OBJECTIVETo clone the glial cell line-derived neurotrophic factor (GDNF) from the mouse testis, construct the eukaryotic expression vector and transfect this vector into Sertoli cells in order to use the gdnf-transfected Sertoli cells as the feeder layer to cultivate spermatogonial stem cells (SSCs).

METHODSTotal RNA was extracted from the testes of normal mature mice and gdnf was cloned and amplified using RT-PCR, inserted into the eukaryotic expression vector and transfected into sertoli cells (TM4 cell line). Immunofluorescence with anti-GDNF antibodies was performed at 40 h following the transfection.

RESULTSgdnf cDNA was cloned successfully, and GDNF expressed after transfected into Sertoli cells.

CONCLUSIONThis study provides a basis for culturing SSCs with gdnf-transfected Sertoli cells as the feeder layer.

Animals ; Cloning, Molecular ; Gene Expression ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Male ; Mice ; Mice, Inbred Strains ; RNA ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; metabolism ; Testis ; cytology ; metabolism ; Transfection

OBJECTIVETo study the isolation of human bone marrow mesenchymal stem cells (MSCs) and in vitro differentiation into chondrocytes as potential seed cell for condyle cartilage tissue engineering.

METHODSHuman MSCs were isolated by percoll solution from normal human bone marrow sample and cultured in flasks. Specific cell surface markers were identified by flow-cytometry. After the cells were treated with inductive medium containing insulin, transferrin, pyruvate, dexathemesone and TGF-beta for 7 - 14 days, microscopic, histological and immuno-histo-chemical studies were performed for chondrogenic phenotype identification.

RESULTSPrimary cultures of human MSCs express CD29 and CD44 positively and meanly, but CD34, CD45 and HLA-DR negatively. After 14 days of induction, the cells were positively stained by safranin O. Immunohistochemical analysis proved strong type II collagen expression.

CONCLUSIONSPercoll helps to generate a better isolation of MSCs from human bone marrow aspirates with a purity more above 95%. The isolated MSCs can be expanded and induced in vitro to differentiate into chondrocytes by inductive medium.

Bone Marrow Cells ; cytology ; Cartilage, Articular ; chemistry ; cytology ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrocytes ; chemistry ; cytology ; Collagen Type II ; analysis ; Dexamethasone ; pharmacology ; Humans ; Immunohistochemistry ; Insulin ; pharmacology ; Mesoderm ; cytology ; Pyruvates ; pharmacology ; Stem Cells ; cytology ; Tissue Engineering ; methods ; Transferrin ; pharmacology ; Transforming Growth Factor beta ; pharmacology

This study was aimed to evaluate the effect of lentinan on the immune function of splenic lymphocytes in rotary cell culture system (RCCS) microgravity environment. The splenic lymphocytes from mice were separated and cultured in the normal gravity and the microgravity environments. The cells were treated with lentinan solution (0, 10, 20 and 40 µg/ml). After incubated with lentinan for indicated times (24, 48 and 72 h), the cell proliferation, secretion of cytokine and the expression of cell surface markers were detected by MTT method, ELISA and flow cytometry respectively. The results indicated that lentinan of above mentioned concentrations did not obviously promote the lymphocyte proliferation, but increased the secretion of IL-2 and IFN-γ and enhanced the expression of lymphocyte surface markers CD4 and CD8 in microgravity environment. It is concluded that lentinan has the ability to enhance the lymphocyte immune function in microgravity environment.
Animals ; Cells, Cultured ; Cytokines ; secretion ; Immune Tolerance ; drug effects ; Immunosuppression ; Lentinan ; pharmacology ; Lymphocyte Activation ; drug effects ; immunology ; Lymphocytes ; cytology ; drug effects ; immunology ; Mice ; Mice, Inbred C57BL ; Spleen ; cytology ; Weightlessness Simulation

AIMTo investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice.

METHODSMice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured.

RESULTSLow doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia.

CONCLUSIONE2 has a dose-related mitogenic effect on spermatogonia.

Animals ; Cell Division ; drug effects ; Cryptorchidism ; physiopathology ; Disease Models, Animal ; Estradiol ; blood ; pharmacology ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Mice ; Spermatogonia ; cytology ; drug effects ; pathology ; Testosterone ; blood

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.
Animals ; CD4 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cordyceps ; Immune Tolerance ; Lymphocyte Activation ; drug effects ; Lymphocytes ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Polysaccharides ; pharmacology ; Spleen ; cytology ; Weightlessness Simulation

Objective To investigate the prevalence of pertussis in infants and children with persistent cough in Beijing during 2011-2016.Methods The eligible infants and children from over ten hospitals who were suspected to have pertussis from 2011 to 2016 were enrolled for detection.Nasopharyngeal secretions and blood samples were collected.Multiplex-PCR was performed for Bordetella pertussis and real-time PCR was performed for nucleic acid of Bordetella pertussis.Results A total of 1 318 eligible cases were enrolled,including 820 males and 498 females.Pertussis was detected positive in 534 cases,including 81.3％ (434/534) of B.pertussis positive cases and 31.8％ (170/534) of IgG positive cases.There were 13.1 ％ (70/534) had double positive for bacteria and antibodies.From 2011 to 2016,the enrolled patients were increased from 103 cases per year to 460 cases per year,and the test positive patients were increased from 29 cases to 194 cases.Among the pertussis patients,466 (87.3 ％) cases were younger than one year old.From the first quarter to the fourth quarter of the year,There were 65 cases,151 cases,205 cases,and 113 cases,respectively.In further analysis of the 268 cases from Children's Hospital affiliated to Capital Institute of Pediatrics,90.7％ of the patients who had whooping cough were scattered children;185 cases (69.0％) of the patients had not begun programmed immunization,71 cases (26.5％) did not complete programmed immunization and 12 cases (4.5％)completed the programmed immunization.Of all the inpatients,21.6％ were critical ill,0.8％ (2 cases) dead,and the remaining patients were recovered and discharged.Conclusions The prevalence of pertussis is increasing,especially in summer.Infants are the most susceptible population.Bordetella pertussis is one of the most important pathogen that can induce persistent and chronic cough.

Objective To investigate the difference between mammary gland tissues and breast cancer tissues.Methods Monoclonal antibodies against Mam-A immunized epitopes were screened for immunohistochemical staining of normal breast tissues and breast cancer tissues.The average optical density was used as an index to identify the quantitative data by computer-aided technology to screen epitope-specific antibodies with significant difference in staining characteristics between two types of tissues.Furthermore the feasibility and effectiveness of breast cancer diagnosis were evaluated.Results Four anti-Mam-A epitope-specific monoclonal antibodies,mAb1152,mAb11617,mAb995 and mAb656,were obtained.Immunohistochemical staining showed that the average density of mAb1152,mAb11617 and mAb995 was significantly different between the two types of tissues.The difference was significant between normal breast tissues and breast cancer tissues under the same conditions.The results showed that mAb11617 was better than mAb1152 and mAb995.At the best working point,mAb11617 was the best,the specificity was 90％ and the sensitivity was 59.62％.Further analysis showed that the sensitivity of mAb11617 combined with mAb995 in the diagnosis of in situ breast cancer was 81.48％ and the specificity was 90％,which was of great diagnostic significance.Conclusion There is significant difference between breast tissues and breast cancer tissues in Mam-A protein immunological activity or expression.This difference,which can be recognized by the specific antibody staining and computer aided technology,is of important diagnostic value.

Embryonic stem (ES) cells are pluripotent cells capable of extensive proliferation while maintaining their potential to differentiate into any cell type in the body. ES cells can therefore be considered a renewable source of therapeutically useful cells. While ES-derived cells have tremendous potential in many experimental and therapeutic applications, the scope of their utility is dependent on the availability of relevant cell quantities. Therefore, most of the researches are being focused on the differentiation of ES cells. ES cell aggregation is important for embryoid body (EB) formation and the subsequent generation of ES cell derivatives. EB has been shown to recapitulate aspect of early embryogenesis, including the formation of a complex three-dimensional architecture wherein cell-cell and cell-matrix interactions are thought to support the development of the three embryonic germ layers and their derivatives. Standard methods of EB formation include hanging drop and liquid suspension culture. Both culture systems maintain a balance between allowing ES cell aggregation necessary for EB formation and preventing EB agglomeration for efficient cell growth and differentiation. However, they are limited in their production capacity. In this paper, we established a new approach for the mass production of EBs in a scalable culture system. The rotary cell culture system (RCCS, STLV type) was adopted to produce EBs. The vessel was placed on its rotary base and the experiment started with a beginning rotation rate of approximately 8 r/min which has been previously determined empirically as the optimal initial speed to yield randomized gravitational vectors while minimizing fluid shear stress. To keep the aggregations pfloating in simulated microgravityq, the rotation rate was increased as the EBs visibly grew. The EB production efficiency was calculated when different cell densities were inoculated. The kinetic change of EBs was measured during the time course of EB formation. Compared with the traditional method of producing EBs with hanging drop, the multi-potential of the resulting EBs in RCCS was analyzed by the capability of cardiomyocyte genesis. The results showed that EBs could be produced by RCCS with high efficiency. The optimal cell density inoculated in RCCS was 10000 cells/ml, in which EB production was about twice higher than that in the suspending culture. Day 4-5 was the optimal time point for harvesting EBs. To clarify whether the differentiated potential of EBs might be affected by the microgravity produced by the rotary cell culture system, cardiogenic induction during ES cell differentiation was evaluated in our study. It was manifested by appearance of spontaneously and rhythmically contracting myocytes. In addition, immuno-histological and RT-PCR detection showed that the harvested EBs in RCCS exhibited the expected cardiac genesis and morphology. So, scalable production of EBs is obtained by RCCS. It will provide a useful approach to generate a large quantity of ES-derived cells for further research or application.