To screen efficient and specific new drugs against hepatitis C virus (HCV) and find the best target sequence of antisense oligodeoxynucleotides (ASODNs) targeting at HCV gene.

To duplicating the regulatory subunit p35Nck5a gene of mouse neuronal cdc2-like kinase in embryonic stem (ES) cells, about 12.2 kb of pGDTV vector for p35Nck5a gene duplication was constructed. The linearized pGDTV vectors were transfected into ES cells by electroporation. 245 drug-resistant cell clones were obtained in both G418 and GANC medium and the frequency of surviving cells was 6.22 x 10(-5). The ES cell clones were identified to have duplicated the p35Nck5a gene by use of both PCR and genomic Southern blotting, and the frequency of homologous recombination is 5.08 x 10(-7). The use of negative selection gene (HSV-tk) results in 7-fold increase at selection efficiency. This study lays the foundations of preparing mouse models of Alzheimer's disease.

Human CD 34+ hematopoietic cells, a distinctive cell population containing hematopoietic stem/progenitor cells, have the capability to highly self-renewal, differentiation into all lineages of committed progenitor cells and reconstitution of both long-term hematopoiesis and immune functions after transplantation. In our previous study, the morphological, ultrastructural and cytochemical features concerning CD 34+ hematopoietic cells obtained by a two-step isolated systems of CIMS-100/FACS 440 with 100% purity were depicted. Based on these observations on CD 34+ hematopoietic cells under light microscope, SEM and TEM, we recently analyze the sterological features of CD 34+ hematopoietic cells with the aid of Quantimet 970 automatic image analyser so that the three-dimension structures regarding CD 34+ hematopoietic cells could be further made clear. By a series of measures including image scan-->modulus transform-->shadow correction-->image store-->statistical analysis, some morphometric parameters of CD 34+ hematopoietic cells were obtained as the followings: diametrs 3.490-6.741 microns, perimeters 11.776-26.240 microns, surface area 9.565-35.686 microns2, form factors 1.048-1.840, nucleus-plasma ratio 0.55-0.72, mean light density 0.17675-0.65100, integral light density 2717.217-46661.000. These morphometric data combined with our previous results from morphology, ultrastructure and functional subsets of CD 34+ hematopoietic cells strongly demonstrate that CD 34+ hematopoietic cells are really a heterogenous population. The possible reasons causing heterogeneous are close associated with either different functional subsets or differentiation lineages. To our knowledge, this is the first identification of sterological features of CD 34+ hematopoietic cells.

About 30%-40% of hematopoietic stem cells in human fetal liver of 3-5 months are in S phase of cell cycle, much higher than the ratio of 10% of that in adult bone marrow. The existance of highly active hematopoietic stem cell proliferation stimulators is probably its molecular basis. CFU-S "suicide rate" in rats was adopted to detect the effective substance. Through several steps of separation, we obtained a relatively purified substance of 35 kDa, termed it as FLS-4. CD 34 positive cord blood cells were sorted and assayed for their response to FLS-4 in 3H-TdR incorporation assay. The response to FLS-4 alone was approximately 1 times the background response seen with no factor added. In combination with IL-6 and IL-3 produced response that was 2.9 and 6.5 fold respectively greater than that observed with no factor added, but was weakly in comparison with the effects of SCF. In combination with GM-CSF or IL-3, FLS-4 can stimulate the formation of blast-colonies. The results indicate that the FLS-4 is very likely to be a novel hematopoietic stem cell proliferation stimulator. In physical or biological characteristics, it exhibited a unique character different from IL-3, IL-6, GM-CSF, SCF or FLT3 ligand those are known to have hematopoietic stem cell proliferation stimulating activity. During the period of active hematopoiesis in fetal liver, FLS-4 might be the candidate in triggering hematopoietic stem cells from resting G0 to S phase.

While producing transgenic mice by ES cell route, it is important to know whether ES cells used have a strong ability to produce chimeras or not. In this report, two methods were used for studying the chimeric ability of two ES cell lines (HDC and MESPU-13). One method was the blastocyst microinjection, that is, chimeras were produced by injecting 15 ES cells into the cavity of C57BL/6J blastocysts. Another was GPI electrophoresis for examinizing the chimerism of ES cells in internal tisssues and organs of chimeras. The results showed that the ability of producing chimeras of MESPU-13 cells was stronger than that of HDC cells. The reason was discussed in detail.

To study the proliferative effect of low energy laser on human cord blood hematopoietic cells in vitro and the relationship between the effect of low energy laser and colony stimulating factor (CSF).

To investigate the cellular biological mechanism of leukocytosis produced by all-trans retinoic acid (ATRA).

To study the clonogenic potential of circulating malignant precursors in the peripheral blood of patients with multiple myeloma (MM).

To determine the effects of recombinant human FLT3 ligand (rhFL) on in vitro expansion of human cord blood CD34+ cells.

To determine the differences of the effect of ultraviolet-B(UVB) on lymphocyte and hematopoietic progenitor cell in cord blood.

To investigate the effects of hematopoietic growth factors (HGFs) on apoptosis of hematopoietic cells in myelodysplastic syndromes (MDS) patients.

To explore an efficient approach to enhance antitumor effect of suicide gene therapy by improving in vivo antigen presenting function and their immunological mechanisms.

To evaluate the hematopoietic potential of long-term cryopreserved human umbilical cord blood as well as the migration of the stem cells in nude mice.

To examine the feasibility of bone marrow hematopoietic stem cell transplantation for thalassemia major.

This paper reports 3 cases of allogeneic peripheral blood stem cell transplantation (Allo-PBSCT) for the patients with chronic myeloid leukemia (CML). The patients received MMC preparative regimen with high dose chemotherapy (Melphalan 170 mg/m2, p.o. on Day-5, MeCCNU 400 mg/m2, p.o. on Day-4, and Cyclophosphomide 60 mg/kg/day, i.v. on Days-3 and -2). The HLA-identical sibling donors received filgrastim (rhG-CSF) for mobilization at a dose of 300 micrograms/day for 6 days. Leukaphereses were done at the 6th day of mobilization. A median of 8000 ml (2 times total blood volume) of blood was processed the collecting: 2.5-4.5 x 10(8)/kg MNC, 12.8-20.0 x 10(6)/kg CD34+ cells (including 4.8-7.5 x 10(6)/kg CD34+CD33-, 8.0-13.0 x 10(6)/kg CD34+CD33+), and 3.5-4.3 x 10(5)/kg CFU-GM. Cyclosporin A and methotrexate were used for GVHD prophylaxis. Hematopoitic function recovered as for 14-20 days to > 0.5 x 10(9)/L of neutrophil count, and for 16-34 days to > 20 x 10(9)/L of platelet count. At day + 100, chromosome analysis of bone marrow cells showed that complete chimera without ph1 positive chromosome in Cases 1 and 3, and a partial chimera with 73% donor karyotype in Case 2. All patients now are in disease free survival. No episode of acute graft versus host disease (GVHD) developed. It was concluded that HLA matched sibling allogeneic PBSCT result in rapid hematopoitic reconstitution and the MMC conditioning regimen is effective both in leukemic cells eradication and in immunosuppression for stem cells engraftment, and the drug related toxicity could be tolerated by patients.

To study the clonal growth on single layer, agar of high proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from both normal mice (NBM) and mice treated 2 days earlier with 5-fluorouracil (FU,BM) using conditioned media.

Eleven ES cell lines from C57BL/6J mice were established, with the primary culture of mice embryos as feeder cells and 1 x 10(3) units Leukemia Inhibitory Factor (LIF) in the DMEM (high glucose) medium. The frequency of establishment was 9.6%. Five of these ES cell lines were demonstrated karyotypically normal with diploid composition of over 70%. They shared the characteristics of early embryonic cells: positive for alkaline phosphatase activity and oct4 gene expression. They also showed high potency to differentiate into wide types of cells in vivo. Following injection of the blastocysts, three of them showed abilities to give rise to chimeras and MESPU35 cell line was identified to have high efficiency of colonization into the germline. The clones of MESPU35 cells maintained the germline competence and the mutant clone also retained the high potency to participate into the development of embryos. MESPU35 cells can serve as a valuable vehicle for the production of mutant mice.

Nine embryonic stem cell lines have been established from mouse strain 129/ter. Three of the nine ES cell lines were karyotypically normal. The nine cell lines showed some difference in the growth rate and the differential competence. Chimeras were made by injecting the ES cells into C57BL/6J blastocysts, and the germline compositions of the chimeras were detected by mating them with albino ICR mice. The results indicated that ES cell line MESPU21 and MESPU22 were both highly germline-competent. Comparing with other ones, these two cell lines both were karyotypically normal and propagated fast. The tissue composition of the teratocarcinomas derived from these cell lines appeared paralle to the results of chimera production. Careful manipulation during the process of ES cell establishment was helpful to obtain good cell lines.

To construct the recombinant vectors for embryonic stem(ES) cell gene targeting which contain the mouse coagulation factor IX (F IX) gene modified by PCR site-directed mutagenesis.

A MT-II gene targeting vector--pMT-II6.7 with 6.7kb sequences homolog to mMT-II and its flanking region has been transfected into Mespu22 with electroporation. We have got 26 positive clones from 104 G418 and Ganc clones with PCR method. From the karyotype analysis, we found two clones with 84% and 88% normal karyotype. They are clone 5-2 and 8-4. Using Southern analysis, we confirm that the two clones are homologous recombinants. These cell lines still have the pluropotential ability to differentiate both in vitro and in vivo. Now we have got a chimera mouse with cells from clone 8-4.