BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) of mice are different from human and rats.The difficulties of culture,sustaining impermanency activity of BMSCs after passage and short-term effect of stem cells tracking limited the study of mice.OBJECTIVE:To obverse modified isolated culture method of BMSCs of mice and the feasibility of long-term fluorescent labeling stem cells.DESIGN,TIME AND SETTING:The experiments were conducted at the Affiliated Hospital of Academy of Military Medicine Science from June to December in 2008.MATERIALS:C57BL/6 mice,males and females,4-6 weeks of age,mean weighing 20 g,were used.METHODS:Stem cell culture fluid serum and liquid change manner were optimized using adherent screening and Percoll separation method.Rat BMSCs were incubated.In accordance with previous experiences of MSCs between human and macaque,Hyclonehigh-grade fetal bovine serum was selected for mouse MSC incubation.Serum was 10% of the medium.Bone marrow cells were washed out using LG-DMEM to filter bone dregs and small muscle blocks.Subsequently,samples were added on the Percoll separating medium at relative density of 1.082,and then incubated in 75 cm~2 culture flask at the concentration of 1.5×10~6/cm~2.BMSCs at the second passage were labeled with CM-Dil.MAIN OUTCOME MEASURES:Morphological changes in primary cultured and subcultured mouse BMSCs were measured.BMSC surface antigen of the second passage of mice (CD105,CD44,CD25,CD34) were determined using flow cytometry.The modified method was assessed to harvest the purity of stem cells.Activity of mouse BMSCs was identified using adipogenic and osteoplastic differentiation.The strength of fluorescent cells following multiple passage was observed.RESULTS:①The attached cells were observed 48 hours after primary cells culture and changed shuttles,triangles and flats at 7 days after culture.The figure become bunches and radial pattam at 3 passages.②MSCs highly expressed CD105,CD44 phenotypes and seldom expressed CD34 and CD45.③Spindle shape of cells gradually disappeared,with increased cell body.Some cells grew in cluster.MSCs changed figures to multilayer knots 10 days after inducing osteoplastic differentiation.MSCs became roundness and appeared fat drops in cells 9 days after inducing adipogenic differentiation.Red fat particles were shown following oil O staining.④The labeling cells gave out oranges light,and marked rates were over 80% in MSCs under the fluorescent microscope.The labeling cells were over 47% in 4 passages MSCs using flow cytometry.CONCLUSION:The modified method gained high-dosage cells in shorten culture time at passage 2 and made CM-Dil long lime labeling cells,which made more convenient for MSCs experiment on mice and stem cells tracer experiment in vivo.

10.3969/j.issn.2095-4344.2013.24.014

Objective The patients with lethal irradiation after sucessful hematopoietic stem cells transplan-tation had blood recovery, but did not avoid to died of multiple organ failure(MOF). To overcome the block, the article investigated mechanisms of mesenchymal stem cells (MSCs) protecting lethal radiated mice from multiple organ failure after haploid bone marrow cells transplantation. Method BALB/c mice irradiated with 8Gy60COγ-rays were randomly divided into two groups: MSCs group, infused MSCs labeled with cm-DiI and bone marrow monocytes of CB6F1 mice; Control group, only infused bone marrow monocytes; normal group, mice were infused cm-DiI marked MSCs without irradiation. The distribution of MSCs and the serous densities of Il-2, Il-10 and TNF-α in the recipients were observed after transplantation. Results MSCs collected in the bone marrow and the intes-tine in normal group at 15 d,in MSCs group MSCs enriched the different organs at 3,15 and 30 d. MSCs regulated down the secretion of IL-2 and TNF-α,and up the IL-10 density. Conclusions MSCs protected mice from multiple organ failure through above effects and may be open a new treatment strategy on acute radiation syndrome by stem cells.

Objective To investigate the effects of recombinant human granulocyte colonystimulating factor(G-CSF) on central and peripheral lymphocyte subset reconstitution after a sublethal dose of irradiation. Methods Sixty female BALB/c mice were given a 6.0 Gy γ-ray total body irradiation (TBI) and randomly divided into 2 equal groups. The mice in G-CSF + TBI group were injected subcutaneously with recombinant human G-CSF 100 μg·kg-1·d-1 for 14 d and the mice in TBI group were injected subcutaneously with the same volume of phosphate buffered solution (PBS) once daily for 14 d. 7,14,21, and 28 d later the mice were killed and their thymus were taken out to prepare of the mononuclear cell suspension to analysis the percentage of thymic CD4 + CD8 + double positive, CD4 +CD8 - single positive, CD4 - CD8 + single positive and CD4 - CD8 - double negtive cells by flow cytometry. Peripheral blood samples were collected from the caudal vein twice a week, and the white blood cell(WBC) counts and absolute number of lymphocytes were assessed by automatic hemocyte analyzer. 14,28, and 60 d later blood samples were collected from angular vein to examine the peripheral lymphocyte subsets by flow cytometry. Cell counting kit-8 was used to detect lipopolysaccharide (LPS) or concanavalin A (ConA) stimulated splenic lymphocyte proliferation. Results The percentage of thymic CD4 + CD8 +double positive cells decreased 7 d after irradiation, rebounded at 14 d, decreased again at 21 d, and then got a permanent recovery. 28 d after irradiation the percentage of thymic CD4 + CD8 + double positive cells in the G-CSF + TBI group recovered to normal and was significantly higher than that of the TBI group (t =12. 22, P < 0. 05). 21d after irradiation the percentage of thymic CD4-CD8 + single positive cells of the G-CSF + TBI group was significantly higher than that of the TBI group (t = 3.77, P < 0. 05). The peripheral WBCs and lymphocytes decreased to the lowest levels 7 d after irradiation and then gradually increased, however, WBCs and lymphoeytes of the G-CSF + TBI group began to recover earlier and faster than the TBI group. The proportion of CD3 + CD8 + T cells of the G-CSF + TBI group was significantly higher than that of the TBI group 14 and 60 d after irradiation (t =4. 31,5.78, P <0.05). But there was no significant difference in the proportion of CD3 + CD4 + T cells between the two groups. The proportion of B lymphoeytes of the G-CSF + TBI group was significantly lower than that of the TBI group 14 d after irradiation(t =7.30, P <0.05), but it recovered quickly, and there were no significant differences in the proportion of B lymphoeytes between the two groups 28 and 60 d after irradiation. The proliferation indexes of splenic lymphocytes in response to LPS and ConA in the G-CSF + TBI group were 4. 37 and 2.98 times higher than those in the TBI group 14 d after irradiation. Conclusions G-CSF could accelerate the recovery of central and peripheral lymphocyte subsets, raise the absolute number of lymphocytes, and enhance their proliferative function, which contributes to the central and peripheral immune reconstitution after acute irradiation.

Objective To investigate the mechanism of mesenchymal stem cells in enhancing the effects of haploid matched bone marrow cells transplantation in mice with acute radiation syndrome(ARS).Methods The survival of mice infused with difierent levels of MSCs and bone marrow cells after 8 Gy TBl were examined.BALB/c female mice irradiated with 8 Gy of 60Co γ-rays were randomly divided into two groups,MSCs group,infused with MSCs of female CB6F1 mice labeled with cm-DiI and bone marrow monocytes of male CB6F1,Control group,only infused with bone marrow monocytes.Peripheral blood counts,T-lymphocyte subpopulation of peripheral blood cells,the sry-gene chimerism of bone marrow of the receiptors,the distribution of MSCs in the receiptors,the occurrence time of cGVHD,pathologic variety of medulla were observed.Resuits MSCs improved the survival of mice after 8 TBI,but 1.5×108/kg of MSCs increased the mortality of irradiated mice.In comparison with the control group,leukocytes and plastocytes recovered rapidly in MSCs group.Megacaryocytes in sternum marrows grew fastly in MSC group.The percent of CD3 and CD4 positive cells in the MSCs group were hisher than those in control post-transplantation.The sry-gene chimerism of bone marrow of the receiptors was higher in the MSCs group than that in the control at 30 d.The MSCs were distributed in intestine,thymus,bone marrow,liver,heart of the receiptors at 30 d.The cGVHD occurrence was 30 d later in MSCs group than that of the control.Conclusions MSCs could improve stem cell engraftment,enhance T-lymphocyte and plastocytes recevery,delay occurrence of cGVHD,repair injured organs and increase survivals.It is indicated that MSCs can enhance the treatment effects of haploid hematopoietic stem cells transplant for ARS.

ObjectiveTo study the changes of cellular immunity caused by intravenous infusion of allogenic rhesus mesenchymal stem cells (MSCs).MethodsMSCs were isolated and cultured.Then the immunomodulatory effects after MSCs infusion were evaluated by means of peripheral blood counts,mixed lymphocyte reaction (MLR) and analysis of lymphocytic subgroup. ResultsMSCs of rehsus were successfully cultivated. No acute toxicities or GVHD were observed in recipients. No obvious changes of peripheral blood counts were present. Recipients A2, A3, A4 were administered with MSC by 4.0 ×105/kg, 1.0 ×106/kg, 2.0×106/kg respectively and relative reaction (RR) of MLR decreased 14 days post MSCs infusion: from 46±2.6 ％to 40.4±1.73 ％ (F =10.19, P =0.023), from (40.9±2.3) ％ to (33±2.1) ％ (F =2.593, P =0.013), from 48.3±2.0 ％ to 39±1.0 ％ (F =28.431, P =0.003) respectively. The decrease degree (ARR) was positively related to the amount of MSCs(F =27.413, P =0.038). RR was restored within 30 days post MSCs infusion. After MSCs infusion, CD3+ CD3+CD4+ and CD3+CD8+ T-lymphocytes decreased in recipient A4, who was administered with the largest number of MSCs, and restored within 30 days. ConclusionMSCs infusion without any other treatment could temporarily inhibit immunity of T lymphocytes in MLR and the immunity inhibition was positively related to the amount of MSCs.The specific immunological characteristics of MSCs were demonstrated with extensive prospect in clinical research.

BACKGROUND: Immunotherapies such as adoptive immune cell infusion and immune-modulating agents are widely used for cancer treatment, and the concomitant symptoms, including cytokine release syndrome (CRS) or immune-related adverse events (irAEs), are frequently reported. However, clinical manifestations induced by mismatched donor granulocyte colony-stimulating factor mobilized peripheral blood mononuclear cell (GPBMC) infusion in patients receiving microtransplant (MST) have not yet been well depicted. METHODS: We analyzed 88 cycles of mismatched GPBMC infusion in patients with acute myeloid leukemia receiving MST and 54 cycles of chemotherapy without GPBMC infusion as a comparison. Clinical symptoms and their correlation with clinical features, laboratory findings, and clinical response were explored. RESULTS: Fever (58.0% [51/88]) and chills (43.2% [38/88]) were the significant early-onset symptoms after GPBMC infusion. Patients possessing less human leukocyte antigen-matching loci with the donor or those with unrelated donors experienced more chills (3 [2-5] loci vs. 5 [3-5] loci, P  = 0.043 and 66.7% [12/18] vs. 37.1% [26/70], P  = 0.024). On the other hand, those with decreased CD4 + /CD8 + T-cell ratio developed more fever (0.8 [0.7-1.2] vs. 1.4 [1.1-2.2], P  = 0.007). Multivariable analysis demonstrated that younger patients experienced more fever (odds ratio [OR] = 0.963, 95% confidence interval [CI]: 0.932-0.995, P  = 0.022), while patients with younger donors experienced more chills (OR = 0.915, 95% CI: 0.859-0.975, P  = 0.006). Elevated ultra-sensitive C-reactive protein levels in the absence of cytokine storm were observed following GPBMC infusion, which indicated mild and transient inflammatory response. Although no predictive value of infusion-related syndrome to leukemia burden change was found, the proportion of host pre-treatment activated T cells was positively correlated with leukemia control. CONCLUSIONS Mismatched GPBMC infusion in MST induced unique infusion-related symptoms and laboratory changes, which were associated with donor- or recipient-derived risk factors, with less safety and tolerance concerns than reported CRS or irAEs.
Humans ; Leukocytes, Mononuclear ; Hematopoietic Stem Cell Transplantation/adverse effects* ; Leukemia, Myeloid, Acute/therapy* ; Unrelated Donors ; Granulocyte Colony-Stimulating Factor ; Graft vs Host Disease