Effect of lentinan against immunosuppression of lymphocytes cultured in simulated microgravity environment / 中国实验血液学杂志

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.

Cordyceps sinensis enhances lymphocyte proliferation and CD markers expression in simulated microgravity environment / 中国实验血液学杂志

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.

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice.</p><p><b>METHODS</b>Mice 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.</p><p><b>RESULTS</b>Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia.</p><p><b>CONCLUSION</b>E2 has a dose-related mitogenic effect on spermatogonia.</p>

Cloning of gdnf in the mouse testis and its expression in sertoli cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>Total 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.</p><p><b>RESULTS</b>gdnf cDNA was cloned successfully, and GDNF expressed after transfected into Sertoli cells.</p><p><b>CONCLUSION</b>This study provides a basis for culturing SSCs with gdnf-transfected Sertoli cells as the feeder layer.</p>

Differential analysis of proteomic profiles between cryptorchid and normal mouse testes / 生理学报

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.

The study of tissue-engineering bone for repair of segmental bone defects / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of tissue-engineering bone on repair of segmental bone defects.</p><p><b>METHODS</b>Segmental bone defect of 21mm was created at sheep left metatarsus, which was then implanted with tissue-engineering bone (the experimental group) and pure porous beta-TCP (the control group) respectively. The bone defect in the blank group was left without treatment. After the sheep were sacrificed at the 1st, 3rd, or 6th month postoperatively, the samples were taken and examined by radiological, histological and biomechanical methods as well as scanning electron microscopy. The sheep in the blank group were sacrificed at the 6th month postoperatively.</p><p><b>RESULTS</b>The osteoid tissue, woven bone and lamellar bone in the defect of the experimental group occurred earlier than in the control group. The new bone formed directly without through a cartilaginous intermediate in the experimental group, while the defect was repaired in a "creep substitution" way in the control group. At the 6th month, radiological and biomechanical tests revealed nearly complete repair of the bone defect of the experimental group, partial repair in the control group and non-healing in the blank group.</p><p><b>CONCLUSIONS</b>Tissue-engineering bone can repair bone defect, accelerating healing and without "creep substitution", which is a good option in repair of critical segmental bone defects. This study set up a basis for clinical applications in the future.</p>

Cloning of Bmi1 cDNA from mouse testis and its expression in E. coli BL21 / 中华男科学杂志

<p><b>OBJECTIVE</b>To produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21.</p><p><b>METHODS</b>Bmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET-28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot.</p><p><b>RESULTS</b>Mouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody.</p><p><b>CONCLUSION</b>There was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly.</p>

Reconstruction of dentin-pulp complex structure by tissue engineering technology / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the possibility of reconstruction of dentin-pulp complex by tissue engineering technology.</p><p><b>METHODS</b>Rat dental pulp stem cells were seeded into HA-TCP scaffold and incubated for 20 hours in vitro. Then the cell-scaffold complex was implanted subcutaneously into the dorsal side of nude mice. 8 weeks postimplantation, the samples were extracted for histological and immunohistochemical examinations.</p><p><b>RESULTS</b>Three strata of tissue were observed in the hole of HA-TCP scaffold. They were dentin-like tissue, predentin-like tissue and pulp-like tissue respectively from the inner surface of the pore to the center. Dentin tubules were obvious in predentin-like and dentin-like tissue lining from the pulp-like tissue through predentin-like tissue and dentin-like tissue. Cells localized along the edge of pulp-like tissue were dense and polarized, resembling odontoblasts. Immunohistochemical study demonstrated DSP and DMP1 expression in these odontoblast-like cells and in the area of predentin-like tissue.</p><p><b>CONCLUSIONS</b>Tissue-engineered rat dentin-pulp complex was reconstructed by seeding HA-TCP scaffold with rat dental pulp stem cells.</p>

Scalable production of embryoid bodies with the rotay cell culture system / 生理学报

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.

Experimental study of cardiac muscle tissue engineering in bioreactor / 中国医学科学院学报

<p><b>OBJECTIVE</b>This study investigates construction of cardiac muscle cell-porous collagen scaffold complex in a bioreactor so as to unveil the possibility of generating 3-dimensional cardiac muscle tissue under the environment that mimics microgravity in vitro.</p><p><b>METHODS</b>1-2-day old neonatal rat cardiac muscle cells were isolated by sequential digestion and pre-plating methods, then seeded onto porous collagen scaffold. The cell-collagen complex was transferred into rotary cell culture system (RCCS) and incubated for 7 days. Cells cultured in 75 ml flasks and constructs cultures on plates served as control. Morphological changes of the cells were observed by light microscope and metabolic rate was recorded. Ultrastructure of the cells growing in porous collagen was observed by transmission electron microscopy. Content of total DNA and protein in the newly-formed tissue were analyzed. H-E and anti-sarcomeric alpha-actin stains were performed in comparison with native cardiac muscle.</p><p><b>RESULTS</b>The isolated cardiac muscle cells adhered to the bottom of the flasks 24 hours after plating and began to beat spontaneously. When incubated for 7 days in RCCS, cell-collagen constructs of form a continuous outer tissue layer containing cells aligned with each other. The cell population in the interior of the construct was less in density than the outer part. Transmission electron microscopy demonstrated that subcellular elements characteristic of cardiac myocytes were in the outermost layer of constructs. A strongly positive stains of anti-sarcomeric alpha-actin suggested presence of cell population of differentiated cardiac myocytes in these constructs. Construct biomass was not significantly different from that in neonatal rat ventricle and approximately 40% of that in adult rat ventricles. Construsts in plates contained a few of cells which were less than those in RCCS. Metabolic activity of cells cultured in RCCS was higher than that in flasks and plates.</p><p><b>CONCLUSIONS</b>Dissociated cardiac muscle cells cultured on 3-dimensional scaffolds in RCCS under favorable conditions can form engineered constructs with structural and functional features resembling those of native cardiac tissue.</p>

Recent advances in islet transplantation and pancreatic stem cell research / 中国医学科学院学报

This paper reviewed recent advances in pancreatic islet transplantation research, including islet isolation, purification, culture, cryopreservation and immunoisolation. Latest progresses in induction of pancreatic stem cell and embryonic stem cell to differentiate into insulin-producing islets were also introduced. On the basis of the present situation and future development of islet transplantation-based therapies for diabetes, the author thought that allogeous islet transplantation is a main choice for type I diabetes today and pancreatic stem cell transplantation for tomorrow.

Experimental study of the isolation, culture and in chondrogenic differentiation of human bone mesenchymal stem cell / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Human 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.</p><p><b>RESULTS</b>Primary 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.</p><p><b>CONCLUSIONS</b>Percoll 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.</p>

Effects of bupivacaine and hyaluronidase on the proliferation of muscle satellite cells in adult rats in vivo / 中国应用生理学杂志

<p><b>UNLABELLED</b>To study the effects of Bupivacaine and hyaluronidase on the proliferation of adult rat muscle satellite cells in vivo.</p><p><b>METHODS</b>Immunohistochemistry, hematoxylin and eosin staining, electron micrograph were used.</p><p><b>RESULTS</b>(1) There are few rare desmin positive satellite cells lie in the myofibers of control group and Sterile saline group which are still continual. MMD of control and Sterile saline group is 0.66% +/- 0.57% and 2.48% +/- 1.13% respectively. Sterile saline group has no significant difference than that of the control (P > 0.05). (2) The myofibers of hyaluronidase group are basically continual. The number of desmin positive satellite cells are increased. MMD of Hyaluronidase is 2.52% +/- 1.41% which has no remarkable difference than that of the Sterile saline (P > 0.05). (3) Plentiful necrosis and degeneration myofibers can been seen in Bupivacaine group and Hyaluronidase + Bupivacaine group coinciding with the activation and proliferation of muscle satellite cells. The number of Desmin positive satellite cells are increased significantly and some of which have formed myotubes. MMD of Bupivacaine and Hyaluronidase + Bupivacaine is 19.01% +/- 4.74% and 22.41% +/- 7.64% respectively which have significant change than that of Sterile saline (P < 0.01).</p><p><b>CONCLUSION</b>The local anaesthetic Bupivacaine can induce the significant proliferation of myoblasts and the formation of myotubes in vivo. Hyaluronidase has no significant effect on the proliferation of satellite cells in vivo under this experimental condition.</p>