a novel Raman label-free method to distinguish 2 kinds of acute myeloid leukemia cells so as to provide basis for clinical research.</p><p><b>METHODS</b>Leukemia cells were collected from bone marrow of above-mentioned patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 30-50 cells from each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA) and cluster analysis, and the spectra of leukemia cells from 7 patients were analyzed and classified. Characteristics of Raman spectra were analyzed combining with ultrastructure of leukemia cells.</p><p><b>RESULTS</b>There were significant differences between Raman spectra of 2 kinds of leukemia cells. Compared with acute monoblastic leukemia cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks in 622, 643, 757, 852, 1003, 1033, 1117, 1157, 1173, 1208, 1340, 1551, 1581 cm. The diagnostic models established by PCA-DFA and cluster analysis could successfully classify these Raman spectra of different samples with a high accuracy of 100% (233/233). The model was evaluated by "Leave-one-out" cross-validation and reached a high accuracy of 97% (226/233).</p><p><b>CONCLUSION</b>The level of macromolecules of Mcells is higher than that of M. The diagnostic models established by PCA-DFA can classify these Raman spectra of different cells with a high accuracy. Raman spectra shows consistent result with ultrastructure by TEM.</p>

Applications of Microscopic Imaging and Analysis Technology in Studies of Neutrophil Movement and Phagocytosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analyze and evaluate the application of spinning disk confocal microscopy and imaging analysis software in movement and phagocytosis of neutrophils.</p><p><b>METHODS</b>Neutrophils were isolated from bone marrow by centrifugation on discontinuous Percoll gradient, and then were stained with PE Gr-1 antibody and mixed with FITC-labeled Zymosan A bioparticles. Multichannel time-lapse videos were captured by using the spinning disk confocal microscopy. The result was analyzed by using volocity and ImageJ software, the parameters associated with movement and phagocytosis of neutrophils were analyzed, including morphological changes, cell tracking, pseudopod dynamics, binding and phagocytosis index.</p><p><b>RESULTS</b>Most neutrophils would be polarized in response to Zymosan particles during a short time. Binding and phagocytosis process occured in forty minutes.</p><p><b>CONCLUSION</b>A method of precisely quantifying the movement and phagocytosis of neutrophils using microscopic imaging and imaging analysis technique has been set up successfully. Using this method, biological activity and function of neutrophils can be evaluated visually and rapidly. The physiologically rapid response to Zymosan particles can be applied to the neutrophils function research in the future.</p>

An improved method for generating integration-free human induced pluripotent stem cells / 中国实验血液学杂志

The genome instability and tumorigenicity of induced pluripotent stem cells (iPSC) hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells (CB MNC), to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre-stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV (spleen focus forming virus) promoter under the hypoxic condition (3%), CB MNC could be mostly efficiently reprogrammed with the efficiency 0.12%. Furthermore, the results showed that erythroblasts (CD36(+)CD71(+)CD235a(low)) were the cells that are reprogrammed with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.

Knockdown of Larp4b in Lin(-) cells does not affect the colony forming ability of mouse hematopoietic cells / 中国实验血液学杂志

Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.