Performance and mechanism of amphiphilic polymeric chelator for enhanced removal of high concentrations of Cu(II) from wastewater.

An amphiphilic polymeric chelator (APC16-g-SX) grafted with sodium xanthate (SX) groups was successfully prepared for the efficient removal of high concentrations of Cu(II) from wastewater. The ordinary polymeric chelator (PAM-g-SX) based on linear polyacrylamide (PAM) was also prepared for comparative studies. The polymeric chelators were characterized by Fourier transform infrared spectroscopy (FT-IR), solid-state nuclear magnetic resonance (13C-NMR), gel permeation chromatography (GPC), elemental analyzer, and scanning electron microscope (SEM). The chelating performance of these polymeric chelators was investigated, and the mechanism of APC16-g-SX for enhanced removal of Cu(II) from wastewater was proposed based on fluorescence spectroscopy, cryo-scanning electron microscope (Cryo-SEM), energy-dispersive spectrometer (EDS), and X-ray photoelectron spectroscopy (XPS) tests. The results show that as the initial Cu(II) concentration in the wastewater increases, APC16-g-SX shows more excellent chelating performance than ordinary PAM-g-SX. For the wastewater with an initial Cu(II) concentration of 200 mg/L, the removal rate of Cu(II) was 99.82% and 89.34% for both 500 mg/L APC16-g-SX and PAM-g-SX, respectively. The pH of the system has a very great influence on the chelating performance of the polymeric chelators, and the increase in pH of the system helps to improve the chelating performance. The results of EDS and XPS tests also show that N, O, and S atoms in APC16-g-SX were involved in the chelation of Cu(II). The mechanism of enhanced removal of Cu(II) by APC16-g-SX can be attributed to the spatial network structure constructed by the self-association of hydrophobic groups that enhances the utilization of chelation sites.

Biomechanical and Mechanostat analysis of a titanium layered porous implant for mandibular reconstruction: The effect of the topology optimization design.

A porous scaffold/implant is considered a potential method to repair bone defects, but its mechanical stability and biomechanics during the repair process are not yet clear. A mandibular titanium implant was proposed and designed with layered porous structures similar to that of the bone tissue, both in structure and mechanical properties. Topology was used to optimize the design of the porous implant and fixed structure. The finite element analysis was combined with bone "Mechanostat" theory to evaluate the stress and osteogenic property of the layered porous implant with 3 different fixation layouts (Model I with 4 screws, Model II with 5 screws and Model III with 6 screws) for mandibular reconstruction. The results showed that Model III could effectively reduce the stress shielding effect, stress within the optimized implant, defective mandible, and screws were respectively dropped 48.18%, 44.23%, and 57.27% compared to Model I, and the porous implant had a significant stress transmission effect and maintained the same stress distribution as the intact mandible after the mandibular defect was repaired. The porous implant also showed a significant mechanical stimulation effect on the growth and healing of the bone tissue according to the bone "Mechanostat" theory. The combination of porous structure with the topology technique is a promising option to improve the mechanical stability and osteogenesis of the implant, and could provide a new solution for mandibular reconstruction.

Exosomes from adipose­derived stem cells promote chondrogenesis and suppress inflammation by upregulating miR­145 and miR­221.

Osteoarthritis (OA) is one of the most prevalent joint disorders globally. Patients suffering from OA are often obese and adiposity is linked to chronic inflammation. In the present study, the potential of using exosomes isolated from adipose­derived stem cells (ADSCs) as a therapeutic tool for reducing chronic inflammation and promoting chondrogenesis was investigated using patient­derived primary cells. First, it was tested whether patient­derived ADSCs could differentiate into chondrogenic and osteogenic lineages. The ADSCs were then used as a source of exosomes. It was found that exosomes isolated from ADSCs, when co­cultured with activated synovial fibroblasts, downregulated the expression of pro­inflammatory markers interleukin (IL)­6, NF­κB and tumor necrosis factor­α, while they upregulated the expression of the anti­inflammatory cytokine IL­10; without exosomes, the opposite observations were made. In addition, inflammation­inflicted oxidative stress was induced in vitro by stimulating chondrocytes with H2O2. Treatment with exosomes protected articular chondrocytes from H2O2­induced apoptosis. Furthermore, exosome treatment promoted chondrogenesis in periosteal cells and increased chondrogenic markers, including Collagen type II and ß­catenin; inhibition of Wnt/ß­catenin, using the antagonist ICG­001, prevented exosome­induced chondrogenesis. Periosteal cells treated with exosomes exhibited higher levels of microRNA (miR)­145 and miR­221. The upregulation of miR­145 and miR­221 was associated with the enhanced proliferation of periosteal cells and chondrogenic potential, respectively. The present study provided evidence in support for the use of patient­derived exosomes, produced from ADSCs, for potential chondrogenic regeneration and subsequent amelioration of osteoarthritis.

Bionic mechanical design and 3D printing of novel porous Ti6Al4V implants for biomedical applications.

In maxillofacial surgery, there is a significant need for the design and fabrication of porous scaffolds with customizable bionic structures and mechanical properties suitable for bone tissue engineering. In this paper, we characterize the porous Ti6Al4V implant, which is one of the most promising and attractive biomedical applications due to the similarity of its modulus to human bones. We describe the mechanical properties of this implant, which we suggest is capable of providing important biological functions for bone tissue regeneration. We characterize a novel bionic design and fabrication process for porous implants. A design concept of "reducing dimensions and designing layer by layer" was used to construct layered slice and rod-connected mesh structure (LSRCMS) implants. Porous LSRCMS implants with different parameters and porosities were fabricated by selective laser melting (SLM). Printed samples were evaluated by microstructure characterization, specific mechanical properties were analyzed by mechanical tests, and finite element analysis was used to digitally calculate the stress characteristics of the LSRCMS under loading forces. Our results show that the samples fabricated by SLM had good structure printing quality with reasonable pore sizes. The porosity, pore size, and strut thickness of manufactured samples ranged from (60.95± 0.27)% to (81.23±0.32)%, (480±28) to (685±31) µm, and (263±28) to (265±28) µm, respectively. The compression results show that the Young's modulus and the yield strength ranged from (2.23±0.03) to (6.36±0.06) GPa and (21.36±0.42) to (122.85±3.85) MPa, respectively. We also show that the Young's modulus and yield strength of the LSRCMS samples can be predicted by the Gibson-Ashby model. Further, we prove the structural stability of our novel design by finite element analysis. Our results illustrate that our novel SLM-fabricated porous Ti6Al4V scaffolds based on an LSRCMS are a promising material for bone implants, and are potentially applicable to the field of bone defect repair.

SOCS1 and SOCS3 Target IRF7 Degradation To Suppress TLR7-Mediated Type I IFN Production of Human Plasmacytoid Dendritic Cells.

Type I IFN production of plasmacytoid dendritic cells (pDCs) triggered by TLR-signaling is an essential part of antiviral responses and autoimmune reactions. Although it was well-documented that members of the cytokine signaling (SOCS) family regulate TLR-signaling, the mechanism of how SOCS proteins regulate TLR7-mediated type I IFN production has not been elucidated yet. In this article, we show that TLR7 activation in human pDCs induced the expression of SOCS1 and SOCS3. SOCS1 and SOCS3 strongly suppressed TLR7-mediated type I IFN production. Furthermore, we demonstrated that SOCS1- and SOCS3-bound IFN regulatory factor 7, a pivotal transcription factor of the TLR7 pathway, through the SH2 domain to promote its proteasomal degradation by lysine 48-linked polyubiquitination. Together, our results demonstrate that SOCS1/3-mediated degradation of IFN regulatory factor 7 directly regulates TLR7 signaling and type I IFN production in pDCs. This mechanism might be targeted by therapeutic approaches to either enhance type I IFN production in antiviral treatment or decrease type I IFN production in the treatment of autoimmune diseases.

Increased circulating levels of neurotrophins and elevated expression of their high-affinity receptors on skin and gut mast cells in mastocytosis.

Mastocytosis is a rare heterogeneous disease characterized by increase of mast cells (MCs) in different organs. Neurotrophins (NTs) have been shown to promote differentiation and survival of MCs, which in turn represent a major source of NTs. Thus, a contribution of NTs to mastocytosis seems highly conceivable but has not yet been investigated. We could demonstrate expression of high-affinity NT receptors tropomyosin-related kinase A (TrkA) for nerve growth factor (NGF)-ß, TrkB for brain-derived neurotrophic factor, and NT-4 and TrkC for NT-3 on skin MCs; and of TrkA and TrkC on intestinal MCs of patients with mastocytosis. Moreover, increased expression of NGF-ß; NT-3; TrkA, TrkB, and TrkC; and isoforms truncated TrkB-T1 and truncated TrkC were observed on skin MCs. Patients with mastocytosis featured elevated serum levels of NGF, NT-3, and NT-4. Levels of NGF-ß and NT-4 correlated with tryptase levels, suggesting a link between MC load and blood levels of NGF and NT-4. Migration of CD117+ progenitor cells from the blood was enhanced toward NGF-ß gradient in both mastocytosis and controls. Together with enhanced NT levels, the elevated expression of modified Trk receptors on skin and gut MCs might contribute to the pathophysiology of mastocytosis in autocrine and paracrine loops.

[Analysis of reason and strategy for the failure of posterior pedicle screw short-segment internal fixation on thoracolumbar fractures].

OBJECTIVE: To analyze the reason and strategy for failure of posterior pedicle screw short-segment internal fixation on thoracolumbar fractures. METHODS: From March 2008 to December 2010,the clinical data of 18 patients with thoracolumbar fracture failed in posterior pedicle screw short-segment internal fixation were retrospectively analyzed. There were 11 males and 7 females with an average age of 37.2 years (ranged, 19 to 63). The time from the first operation to complication occurrence was from 6 to 44 months with an average of 14.3 months. Of them,fusion failure was in 7 cases (combined with screw breakage in 4 cases), the progressive neuro-dysfunction was in 5 cases,the progressive lumbodorsal pain was in 6 cases. All 18 patients with kyphosis were treated with anterior internal fixation remaining posterior fixation (9 cases) and anterior internal fixation after posterior fixation removal (9 cases). RESULTS: All the patients were followed up from 18 to 50 months with an average of 30.5 months. No intetnal fixation loosening and breakage were found, moreover, X-ray and lamellar CT showed bone healing well. Preoperative, postoperative at 3 months and at final follow-up, ODI score was respectively 31.6+/-5.1, 8.6+/-5.7, 8.3+/-3.2; VAS score was respectively 7.2+/-2.3, 2.3+/-0.7, 2.1+/-1.1; kyphosis angle was respectively (-21.2/-+7.8 degreeso, (-5.3+/-6.8 degrees ), (-5.8+/-7.8 )degrees. Compared with preoperative data ,above-listed items had obviously ameliorated(P<0.05). CONCLUSION: Treatment of thoracolumbar fracture with posterior pedicle screw short-segment internal fixation may result in the complications such as bone nonunion ,internal fixation breakage and progressive kyphosis. Anterior reconstruction may be a good strategy for the failure of posterior operation.

Attenuated TGF-ß1 responsiveness of dendritic cells and their precursors in atopic dermatitis.

The responsiveness of DCs and their precursors to transforming growth factor beta1 (TGF-ß1) affects the nature of differentiating DC subsets, which are essential for the severity of atopic dermatitis (AD). To evaluate TGF-ß signaling in monocytes and monocyte-derived DCs of AD patients compared with that of controls, in vitro generated Langerhans cell (LC) like DCs, expression of TGF-ß receptors, phospho-Smad2/3 and Smad7 were evaluated. Furthermore, TNF-α expression and synergistic effects of TNF-α upon TGF-ß signaling and DC generation were evaluated. We found LC-like DC differentiation of monocytes from AD patients in response to TGF-ß1 was remarkably reduced and TGF-ß1 receptor expression was significantly lower compared with that of healthy controls. Attenuated TGF-ß1 responsiveness mirrored by lower phospho-Smad2/3 expression after TGF-ß1 stimulation and higher expression of inhibitory Smad7 was observed in monocytes from AD patients. During DC generation, mRNA expression of Smad7 was relatively higher in LC-like DCs of AD patients. Lower TNF-α expression of monocytes from AD patients might further contribute to attenuated TGF-ß signaling in the disease since TNF-α had synergistic effects on TGF-ß1 signaling and LC generation through mediating the degradation of Smad7. Our results demonstrate alleviated TGF-ß1 signaling together with the amount of soluble co-factors might direct the nature of differentiating DCs.

The immunoglobulin E-Toll-like receptor network.

Allergens and microbial antigens impact on effector cells and antigen-presenting cells in allergic diseases. Allergens bind specifically to immunoglobulin E (IgE) linked to the high-affinity receptor for IgE (FcepsilonRI) and stimulate a cascade of cellular events. This leads to the release of mediators of allergic reactions by effector cells on the one hand and antigen uptake, presentation and T cell priming by antigen-presenting cells on the other hand. In contrast, microbial antigens are recognized by pattern-recognition receptors (PRRs) of the innate immune system, to which Toll-like receptors (TLRs) belong. In view of the high number of microbial antigens, allergens and other soluble ligands in the cellular microenvironment in vivo, it is very likely that not only separate, but also concomitant stimulation of both receptor types, i.e. FcepsilonRI and TLRs, occurs frequently under physiological conditions and in particular in the context of allergic and infectious disorders. Thus, interaction of TLRs with FcepsilonRI and regulation of the IgE synthesis is of critical immunological importance, since it might profoundly modify the activation state of cells and the nature of the evolving immune responses. Current knowledge about the cross talk of TLRs with FcepsilonRI- and IgE-related immune responses is discussed herein.

Human plasmacytoid dendritic cells support Th17 cell effector function in response to TLR7 ligation.

Signals involved in the commitment of Th17 differentiation are of substantial interest for our understanding of antimicrobial defense mechanisms and autoimmune disorders. Various ways in which myeloid dendritic cells modulate Th17 differentiation have been identified. However, although plasmacytoid dendritic cells (PDCs) are regarded as important players in antiviral/antimicrobial host defense and autoimmune diseases, a putative modulatory role of PDCs in Th17 differentiation has not yet been elucidated in detail. We demonstrated that PDCs are capable of promoting Th17 differentiation in response to TLR7 stimulation. Further, both the differentiation of Th17 cells from naive T cells and the amplification of Th17 effector functions of memory T cells are promoted by PDCs after TLR7 activation. Our data are of strong clinical relevance because TLR7 activation in PDCs might represent one of the missing links between innate and adaptive immune mechanisms and contribute to the amplification of Th17-driven autoimmune disorders as well as viral host defense.

Molecular pathogenesis and clinical implications of eczema herpeticum.

A subgroup of patients with atopic dermatitis develops one or more episodes of a severe viral skin infection caused by herpes simplex virus superimposed on eczematous skin lesions. This condition is named atopic dermatitis complicated by eczema herpeticum. Characteristic features of patients developing eczema herpeticum include an early age of onset of atopic dermatitis with a persistent and severe course into adulthood, predilection for eczematous skin lesions in the head and neck area, elevated total serum IgE levels and increased allergen sensitisation. Deficiencies at the level of both the innate and the adaptive immune system, which have been identified in atopic dermatitis, are much more pronounced in this subgroup. Predisposing cellular factors include a reduced number of plasmacytoid dendritic cells in the epidermis and a modified capacity of these cells to produce type I interferons after allergen challenge. In addition, lower levels of antimicrobial peptides in the skin of atopic dermatitis patients, resulting in part from a Th2-prone micromilieu, contribute to the lack of an effective defence against viral attack. In this review, we summarise the current knowledge of the molecular pathogenesis of eczema herpeticum.

Tacrolimus and TGF-beta act synergistically on the generation of Langerhans cells.

BACKGROUND: The proportion of dendritic cell subpopulations in the skin is important for the severity of atopic dermatitis because topical treatment with tacrolimus leads to rapid depletion of inflammatory dendritic epidermal cells, whereas Langerhans cells (LCs) predominate in cured sites. OBJECTIVES: The effects of tacrolimus and TGF-beta1 on LC differentiation and the idea of tacrolimus skewing the differentiation of epidermal precursors to LCs were evaluated. METHODS: The presence of LC markers, MHC, and costimulatory molecules and stimulatory capacity toward T cells of monocyte-derived LCs were analyzed. Skin samples of patients with atopic dermatitis were assessed by means of immunofluorescence microscopy before and after tacrolimus treatment. TGF-beta production of skin cells was analyzed. RESULTS: Tacrolimus and TGF-beta1 act synergistically on the generation of LCs and the expression of CD40, CD80, CD86, CD83, and MHC II; stabilize TGF-beta receptor II expression; and decrease the stimulatory capacity of LCs toward T cells. In vivo the number of epidermal LCs in tacrolimus-treated skin increased. CONCLUSION: The synergism between TGF-beta1 and tacrolimus leads to the generation of LCs, reduced expression of costimulatory and MHC II molecules, and reduced stimulatory activity. Shifting the balance of the dendritic cell population to LCs might be of major importance for the therapeutic effect of tacrolimus.

Toll-like receptor 4 ligation enforces tolerogenic properties of oral mucosal Langerhans cells.

BACKGROUND: Despite high bacterial colonization, acute infections are rare in the oral mucosa, implicating tolerogenic predominance. Bacterial antigens like LPSs are recognized by innate immunity receptors such as Toll-like receptor 4 (TLR4), associated with LPS receptor (CD14). OBJECTIVES: Toll-like receptor 4 agonist monosphoryl lipid A has been successfully used as adjuvant in subcutaneous immunotherapy, suggesting reinforcement of allergen-specific tolerance. Recently sublingual immunotherapy (SLIT) has been shown to be an effective alternative to subcutaneous immunotherapy. We observed CD14 expression on human oral Langerhans cells (oLCs), representing a major target of SLIT. However, not much is known about TLR4 expression and its effect on oLCs. METHODS: Cell suspensions were obtained by trypsinization of human oral mucosa and analyzed by flow cytometry, RT-PCR, cytometric bead arrays, ELISA, and mixed lymphocyte reactions. RESULTS: We could show that oLCs express TLR4, and its ligation by monosphoryl lipid A upregulated expression of coinhibitory molecules B7-H1 and B7-H3 while surface expression of costimulatory molecule CD86 was concomitantly decreased. Furthermore, TLR4 ligation on oLCs increased their release of the anti-inflammatory cytokine IL-10 and decreased their stimulatory capacity toward T cells. Moreover, TLR4-ligation on oLCs induced IL-10, TGF-beta1, Forkhead box protein 3, IFN-gamma, and IL-2 production in T cells. CONCLUSION: In view of these data, TLR4-ligation on oLCs might not only play a role in pathogen recognition for efficient immunity but also contribute to the tolerogenic state predominating in the oral cavity.

Inhibitory oligodeoxynucleotides downregulate herpes simplex virus-induced plasmacytoid dendritic cell type I interferon production and modulate cell function.

Recognition of nucleic acids by TLR9 expressed by human plasmacytoid dendritic cells (PDC) plays a key role in the defense against viral infections. Upon microbial pathogen stimulation, PDC secrete large amounts of type I interferon and arbitrate thereby both innate and adaptive immune mechanisms. Unmethylated CpG motifs, which are an integral part of bacterial or viral DNA, are used in vitro and in vivo to activate the TLR9 pathway, whereas inhibitory oligodeoxynucleotide (iODN) are capable of depressing TLR9 signaling. In this study we demonstrate that TTAGGG motifs containing iODN efficiently block the TLR9 signaling in terms of herpes simplex virus (HSV)-induced type I interferon production by PDC. However, iODN, as well as control ODN, still promote PDC maturation with upregulated expression of costimulatory molecules, major histocompatibility complex molecules, and other signs for PDC maturation. Furthermore, iODN and control ODN incubated PDC demonstrate increased T-cell stimulatory functions. Coculture experiments with autologous T cells indicate that iODN-treated PDC induce more CD4(+)CD25(+)Foxp3(+) T regulatory cells from naive CD4(+) T cells and preincubation of HSV-stimulated PDC with iODN upregulated T cells' IFN-gamma production. These data indicate that iODN, while blocking type I interferon production by PDC, modify PDC activation and maturation as well as T-cell priming and stimulation. Knowledge about the different functions of iODN on PDC elucidated might be crucial for immunotherapeutic strategies in which iODN motifs are used to prevent the interaction of CpG-DNA with TLR9 to calm down specific immunological responses, because our data indicate that iODN might not only have inhibitory functions but also be effective activators of immune cells.

[Mesenchymal stem cells do not differentiate into "quasi-sperm"].

OBJECTIVE: To determine whether the surviving mesenchymal stem cells (MSCs) in the testis after transplantation can differentiate into quasi-sperm. METHODS: (1) Making an animal model with sterilized testes. Forty 4-week old white male BASB/C mice were used to establish an animal model with sterilized testes and divided randomly into an experimental and a control group. (2) Cell preparation. The MSCs from 10 gray male 129-mice were isolated, cultured and purified by Percoll density gradient centrifugation combined with the adherent method. When the MSCs grew to an adequate number, they were made into a cell suspension with NS at a concentration of 1 million cells/ml. (3) Xenogeneic transplantation of the MSCs into the testis. The MSC suspension was blindly injected into the testes of the mice in the experimental group and NS into the testes of the controls. (4) Post-transplantation observation. Forty white female BASB/C mice were adopted, each put into a box with a male mouse from the experimental group or the control group, and then observed for pregnancy. RESULTS: In the experimental group, 8 cases of pregnancy (40%) were observed at 31-46 d (38.5 d on average), the offspring all white. In the control group, only 1 case of pregnancy (5%) was seen at 45 d, the offspring all white, too. It was suggested that the MSCs of the 129-mice failed to differentiate into functional quasi-sperm and pass their genes to their offspring, as would expectedly have been presented by a mixture of black and white. The pregnancy rates of the two groups were significantly different (P < 0.05), which indicated that MSCs could promote the healing of the testis damage. CONCLUSION: MSCs cannot differentiate into quasi-sperm after heterogeneity transplantation into the testis, but can promote the healing of the testis damage.

[Making of the animal model with sterilized testes].

OBJECTIVE: To explore the methods of making an animal model with sterilized testes. METHODS: (1) X-ray local irradiation. Seventy 8-10-week-old male mice were equally divided into 6 experiment groups and a control group. The testes of the mice in the 6 experiment groups were irradiated sequentially by 1000, 1200, 1400, 1600, 1800 and 2000 cGy X-ray for 10 minutes, while those in the control group remained untreated. And then the pregnancy test was performed. (2) Cyclophosphamide injection. Forty 4-5-week-old male mice were divided into 3 experiment groups and a control group, the former treated with different doses of Cyclophosphamide via ip and the latter Natiichloridi Saline (N.S.) via i.p., followed by the pregnancy test. (3) Diphereline injection. Twenty 8-10-week-old male mice were equally divided into an experiment group and a control group, the former treated with Diphereline via ip and the latter N.S. via i.p., followed by the pregnancy test. (4) Identification by such pathologic examinations as TUNE1. technology, HE staining and immunohistochemical staining. RESULTS: (1) X-ray local irradiation. The male mice of Group 1 and 2 made their female partners pregnant respectively 10 and 15 days after the X-ray irradiation, but not those of Group 3 and 4 in our 3-month observation, and those of Group 5 and 6 died respectively 2 and 5 days after the X-ray irradiation. By comparison, the controls got their female partners pregnant within 3 days after placed together. (2) Cyclophosphamide injection. The male mice of Group 1 gained weight about 7 g and achieved pregnancy 9-14 days after drug termination, those of Group 2 gained around 4 g but failed to effect pregnancy, and those in Group 3 lost weight and died respective at 3, 4 and 5 weeks during the medication, while the controls all got their female partners pregnant within 3 days after put together. (3) Diphereline injection. The 10 male mice of the experiment group effected pregnancy 3 weeks after drug termination, while the 10 controls achieved the same result with 3 days after placed together. (4) Pathologic identification: TUNEL technology showed that apoptotic cells were occasionally seen (0.71 +/- 0.12)% in the testis tissue of the control group and remarkably increased (10.36 +/- 1.48)% in the model group, with significant difference between the two groups (P < 0.05). HE staining revealed normal testis tissues and convoluted seminiferous tubules with large numbers of germ cells in the control group, but atrophied convoluted seminiferous tubules and estranged cell linkage with only Ledig's cells but no germ cells in the model group. Immunohistochemical staining showed that the positive expression rates of CD29, Hsp90alpha and CD117 were respectively (50.30 +/- 5.2)%, (41.6 +/- 3.5)% and (73.6 +/- 3.7)% in the control group, as compared with (1.3 +/- 0.2)%, 0% and (1.6 +/- 0.3)% in the model group, with significant difference (P < 0.01). The positive expression rate of p53 was (19.7 +/- 0.8)% in the control group, significantly different from that of the model group, which was (39.4 +/- 2.9)% (P < 0.01). CONCLUSION: The animal model with sterilized testes can be made either by X-ray local irradiation of the testis or by Cyclophosphamide injection via i.p..

[Transplantation of mouse bone marrow mesenchymal stem cells into the xenogeneic testis].

OBJECTIVE: To study transplantation of mouse bone marrow mesenchymal stem cells (MSCs) into the xenogeneic testis. METHODS: (1) The tibias and femurs were dissected from 5-6-week-old mice. The marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured and purified in vitro by Percoll density gradient centrifugation combined with adherent method. (2) MSCs of the third generation were adopted and marked with Hoechest33342 for observation, and then made into cell-suspending fluid. (3) The marked MSCs were transplanted into the testis of the xenogeneic mouse by testis net injection. The biopsies of the testis tissues were carried out at different time and made into frost slices at three sites for observation. RESULTS: (1) A lot of purified MSCs were obtained at the third generation. (2) The nucleoli of the marked MSCs showed light-yellow under the fluoroscope. (3) Xenogeneic transplantation of mouse bone marrow MSCs by testis net injection was successful, without immunoreaction. On the 1 st day after transplantation, MSCs only concentratively distributed in the medial slices, the nucleoli being light-yellow; On the 1 st and 3 rd day, MSCs dispersively distributed in the medial slices; On the 6th, 9th and 12th day, MSCs presented in all the slices of the three sites, some ranging tubally; On the 15th and 18th day, the fluorescence of MSCs weakened; On the 21 st day, the fluorescence of MSCs disappeared. CONCLUSION: Transplantation of mouse bone marrow MSCs into the xenogeneic testis by testis net injection is effective and feasible, without immunoreaction. MSCs can survive after transplantation.

[Isolation and culture of mouse marrow mesenchymal stem cells (MSCs) and biological characteristics of MSCs cultured in conditions for spermatogonia in vitro].

OBJECTIVE: To isolate, culture and purify mouse bone marrow mesenchymal stem cells (MSCs) and observe the main biological characteristics of MSCs cultured in conditions for spermatogonia in vitro. METHODS: The tibias and femurs were dissected from 5 - 6-week old mice and the marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured, purified in vitro by Percoll density gradient centrifugation combined with adherent method and identified by dynamic observation of stem cell characteristics by transmission electron microscope, HE staining, and immunohistochemical detection of cell markers. The quantities of such cytokines as IL-6, IL-8, G-CSF and SCF in culture liquid with MSCs were measured by ELISA, and compared with those of the control group. MSCs of the third generation were divided into two groups to be induced and cultured. MSCs of the control group were cultured with basal medium, while those of the experimental group with conditional medium. The results were analysed by microscopic observation, HE staining and immunohistochemical methods. RESULTS: Pure MSCs were obtained. The cultured cells, with stem cell characteristics, shuttle-shaped at HE staining, immature under the transmission electron microscope and CD44 and CD90 positive by immunohistochemical detection, could be identified as MSCs. Compared with the control group, the quantities of IL-6, IL-8, G-CSF and SCF in the experimental group increased significantly (P < 0.05). The shapes of MSCs changed and immunohistochemical staining for CD27, CD119 and Oct-4 was positive in the experimental group, but both were just the opposite in the control group. CONCLUSION: Pure MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method and identified by dynamically observing stem cell characteristics, HE staining, observation under the transmission electron microscope and immunohistochemical detection of cell markers. MSCs can secrete cytokines such as IL-6, IL-8, G-CSF, SCF, and so on. MSCs cultured in conditions for spermatogonia may show some biological characteristics of spermatogonia.

[Study on the animal model for severe acute respiratory syndrome].

To screen small animals susceptible to SARS-CoV, five species of animals, including guinea pig, hamster, albino hamster, chicken and rat, were experimentally infected with SARS-CoV strain BJ-01 by different routes. On the basis of this, further cynomolgus and rhesus macaques were selected and experimentally inoculated SARS-CoV, the quality they serve as animal model for SARS was evaluated. The results showed that, all five species of small animals chosed were not susceptible to SARS-CoV, no characterized changes in clinical sign and histopathology were observed after infection, but from the lung samples of large rat and pig guinea, the genomic RNA of SARS-CoV could be detected by RT-PCR at day 14 post infection, this suggested that SARS-CoV could replicate in these animals. After inoculated with SARS-CoV, all inoculated cynomolgus and rhesus macaques had developed interstitial pneumonia of differing severity. These changes on histopathology were similar to that seen in SARS patients, but the pathological lesions were less severe than that of human. Except interstitial pneumonia, no other characterized pathological changes were observed. This suggested cynomolgus and rhesus macaques were not the ideal animal model for SARS in fact, but they could serve as animal model for SARS when a more ideal animal model is absent.