The aim of this study was to explore effect of CD8+CD28- T-lymphocyte in the inhibition of mesenchymal stem cells (MSC) on T-lymphocyte proliferation. T cells were harvested by using nylon column and CD8+ T cells were sorted by magnetic beads; the T-lymphocyte proliferation in the presence of PHA was evaluated by MTT; the proportion of CD8+CD28- T cells was assayed by fluorescence-activated cell sorter (FACS). The results showed that MSC inhibited T-lymphocyte proliferation and the inhibitory effect depended on the amount of MSC; the data of FACS indicated that in the CD8+ T cells co-cultured with MSC, CD8+CD28- T cells were up-regulated significantly, compared with the non-treated CD8+ T cells. In conclusion, MSC perform their immunosuppressive function by up-regulation of CD8+CD28- T cells.

To explore the feasibility of nonmyeloablative conditioning regimens, hematopoietic reconstitution, chimera level and the occurrence of GVHD after nonmyeloablative allogeneic stem cell transplantation in H-2 haploidentical mice, CB6F1 mice were used as the recipient and were divided into 3 groups, mice were pretreated five days before transplantation. Group A was pretreated with myeloablative conditioning regimens (TBI with 10.5 Gy), group B was pretreated by TBI (2 Gy) + Ara-C + Cy and group C-TBI (2 Gy) + Ara-C + CY + Flu, respectively. For all recipient mice, the prevention of GVHD was not given, and 2 x 10(7) bone marrow cells mixed 1 x 10(7) spleen cells from C57BL/6 mice were injected through tail vein on day 0, and then hematopoietic recovery, engraftment and GVHD of recipients were observed. The results of chimera detection after transplantation showed that the engraftment of group A remained full donor chimerism, and engraftments of group B and group C were associated with mixed chimerism or full donor chimerism, but the chimerism of group B remained below 80% and tended to decrease after 50 days whereas chimerism of group C was above 80% (chimerism close to or being full donor type) and preserved even after 50 days. GVHD occurred in all the recipient mice due to that prevention was not given, wherein the occurrence and death rate of GVHD in group A was obviously higher than that of group B and group C (P <0.01), but there was no statistical difference between group B and group C. In conclusion, the nonmyeloablative conditioning regimens mainly based on fludarabine can form stable and lasting engraftment in the body of recipients. The mixed chimerism established in recipients induce tolerance of transplantation and decrease or avoid the occurrence of GVHD.

The aims of this study were to analyze the composition of umbilical cord blood cells (UCBC), to examine the characteristics of dendritic cells (DC) before and after culture, to search the method of differentiation and increase of DC in vitro and to appraise surface antigen from UCBC. Twelve units of umbilical cord blood were collected from May 2002 to September 2002. Peripheral blood mononuclear cells of 9 cases were collected from healthy adult donors. The nature of UCBC was freshly determined and then UCBC were cultured for 1, 2, 3 and 4 weeks with granulocyte-monocyte colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and EPO. Method of flow cytometry was used to determine the number of DC and cell surface antigens before and after culture by using monoclonal antibodies. The monoclonal antibodies included CD4, CD8, CD19, CD34, CD38, CD83, CD1a, CD11c and CDw123. The results showed that amounts of CD34+ progenitors in peripheral blood cells were 0.02 x 10(5)/ml, and amounts of CD34+ progenitors in human UCBC were 0.22 x 10(5)/ml. UCBC cultured for 1, 2, 3 and 4 weeks with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a+ CD11c+ CD83+ CDw123+ DC. Numbers of DC from UCBC remarkably generated in 2-4 weeks and then decreased in number. By culture with cytokines DC increased up to (10.6 - 28.2) x 10(5)/ml in actual numbers. It is concluded that the mononuclear cells of UCB are able to differentiate into CD1a+, CD83+, CD11c+ and CDw123+ DC when UCBC are cultured with proper cytokines of GM-CSF, SCF, EPO and IL-3 for 2-4 weeks. These DCs as antigen presenting cells are possibly effective in cancer immunotherapy.

It is one of the core questions that how human embryonic stem cell (hESC) maintain the undifferentiated state during the propagation process. The current advance in answering this question included the studies on LIF signal transduction, Oct-4 factor and Nanog gene. But the question is far from being elucidated. In order to further understand the mechanism of maintaining the undifferentiated state of hESC, we screened out the expressed sequence tags (ESTs) which expressed highly in hESC but expressed lowly or unexpressed in the differentiated hESC's clones (dhESC) by suppression subtractive hybridization (SSH) and cDNA dot blot techniques. One hundred and five putative positive clones were screened out to be sequenced and were compared with the GenBank. Among them, seventy six clones represented sixty one identified genes that shared high homology with sequences in GenBank. Eighteen clones represented fifteen hypothetic genes, and eleven clones were function-unknown. Eight clones were picked out to analyze using semi-quantitative RT-PCR, the results confirmed that the expression of seven clones among them were higher in the hESC, and lower or no expression in the dhESC. The results showed that many differentiated expressed genes were involved before and after hESC's differentiation. Further functional analysis to those genes that expressed highly in the undifferentiated hESC and lowly in the dhESC might provide a basis to understand the mechanism of maintaining the undifferentiated state of hESC.

To study the effect of catechin, the active component of Spatholobus suberectus Dunn, on bone marrow cell cycle and the expression of IL-6 and GM-CSF mRNA in spleen cells of normal and marrow-depressed mice in order to clarify the mechanism of hematopoietic-supportive effect of catechin.

A murine beta-casein gene targeting vector was constructed using the cloned genomic sequence. The short arm was 2.7 kb including mouse beta-casein gene 5' flanking sequence, exon1, intron1 and partial exon2. The long arm is a 3.4 kb fragment including partial intron2, exon3 approximately 7, intron3 approximately 6 and partial intron7. The human t-PA mutant cDNA was subcloned in the exon2 and fused with the mice beta-casein signal peptide sequence. The positive selective marker neo was placed in the middle of intron2. A tk negative selective marker was just outside the short arm. TC-1 ES cells were cultured and amplified on G418 resistant feeder layer. The linearized targeting construct DNAs of 45 microg were introduced into 2 x 10(7) ES cells by electroporation. Totally 192 ES clones were picked up after cultured in G418 and Gancyclovir for 7 days. The colonies were amplified and subjected to genomic DNA preparation. The genomic DNAs were digested with EcoR I and used for Southern blot analysis. A probe inside the 5' homologous arm was used for hybridization. A 9.8 kb band was found in wild type, but the band was shift down from 9.8 kb to 6.6 kb in the beta-casein gene targeted allele because a new EcoR I site was introduced into the exon2 along with the human t-PA mutant gene. There were 9.8 kb and 6.6 kb bands in targeted ES cells. One clone of targeted ES cells with correct homologous recombination events was obtained among 78 analyzed clones. It lays foundation for gene targeted mice making.

To review the operations of neural stem cells transplantation in patients with brain trauma or spinal cord injury and their postoperative management.

To explore the feasibility of the isolation, culture and identification of the neural stem cells originated from the hippocampi of neonatal rats (1-3 d) and adult rats respectively.

To investigate the differentiation of bone marrow-derived stem cells (BMDSCs) migrated from blood circulation and resided in the injured brain tissue.

To establish human colorectal crypt cell line.

Embryonic stem cells are pluripotent cells derived from blastocyst-stage embryos. They are characterized by their capacity for self-renewal and by the ability to differentiate into a variety of cell types. In vitro, ES cells can spontaneously aggregate into the formation of embryoid bodies (EBs) comprised of a range of differentiated cells. When injected into severe combined immunodeficient (SCID) mice, ES cells will induce the generation of teratomas, which include differentiated cells from three embryonic germ layers. We can induce the differentiation of ES cells into cardiocytes by treatment with several growth factors or co-culture with certain other cells. The pure differentiated cardiocytes from ES cells are then selected and implanted into the infarcted hearts. The hearts' function will be improved. ES cells implantation is a novel and potential method to treat infarcted hearts.

The 3.0 kb 5' arm (long arm, LA) of rat HPRT gene knock-out vector was cut by Sal I from rat HPRT gene genomic bacterial artificial chromosome (BAC) , and the 1.7 kb 3' arm (short arm, SA) was proliferated by PCR. Neo', 5' arm, 3' arm were sequentially cloned into pBS vector's relative restriction enzyme sites. For acquirement of tk gene, the 5' arm -Neo' -3' arm fragment inserted into pKO vector to construct pKO-HPRT. The pKO-HPRT was linearized by Not I, extracted by ethidium bromide, butanol and phenol/chloroform, and dialyzed by 0.025 microlmol/L Millipore. At the same time, rat neural stem cells cultured from E14.5-16.5 rat fetal brain. Passage 2 rFNSCs was tranfected by linearized pKO-HPRT with Fugene-6t transfection reagents. After 80 microg/ml G418 and 0.2 micromol/L ganciclovir selection, the survived cells was cultured in suspension to form neural spheres. The spheres can be picked up under the microscopy, and proliferated in 96- ,48- and 24-well plates sequentially. When the cell number reached 4 x 10(3)/well, half cells was lysed by lysis buffer to extract DNA, the other half was kept on growing to freeze and extract RNA. The knock-out cell colonies first detected by PCR, then confirmed by Southern blot and RT-PCR. All the results show that we have knocked out HPRT gene in three rat fetal neural stem cell colonies from 32 colonies.

To probe into the possibility of obtaining dendritic cells (DCs) from human cord blood with rhIFN-alpha and rhGM-CSF.

To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro and to investigate the possibility of using cultured cartilage as a substitute for meniscus.

To study the conditions to induce differentiation of human embryonic retinal cells (HRCs) in vitro.

To isolate and chondro-inductive culture of human adipose tissue-derived stromal cells and to study their heterotopic chondrogenesis by loading them on alginate gel.

To study the efficacy of combined use of low-dose granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in mobilizing hematopoietic stem/progenitor cells.

From our over a decade-long experience in experiments and clinical applications of human hair keratin (HHK) artificial tendon, a conclusion was drawn that HHK artificial tendon components and their degradation products could stimulate the proliferation and differentiation of tenoblasts in the neighboring tissues into tenocytes. With the regulation of this process by certain cytokines secreted by other cells, autotendons can be finally formed. We also found that after grafting for the tissue defects such as in the bone, nerve and muscle, "in situ construction" of the tissue/organ substitutes occurred, which inspired us to propose a wholly new theoretical system--in vivo tissue engineering, defined as in vivo reconstruction of the defected tissues or organs. The grafted absorbable scaffold biomaterial itself and its degradation products can activate the mitosis, proliferation, and differentiation of adult stem cells in the surrounding tissues, which organically interact with the material to form an organic complex under in vivo physiological conditions. Finally the matrix material is completely replaced by the complex, an almost identical structure with the normal tissue in terms of anatomy and histology. One of the advantages of in vivo tissue engineering is "in vivo construction" of the tissue/organ substitutes in anatomy, histology and function way of "in vivo cultivation" of the seed cells under in vivo microenvironment and in vivo precise regulation mechanism. It solves such problems as immune rejection against seed cells, variation and functional deterioration of the seed cells, complicated preservation procedure, transportation and high cost. The most prominent advantage of this technique is that it best meets the clinical need, not only in the sense of its powerful clinical potential, but also in its significant theoretical value.