The setting up of morphologic experimental centre has brougt experimental methods and means to a higher level. However, it is worth considering how to share the resource furthest,and how to exert experimental technicians’ efforts. This article intends to discuss those problems.

Object To study the effect of cryopreservation on the ability of hematopoietic stem cells from human bone marrow and the action of total saponins of Panax ginseng (TSPG) to reduce their freezing injury. Methods Gradual cooling methods were used to place mononuclear cells (MNC) in liquid nitrogen with 5% dimethylsulfoxide (DMSO) for 3—5 months. After thawed, the biological properties of MNC were monitored, which included the mean trypan blue exclusion rate and the mean recovery rate of MNC, CFU-Mix, CD34+ cell, and total cells; Thawed MNC were cultured with various concentrations of TSPG, the effect of TSPG on the recoverable ability from cryopreservation damage were detected by colony-forming assay, colorimetric MTT assay for cell proliferation and flow cytometry. Results A fraction of MNC lost their proliferatory ability after thawed, but the damage was not deteriorated with freezing time. TSPG (25 ?g/mL) could raise the colony production rate of thawed hematopoietic stem cells; TSPG (25—50 ?g/mL) could raise the colony production rate of thawed hematopoietic stem cells; TSPG (25—50 ?g/mL) could improve their proliferation; TSPG (25 ?g/mL) could also promote the entray of them into cell proliferatory cycle. Conclusion TSPG could induce the proliferation of thawed hemato- poietic stem cells and raise the post-freezing recoverable ability of hematopoietic stem cells.

BACKGROUND: Most of hematopoietic growth factor regulates proliferation and differentiation of blood cells through JAKs-STATs signal transduction pathway. Total saponins of Panax ginseng (TSPG) can promote in vitro differentiation of CD34+ hematopoietic progenitor cells into erythroid cells, with similar effectiveness of hematopoietic growth factor.Erythropoietin receptor (EpoR) expression on the cell membrane of progenitor cells is critical during the erythroid differentiation process.OBJECTIVE: To investigate the molecular mechanism of TSPG to induce erythroid cells through erythropoiesis and its receptor-mediated JAK2/STAT5 signal transduction.DESIGN, TIME AND SETTING: An in vitro cytological observation. The study was performed at the Department of Histology and Embryology, Institute of Basic Medicine, Chongqing Medical University from May 2006 to October 2008.MATERIALS: Umbilical cord blood of normal full-term pregnancy was provided by the First Hospital of Chongqing Medical University. TSPG, purity＞95%, provided by Chongqing Institute of Traditional Chinese Medicine, was diluted in RPMI-1640 for work concentration of 1 g/L and degermed by positive pressure filtration.in RPMI-1640 culture solution containing horse serum, with various dilutions of TSPG (0 as blank control, 10, 25, 50, 75,100 mg/L). The MNCs were cultured on 96-well culture plate, with 0.2 mL in each well. Early erythroid cells were counted on were harvested and cultured separately in RPMI-1640 culture solution containing 10% horse serum as control group and in TSPG (25 mg/L)- conditioned culture system as experimental group. 5 U/mL Epo was added for 0, 2, 5 and 30 minutes.Immunoprecipitation of JAK2/STAT5 was used for the effect of TSPG on Epo/EpoR-induced tyrosine phosphorylation of JAK2/STAT5.MAIN OUTCOME MEASURES: Effect of TSPG on proliferation of erythroid progenitor cells from human umbilical cord blood;Effect of Epo on the proliferation of hematopoietic cells; Effect of TSPG on EpoR expression of the umbilical blood cells; tyrosine phosphorylations of JAK2 and STAT5.RERULTS: TSPG (10-75 mg/L) promoted the colony formation of BEU-E, CFU-E, and the preferential differentiation into erythroid lineage cells was most induced from 25 mg/L of TSPG. Using the colorimetric MTT assay, MNCs exhibited proliferative responses to Epo (2-50 U/mL) reaching maximum at 5 U/mL Epo. The addition of TSPG did not increase the expression of EpoR after MNCs were incubated in the presence of with or without TSPG for 24 hours. The pretreatment with TSPG for 24 hours enhanced Epo-induced tyrosine phosphorylation of JAK2 and STAT5 (STAT5a and STAT5b).

Objective To study the temporal and spatial expression of Nanog in developing primordial germ cells(PGCs) of mouse.Methods Immunofluorescence technique was taken to qualitatively analyze the expression of Nanog in PGCs of 7.5days(post-coitus days)-15.5days mouse embryo.A further quantitative analysis of Nanog expression change in PGCs of 11.5days-15.5days mouse embryo was done by using SSEA-1 and Nanog double labelling immunofluorescence staining with confocal microscopy.Results The PGCs in mouse allantois,hindgut,dorsal mesentery and genital ridge were Nanog positive.Both of the highest positive ratio and the highest fluorescence intensity appeared in PGCs of 11.5days mouse embryo.For the female mice,Nanog expression drops dramatically after 12.5days,and for the male mice,a noticeable decline of Nanog expression was occurred after 13.5 postcoitus days.Conclusion\ Nanog expresses stably in the proliferating PGCs.The down-regulation of its expression may be related with the differentiation of PGCs.

AIM:Studies on totipotential stem cells using primordial germ cells (PGCs) have the same application prospect as embryonic stem cells, especially for the animals difficult to isolate embryonic stem cells from blastula. In vitro culture condition of embryo germ cells is the key to control this. This study designs a method to effectively amplify PGCs of mice in vitro and establishes an effective culture method of PGCs. METHODS: Experiments were carried out in the Department of Histology and Embryology of Chongqing Medical University and Key Laboratory of Biochemistry and Molecular Pharmacology from October 2006 to August 2007. ①Clean Kunming pregnant mice (embryos 0.5 dpc) were provided by Animal Experimental Center of Chongqing Medical University. Experimental procedures met the animal ethical standards. ②Allantois and surrounding tissues of 8.5 day post coitum (dpc) embryos, the hindhut and surrounding tissues of 9.5 dpc and 10.5 dpc embryos, the gonadal ridges and surrounding tissues of 11.5 dpc and 12.5 dpc embryos were collected and digested with 0.25%pancreatin-0.02%EDTA, then the cells were cultured in the plastic petridishes which are pretreated with 0.1% gelatine. The formation ratio of primary and the first passaged Embryonic germ (EG) clones were compared among those different time points. The SSEA-1 positive ratio of isolated cells was compared between the two days by immunohistochemical method. The assays of clone numbers counting and MTT were used to compare the different proliferation ability of PGCs from the 11.5 dpc and 12.5 dpc embryos. The effects of expanding PGCs from 11.5 dpc and 12.5 dpc embryos by those two ways above were compared. The EG clones were analyzed by the expression of alkaline phosphatase and the immunologic marker SSEA-1,the undifferentiated marker Nanog and the differentiation ability in vitro were also tested. RESULTS:①The formation ratio of primary and the first passaged EG clone from 11.5 and 12.5 dpc embryos was higher than that from 8.5 to 10.5 dpc embryos and efficiency of expansion was increased (P 0.05). On the third day, the number of the primary EG clones from 11.5 dpc embryos was higher than that from 12.5 dpc embryos (P 0.05), but the two groups had different characteristics. ④The EG clones have representative morphology with high level of alkaline phosphatase activity and SSEA-1, Nanog expression, which can differentiate into the cystic embryoid body. CONCLUSION: 11.5 dpc and 12.5 dpc mouse embryos, especially 11.5 dpc embryos are the optional time points to expand PGCs efficiently. Co-culturing with the tissue and isolating the gonadal ridges both of these two ways are practicable.

BACKGROUND: Mesenchymal stem cells have ability of multi-directional differentiation, and can be induced to differentiate into epithelial cells in vitro. The differentiation of epithelial cells in fetal primitive gut is induced by mesochymal cells of intestines. The report of bone marrow mesenchymal stem cells (BMSCs)differentiate into epithelial cells induced by intestinal connective tissue has not been identified. OBJECTIVE: To observe the possibility of BMSCs differentiate into epithelial cells induced by fetal intestinal connective tissue. DESIGN: Culture in "vitro and comparative observation. SETTING: The experiment was carried out in the Department of Histology and Embryology, Chongqing Medical University from July 2004 to July 2006. MATERIALS: Epidermal growth factor (EGF, Sigma); CK, CK20 (Zhongshan Bio-Tech, Co.,Ltd). Bone marrow of limbs was collected from 6 aborted fetus samples aged 4-5 months. Adding Ficoll to centrifugalize, BMSCs were isolated, cultured and proliferated. The intestinal segment about 15 mmx5 mm was obtained sterilely from fetal duodenal papilla to colon, then muscular tunic and adventitia were peeled. Enzymatic digestion was used to remove the epithelial cells on the mucosa surface. The lump of intestinal connective tissue was cut into 15 minx5 nun. Fetus samples were provided by Department of Gynaecology and Obstetrics in Clinical College, all the parturients agreed to the offer, and the experiment was approved by the hospital ethical committee. METHODS: The experiment was assigned into 4 groups. In groups A and B, the DAPI labelled BMSCs (3x104) at the third generation were transplanted on the submucosa of intestinal connective tissue;.In groups C and D, the DAPI labelled BMSCs were only cultured on the cover glass; In groups B and D, EGF in final concentration of 10 ng/mL was added. MAIN OUTCOME MEASURES: After cultured for 12 days, the morphous and distribution of DAPI labelled BMSCs were observed under fluorescence microscope; the cell morphous on surface of the same intestinal connective tissue was observed with hematoxylin-eosin stain. Expressions of CK and CK20 as well as whether the intestinal connective tissue express EGF were all detected by immnnohistochemical stain. Production of basal lamina between the DAPI labelled cells and connective tissue was assayed with periodic acid-Schiff reaction.RESULTS: On the surface of the tissue lump in groups A and B, the DAPI labelled BMSCs could be seen under fluorescenc microscope, in the same section stained by hematoxylin-eosin, there were some epithelioid cells. During culture, the medium were changed several times, the marked cells that were not eluted, grew on the surface of connective tissue.Immunohistochemistry revealed, in groups A and B, cells on the surface of the tissue lump both expressed CK and CK20. Immunofluorescence stain shown that some of cells on the tissue lump had the DAPI marked nncelus (sapphirine fluorescence) and CK (red fluorescence) in cytoplasm simultaneously. It indicated that the 3rd generation of DAPI labelled BMSCs had differentiated into epithelial cells. Cells in group C were negative for CK and CK20, but those in group D were positive, which indicated that EGF could induce BMSCs differentiate into epithelial cells. There were glycosidoprotein with basal membrane-like structures appeared between the cells and connective tissues in periodic acid-schiff stained section. The uncultured connective tissue expressed EGF. CONCLUSION: Both EGF and fetal intestinal connective tissues can induce BMSCs differentiate to epithelial cells in vitro; the EGF presented in the intestinal connective tissues may be one of factors in this induction process.

BACKGROUND: Hematopoietic growth factors (HGFs) can be involved in different hematopoietic cells and different phase of differentiation. Erythropoietin (EPO) can promote he proliferation and differentiation of erythroid progenitor cells in vivo and in vitro, Interleukin-3 (IL-3) appears to have both the characteristics of a differentiating hormone and the ability to generate proliferation of relatively early stem cells. IL-3 acts to enhance the effect on the proliferation and differentiation of various hematopoietic cells.OBJECTIVE: To investigate the effect of HGFs and total saponins of panax ginseng (TSPG) on erythropoietic differentiation of purified CD34+ cells from human bone marrow.DESIGN: An observational comparative experiment.SETTING: Chongqing Medical University.MATERIALS: This experiment was performed in the Department of Histology and Embryology, Institute of Basic Medical Research and Key Laboratory of Biochemistry and Molecular Pharmacology in Chongqing Medical University from July 2002 to January 2004. Human bone marrow was collected from extracted ribs of patients without hematopoietic system diseases in the Department of Chest Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA. Informed contents were obtained from all the patients. TSPG (purified to more than 95%) was provided by Chongqing Institute of Chinese Traditional Medicine; Fetal bovine serum (FBS), fluorescein isothiocyanate (FITC)-conjugated anti-CD34 Ab from Becton Dickinson; FITC isotype-matched mouse IgG1 and rabbit anti-CD71 Ab from Santa Cruz Biotechnology Inc; Recombinant human erythropoietin (EPO) from Amgen. rHu Flt3 Ligand (FL), rHu thrombopoietin (TPO), rHu stem cell factor (SCF) and rHu interleukin-3 (IL-3) from Santa Cruz Biotechnology Inc.METHODS: CD34+ hematopoietic stem/progenitor cells (HSC/HPC) were isolated by StemsepTM according to the strategy of negative selection on immunomagnetic separation. The sorted CD34+ cells were incubated in various combinations with IL-3+EPO, SCF+IL-3+EPO and FL+TPO+SCF+IL-3+EPO. SCF+IL-3+EPO was taken as the control group, TSPG of 10, 20, 50, 70 and 100 mg/L, then the total cell number and ratio of CD71+ cells were detected. CD34+ culture systems were added by TSPG of different dosages, those un-added by TSPG as control group. Methylcellulose culture method was used to detect the capacity of TSPG in inducing the proliferation and differentiation of CD34+ HSC/HPC [burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E)].MAIN OUTCOME MEASURES: ① CD34+ cell proliferation in vitro in different cytokine groups; ② Effects of TSPG on inducing CD34+ cells to differentiation into CD34+ erythroid lineage hemocytes; ③ Effects of TSPG on the ability of CD34+ cells in forming erythroid lineage progenitor cells.RESULTS: ①For FL+TPO+SCF+IL-3+EPO, the mean increase in overall cell number corresponded to 546.38-fold expansion, and the ratio of CD71+ cells was increased to (61.20±5.31)%. ② TSPG 20 mg/L was identified as the most potent concentration of those tested, and the ratio of CD71+ cells was increased from (48.39±5.07)% to (56.20±1.40)%. ③ The addition of TSPG(10-50 mg/L) increased the colony production rates of BFU-E and CFU-E.CONCLUSION: A combination containing FL+TPO+SCF+IL-3+EPO may obviously induce CD34+ cells to proliferate and differentiate to erythroid lineage; TSPG may promote CD34+ cells to differentiate into erythroid lineage by cooperating with HGFs.

Objective To investigate the clinical efficacy of electroacupuncture in treating mild and moderate female stress incontinence.Methods One hundred and eighty female patients with mild or moderate stress incontinence were randomly allocated to treatment and control groups, 90 cases each. The treatment group received electroacupuncture and the control group took midodrine hydrochloride tablets. The International Consultation on Incontinence Questionnaire Urinary Incontinence Short Form (ICIQ-UI SF) score was recorded and 1-hour pad test leakage was measured in the two groups before and after treatment. The clinical therapeutic effects were compared between the two groups.Results There were statistically significant pre-/post-treatment differences in the ICIQ-UI SF score and pad test leakage in both groups (P<0.01). There were statistically significant post-treatment differences in the ICIQ-UI SF score and pad test leakage between the treatment and control groups (P<0.01). The total efficacy rate was 86.7% in the treatment group and 68.9% in the control group; there was a statistically significant difference between the two groups (P<0.05).Conclusions Electroacupuncture is an effective way to treat mild and moderate female stress incontinence. It can improve urinary continence and reduce urine leakage in the patients.

Objective To explore the protecting mechanism of erythropoietin (EPO) on learning and memory of Alzheimer disease (AD) rats. Methods The AD model was made by injected into rat hippocampal CA1 subregion with amyloid beta-protein(Aβ). Male Spraque-Dawley rats were as the experimental objects, which were randomly separated into 3 groups including Sham, Saline control and EPO treatment. After Aβ was injected into rats hippocampal CA1 subregion ,saline or EPO was respectively injected into the lateral ventricle of rats,with help of stereotaxic coordinates, upon the designed conditions. Hippocampal CA1 subregion Bcl-xl expression changes were observed 24 hours after the operation, and learning and memory abilities were checked 4 weeks after the operation. Results 24 hours after the operation Bcl-xl expression in the EPO group and the Saline group was less than the Sham control ,while Bcl-xl positive cells( 100.42 ± 12.43/field) in the EPO group were more than in the Saline group( 82.06 ± 19.68/field ) (P ＜ 0. 05 ). 4 weeks after the operation learning ability in the EPO group ( 20.38 ± 5.88 ) was better than Saline group ( 25.50 - 3.25 ) (P ＜ 0. 05 ), and memory ability in the EPO group (4.75 ± 1.75 ) was better than the Saline group(2.88 ± 1.55 )(P ＜ 0.05 ). Conclusion EPO could improve the learning and memory abilities in the model rats,and it could be related with EPO restraining Bcl-xl expression decreasing.

Objective To study the effects and related mechanism of low-dosed methyl-mercury chloride (MMC) on the epithelial apoptosis of fetal mice intestine. Methods Pregnant mice on E12.5 d and E13.5 d were injected with different doses of MMC (0, 1, 2, 4 mg/kg) intraperitoneally. After 48 h, their duodenum and colon were dissected out and sliced into the paraffin sections. Some sections were stained with HE to count the numbers of apoptotic bodies (NAB) with stereological method. The others were stained with the immunohistochemical method to observe the expression of apoptosis-associated proteins Bcl-2, Bax and immediate early gene c-fos. Results ①In all experimental groups, the NAB in the epithelium were higher than that of control groups (P