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BACKGROUND: Skeletal muscle satellite cells are muscle-derived stem cells with proliferation and differentiation potential. Recently, foreign researches have reported that skeletal muscle satellite cells can be activated by some definite microenvironmental factors and differentiate into hematopoietic stem cells and thereby they will have the potential of hematopoietic reconstruction.OBJECTIVE: To initially validate the potential of adult muscle-derived stem cells- skeletal muscle satellite cells differentiating into hematopoietic stem cells.DESIGN: Validation animal experiment.SETTING: Department of Histology and Embryology, College of Basic Medical Science, Tianjin University of Traditional Chinese Medicine.MATERIALS: Sixty-five male Kunming adult mice, weighing 25-28 g, were involved in this study. Five Kunming neonate rats, aged 5 days, were provided by the Laboratory Animal Center, Department of Medicine, Peking University.METHODS: This experiment was carried out in the Laboratory for Cell Culture, Department of Human Anatomy and Histo-embryology, School of Basic Medical Sciences, Peking University Health Science Center between August 2001 and August 2003. Skeletal muscle satellite cells of 5 neonate rats were isolated by collagenase and trypsin digestion. Bone marrow mononuclear cells of 5 adult Kunming mice were isolated. Sixty adult female mice were used as recipients, irradiated with 60Coγ 8.0 Gy and then randomized into 4 groups: control group, in which, the mice were untouched; culture fluid infusion group, in which, the mice were injected with DMEM/F-12 medium through caudal vein; satellite cell infusion group, in which, the mice were injected with 0.3 mL satellite cell suspension through caudal vein (cell concentration 1×109 L-1); bone marrow-derived cell infusion group, in which, the mice were injected with 0.3 mL bone marrow-derived cell suspension (cell concentration 1×109 L-1) through caudal vein.MAIN OUTCOME MEASURES:①The survival rate of 14-day-old mice in each group. ②The surviving recipient mice were euthanized 14 days after irradiation, and tubercles on the surface of spleen were counted by naked observation; Bone marrow mononuclear cell smear was stained by Wright-Gimesa.RESULTS:① Determination of colony forming unit-spleen (CFU-S): No significant difference in the number of spleen tubercles of mice existed between satellite cell infusion group and bone marrow-derived cell infusion group 14 days after irradiation (P＞0.05). ②Histological identification of bone marrow-derived mononuclear cells: Many hematopoietic cells appeared at the early stage in the bone marrow-derived mononuclear cell smears between satellite cell infusion group and bone marrow-derived cell infusion group. Their morphology meets the biological characteristics of hematopoietic cells at the early stage. ③ The survival condition of irradiated mice: All the mice in the control group and culture fluid infusion group died 9 to 13 days after irradiation. In contrast, 8 mice from the satellite cell infusion group and 13 the bone marrow-derived cell infusion group survived 14 days after irradiation.CONCLUSION: Skeletal muscle satellite cells have the function of differentiating into hematopoietic stem cells.
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Objective To investigate the histochemical changes of 3?-hydroxysteroid dyhydrogenase(3?-HSD), sucinic dehydrogenase(SDH) and lipid on rat adrenal cortex in the carcinogenesis-resistance with selentum. Methods Wistar rats stomach carcinogenesis (formation of aneuploid cells in the glandular stomach mucosa) was induced by MNNG(N-methy1-N-nitroso-guanidine,20 mg/kg)gavage. The changes of the adrenal cortex were studied by histochemistry and image analysis during preventing glandular stomach cancer with selenium(0.1 mg/kg and 2.0 mg/kg) Results 1.Dietary Se inhibited the aneuploid cell formation,which induced by MNNG gavage in Wistar rat stomach; 2.The histochemical reactions of 3?-HSD and SDH were both significantly stronger in experimental group than that in normal control group in the course of the rat stomach carcinogenesis(P
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lymphocyte,and especially the small lymphocytes were moreheavily labeled than the larger ones,and the Con A receptor of NK cell exhibitedpolarization along the cell surface.These results suggest that:1.the extent of ConA receptors was related to the functional activities of the ceils,the more active thecell is,the more the Con A receptors will be;2.the receptors of Con A wereprobably related to the cell recognition,for instance,on the NK cell Con A rece-ptors showed polar distribution;3.the number of receptors of Con A was dependenton the cell differentiation,the more mature the cell is,the more the Con Areceptors are.
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Objective To observe the effect of selenium on the ACTH positive cells in the pars distalis of pituitary of rat with colon mucosa cancer induced by azoxymethane(AOM).Methods Twenty 3 weeks old Sprague Dawley male rats were randomly divided into 4 groups: the normal control group;the experimental control group;the selenium before AOM induced colon mucosa cancer group;the selenium after AOM induced colon mucosa cancer group.To induce rat colon mucosa cancer,3 weeks old SD male rats received(i.p.) injections of AOM at a dose of 15mg/kg once a week for two weeks.Selenium(Na_2SeO_3,4mg/L) began to be provided in the drinking water before and after AOM administration,and lasted for the whole experiment period.The rat pituitary tissues were excised from the 34 weeks old rats.The morphological changes of the ACTH positive cells in the pars distalis of pituitary were observed using immunohistochemical methods,and then the results were analyzed by image analysis system.Results Aberrant crypt foci(ACF) and aberrant crypt(AC) were showed in the colon mucosa of rats that received two weekly i.p.injections of AOM by light microscopy with methylene blue staining.The ACTH positive cells in the experimental group rat displayed a significant increase compared with those in the normal control group rat(P
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Objective To investigate the distribution and the number of splenic lymphocytes in the patients with severe acute respiratory syndrome(SRAS).Methods Immunohistochemical method with four specific antibodies(CD3,CD4,CD8,CD20) was used to detect the distribution of lymphocyte special antigens in the spleens of six cases who died of SARS and six normal cases as the controls.The numbers of lymphocytes were analyzed with image analysis system.Results In the spleen of SARS patients,splenic corpuscle and periarterial lymphatic sheath (white pulp) were damaged severely. In the number of periarterial lymphatic sheath decreased 93.39% and in the number of splenic capsule decreased about 80% even disappeared at all. Red pulp hemorrhage necrosis was widely spread. In red pulp the number of CD3~(+)T cells decreased 71.76%,even disappeared at all,in the number of CD4~(+) and CD8~(+)T cells markedly decreased 86% and 84% respectively.The number of CD20~+ B cell decreased more than 80%. Conclusion T cells and B cells in spleens of SARS patients decreased widely spread and markedly in number,indicating that the immune system had been damaged severely,and the destroyment of the immunosystem may be the primary lesion of SARS.
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Objective To investigate the effect of macrophage-conditioned medium on the growth of mouse myoblasts. Methods After a 3-day intraperitoneal injection with a mixture of muscle homogenate and starch,macrophages were obtained from mouse peritoneal cavity and cultured for 48 hours in serum-free DMEM/F12.Then the cultured medium was harvested.Myoblasts were obtained from newborn mice skeletal muscle and grown in DMEM/F12 supplied with 10% FBS for 36-48 hours,and then exposed to 0.5% FBS medium with or without macrophage-conditioned medium.Cells were harvested at 72 hours later,their growth was observed with Wright staining,LDH cytochemistry and immunocytochemistry. Results Macrophage-conditioned medium can maintain the normal morphology and the activity of myoblasts grown in low concentration of serum and promote their proliferation.There was a significant difference between average absorbance and integral absorbance of LDH positive cells of the two groups(P
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Objective To observe the morphologic changes of immature dendritic cells(imDCs) before and after the electransfection of human hepatic cancer cell RNA. Methods Monocytes were purified from human peripheral blood,and induced into imDCs.Then human hepatic cancer cell RNA was electransfected into monocyte-derived imDCs.ImDCs were identified by the immunocytochemical method with 7 specific antibodies before and after electransfection.These dendritic cells were observed by scanning electron microscopy. Results After electransfection of human hepatic cancer cell RNA there were few changes of molecule expressions in imDCs.ImDCs were in round,oval and irregular shapes before electransfection.Their sizes were not identical but all bigger than monocytes.There were many protrusions in different shapes which looked like dendrite,or/and bubble,veil cloud on the surface of these imDCs.Although there were some cell fusions and cell deaths after electransfection,most imDCs recovered from the damage.Electransfecting human hepatic cancer cell RNA into imDCs would make pores on cell membrane.Conclusions The pores on cell membrane make it possible that the exogenous material enters imDCs.This study can prove the possibility of electransfecting human hepatic cancer cell RNA into imDCs to make cancer vaccine,which provides a new way for tumor biologic therapy.
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Objective To observe the distribution and the effect of the quantum dots(QDs) on mouse abdominal cavity macrophages.Methods The QDs were co-cultured with mouse abdominal cavity macrophages in vitro.The differentiation and effect of the QDs on macrophage ultrastructures were observed under electronic microscope. Results The QDs were enveloped with unit membrane and internalized in the cytoplasm of the macrophage under transmission electron microscope.And it formed vacuolelike structures in the macrophage.There were many lamellar processes on the surface of the macrophage under scanning electron microscope.Conclusion The QDs can promote macrophage activation,and make its surface projection increased.The QDs were internalized by the macrophage,distributed in the cytoplasm,and formed vacuolelike structures enveloped with unit membrane.
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In order to observe the effects of the supernatant of WEHI-3 cell culture which contains IL-3 (interleukin 3), we have Performed some experiments by adding WEHI-3 supernatant to murine bone marrow cells culture. Cell culture of six days shows that the supernatant of WEHI-3 can promote adherence and proliferation of the stromal cells. There are 1.334?10~6 stromal cells/L in the culture which contained 20% WEHI-3 supernatant. Four kinds of stromal cells can be identified under both light microscope and SEM. Fibroblast-like cells which tend to be spindle-shaped with many filament-like and microvilli-like dendrites on the cell surface; Riticular-like cells which are irregular with some folds on the dendrites surface occasionally; Macrophage-like cells which are round with many phagocytic granules in the cytoplasm, and are characterized by positive ANAE staining, and many lamina-like folds and long filament-like dendrites on the cell sufrace; Fat cells are few in number, the cell body is ovoid with lipid droplet, and show strong cytoplasmic staining for Sudan black B, the cell surface is smooth. At the same time we have also observed the close contact between the hematopoietic cells and the stromal cells. In the culture without WEHI-3 supernatant, we have found few small adhered cells with less cytoplasm and weak enzyme activaties under light microscope, and with atrophied cell body and flat surface under SEM. The cell count of the stromal cells in normal culture is 58.83/mm~2. The results showed that WEHI-3 supernatant can promote the growth and proliferation of the stromal cells of murine bone marrow with some histochemical changes.
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The effects of the Biological Suppressor of Cancer(BSG)on human normal hone marrow cells and leukaemic cells were studied.There are some evidences that the BSC possesses a biological activities which apparently kill leukaemic cells and inhibit their DNA synthesis,but it has no such effects on erythroblast.The BSC extracted from ascites of ascitic tumors shows no cancer lines specificity,nor tissue specificity.
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Objective To study the effect of Na-2SeO-3 on experimental carcinogenesis of stomach and on p53 and p16 expression. Methods Weaning male Wistar rats were divided into four groups randomly:high selenium group(4mg/L),low selenium(2mg/L)group,experiment control group and normal control group.Wistar rat gastric cancer was induced by N-methyl-N′-nitro-N-nitrosoguanidine(MNNG,20mg/kg)given daily for 10days.Na 2SeO 3 was given by piped drinking before one week of MNNG administration.Rats were killed at the 43th week.The surface characters of gastric mucosa were observed with noked eyes.Histopathologic changes were observed by methods of HE staining and Alcian Blue Periodic acid Schiff reaction(AB-PAS).Changes of p53 and p16 were detected by SP immunohistochemical method.The immunohistochemical results were quantitatively analysed by image analysis.Statistical analysis was taken by SPSS. Results Dietary Na-2SeO-3(2mg/L,4mg/L)aggravated gastric erosion and hemorrhage and promoted intestinal metaphasia of gastric cancer (P0.05).Conclusion These results suggest that dietary Na-2SeO-3 by piped drinking might not decrease incidence of Wistar rat gastric cancer induced by MNNG.The mechanism may be associated with the mutations of p53 and abnormal expression of p16 gene.
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Objective To analyse the number of dendritic cells and macrophages and the size of macrophages in the spleen of SARS patients so as to provide evident for the study of pathology and pathogenesis of SARS. Methods Immunohistochemical method with four specific antibodies(S-100,CD68,HLA-DR,CD83) were used to detect the dendritic cells and macrophages in the spleens of six dead patients of SARS and six accidental deaths as the controls,The number or the size of these positive cells was analysed with image analysis system. Results In the spleens of SARS patients,the number of S-100~+ dendritic cells in the white pulp was reduced by 80.4% on average,and even disappeard.The number of CD68~+ macrophages in the red pulp was reduced by 39.48% in SARS spleens,and the average size of individual macrophages was increased by 2.21 times.HLA-DR~+ antigen presenting cells(APC) reduced remarkably in the SARS spleen white pulp.CD83~+ mature dendritic cells did not exist in either SARS spleens or the control spleens.Conclusion The function of antigen presentation had been damaged severely,which supports that SARS should be categorized as a viral immune deficiency disease.SARS virus doesn't induce the maturation of DC.The increase in the size of macrophages indicated that they were in an activated state and may play a role in the pathogenesis of SARS.
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Objective To investigate the method and optimum conditions of electric transfection,and the major influential factors of electransfection efficiency and the survival rate of dendritic cells. Methods RNA was extracted from human hepatocarcinoma cell line(Bel 7402).Purified monocytes as precursor DC-s(pDC-s) were separated from human peripheral blood cells(PBMCs) by density gradient centrifugalization with lymphocyte gradation fluid and adherence method,pDCs were incubated in RPMI-1640 medium containing rhGM-CSF(8?10~5IU/L) and rIL-4(5?10~5IU/L) for 7 days and made them fully differentiate into immature DCs(imDCs).The total RNA human hepatocarcinoma cell and green fluorescent protein(GFP) were electransfected respectively into imDCs by electroporation apparatus with different electric voltages,times of impulse,cell concentrations,temperatures and electroporation buffers.Numbers of green fluorescence positive cells and the total cell number were counted respectively under fluorescent microscope,and visible light microscope.One day after the electric transfection,the cells were stained with 0.4% trypan blue,and electransfection efficiency and the cell survival rate were counted. Results Electransfection efficiency was increased to the highest value,up to about 49.7% when imDCs with the concentration of 5?10~6 cells/ml were mixed with 40?g-total RNA of human hepatocarcinoma cell,the electric voltage of electroporation apparatus was set at 300V,and the time of impulse was 500Us.Conclusion Electric transfection provides a technical possibility to make human hepatocarcinoma RNA into imDCs.The major influential factors of the electransfection efficiency were electric voltage and impulsing time.As receptor cells,the imDCs growing condition was also an important influential factor.
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Objective To investigate the distribution and significance of CD20 positive cells in the early human embryo. Methods Distribution and morphology of CD20 positive cells in 10 specimens of human embryo aged from 6 to 7 weeks were brown,and studied with immunohistochemical method. Results 1.CD20 positive cells appeared in the liver of early human embryo.2.The immunohistochemical positive substances were brown,and found chiefly in the nuclei of B cells as unevenly-distributed granules,and were not detected in the cytoplasma and on the cell membrane.Conclusion The accuracy of location and distribution of CD20 positive cells in the nuclei of B cells of early human embryo liver may provide an important clue for further exploration of the functional mechanism of CD20 in the process of B cell proliferation and differentiation.
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Objective To study the development of hereditary dystrophic retina of rd mice and photoreceptors apoptosis. Methods Retinal sections of rd mice and their controls at different ages ranging from postnatal days 5 to 40 were examined by morphological(light and electronic microscope), morphometric and TUNEL analysis. Results Compared with age-matched control mice, the retinal dystrophy of rd mice began at postnatal days 10, resulting in rapid loss of photoreceptors and reaching a peak at postnatal days 18. TUNEL postitive nucleus of photoreceptor cells emerged from postnatal days 10 and reached a peak at postnatal days 14 and 16. Ultrastructure of photoreceptor cell layer showed marked nuclear pyknotosis and chromatin margination. Apoptotic bodies of photoreceptor cells were observed.Conclusion Rd mice retina degenerated during the process of maturity. Photoreceptor cell death occurred through apoptosis.
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Objective To investigate the number,distribution and expression intensity of dendritic cells and T lymphocytes in human cervical intraepithelial neoplasia and invasive carcinoma for seeking biotherapeutic evidence of cervical carcinoma. Methods The number,distribution and expression intensity of CD-(1a),S-100,CD-3, CD-8 positive cells were determined in cervical intraepithelial neoplasia and invasive carcinoma and human normal cervix tissue as the control,by using immunohistochemical technique and computerized imaging analysis system. Results Compared with human normal cervical tissue,the number and the expression intensity of CD~+-(1a),S-100~+,CD~+-3,CD~+-8 cells in cervical intraepithelial neoplasia significantly increased(P