Colony forming unit of granulocytes and macrophages from peripheral blood (PB CFU-GM) represents stem and/or progenitor cells from human blood. In this paper, the effects of histamine H2 receptor agonist 4-methylhistamine (4-MH) and its antagonist ranitidine (Ranit) on the growth of PB CFU-GM cultured in methylcellulose system were studied and their differential effects on normal PB CFU-GM and leukemic HL-60 cells were compared with the effect of the antineoplastic agent cytosine arabinoside (Ara-C). It was found that the histamine H2 receptor agonist 4-MH stimulated the growth of PB CFU-GM when 4-MH was added at the concentrations from 10(-9) mol.L-1 to 10(-6) mol.L-1 among which the dose 10(-8) mol.L-1 exerted most potent stimulating effect (the PB CFU-GM colony numbers was 137.68% +/- 8.20% vs the control, P < 0.01). In contrast, the antagonist Ranit showed inhibitory effect on the growth of PB CFU-GM when at the concentrations 10(-9)-10(-5) mol.L-1 cultured for 14 d in the same methylcellulose system. The inhibition rate was 23.73% +/- 1.16% (10(-9) mol.L-1) and 41.42% +/- 6.75% (10(-6) mol.L-1), respectively. Although both Ranit and Ara-C could inhibit the growth of PB CFU-GM in vitro, Ranit exerted much greater inhibition on HL-60 leukemic cells than on normal PB CFU-GM at the dose of 10(-6) mol.L-1 (100% inhibition for HL-60 and < 50% inhibition for PB CFU-GM). However, the inhibition rate of Ara-C for both HL-60 and PB CFU-GM was 100% at the intensive chemotherapeutic dose of 10(-5) mol.L-1. It would appear that the histamine H2 receptor agonist 4-MH possesses stimulating effect on the growth of PB CFU-GM similar to its effect on CFU-GM from bone marrow as documented before. It is suggested that the histamine H2 receptor antagonist Ranit has, to some extent, potential in the treatment of myeloid leukemia, especially when combined with antineoplastic agent Ara-C at suboptimal doses.

DDB is a hepatoprotectant and has been widely used for the treatment of chronic viral hepatitis in China. The drug markedly improved the abnormal liver function particularly in lowering the elevated serum transaminases in patients. It is known that there is a close correlation between primary hepatocarcinoma and chronic viral hepatitis. The aim of the present study is to evaluate the effect of DDB on hepatocarcinoma cell line. The results showed that the growth and clonogenicity of Bel-7402 human hepatocarcinoma cell line cultured with DDB were markedly inhibited. The nucleoles of the cells treated with DDB disappeared or their numbers and nucleus/cytoplasm ratio decreased under electron microscopic observation. DDB at the concentration of 10(-4) mol.L-1 significantly increased the contents of cAMP and calmodulin (CaM) in Bel-7402 hepatocarcinoma cells. DDB was also found to inhibit topoisomerase II activity of Bel-7402 hepatocarcinoma cells. These results suggest that the mechanism of inhibition of DDB on several phenotypes of Bel-7402 cell line may be related to its effect on cAMP and CaM content as well as topoisomerase II activity.

The methods of cord blood collection was investigated in this study. The purpose of this study was to evaluate human umbilical cord blood as a alternative to bone marrow in the provision of transplantable progenitor cells for hematopoietic reconstitution. The results showed that using modified technique with a sterile and closed system, the volume of cord and placental blood would all exceed 120 ml. The average amount of blood collected was 132.2 +/- 12.13 ml and the highest was 158 ml. Cord blood culture for bacteria and fungi were negative. Blood clots were not present. And in the examination of each single cord blood, the contents of nucleated cells (NS) and mono-nucleated (MNS) were, respectively, 18.9 X 10 +/- 1.7 X 10, 8.2 X 10 +/- 0.8 X 10. The result suggested that the collection of placental blood with the modified technique in a sterile and closed system is a simple, safe and efficient procedure. The average amount of blood collected and hematopoietic cells would satisfy the needs of cord blood transplantation. Immediate cut of the umbilical cord followed by a prompt puncturing is critical for the volume and quality of the sample.

We have previously shown that platelet factor 4 (PF 4) is a potent inhibitor of megakaryocytopoiesis and that it may protect stem cells from 5-fluorouracil (5-FU) cytotoxicity. In the present work, the effects of human PF 4 on megakaryocyte (MK) growth from human CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta 1 (TGF-beta 1). Development of MK from CD34+ cells in both plasma clot culture and liquid culture was significantly inhibited by PF 4 (5 micrograms/ml) and TGF beta 1 (1 ng/ml). Inhibition of cell growth by PF 4 was reversible judging from the fact that the CD34+ cells preincubated with PF 4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF-beta 1 pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF 4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF 4 at 40 micrograms/kg with an interval of 6 h followed by one injection of 5-FU at 150 mg/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF 4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF 4, TGF-beta 1 blocked more cells in G 1 phase. These results demonstrate that PF 4 and TFG-beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF 4, unlike TGF-beta 1, exerts its inhibitory effect on cell growth in a reversible and S phasespecific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.

It is generally held that control of viability, proliferation, differentiation, maturation and programmed cell death events in hematopoietic stem/progenitor cells existing in a hierarchical fashion in vivo is a successively developmental process. The CD34+ antigen preferentialy expressed on hematopoietic stem/progenitor cells has so far been recognized as one of the earliest known antigens. Many advances on molecular structure of CD34 antigen would help us to understand deeply a lot of important questions regarding CD34+ hematopoietic stem/progenitor cells. These questions are, for instances, functional subclass, expression range and other biological properties including cell cycle state, fluorescent light scatter, ect. All of these studies would renew and consummate the modern concept of hematopoietic stem cells and the fundamental model of hematopoiesis and its regulation.

In order to explore the clonal origin and malignant pathway of multiple myeloma (MM), a study has been made to investigate the differentiation stage of malignant progenitor cells in peripheral blood (PB) of MM. The immnophenotype of mononuclear cells (MC) in PB and bone marrow (BM) from 17 patients with MM were studied with APAAP method by using monoclonal antibodies directed to a series of B-cell markers (CD10, CD19, CD20, CD45RA, CD45RO, CD38). There were no plasma cells in samples of PB examined. Out of the 17 patients, MC of PB from 10 expressed plasma cell antigen CD38. This expression was significantly different from normal control (P < 0.001) and identical with that of BM. MC of 9 patients were CD38+ and CD45RO+, 3 were CD38+, CD45RO+ and CD45RA+ and 1 was CD38+, CD45RA+, CD45RO+ and CD10+ in PB. The results indicate that while the B-cell marker's expression of PB matches with that of BM, its forms are diversified. This suggests that the PB of MM patients contains precursors at multiple differentiation stages of myeloma cells, from early B cell to active B cell, late B cell and preplasma cell. MC of PB from 10 patients expressed CD45RO while that of BM from 5 out of them expressed the same. This indicates that preplasma cells which are CD45RO+ in PB may eventually return to BM for homing and differentiating into plasma cells, thus losing CD45RO and expressing CD38 only. This study has proved the presence of malignant cells in PB of MM, confirmed the speculation of the malignant pattern and provided the theoretical basis for purging of PB in autologous peripheral blood stem cell transplantation.

The methods of mobilization and collection of stem cells in peripheral blood stem cells transplantation (PBSCT) and the association between the number of stem cells transplanted and hematopoietic recovery were studied. The investigation was carried in 22 patients (11 acute leukemia, 6 multiple myeloma, 4 non-Hodgkin's lymphoma, 1 breast cancer). Three regimens for mobilization were carried out as follows: 1) chemotherapy + tetrahydrofolic acid + dexamethasone, 2) chemotherapy + rhGM-CSF + dexamethasone, 3) chemotherapy + rhG-CSF + dexamethasone. Besides, CD34/CD33 dual-color direct immunofluorescence flow cytometry assay was performed in 7 cases in the rhG-CSF group. The results showed: 1) The mean number of collected cells (MNC) in the rhG-CSF group was MNC (8.29 +/- 6.14) x 10(8)/kg and CFU-GM (21.35 +/- 17.24) x 10(4)/kg, being highest among the 3 groups. 2) The number of CD34+ cells correlated with MNC and CFU-GM. CD34+ cells in the peripheral blood were 0 or < 0.5% before mobilization and increased markedly 6-8 days after rhG-CSF administration. Harvesting should be started at that time and carried out every day until CD34+ cells reached 5 x 10(6)/kg. 3) The number of PBSC transplanted was the key to hematopoietic recovery.

CD34+ cells were isolated from human umbilical cord blood by using a high-gradient magnetic cell sorting system with anti-CD34 monoclonal antibody coated with microbeads, and then inoculated onto a pre-formed irradiated bone marrow stroma to evaluate the grafting efficiency of CD34+ cells with rhGM-CSF or IL-3, alone or in combination. The results showed that only 36% CD34+ cells in the control, and 68 - 89.6% in the groups with growth factors treatment seeded to the stroma. A short incubation of CD34+ cells with growth factors could rapidly reconstitute a long-term hemopoiesis in the long-term liquid culture system. This demonstrates that a brief treatment of CD34+ cells with GM-CSF or IL-3 can improve the grafting efficiency of transplanted umbilical cord blood cells.

Using single layer agar culture method, we make a study on culture of human hematopoietic progenitor cell from umbilical cord blood. The result showed the mean value of the granulocyte macrophage progenitor cells (CFU-GM) from twenty neonates cord blood was 95.6 +/- 36.5/10(6) MNC. We compared the mixed culture of two different blood types neonates cord blood hematopoietic progenitors with single sample and found little difference between them in number and growth characteristics. This may provide a theoretical basis for clinical transplantation of mixed human cord progenitor cells.

The APC gene was identified in 1991 at chromosome 5 q 21, which is responsible for the familial adenomatous polyposis (FAP). The gene has been classified as one of the tumor suppressor genes. The APC gene mutations were suggested to initiate sporadic as well as inherited colorectal neoplasia and to be related to mental retardation. The different forms of APC gene expression and their association to carcinogenesis have been carefully studied. However, the function of APC gene in the central nervous system has not been known. In this study, on the basis of the cDNA cloning of APC homologue in the guinea pig by Dr. Fan Meng, we rescued this fragment including the full length encoding region from plasmid pMe 18s and then subcloned it into the polylink site of the plasmid pBluscript KS. Both digoxigenin labeled sense and anti-sense RNA were synthesized by in vitro transcription. RNase protection assay and in situ hybridization enable us to examine the distribution of APC transcripts in guinea pig brain. Strong signals were detected in hippocampus. APC mRNA was mainly localized in the pyramidal neurons of CA 1, CA 3, as well as in the dentate granule cells; the cerebellum granular cells also showed strong staining; in the cerebrum, the parietal and primary olfactory cortex showed stronger signals than the frontal cortex; in olfactory bulb, positive cells with strong signals were observed: the brain stem showed a relatively weaker staining. Very similar expression pattern was also shown in embryonic guinea pig brain; except that the expression of APC gene in frontal cortex and olfactory bulb was stronger than that in adult animals. The results suggest that the APC transcripts in brain may play an important role during the early development of the central nervous system. Further study may enable us to take a deeper insight into the mechanism underlying inherited mental deficiency.

In order to prevent the ototoxicity of kanamycin (KM), the effects of Poria Cocos (PC) on the ototoxicity induced by KM in guinea-pigs was observed by infusing the PC decoction into the guinea-pigs with comparing the difference in the general intoxicating symptom, prayer's reflex (PR) threshold, brainstem auditory evoked potentials (BAEPs) and the absence rate of outer hair cells in the first turn of cochlea. The results suggested that the ototoxicity of KM was retarded by giving the PC decoction. On 7th day after given KM, PR threshold of 4,6,8 kHz in the medicated group and control group were 4.7 +/- 1.5 dB, 5.7 +/- 2.4 dB, 2.7 +/- 1.2 dB and 10.5 +/- 3.2 dB, 12.1 +/- 3.7 dB, 8.5 +/- 2.7 dB respectively. BAEPs threshold on 13th day given KM raised 22.7 +/- 9.7 dB in the medicated group and 51.3 +/- 14.4 dB in control group. The absence rate of outer hair cells in first turn of cochlea were 39.4% and 67.4% respectively. The results suggested that PC might be antagonistic to the ototoxicity of KM in guinea-pigs.

The antitumor action of extracts from Fructus Mume and the main triterpenoid component ursolic acid on HIMeg and HL-60 cells in vitro was tested. The immuno-response in rats was also studied. The result showed that Fructus Mume had inhibiting effect on proliferation of HIMeg and HL-60 cells.

A TGF-beta 1 gene expression plasmid was constructed by inserting the porcine 1.7 Kb TGF-beta 1 cDNA into BamHI site of retrovirus vector Dol. The plasmid DNA was introduced into mouse embryonic stem cells (ES-5 line) by calcium phosphate mediated transfection, and transfected ES-5 cells were then selected by stepwise increase in G418 concentration. Finally, we obtained 21 clones that could be stably grown in culture medium with G418 at 500 micrograms/ml and were designated as ES-T cells. Dot blot and Northern analysis of total RNA and polyA+ RNA extracted from those ES-T cells were shown in FIg. 2 and 3, demonstrating that 6 clones could express exogenous porcine TGF-beta 1 mRNA. The stronger hybridized signal in two clones (ES-T6 and ES-T 16) of them were further proved by southern hybridization of genomic DNA from these ES-T cells with 1.7 Kb TGF-beta 1 cDNA probe (Fig. 4). The product of TGF-beta 1 gene overexpression in ES-T 6 cells was shown in Fig. 5 and 6 by SE-LISA for TGF-beta 1 immunoreactivity to TGF-beta 1 antibodies and biological assay for CCL/64 cell growth inhibition, respectively. With respect to some biological characteristics, ES-T 6 cells, like their parent ES-5 cells, retained their pluripotent properties and positive SSEA-1 antigen (Plate I, Fig. 1). ES-T6 cells were expanded and used for studies of in vitro differentiation. Both of ES-T 6 cells and control ES-5 cells could form a lot of simple aggregates and differentiate into embryoid bodies by hanging drop culture for 3 days in the presence of retinoic acid (RA) at 10(-9) mol/L. Then individual embryoid bodies were plated on gelatinized tissue culture wells. On the third day of further culture without RA, a large amounts of epithelial-like and round cells occurred around the embryoid bodies formed either from ES-T 6 cells or ES-5 cells (Plate I, Fig. 4). However, with further culture of embryoid bodies, only the cells differentiated from ES-T6 embryoid bodies could arrange themselves and differentiate into a lot of radially arranged tubular structures (Plate II, Fig. 5). The frequency of tubular structures present in ES-T6 embryoid bodies were about 95.5%, but in ES-5 group there was only about 17.8% cases giving less defined tubular structures (Plate III, Fig. 8).(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-6 (IL-6), a pleiotropic cytokine, is involved in extensive immune regulation and hematopoietic regulation. We observed the effect of fibroblast mediated human IL-6 gene therapy on hematopoiesis. The platelet counts started to increase at day 4 after implantation of IL-6 highly secreting fibroblast cells and peaked at day 10 and lasted at high level for 22 days. The neutrophil counts were elevated after their implantation, but WBC did not show any remarkable increase. The CFU-GM and CFU-MK in bone marrow and spleen were also increased significantly. The results demonstrated that fibroblasts mediated human IL-6 gene therapy can significantly augment in vivo hematopoietic functions in bone marrow and spleen and elevate the number of nuetrophils and platelets. This study provides a new approach to treat thrombopenia and chemotherapy or radiotherapy-induced hematopoietic suppression.

The human wild-type Rb gene cDNA has been cis- or trans inserted into the retrovirus vector DOL, resulting in a sense-expression vector DOLRB and an antisense-expression vector DOLRBAS of Rb gene. By eletroporation transfection techniques, the vector DOLRB has been introduced into the human breast carcinoma cell line MDAMB468 and human hepatocellular carcinoma cell line SMMC7721 both of which have an inactivated Rb gene and the vector DOLBAS, into normal human embryonic lung fibroblasts HEL cells. With the expression of Rb protein, the growth rate of the MDAMB468 cells is decreased by about 50%, their colony formation ability in soft agar is repressed completely, and their tumorigenicity in nude mice is repressed partially. Meanwhile, the cell population of G1 phase of Rb+ MDAMB468 cells is increased markedly. About 75% of transfected SMMC7721 cells have been killed by Rb gene product. For HEL cells, with the transient expression of antisense Rb gene, the Rb protein synthesis is reduced and the growth rate of those cells increased, but no colonies of HEL cells are formed in soft agar.

The behaviour of ICMs isolated from early blastocysts of rabbit by microsurgery and incubated in cultural system in vitro, are varied according to the growing state of ICM. The band type growing extends from the proximal to the distal end, while the differentiation of cells in this kind initiates from the distal toward the proximal and gradually. The band type ICM possesses obvious polarity. The differentiation of different kind of cells appear one after another in order and the arrangement of differentiated germinal layer are clearcut. Therefore, the band type ICM is a good model for the investigation of differentiation cell and cell lineage. Whereas the ball type of growing ICM possesses no polarity. The cell in this type of ICM appears to differentiate from the outer surface of the ball toward the center gradually. The cellular differentiation starts later and the rate of proliferation of differentiated cells are lower than those of the band type ICM. After 7 days of incubation in vitro most of the ICM remain undifferentiation. The ball type ICM is a good model for the isolation of the embryonic stem cell line. There are two steps that the extraembryonic endoderm of rabbit are differentiated from ICM. The first kind of extraembryonic endoderm formed from ICM after 3 days of incubation in vitro. It is the parietal extraembryonic endoderm which migrate so far from the primitive ectoderm. The second kind of extraembryonic endoderm differentiates from ICM after 4 days of incubation in vitro. It is the visceral endoderm, most of which followed the migration of the first endoderm and the rest of them invade into the trophectoderm near the primitive ectoderm.

The afferent projections to the central superior raphe nuclus (CS) in the rat were investigated by means of HRP retrograte tracing method. 3% WGA-HRP 0.05-0.1 microliters was injected into the CS in each of 11 rats. The labeled cells were found in the medial part of lateral habenular nucleus, posterior hypothalamic area, lateral subnucleus of interpeduncular nucleus, dorsal raphe nucleus, periaqueductal gray matter, dorsal paramedial nucleus, preposital hypoglossal nucleus, dorsal paragiganto-cellular nucleus, magnum raphe nucleus, and fastigatum of cerebellum. The results provide morphological basis for researching function of the CS in acupuncture anesthesia.

In order to study the expression of growth hormone gene in heterologous cells, the retroviral vector pINS was used to construct the recombinant plasmid carrying the human growth hormone gene (hGH), and then it was introduced into mouse fibroblast cell line 3T3 and mouse embryonic stem cell line CCE. Levels of accumulation of hGH secreted into medium was quantified by a radioimmunoassay. Expression level of hGH gene varied considerably from one clone to another. Several 3T3 clones expressing hGH gene at high level were obtained, such as GHSNC 20, in which hGH concentration in medium was 3784ng/ml, Integration of exogenous gene was stable. Southern blot result showed that such kind of difference was not related to the number of integrated copies of the foreign gene. The expression of ES clones transfected with pINS-GH was undetectable, and integration of the foreign gene was unstable. Southern blot result showed that hGH gene really integrated into the cellular genome, and no rearrangement of foreign gene is found at present.

To estimate the differential potentiality of ES (Embryonic Stem) cell line MESPU 13, the heart, liver, spleen, lung, kidney, pancreas, gonad, muscle and blood of 19 chimeric mice were analyzed for GPI (Glucose Phosphate Isomeras) marker, in these studies, type A band from the ES cells, appeared parallel to the coat color chimerism of the mice. When coat color chimerism is below 40%, type A band was not seen except in the kidney of a few mice. Type A band was detected in nearly all the organs and tissues, when coat color chimerism was over 85%, indicated the ES cell has a fairly high potential to differentiate into cells of endoderm, mesoderm and ectoderm. In addition, only type A band was observed in the muscles of 6 mice which coat color chimerism is over 85%, the results indicated that these muscles were differentiated only from ES cells.