Transforming growth factor-α promotes mouse blastocyst outgrowth and secreti on of matrix metalloproteinases

Objective To study the effect of transforming growth factor-α (TGF-α) on early stage of embryo implantation.Methods Mouse blastocysts were cultured in vitro in medium containing various concentrat ions of TGF-α. Blastocyst implantation capacity was evaluated by calculating the percentage of embryos with attachment or outgrowth. Matrix metalloprotein ases (MMPs) secretion of blastocysts was observed using gelatin zymography. Results There was no significant difference in the percentage of attachment betwee n control and TGF-α treated groups, but the percentage of outgrowth of TGF-α treated groups was significantly higher than that of the control group after 24 h culturing. Gelatin zymography showed that blastocysts cultured in TGF-α treated groups started secreting MMPs earlier than those in the control group.Conclusion TGF-α is involved in regulating the mouse embryo implantation process by promo ting blastocyst outgrowth and secreting matrix matalloproteinases.     

Early Embryonic Development in Vitro Co-cultured with Different Somatic Cells in Mice

The effect of different somatic cell cultures on mouse embryo development at early cleavage stage in vitro was studied.Oviductal epithelial,cumulus,uterine of fibroblast cells were co-cultured with mouse embryos respectively for 4 days,Embryos at 1-cell stage were collected from swollen ampullae of the oviducts.A total number of 121,111,132 and 89 embyos were co-cultured with oviduct epithelial,cumulus,uterine and fibroblast cells respectively,Among them,109,88,101 and 20 embryos developed regularly to the blastocyst stage respectively.The blastocysts were transferred to the uteri of synchronized recipient mice,only 9 blastocysts co-cultured with oviductal epithelial cells and 6 blastocysts with cumulus cells had developed normally and been implanted.These data indicated that oviductal epithelial cells and cumulus cells exert a specific promoting action on early embryonic development in vitro.     

Establishment of a novel method for primary culture of normal human cervical keratinocytes

Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum （FBS）. However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro. Methods Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium （K-SFM） supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity. Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%. Conclusion A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.     

Expression of Cytokeratin,Actin and Endocrine Hormones in Human Hatched Blastocyst Implantation Model in vitro

Objective To investigate the expression of cytokeratin,actin and hCG,E2 and P of human hacthed blastocysts in the model.Methods Human hatched blastocysts were co-cultured with human endometrial decidualization stromal cell monolayer.The process of orientation,attachment,outgrowth and invasion in morphology were observed.Immunofluorescence staining for cytokeratin and actin,immunofluorescence measurement of hCG and radioimmunoassay measurement of E2 and P were performed.Results Blastocysts attached to stromal cell layer after 5h in co-culture.After 24h in co-culture,the trophoblast protruded from two opposite poles of the blastocyst and underwent outgrowth into the stromal cell monolayer,blastocyst became bigger and invaded finally into the stromal cells.After 48h in co-culture,cytokeratin staining was only visible in trophoblast but not stromal cells,actin staining was visible in both of trophoblast and stromal cells with distinct conformation and structure.Stromal cells had prominent linear actin filaments,aligned along the long axis of the cells.Cytokeratin staining in trophoblast cells was localized in short filaments arranged in a mesh.hCG,E2 and P levels in the supernate of stromal cell-blastocyst co-culture were higher than the supernate from blastocyst cultured only （P〈0.01）.Conclusion An implantation model for the reflection of the process of human blastocysts attachment,outgrowth and invasion into stromal cells has been established in vitro.Cytokeratin,actin and hCG,E2 and P take place corresponding changes in the human implantation blastocyst cells.     

A Comparative Study on Rat Intestinal Epithelial Cells and Resident Gut Bacteria （ii） Effect of Arsenite

Objective In order to use facultative gut bacteria as an alternate to animals for the initial gastrointestinal toxicity screening of heavy metals, a comparative study on rat intestinal epithelial cells and resident gut bacteria was undertaken. Methods in vitro growth rate of four gut bacteria, dehydrogenase （DHA） and esterase （EA） activity test, intestinal epithelial and bacterial cell membrane enzymes and in situ effect of arsenite were analysed. Results Growth profile of mixed resident population of gut bacteria and pure isolates of Escherichia coli, Pseudomonas sp., Lactobacillus sp., and Staphylococcus sp. revealed an arsenite （2-20 ppm） concentration-dependent inhibition. The viability pattern of epithelial cells also showed similar changes. DHA and EA tests revealed significant inhibition （40%-72%） with arsenite exposure of 5 and 10 ppm in isolated gut bacteria and epithelial cells. Decrease in membrane alkaline phosphatase and Ca^2＋-Mg^2＋-ATPase activities was in the range of 33%-55% in four bacteria at the arsenite exposure of 10 ppm, whereas it was 60%-65% in intestinal epithelial villus cells, in situ incubation of arsenite using intestinal loops also showed more or less similar changes in membrane enzymes of resident gut bacterial population and epithelial cells. Conclusion The results indicate that facultative gut bacteria can be used as suitable in vitro model for the preliminary screening of arsenical gastrointestinal cytotoxic effects.     

The anti-HSV-2 effect of alumen: In vitro and in vivo experimental studies

This study investigated the anti-HSV-2 effect of alumen through in vitro and in vivo expe-riments.Viable cell counting was employed to assess the toxicity of alumen on Vero cells.The inhibition rate of HSV-2 was defined as the cytopathic effect(CPE)of the cells infected with the virus.Alumen suppositories of different concentrations were vaginally applied to the guinea pigs which were then infected with HSV-2 via a vaginal route.The clinical symptoms were observed and the local virus titer calculated.The results showed that alumen had an in vitro anti-HSV-2 effect by means of antiviral dup-lication,direct killing of the virus,and antiviral adsorption.Alumen suppositories of different concen-trations could reduce or completely inhibit HSV-2 infection in guinea pigs.It was concluded that alumen had an in vitro anti-HSV-2 effect through multiple approaches and it could suppress in vivo vaginal HSV-2 infection of guinea pig to some extent.     

Effect of Cigarette Smoke Extract on the Proliferation of Human Airway Epithelial Cells and Expression and Activation of FAK

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases； Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.     

Study on effect and mechanism of sodium ferulate in preventing and treating ozone induced lung injury in mice

Objective:To study the effect and mechanism of sodium ferulate (SF) in preventing and treating ozone (O_3) induced lung oxidative injury in mice.Methods:Lung oxidative injury model mice were established by making them inhale O_3.The activity of anti-oxidase and membranous microviscosity in epithelial cells in the lung of mice were determined,and the ultrastructural change of lung tissues was observed with electromicroscopy.Results:Activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were reduced,while membranous lipo-microviscosity significantly increased in the pulmonary epithelial cells of model mice,revealing ultrastructural change.These abnormal changes were reversed by SF treatment,which was manifested as the significantly raised activities of SOD and GSH-Px after treatment with high and moderate doses of SF,showing a significant difference compared with those in the model group (P<0.01).Membranous lipo-microviscosity basically approached that in the control group (P>0.05);electron microscopic examination showed a basically normal morphological structure of pulmonary epithelial cells,with the change in lung injury significantly milder than that in the model group.Conclusion:O_3 could induce oxidative injury of lungs in mice,and SF could enhance the anti-oxidation capacity of mice and scavenge the oxygen free radicals so as to alleviate the injury.     

Effects of the Extract of Ammopiptanthus mongolicus cheng f. （JA1） on Induction of Apoptosis of HepG2 in vitro and Its Molecular Mechanisms

Objective To study the effects and the mechanisms of extract from a leguminous plant （Ammopiptanthus mongolicus cheng f.） （JAl ） in northwest China on inducing apoptosis and inhibiting proliferation of HepG2 hepatocarcinoma cell in vitro. Methods The HepG2 cell line was used as target cells. The effect of 3A 1 on HepG2 cell growth was detected by microculture tetrazolium assay （MTr）, flow cytometry assay, DNA agarose gel electrophoresis and transmission electronic microscopy. The expressive effect of the wt-p53 in HepG2 cells was analyzed with p53 protein test-reagent. Results JAl not only had significant anti-proliferative effects depending upon time and dosage, but also induced apoptosis of HepG2 cells. Apoptotic typical morphological changes were observed in JAl-treated HepG2 cells under transmission electronic microscope, ＂Sub-G 1＂ phase peak occurred in flow cytometry and DNA ＂ladder＂ was found in DNA agarose gel electrophoresis. The expression of the wt-p53 increased in vitro, and 3Al-treated HepG2 and the positive cell percentage of the wt-p53 protein also increased. Conclusions JAl could obviously induce apoptosis and inhibit proliferation of HepG2 cells in vitro, and these effects are closely related with the increase of wt-p53 expression. JAl can be used as a good source of medicinal plant for the treatment of hepatocarcinoma.     

Effect of Antisense Oligonucleotide to Annexin Ⅱ on the t—PA—mediated Plasminogen Activation in vitro

Summary: In order to study the role of annexin Ⅱ , a recombinant expression vector, pZeoSV2 (+)ANN Ⅱ , containing the annexin Ⅱ cDNA, was developed. The 1.1-kb-length annexin ⅡeDNA was inserted into a expression vector, PZeoSV(+) and transfected into HL-60 cells which had low baseline expression of Ann- I. pZeoSV(+) ANN I was analyzed by restriction mapping and the Ann-I sequence identified. The ability of the transfected cells, non-transfected and mock-transfected cells to stimulate t-PA-depend plasminogen activation was compared. The results showed that HL-60 with pZeoSV (+)ANN I transfection could significantly increase the plasminogen activation (8. 9± 1.2 U) in vitro with the difference being significant as compared with non-transfected (1.5±0. 4 U) and mock-transfected cells (4.2±0. 9 U), respectively. Antiannexin Ⅱ oligonucleotides significantly inhibited the binding ability of t-PA and plasminogen to annexin Ⅱ , and obviously reduced the plasminogen activation in vitro. The above findings showed human umbilical vein endothelial cells (HUVECs) treated with sense or missense oligonucleotides indicated no significant change in binding of t-PA and PLG. Treatment of HUVECs with antian nexin Ⅱ oligonucleotides could significantly reduce the plasminogen activation by 2.4± 0. 3 U as compared with sense oligonucleotide group in binding of t-PA and PLG. These results, therefore,suggest that Ann- Ⅱ can bind plasminogen and participate in the stimulation of t-PA-dependent ac tivation of plasminogen, and that interference with Ann- Ⅱ mRNA by antisense oligonucleotidemay be a new strategy for the therapy of bleeding in patients with hyperfibrinolysis.