Objective Prepare biomimetic muitilayered scaffold which has similar structure of natural cartilage.Method By lyophilizing the scaffolds which were prefrozen at-20℃ and in liquid nitrogen successively,we prepared double-layered spongy scaffolds.By partially thawing the prefrozen samples and refreezing them in liquid nitrogen before the final liyophilization,we prepared biomimetic multilayered scaffolds with about 2mm thickness.XRD and FT-IR were used to confirm the interaction between collagen and chitosan.SEM was used to observe the morphologies of the scaffolds.The mechanical properties of pure chitosan scaffolds,pure collagen scaffolds,composite single-layered scaffolds and biomimetic multilayered scaffolds were compared both in dry and wet conditions.Results There was chemical interaction between collagen and chitosan.Composite materials will form better pore structure.The biomimetic multilayered scaffolds have upright pores,round pores and a dense layer from bottom to top of the scaffolds.The scaffolds have quite different mechanical properties between dry and wet state.Under wet state,the different layers of the biomimetic muitilayered scaffold have different mechanical properties.Results The biomimetic structure of the multilayered scaffold is very close to that of the natural articular cartilage,and the different layers of the biomimetic muitilayered scaffold had different mechanical properties under wet state.These are hopefully beneficial to help maintain the phenotypes of chondrocytes and promote the repairing effect of cartilage defects.

BACKGROUND: Researches indicated that bone marrow stem cells (BMSCs) could differentiated into neural stem cells in vitro, but what was the role of neural stem cells(NSCs) in the recovery of cortical injury,whether the NSC is capable of growing and migration in injured still remained unknown.OBJECTIVE: To explore the growing state of autograft NSC derived from crab-eating macaque BMSC transplanted in brain.DESIGN: Prospective case control study based on experimental animals.SETTING: Department of neurosurgery in a hospital of a military medical university.MATERIALS: This study was carried out at Center Laboratory of Neurological Research Institute, Zhujiang Hospital affiliated to the First Military Medical University of Chinese PLA. Six healthy adult crab-eating macaques were purchased from the South China Primate Animal Center.INTERVENTIONS: BMSCs harvested from six crab-eating macaques were cultured in vitro and induced to differentiate into neural stem cells, which then labeled by bromodeoxyuridine(BrdU) and autografted into brains.MAIN OUTCOME MEASURES: Brain tissues underwent hematoxylin and eosin(HE) staining and immunohistochemical staining before observed under optical microscope.RESULTS: The results of HE staining showed that the cell number in injured brain vas obviously higher in both instant and delayed transplanting groups than sham-transplanting group; moreover cells were proved reacting to BrdU by immunohistochemical staining in cortical injuries of both groups at 1-6 months following stem cells autograft, as well as at neighboring white matters at half year later, but no BrdU positive cells could be found in traumatic controls, sham-transplanting group and normal brains.CONCLUSION: NSCs derived from in vitro cultured BMSCs were proved capable of surviving, proliferating, differentiating and migrating in cortex after autograft, so that BMSCs is considered as replacing cells or the source of NSCs; moreover autograft stem cells could survive, proliferate and migrate in old cortical traumatic focus.

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Objective To evaluate the efficacy of Gant-Miwa and anal encircling procedure with Xiaozhiling injection for complete rectal prolapse.Methods Clinical data of 16 patients with complete rectal prolapse undergoing Gant-Miwa and anal encircling procedure with Xiaozhiling injection between Feb.2011 and Nov.2014 were retrospectively analyzed.Results All patients were operated successufully and theirs prolapse symptons disappeared.Urinary retention was found in 3 cases,2 cases were observed a replase who were infected in the area of the anal operation,1 cases was found delayed healing of perianal incision,and 2 cases were happened constipation duing to the rectal stenosis.Complications wasn't happened such as bleeding,perianal abscess,anal fistula,and so on.Thirteen cases were followed-up of average 2.2 years and 2 cases were observed postoperative recurrance.Conclusions Gant-Miwa and anal encircling procedure with Xiaozhiling injection for the treatment of complete rectal prolapse is a procedure with minimally trauma,fewer complications and low recurrence rate,which is worth promoting after innovation and improvement.

BACKGROUND: Bone marrow mesenchymal cells, multiple-potential non-hematopoiefic stem cells adhering to the wall in vitro culture, can be induced to proliferate and differentiate towards neurons and glia cells.OBJECTIVE: To investigate the growth state of cat bone marrow mesenchymal cells in vitro culture, as well as the capability to differentiate towards neural stem cells.DESIGN: A randomized sampling study.SETTING: Department of Neurosurgery, Zhujiang Hospital, Southern Medical University.MATERIALS: This study was carried out at the Central Laboratory of the General Military Neurological Research Institute, Zhujiang Hospital, Southern Medical University between January and December 2002. Twenty healthy home-raised cats, aged 1.0 - 2.0 years and weighing 2. 5 - 4.0 kg, male and female in half, were provided by the Animal Center of the First Military Medical University of Chinese PLA.INTERVENTIONS: Bone marrows were randomly aspirated from the left or right hindlimbs in order to separate bone marrow mesenchymal cells, then the bone marrow mesenchymal cells single cell suspension was co-cultured with neural stem cell culture media in vitro so as to induce differentiation to neural stem cells with tretinoin. CK2 type inverted optical microscope(Olympus,Japan) was used to observe the growth of bone marrow mesenchymal cells in vitro culture, as well as 4, 12, 24, 48 hours of induction upon eliminating or not eliminating the wall-adhering cells. Bone marrow mesenchymal cells in stem cell stage were identified under Olympus optical microscope with modified immunohistochemical staining.MAIN OUTCOME MEASURES: The growth state and the immunocytochemical staining of living bone marrow mesenchymal cells exposed to experimental intervention were observed under the Olympus inverted optical microscope.RESULTS: Data from the 20 cats were analyzed without loss. Reversed microscopic observation revealed that cat bone marrow mesenchymal cells becrame larger when cultured in vitro, which were rich in plasmic granules with prominence projecting, adhering to the wall and forming cell clones. These cells were then successively cultured, and imnunohistochemical staining analysis suggested that the passaged bone marrow mesenchymal cells could express neural stem cells-specific antigen Nestin and differentiate towards glia-like cells and neuron-like cells.CONCLUSION: Cat bone marrow mesenchymal cells possess the characteristics of stem cells; they can be amplified into cell clones and induced to express the property of neural glia cells and neuron-like cells under proper condition.

We investigated the roles of the crude antigen(CA) of Clonorchis sinensis and excretory secretory products (ESPs) in the polarization of Th1 and Th2 cells.Bone marrow-derived cells were generated from BALB/c mice and isolated into immature DCs;immature DCs were then treated with either CA (CA stimulated group),ESPs (ESPs stimulated group),LPS (positive control group) or PBS (negative control group) for 24 hours.Then the CD4+T cells were isolated from mouse spleen by using anti mouse-CD4 Microbeads,and further cocultured with stimulated DCs for another 72 hours.The purities of DCs and CD4+ T cells were evaluated by flow cytometry and the expressing levels of T-bet mRNA and GATA-3 mRNA were detected by real-time PCR.ELISA was used to detect the levels of IFN-γ and IL-4 cytokines in the supernatant.mRNA levels of T-bet and GATA-3 in the ESPs group were higher than those in PBS-stimulated group (P＜0.05).The concentrations of IFN-γ and IL-4 cytokines in the culture were increased in the ESPs group,compared with PBS stimulated group(P＜0.05).IFN-γ but not IL-4 was increased in CA group (P＜0.05).The results implied that CA might play a role in Th1 type immune response,and ESPs likely play roles in both Th1 and Th2 immune responses.

Objective To isolate and identify exosomes from serum samples of the patients with polymyositis / dermatomyositis (PM/ DM),and analyze their protein composition preliminarily.Methods Exosomes from serum samples of the patients with PM/DM were isolated and purified by the ExoQuickTM kit.The morphological characteristics and particle size of exosomes were determined by transmission electron microscope (TEM) and NanoSight analyzer,respectively.The surface markers of exosomes such as CD9,CD81 and Flotillin-2 were identified by western blot.The concentration and composition of exosome protein were determined by the BCA method and SDS-PAGE,respectively.Results The exosomes from serum samples of PM/DM patients displayed round or oval vesicles with membrane structure under TEM,and their diameter range was about (92 ± 67) nm.western blot showed that these exosomes expressed CD9,CD81 and Flotillin-2.The total protein concentrations of exosomes in the patients with PM/DM and healthy controls were 14.68 (6.00,32.55) μg/μL and 14.09 (8.00,23.28) μg/μL,respectively.SDS-PAGE showed that high-abundance proteins enriched in 55-70 kD in both PM/DM patients and healthy controls,and that there were different bands in 40-55 kD between them.Conclusion Exosomes are isolated from serum samples of the patients with PM/DM successfully,and their protein concentration and composition are analyzed preliminarily,which provides the experimental evidences for further finding differential proteins.

Objective To evaluate the effect of military performance stress on the changes of concentrations in salivary chromogranin A ( CgA) ,β-endorphin (β-EP) and salivary IgA ( sIgA) of submariners.Methods Twenty-nine submarine soldiers were selected, and their saliva samples were collected separately at the end of a long dive trip and nine months after relaxation ashore.In addition,the saliva samples of twenty-eight graduate students were collected as the normal control.The method of ELISA was used to detect the level of salivary CgA,β-EP and sIgA.Results After long-term dive training, the submariners showed significantly decreased CgA,β-EP and sIgA.Conclusion After a long term dive trip, chronic military performance stress is associated with the decline of salivary CgA,β-EP and sIgA, indicating that the function of sympathet-ic adrenal medulla is suppressed.The biological significance of these changes needs to be assessed in the future.

Objective To investigate the clinical efficacy and adverse events of methimazole ( MMI ) treatment for children with hyperthyroidism, and to identify the predictors of remission and relapse. Methods A total of379children(260girlsand119boys)diagnosedwithhyperthyroidismandtreatedbyMMIinGuangzhouWomenand Children's Medical Center from March, 2004 to July, 2014 were retrospectively analyzed. The average age at diagnosiswas(9.3±2.3)years(range2.0~15.9years). Results AftertreatmentwithMMIfor3and6months, the thyroid functions of 96. 3%(365/379) and 98. 9%(375/379) patients returned to normal, respectively. By the end of this study, 256(67. 5%) patients continued to use MMI treatment and 44 patients(11. 6%) dropped out. 79 patients(20. 8%) achieved remission, 35 patients (44. 3%) of whom experienced a later relapse. Children who achieved constant remission had significantly lower FT3 and FT4 levels at diagnosis compared with the relapsed children(P<0. 05 or P<0. 01). It was more likely to remain long-term remission for children turned to be euthyroid within 3 months after initiating MMI treatment(P<0. 05). The relieved patients with family history of thyroid diseases weremorelikelytoberelapsed(P<0.05). Therewerenosignificantdifferencesinage,gender,exophthalmos, initial goiter size, thyroid peroxidase autoantibody, and thyroglobulin antibody levels between the relieved and relapsed patients. The overall incidence of adverse events associated with MMI was 27. 7%, mainly elevated alanine aminotransferase, bilirubin, and neutropenia. Most(66. 7%) of adverse events occurred within the first three months of MMI treatment. Conclusion MMI has a good effect on pediatric hyperthyroidism, with low remission and high relapse rate. The low thyroid hormone concentrations at diagnosis and normalization of thyroid function within three months seem to be useful predictors of remission. Vigilance is needed concerning MMI-associated adverse events throughout the MMI treatment period, especially during the first trimester of MMI initiation.

Objective To assess the value of contrast-enhanced ultrasound (CEUS) on quantitative analysis of re?nal cortex perfusion in hypertensive rabbits model. Methods Hypertensive rabbit modal (n=10) were established by inject?ing N-nitro-L-arginin methylester (L-NAME). CEUS and Cystatin C (CysC) serum level analysis were performed at differ?ent time points:before and the 2nd, 4th, 6th and 8th week after injecting L-NAME. Time-intensity curve and area under curve (AUC) were analyzed quantatively while correlation of AUC and CysC were also analyzed. Results Serum level of Cys C in?creased significantly at the 6th week after L-NAME administration which is earlier than the increase of serum levels of Scr and BUN. AUC decreased at first then increased after L-NAME administration. Upon addition of L-NAME, rise time (RT) and peak intensity (PI) decreased while mean transit time (MTT), time from peak to one half (HPT) and time to peak (TTP) in?creased. Our study confirmed a positive correlation between AUC and Cys C (r=0.950, P<0.001). Conclusion Setting up rabbits model by L-NAME is convenient and reproducible, which is an useful tool in experimental study of preclinical and clinical phase of hypertensive renal injury. CEUS combining with CysC serum level analysis is considered as an effective technology for evaluating renal function in hypertensive patients.