To develop the methodology to purify and culture expand mesenchymal stem cells (MSC) from human bone marrow and investigate the optimal system for MSC differentiation into chondrocytes mononuclear cells were harvested by gradient centrifugation on Percoll at density of 1 073g/ml and seeded in low glucose DMEM containing 10% fetal calf serum. The purity of cells was evaluated by flow cytometric technique. MSC of 2 passages thereafter were absorbed into a biomaterial of gelatin sponge and induced to differentiate in to chondrocytes for one week under the influence of TGF ? and other inductive agents. The feature of chondrocytes in engineered tissues was identified by toluidine blue staining at various time points. The results showed that human mesenchymal stem cells culture expanded were positive for CD166, CD29, and CD44, but negative for CD34, CD45, and HLA DR. Furthermore, when treated cells absorbed into the biomaterial were implanted subcutaneously into BALB c nude mice, they formed cartilage like tissues after 4 weeks.Our conclusions is that cultured marrow MSC have unique biological features and the capacity to differentiate into chondrocytes in vivo , so they are useful for cartilage engineering by serving as the seed cells.

To explore the effect of icariside ? on osteoprotegerin(OPG) expression in osteoblasts, primary osteoblasts were cultured with different concentration of icariside ? and 17?estradiol. As a major metabolite of icarrin in vivo, icariside ? exerted a better effect on OPG expression which plays a key role in osteoporosis. 20 ng/dl icariside ? also exerted better effect on OPG secretion than that of positive control group. Consequently, icariside ?could be a candidate for osteoporosis treatment.

Objective To evaluate US, CT and 99mTc-MIBI in the localization of hyperfunctioning parathyroid tumors. Methods Among the 47 patients with primary hyperparathyroidism 45 underwent ultrasound, 47 did CT scan and 36 did double phase imagings. Results Forty-six adenomas, 2 adenocarcinomas and 2 hyperplastic glands were removed from 47 patients. The results showed that the sensitivities were 43%,78%,92% for B-utrasound; CT and isotope imaing respectively.The specificities were 96%,97%,100%;and the accuracies were 82%,92%,98%. There was significant difference between 99mTc- MIBI and CT (? 2=6.627,? 2=4.884,P

Transfer of drug resistance genes into hematopoietic cells is an attractive approach to protect hematopoietic system from the toxic effects by chemotherapeutic agents in cancer patients. In this study, transduction of mdr-1 in combination with dihydrofolate reductase (dhfr) gene was performed, and the expression of exogenous genes and chemoprotection capacity in mouse bone marrow cells were observed. The results showed that approximately 15% of bone marrow cells transfected with the retroviral vector expressed mdr-1 as assayed by flow cytometry. Gene transfer resulted in about 0.9 - 13 fold and 0.5 - 2.6 fold increase in the resistance of CFU-GM to taxol and methotrexate in vitro, respectively (P < 0.05). Moreover, seven months after transplantation to syngeneic mice with mdr-1 and dhfr-transfected bone marrow cells, peripheral blood cells in recipients were still positive for gp170 as evaluated by FACS as well as for mdr-1 and dhfr by PCR amplification. These results indicate that hematopoietic progenitors can be transfected by retrovirus containing mdr-1 and dhfr genes, and that functional drug resistance accompanies their expressions. Furthermore, genetic chimerism might exist in hematopoietic stem cells. In conclusion, transfer and expression of mdr-1 and dhfr genes in bone marrow cells might be applicable in gene therapy research in cancer patients.

The generation of large quantities of novel human T cell clones ex vivo would make a wide range of gene-and immuno-therapies for tumor and AIDS possibly. Although it is well established that T cells are derived from CD34(+) cells, the involvement of thymic fragments from either human or murine fetus makes the in vitro T cell perliferation very cumbersome. In this report, cord blood mononuclear cells were used as accessory cells to support the differentiation of CD34(+) cells into naive T cells stimulated with SCF and IL-2. CD4(+) and CD8(+) T cells, under the cultural conditions, were continuously produced in vitro at least over a period of 3 weeks and their ratios changed gradually. CD4/CD8 double positive T cells and RAG-2 gene were existed, and RAG-2 gene, reponsible for TCR rearrangement, was expressed during the cell proliferation. Our study presents a simple culture system in vitro to acquire large quantities of naive T cell clones.

Bone marrow mesenchymal stem cells (MSCs) are multipotential progenitors of connective tissues and bone marrow stroma as well, which implies the modulatory function of MSCs in hematopoiesis. To clarify the contributions of MSCs to hematopoiesis, the methods for isolation and expansion of MSCs were established and long-term bone marrow cultures were performed using irradiated MSCs as the feeder layer. The results here showed that CD34(+) cells from cord blood formed hematopoietic foci adherent to the monolayer. Furthermore, colony-forming cells remained in the coculture of 5 weeks, indicating the maintenance of long-term culture-initiating cells (LTC-IC). Flow cytometry analysis showed that about 1% of the hematopoietic cells in the culture were positive for CD34 and around 15% were CD41a-positive. It is clear that MSCs maintain LTC-IC in vitro and promote differentiation of hematopoietic progenitors especially into megakaryocytic lineage. The preliminary results here demonstrate that MSCs residing in the bone marrow might be a crucial cellular component in the hematopoietic microenvironment.

The purpose of this study was to investigate the function of dendritic cells derived from chronic myeloid leukemia (CML-DC). Mononuclear cells were prepared from bone marrow and peripheral blood of 24 patients with CML, and the DCs were obtained by incubation of MNCs with media containing GM-CSF, IL-4 and TNF-alpha. The phenotype of CML-DCs was identified by flow cytometry. FITC-dextran uptake, (3)H-TdR incorporation or MTT assay and lactate dehydrogenase release assay were used to detect uptake of exogenous antigen in immature DCs, the antigen presenting ability in mature DCs and specific cytotoxicity of CTL to leukemic cells, respectively. The DCs with high expression of CD1a, CD86, CD80, HLA-DR, CD54 and CD4 were obtained from marrow and blood of patients with CML. The uptake of FITC-DX was observed in early DCs. There was a potent stimulation to allo-MLR in DCs cultured for 7 - 10 days, and a lightly lower stimulation to auto-MLR. CML-DCs can induce the generation of specific cytotoxic T cells. These results suggest that CML-DCs are functional DCs with the ability to induce anti-leukemia effect.

Objective This study aimed to establish a reliable primary culture protocol for preparing murine spleen-derived mesenchymal stem cells ( MSCs) by tissue explant culture.Methods Healthy mouse spleens were crushed by syringe handle to harvest spleen mesenchymal tissues.Then the tiny pieces of spleen tissue were digested by collagenase II before seeded into culture flasks.The morphological characteristics of spleen tissue-derived cells were observed under the inverted microscope.Further, the surface antigen profile of the cells was analyzed by flow cytometry (FACS).The cells were induced to differentiate into osteoblasts and adipocytes.Results The murine spleen-derived MSCs exhibited a spindle-shaped appearance.The FACS results showed that the spleen-derived MSCs highly expressed CD29, CD44, CD105 and Sca-1, but weakly expressed CD11b, CD34, CD45 and Ia. In addition, the spleen-derived MSCs steadily differentiated into osteoblasts and adipocytes in the induction medium.Conclusions A method of primary culture of murine spleen-derived MSCs by explant culture is successfully established.The harvested MSCs exhibit high purity and cell proliferation ability, and provide a reliable cell model for related researches.

BACKGROUND:Spinocerebel ar ataxia is a inherited neurodegenerative disease with progressive cerebel ar masonic movement disorders as the main clinical manifestation. So far, no drug is available to control the disease progression. OBJECTIVE:To observe the clinical effect of umbilical cord mesenchymal stem cells in treating spinocerebel ar ataxia by intrathecal injection. METHODS:Thirty-eight cases of spinocerebel ar ataxia were given umbilical cord mesenchymal stem cells by intrathecal injection, 1×106/kg once a week, four times as a course. These 38 cases received 52 courses. Among them, 27 cases received 1 course, 8 cases received 2 courses and 3 cases received 3 courses. International Cooperative Ataxia Rating Scale (ICARS) and Activity of Daily Living Scale (ADL) were used to evaluate patients’ neural functions (the greater scores, the more severe damage) and ability of daily living (the lower score, the stronger the ability of daily living). After treatment, al patients were subjected to fol ow-up visit. RESULTS AND CONCLUSION:The total effective rate of 52 courses of treatment was 84.62%. ICARS and ADL scores were significantly decreased at 1 month after treatment (P<0.01). In most of effective patients, unstable walking and standing, slow movement, upper limb fine motor disorder, writing difficulties, dysarthria, eye movement disorders were improved. After treatment, common adverse effects were dizziness (1 case), low back pain (2 cases), headache (1 case), and fever (2 cases). Al these symptoms disappeared within 1-3 days. No treatment-related adverse events happened in the median fol ow-up of 39 months (11-59 months). The il ness of effective patients had been stable for 1-19 months, average (5.95±4.84) months. Intrathecal injection of umbilical cord mesenchymal stem cells is safe to ameliorate clinical symptoms to some extent within a certain time. It may delay the progression of spinocerebel ar ataxia. Multiple courses of treatment can help to further improve neurological function in most patients.

【Objective】 To explore the methods and safety of autologous peripheral hematopoietic stem cells collection in patients with sequential double transplantation of solid tumors and conduct efficacy analysis. 【Methods】 Peripheral blood stem cells were collected from 27 patients with solid tumors after routine mobilization of rhG-CSF and rhGM-CSF.A specific program was made for the patients.The condition and cooperation degree of children were comprehensively evaluated before cell collection, and a femoral venous catheterization was inserted to ensure the cells collected smoothly.A mononuclear cell collection(MNC) program was selected, and machine parameters were set based on the patient's low body weight.The number of mononuclear cell (MNC) and the CD34⁺ cell was detected by flow cytometry for retrospective analysis. 【Results】 A total of 73 cell collections were performed in 27 patients, and the number of mononuclear cells and CD34⁺ cells was 12.586(10.22~19.586)×10⁸/kg and 13.575(7.275~23.825)×10⁶/kg, respectively, which can meet the requirement of sequential double transplantation. No intoxication of citrate and other serious adverse reactions occurred, and the follow-up was generally in good condition. 【Conclusion】 The method is effective and safe for pediatric patients, even for pediatric patients with low weight. Sufficient stem cells can be collected for patients with solid tumors by this method to meet the requirement of sequential double transplantation.