Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology / 中国实验血液学杂志

OBJECTIVE@#To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology.@*METHODS@#20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings.@*RESULTS@#The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased.@*CONCLUSION@#The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.

Study on Thalassemia in Han Population in Sanya of Hainan Province / 中国实验血液学杂志

OBJECTIVE@#To study the distribution characteristics of thalassemia genotype in Han Population in Sanya of Hainan Province.@*METHODS@#Gap PCR and reverse dot hybridization were used to detect and analyze the thalassemia gene in 572 suspected thalassemia carriers of Han Population in Sanya.@*RESULTS@#Among the 572 Han Population in Sanya, 271 cases of thalassemia gene abnormality were detected, among which 161 cases were founded to be carriers of α-thalassemia gene. A total of 9 genotypes were detected, in the following order of the detection rate was －－SEA/αα，－α3.7/αα，－α4.2/αα，－－SEA/－α3.7，－－SEA/－α4.2，－α4.2/－α4.2，－α3.7/－α4.2，－α3.7/－α3.7，－－SEA/－－SEA. Among them, the deletion type (－－SEA/αα) in southeast Asia was the most common, accounting for 66 cases. 99 cases of β-thalassemia were detected, there were 7 genotypes, all of which were heterozygous. The order of the detection rate was CD41-42/βN, IVS-II-654/βN, CD17/βN, CD71-72/βN, -28/βN, －29/βN, CD27-28/βN. Among them, CD41-42/βN was the most common, accounting for 51 cases. In addition, 11 cases of combined α and β thalassemia were detected. Five kinds of genotypes were checked out, the order of detection rate was －α3.7/αα composite CD41-42/βN, －－SEA/αα composite IVS-II-654/βN, －α4.2/－α4.2 composite CD41-42/βN, －α4.2/αα composite －29/βN , －－SEA/ －α4.2 composite CD41-42/βN.@*CONCLUSION@#Han Population in Sanya of Hainan Province is a high-risk population of thalassemia, the genotype characteristics are different from other areas with high incidence of thalassemia in China. The main type of α-thalassemia is the deficiency mutation of southeast Asia, while CD41-42 heterozygous mutation is the main type of β-thalassemia.

Analysis of Factors Influencing Autologous Peripheral Blood Stem Cells Mobilization in Patients with Lymphoma and Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To analyze the factors influencing the mobilization of autologous peripheral blood stem cells (auto-PBSCs) in patients with lymphoma and multiple myeloma, and provide reference for optimizing the autologous stem cell mobilization regimen.@*METHODS@#Clinical data of 33 multiple myeloma and lymphoma patients received auto-PBSCs mobilization in our center from January 2015 to December 2018 were collected, the correlation of mobilization failure rate with gender, age, courses of chemotherapy before mobilization, does of recombinant human granulocyte colony stimulating factor (rhG-CSF), type of disease, and chemotherapy regimen were retrospectively analyzed.@*RESULTS@#Type of disease and course of pre-mobilization chemotherapy could affect the mobilization failure rate (P0.05).@*CONCLUSION@#Multi-course chemotherapy before collection and lymphoma patients are poor factors negatively impacting on auto-PBSCs mobilization.

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin / 中国实验血液学杂志

OBJECTIVE@#To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms.@*METHODS@#MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot.@*RESULTS@#The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group.@*CONCLUSION@#MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.

Effects of Bone Marrow Mesenchymal Stem Cells Derived from Patients with Newly Diagnosed Acute Myeloid Leukemia on the Cell Proliferation, Cell Cycle and Immunophenotypes of HL-60 Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the role of bone marrow microenvironment(niche) in the development of acute myeloid leukemia (AML) and the effect of AML patients-derived MSC on the proliferation, cell cycle and immuno-phenotypes of HL-60 cells.@*METHODS@#The MSC derived from bone marrow of patients with newly diagnosed AML were isolated and co-cultured with HL-60 cells. The effect of MSC on proliferation of HL-60 cells was detected by using 3H-TdR incorporation method, the cell cycle and immunophenotypes of HL-60 cells were detected by flow cytometry.@*RESULTS@#The results of 3H-TdR incorporation assay showed that both AML-MSCs and normal MSCs remarkably suppressed the HL-60 cell proliferation in a time- and dose-dependent manner. The results of cell cycle analysis demonstrated that AML MSCs and normal MSCs induced arrest of the HL-60 cells in G/G phase. The results of immunophenotyping revealed that MSCs suppressed the expression of CD11a and CD154 on the surface of HL-60 cells. Moreover, AML MSCs exhibited increased inhibitory effects than that of normal MSCs. However, no remarkable effect of MSCs on CD54 expressions of HL-60 cells was observed in the current study.@*CONCLUSION@#AML-MSCs possess effects on HL-60 cell proliferation, cell cycle and immunophenotypes similiar to normal MSCs, but exhibited increased suppressive capacity on the expression of CD11a and CD154.

Bone Marrow Mesenchymal Stem Cells from Newly Diagnosed Acute Myeloid Leukemia Patients Exhibit Enhanced Osteogenic Differentiation Capacity / 中国实验血液学杂志

OBJECTIVE@#To study the influence of acute myeloid leukemia (AML) microenvironment on mesenchymal stem cells (MSCs).@*METHODS@#MSCs were isolated from the bone marrow of newly diagnosed AML patients (AML-MSCs) and were cultured. The morphology of MSC was observed by inverted microscopy, the immunophenotypes of MSC were detected by flow cytometry, the proliferation ability of MSC was detected by using MTT method, the multi-differentation ability of MSC was assayed by osteogenic, lipogenic and chrondrogenic induction. The morphologic features, immunophenotypic characteristics, cell proliferation, and multipotential differentiation capability were compared between the MSC derived from normal healthy donors and AML patients.@*RESULTS@#AML-MSCs presented the morphological features similar to the normal MSCs. In addition, AML-MSCs highly expressed CD29, CD44, CD73, CD105 and HLA-ABC. Meanwhile, they were homogenously negative for CD14，CD31, CD34, CD45, CD80, CD86 and HLA-DR. Further-more, AML-MSCs showed cell proliferation ability similar to normal MSCs. Notably, AML-MSCs exerted increased osteogenic-differentiation capacity as compared with normal MSCs.@*CONCLUSION@#AML-MSCs possess typical MSC phenotypes but displayed enhanced osteogenic-differentiation capacity.

Leptin and its receptor in acute myeloid leukemia / 中国实验血液学杂志

Leptin, the product of obese gene, is a secreting protein that exerts multiple biological functions by binding to its receptor. Leptin regulates nutrient intake and metabolism, and is secreted from adipocytes, which occupy most of the bone marrow cavity and constitute the microenvironment. Leptin not only plays an important role in the control of the proliferation and differentiation of normal primitive hematopoietic cells, but it also stimulates the growth and viability of leukemic cells. Leukemic cells of some patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia, and chronic myeloid leukemia also express the leptin receptor. Furthermore, leptin also stimulates leukemic cell growth in vivo by promoting angiogenesis. These findings suggest the possibility that leptin and its receptor play roles in the pathophysiology of leukemia, and blockage of leptin binding to its receptor might have potential therapeutic benefits in the treatment of certain leukemias. This review discusses the biological characteristics of leptin and its receptor, the relation of leptin and its receptor with normal hematopoiesis, the relation of leptin and its receptor with AML and so on.

Clinical study on anti-leukemia effect mediated by dentritic cells / 中国实验血液学杂志

Immunotherapy of tumor is extensively attentioned as an important part of combined therapy of tumor in recent years. Dendritic cell (DC) is the most powerful antigen presenting cell (APC) by now which not only activates auto-immunity to attack tumor cells, but also does help to enhance antitumor effect for allogenic bodies. To explore the feasibility and safety of clinical therapy application of peripheral blood derived DC cultured ex vivo, and analyze the influence of DC-inducing-immunotherapy upon long-term survival of ANLL patients accepted autologous bone marrow transplantation, peripheral blood mononuclear cells (PBMNC) of 13 ANLL patients after autologous bone marrow transplantation were collected by using CS3000Plus. DC immunotherapy was administered after cultivation of PBMNC ex vivo for 2 weeks, desease-free survival time was observed after therapy for long time follow-up. The results showed that no any severe adverse event associated with DC therapy was observed, the survival analysis of Kaplan-Meier suggested that five year survival rate was 75.52% in DC group while 45.71% in non-DC group. DC group surpassed non-DC group in accumulative survival rate. It is concluded that the ex vivo cultivation and clinical therapy application of DC derived from peripheral blood are feasible and safe, DC immunotherapy in patients with acute non-lymphocytic leukemia after autologous bone marrow transplantation prolongs desease-free survival time and enhances long-term survival rate.

Effect of human bone marrow mesenchymal stem cell on allogeneic T lymphocyte phenotype in vitro / 中国实验血液学杂志

The purpose was to study the effect of mesenchymal stem cell (MSC) on immune function, and to explore the new strategy to prevent graft versus host disease (GVHD) and host versus graft reaction (HVGR) in allogeneic bone marrow transplantation. MSCs from human bone marrow cells were isolated and cultured. The purity of MSCs were identified with the spindle-fibroblastic morphological characterization by microphotography, and the phenotypes were tested by flow cytometry. MSCs were planted in 6-well plates (8 x 10(4)/well for group A, 4 x 10(4)/well for group B and 2 x 10(4)/well for group C), and cocultured for 7 days with T cell isolated from peripheral blood. Peripheral blood T cells non-cocultured with MSC acted as the control group. CD3, CD4, CD8, and CD25 expressed on T cells were analyzed by flow cytometry after coculture with MSCs for 0, 24, 72 hours and 7 days respectively. The results showed that CD4(+)CD25(+) T cells and CD8(+) T cells of allogeneic T lymphocytes cocultured with bone marrow MSCs (group A and group B) increased obviously as compared with control group (T lymphocytes uncocultured with MSCs), and there were no difference between groups A and B. CD3, CD4, CD8 and CD25 expressed on T cells had no significant difference between experimental groups and control group. The result suggested that CD4(+)CD25(+)-regulatory T cells and CD8(+) T cells were increased apparently when cocultured with allogeneic MSCs. It is concluded that MSCs pretreatment may be useful in the prevention of GVHD and HVGR in allogeneic bone marrow transplantation and provides a new strategy to induce transplantation tolerance.

The effect of MSC on cytokine production by naive T cell differentiated in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of mesenchymal stem cell (MSC) on naive T cell and to explore its mechanism of immunoregulation.</p><p><b>METHODS</b>After culturing for 3 passages, MSC was incubated with naive T cells differentiated from cord blood CD34(+) cells in vitro. Variance of cytokine produced by naive T cell in culture supernatant was analyzed by enzyme-linked immunoassays.</p><p><b>RESULTS</b>On the 7th day of co-culture, a mild proliferation of T cells in the co-culture group was observed: (9.15 +/- 0.68) x 10(5)/well in MSCs + naive T cells group versus (4.87 +/- 1.33) x 10(5)/well in naive T cells alone group (P < 0.05). IFN-gamma production was lower in MSCs + naive T cells group than that in naive T cells alone group: (1.147 +/- 0.181) pg/ml versus (4.897 +/- 0.189) pg/ml (P < 0.05), but IL-2 production was higher in the co-culture group: (16.141 +/- 2.729) pg/ml versus (2.551 +/- 0.460) pg/ml (P < 0.05). Neither IL-4 nor IL-10 were detected.</p><p><b>CONCLUSIONS</b>MSC have allogeneic effect on naive T cell, but may suppress alloreactive T lymphocyte and reduce the incidence of GVHD partly by decreased IFN-gamma production. The result may provide new clues for explaining immunoregulatory mechanism of MSC.</p>