Sequential extraction and DGD embedment-free section TEM techniques were used as new methods to study the intermediate filament-lamina-nuclear matrix (IF-L-NM) system of cells. Murine embryonic stem cells (ES-M13) were investigated by means of electron microscopy, immunofluorescence microscopy and Western-blot analysis. There existed a delicate nuclear matrix network in the nucleus domain of detergent-extracted ES cells. The filaments of the nuclear matrix were found in close association with the nuclear lamina. Some intermediate filaments were also observed in the cytoplasm. In immunofluorescence microscopy, a bright, slightly granular cytoplasmic fluorescence, showing no polar concentration, was revealed with keratin monoclonal antibody AF5. We could not detect any significant fibrillar staining in the ES cells by indirect immunofluorescence method. With antibodies to vimentin and desmin, the ES cells showed only a nonspecific, weak fluorescence, similar to that seen in the control. In Western-blot analysis of electrophoretically separated polypeptides, three polypeptides with molecular weight of 65 KD, 62 KD and 52 KD, reactive with keratin monoclonal antibody were detected in the ES cells.

The effects of androgen on cytokeratin (CK) 8 mRNA expression in rat ventral prostate (VP) were studied by in situ hybridization with an antisense RNA-probe. It was found that 1) the CK 8 antisense RNA-probe was accumulated only within prostatic epithelial cells, indicating that the CK 8 mRNA is a specific and sensitive marker for prostate epithelia. 2) After castration, the signals of CK 8 mRNA in VP sections were significantly increased. Administration of androgen to the castrated male host repressed the elevated expression of CK 8 mRNA in VP. This effect can be antagonized by the simultaneous administration of an antiandrogen. 3) Unlike other androgen-repressed gene, the elevated expression of CK 8 mRNA in VP was persisted even 2 months after the process of glandular involution was completed. 4) During prostate development, the strongest CK 8 mRNA staining was found in the early neonatal prostatic epithelia which were composed mainly of prostatic stem cells. Thereafter, when the levels of serum androgen were gradually increased, the shift of CK 8 mRNA staining to peripheral region and decreased level of overall CK 8 mRNA were noted. These data support the notion that the CK 8 gene of prostate epithelia is a new class of androgen-repressed one. The data also indicated that the expression of CK 8 gene is closely related with the proliferation and differentiation of prostate stem cells, and excessive expression of CK 8 mRNA is a characteristic for prostatic stem cells.

The effects of iontophoretic application of H- (acid NS) on the spontaneous discharge of nucleus paragigantocellularis lateralis (PGCL) neurons were studied in brainstem slice in rat with multibarrel microelectrode techniques. Most of the PGCL neurons tested (18/20, 90%) showed an excitatory response to H+ and the response was dose dependent, but none was inhibited. The results suggested that there were H+ sensitive cells in PGCL and these cells may be involved in central chemoreception.

Viscum Coloratum Flavonoid (VCF) is one of the dihydro-flavonoids isolated and purified from the stem and leaf of Viscum Coloratum. It was proved both experimentally and clinically to have the effects of anti-tachyarrhythmias. The mechanism of this effect, however, was unclear. In this study, the effects of the drug on the fast response action potentials (FAP) in canine Purkinje fibers and guinea pig ventricular myocardial cells were investigated by glass-microelectrode technique. The results indicated that 0.1 mg/ml VCF accelerated the repolarizations of 2nd and 3rd phase of FAP, shortened action potential duration (APD), and slightly shortened effective refractory period (ERP) (canine) or unchanged (guinea pig). delta ERP/delta APD ratio was increased, therefore, ERP was relatively prolonged. All effects appeared after using the drug for 2 minutes, reached stable status after 10 minutes, and disappeared after washing off the drug for 15 minutes. It was suggested that VCF was the drug which exerted effects rapidly, maintained effects shortly, and had effects reversibly. The cellular electrophysiologic mechanisms of VCF on anti-tachyarrhythmias should be attributed to prolong the ERP relatively and abolish the reentry. This drug is particularly applicable to the tachyarrhythmias due to reentry.

Using serum-free culture system and liquid/semi-solid dual culture technique, we examined the effect of rhSCF on self-renewal of the stem/progenitor cells of human acute myelogenous leukemia (AML-CFU). The results revealed that the rhSCF as a hematopoietic growth factor (HGF) exerted its effect hierarchically earlier than interleukin-3 and granulocyte/macrophage colony-stimulating factor on AML-CFU. rhSCF, whether used alone or in combination with other HGFs, showed potential maintenance of self-renewal of AML-CFU in most AML patients studied. However, considerable discrepancy existed in maintenance among various FAB types of patients or even in patients with the same FAB type. It is suggested that SCF may play an important role in the pathological development of AML.

The fibrin formation in brain traumas was studied by immunohistochemical method. Eleven cases of brain trauma were examined. The fibrin was demonstrated not only in the areas of brain contusion and the neuronal cytoplasm of hypothalamus, thalamus, brainstem, and cerebellum, but also on the membrane of endothelial cells and red blood cells within some capillaries. The fibrin was also found around capillaries and the areas far from the brain trauma. It was suggested that injuries to the brain occurred not only in the local areas of the brain subjected to the violence, but also in the areas far from the local injury. It meant that the brain injuries were not local, but diffuse. The fibrin observed in the neuronal cytoplasm, and on the membrane of endothelial cells and red blood cells within some capillaries was a sign of ante mortem brain traumas. The combined HE staining and immunohistochemical staining of the fibrin is useful in the demonstration of simple brainstem traumas.

The effect of recombinant human growth factors: rhIL-6, rhGM-CSF and rhEPO on normal human hematopoietic progenitor cells was studied by using liquid and semisolid culture systems. The results showed that the expanding folds of progenitor cells after culturing with the three growth factors were 4.3 +/- 0.6, 9.4 +/- 0.9 and 13.7 +/- 1.0 respectively, being markedly higher than those of control (2.4 +/- 0.7, P < 0.01. In the presence of IL-6 alone, no CFU-E colony was observed and only CFU-GM colonies were formed in the semisolid culture. When IL-6 was added with EPO, the number of CFU-E colonies was significantly higher than that of EPO alone (P < 0.01). In the presence of IL-6 plus GM-CSF or IL-6 plus GM-CSF and EPO, CFU-Mix colonies were observed. Moreover, the total number of colonies was significantly higher than that of IL-6 or GM-CSF alone (P < 0.01). It is suggested that IL-6 is an important hematopoietic regulator. IL-6 may act on hematopoietic progenitor cells to enhance their proliferation. The target cells of IL-6 are the same as those of GM-CSF. IL-6 has synergistic action with EPO and GM-CSF, to enhance erythropoiesis with EPO and proliferation of multipotential progenitor cells with rhGM-CSF and EPO. However, the mechanism of the synergism between IL-6 and other growth factors is still unknown.

Stem cell factor is a recently identified earliest-acting hematopoietic growth factor and a ligand for the c-kit proto-oncogen. Based on our recent observations that recombinant rat interleukin-3 (IL3), human interleukin-6 (IL6) and murine granulocyte-macrophage colony stimulating factor (GM-CSF) possessed different degrees of suppressive activities on the proliferation of LT 12 cell line derived from BNML rat leukemic model, SCF was evaluated alone and in combination with either IL3, IL6 or GM-CSF for effects on leukemopoiesis in vitro. The results indicated that SCF alone had suppressive effect on DNA synthesis and colony forming unit-leukemic blast (CFU-L) in LT12 cells. 100ng/ml of SCF caused substantial reduction in colony number and 3H-TdR uptake although this suppression was of lower magnitude than those induced by IL3, IL6 or GM-CSF. Enhanced suppression on the proliferation of LT12 cells was observed when SCF was used in combination with one of these three factors. Among these combinations, SCF+GM-CSF or SCF+IL6 resulted in more suppression on LT12 cells than SCF+IL3 did. Combination of SCF with two or three factors produced even more suppression. No apparent effect on the size of leukemic colony was seen. Furthermore, in growth kinetics study of LT12 cells in the presence of SCF production of LT12 cells declined. Thus, SCF appears to have divergent hematopoietic activities on BNML rat model: effective stimulation of granulopoiesis and weak suppression of leukemopoiesis.

PCR is an effective method for detecting homologous recombinants from ES cells, because there are different genomic DNA structure among non-recombinants, site specific recombinants and non-site specific recombinants. When we design suitable primers, we can analyze and identify site specific recombinants, non-site specific recombinants and non-recombinants. In this paper, we design two different pairs of primer (according to the genomic DNA patterns) for using PCR to detect the cells which resist to G418 and 6-TG after plasmid pRV4.0 transforming male mouse ES cells, we get the homologous recombinants quickly, simply and specifically. We test the reliability of PCR using southern blot hybridization.

Hemopoietic progenitor cells (BFU-E, CFU-E and CFU-GM) of 69 cases of aplastic anemia were cultured in vitro while the sensitivity of CFU-E and BFU-E to androgen was assayed. The suppressive effect of peripheral blood mononuclear cell (PBMNC) from patients on normal CFU-GM growth was also studied. Hence 69 cases of aplastic anemia were divided into three types: 10 cases of stem cell deficiency type, 30 cases of immuno-mediated type and 29 cases of androgen response type. According to above classification, different treatment programs were practiced using both TCM and western medicine. In the androgen response type, androgen and TCM were used and the effective rate reached 92.3%. In the immuno-mediated type, in addition to TCM. Immunosuppressive agents were cautiously used and the effective rate was 70.6%. As for the stem cell deficiency type, 10 patients' conditions were usually very severe so comprehensive therapeutical means were adopted and 3 cases were improved. The overall response rate of these three was 73.2%. These results showed that the treatment program under the direction of classification have greatly improved the curative effect.

In the study, Na2SeO3 effectively limited the growth and proliferation of Tca8113 cells both in vitro and in vivo. The response was dependent on the dose, the starting administered time, exposed length of selenium and density of inoculated Tca8113 cells. At 1 microgram/ml dose of selenium, there were remarkable inhibitory effects while no detectable inhibitory effect to L929 cells. 1 microgram/ml dose for less than 24 hours, the growth and proliferative ability of Tca8113 cells was reversal whereas more than that period, was irreversal. In vivo experiment, the morbidity of transplanted tumors was remarkably depressed with Se-intraperitoneal injection. At 60 micrograms/ip dose, the weight of nude mice were not reduced and the pathological changes in liver and kidney had not found.

Ginsenosides are the main active component of Panax ginseng. It has been shown that ginsenosides have antineoplastic, antiaging, immunologic function enhancing and other pharmacological actions. In this article, result of experimental studies showed ginsenosides extracted from stem and leaf of Panax ginseng (GSL) has inductive differentiation effect on all types of acute nonlymphocytic leukemia cells in primary culture. The effect on M5, M4 was most potent, followed by M1, M2 and the least, on M3. Through analysis, it was considered that the inductive differentiation effect of ginsenosides might be due to the comprehensive effect of increasing intracellular cAMP and inducing interferon. Since GSL have some other important actions, therefore, if it could be used as a differentiation inducer in clinical practice or combined with other antineoplastic drugs, it would show co-antineoplastic actions in many aspect.

Nuclear transplantation has been proved to be a very powerful method in researching the relationship between nucleus and cytoplasm during embryogenesis. Electrofusion is a new means of cell fusion which is being improved in the past nearly ten years. In this study, we produced interorder nucleocytoplasmic hybrid embryos between mouse and rabbit with these two techniques. Two phenomena, i.e. premature chromosome condensation (PCC) and nuclear swelling, were observed after mouse 8-cell stage blastomeres were fused into rabbit enucleated oocytes. After 4.5-days cultured in mouse oviducts, 5.4% reconstituted embryos developed into blastocysts. By karyotype examination, it was known that only mouse chromosomes were presented in the blastomeres, one blastocyst even had a normal mouse karyotype (2 n = 40, XX). As a conclusion, we consider it is possible to obtain normal early development of interspecies nucleocytoplasmic hybrid embryos between mouse and rabbit.

Human multidrug resistance gene (mdr1) 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 colchicine concentration (30, 50, 100, and 200 ng/ml respectively). Finally, we obtained 4 clones that could be stably grown in culture medium with colchicine at 200 ng/ml and designated as ES-mdr1 clones A, B, C, and D. Southern blot analysis of DNA from ES-mdr1 A and D cells digested by Hind III and hybridized with mdr1 cDNA 5 A probe was shown in Fig. 3. Characteristic 4.8 and 2.4 kb fragments of mdr1 gene were found as expected and their amplification under increased concentration of colchicine in culture medium was also evident from the figure. Slot blot and Northern analysis of total RNA and poly A+ RNA extracted from ES-mdr1 cells were shown in Fig. 4 and 5, demonstrating that ES-mdr1 cells could express mdr1 mRNA. Indirect immunofluorescence analysis with antibodies against p170 glycoprotein indicated that p170 protein translated from mdr1 mRNA was present at the surface of ES-mdr1 cells (Plate I, Fig. 2). The biological characteristics of ES-mdr1 cells cultured in medium containing 200 ng/ml colchicine were investigated. The cells maintained their undifferentiated morphology and grew in nests (Plate I, Fig. 1), like the parental ES-5 cells. When ES-mdr1 cells were cultured in suspension in vitro, these cells were still capable of producing simple and cystic embryoid bodies. ES-mdr1 cells injected subcutaneously into 129 mice formed tumor-like outgrowths giving a great variety of cell types (Plate I, Fig. 4). These results indicated that the integration and expression of human mdr1 gene and selection against colchicine did not affect the pluripotency of ES-mdr1 cells both in vitro and in vivo. However, ES-mdr1 cells, unlike their parental ES-5 cells, could no longer be induced to differentiate by either RA or HMBA (Plate I, Figs. 3a, 3b), indicating that the human mdr1 gene transfected ES cells had changed their competence of inducible response to differentiation in vitro. The details and possible significance of such change require further studies. From the above preliminary data, we are of the opinion that ES-mdr1 cells may serve as a model to study the mode of action of p170 glycoprotein at cellular level and to screen possible means to counteract the action of mdr1 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

An acute promyelocytic leukemic cell line LT12 was transduced with IL6R cDNA by electroporation. The resultant LT12-IL6R+ leukemic cells expressed more than 1000 IL6R per cell. The effect of the recombinant IL6-PE40 on both the LT12-IL6R+ cells and the IL6R- parental cells (LT12-IL6R-) was studied by leukemic progenitor colony (CFU-L) formation and 3H-TdR incorporation assays. It was found that IL6-PE40 at concentrations ranging from 1 to 1,000 ng/ml inhibited CFU-L formation and DNA synthesis of LT12-IL6R+ cells in a dose-dependent manner. No inhibition was seen at the same concentration range in LT12-IL6R- cells. At concentrations of 250-1000ng/ml, IL6-PE40 led to only 40% inhibition of DNA synthesis in LT12-IL6R- cells. Inasmuch as rIL6 at the same concentrations had no significant effect on both IL6R+ and IL6R- cells, we concluded it is IL6-PE40 that exerts its highly specific killing on leukemic cells expressing high levels of IL6R.

When the Tca8113 cells contacted with streptococcal preparation 722 in 0.2-1.0 KE/ml concentration directly and continuously in Vitro, the ability of colony-forming of the cells was lost. Using time lapse microcinematographic study it was confirmed that this agent has a direct cytostatic activity on Tca8113 cells, but it was also confirmed that this activity was reversible after 4 days. Effect of 722 on transplantable tumor of Tca8113 cells in nude mice with different routes (ip, id, it) had no-response. The results suggest that the effect of 722 preparation on antitumor in Vivo must have a complete immune function in the host.

A semi-solid cell culture technique was used to study the sensitivity of K562, HL-60, and Raji leukemic cell lines to the inhibitory effect of psoralen plus ultraviolet irradiation. Results indicated that: 1) the inhibition rate of K562, HL-60, and Raji cell lines were 86%, 35%, and 36%, respectively; and 2) K562, HL-60, and Raji cell lines were treated with psoralen (20 micrograms.ml-1) for 1 h, then irradiated with ultraviolet (1 J/cm2) for 10 min, none of the leukemic cell lines showed colony or cluster formation. These suggested that the cytocidal effect of psoralen plus ultraviolet might be useful to eradicate the residual leukemic cells in the bone marrow transplantation.

Beta-carotene at concentration of 6.25 micrograms/ml was shown to inhibit significantly the colony forming efficiency (CFE) of cultured human lung cancer 801 cell line and at 12.5 micrograms/ml was shown to completely inhibit CFE. A 42%-68% (P < 0.01) decrease in spontaneous lung metastasis of LA795 murine pulmonary adenocarcinoma was observed when T739 inbred strain mice were fed a diet with beta-carotene (25mg/100g diet). Ability of DNA and RNA synthesis of lung cancer cells were decreased (P < 0.05) after treating with beta-carotene (25 micrograms/ml) by 24 hours, but the ability for DNA synthesis of human lymphocytes was not effected by the same treatment. Expression of ras oncogene was proved to be inhibited by beta-carotene because the product of ras p21 proteins of cancer cells was decreased after treating by beta-carotene.