BACKGROUND:Acelular matrix which is decelularized but retains the matrix components in the nature materials can reduce the immunogenicity of natural materials effectively and keep the material mechanical strength. OBJECTIVE:To prepare the natural biological scaffold material by using elution method to remove the cels in the rabbit costicartilage matrix. METHODS:After removal of the surrounding tissues, the costicartilage samples from New Zealand white rabbits were randomly divided them into three groups: costicartilages untreated as normal control group, detergent-enzyme digestion for 48 hours as 48-hour treatment group, detergent-enzyme digestion for 96 hours as 96-hour treatment group. Hematoxylin-eosin staining and electron microscope were used to observe decelularized effects. At 7 days of induction, bone marrow mesenchymal stem cels, 3×109/L, were colected and co-cultured with alogeneic acelular costicartilage matrixin vitro. At 3 and 7 days of co-culture, the composite was taken for observation of cel growth on the cel-free substrate surface under electron microscope. RESULTS AND CONCLUSION:Two or three cartilage cels were closely packed in each cartilage pit, began to depigment using the detergent-nuclease digestion method, and then completely depigmented at 96 hours after treatment. At 3 days of co-culture, there were a large amount of polygon-shaped adherent cels with pseudopodiasobserved on the acelular matrix surfacein vitro, and cels could proliferate and divide in partial regions. At 7 days of co-culture, adherent cels spread mostly throughout the acelular matrix surface, these cels were flat-shaped and extended with multiple interconnected processes. Mass of secreted excelular matrixes were deposited on the matrix surface and showed frost-like changes, indicating the prepared acelular matrix has favorable cytocompatibility.

Objective Taking CNE-2Z multicellular spheroids (MCSs) as the simulation of solid tumors, to investigate the role of SAPK/JNK signaling pathway in multicellular resistance to radiotherapy for human nasopharyngeal carcinoma (NPC). Methods Human NPC cell line CNE-2Z were cultured into multicellular spheroids by using liquid overlay technique, then divided into control MCSs, irradiated MCSs (average dose in one minute: 2 Gy), sp-600125(a specific inhibitor for SAPK/JNK signaling pathway)+irradiated MCSs, sp-600125+MCSs. Western blotting was employed to analyze the activity of SAPK/JNK signaling pathway in MCSs, and the expression of Caspase-3 protein before and after sp-600125 treatment; X-ray induced cell apoptotisis in MCSs before and after sp-600125 treatment was detected by TUNEL. Results The level of SAPK/JNK phosphorylation in MCSs was a dynamic course after radiation, and the phosphorylation peaked at 2 h after irradiation; The apoptotic rate of MCSs (P

AIM:To investigate the immune depressive effect on the reactive T cells and to explore the immunologic injury mechanism of beta cells of islet in type 1 diabetes mellitus(DM-1).METHODS:pAd5/PD-L1-GPI adenovirus vector with target gene was constructed and transfected into NIT cells which are known as a mouse insuloma cell line.The highly expressed membrane protein of PD-L1-GPI was confirmed by Western blotting.The peripheral blood non-adherence lymph leukocytes and target cells were cultured to detect lymph leukocyte proliferation and the T cell function.The level of IL-2,TNF-? and IFN-? were detected in the cell culture fluid.RESULTS:Compared with the control group,the NIT cells modified with PD-L1-GPI inhibited the sensitized lymph leukocyte proliferation effectively and down-regulated the level of some cytokine secretions such as IL-2,IFN-? and TNF-?(P

ObjectiveTo explore the characteristics of traditional Chinese medicine (TCM) syndrome differentiation in chronic complications of type 2 diabetes mellitus (DM).Methods124 patients with chronic complications of type 2 DM were scored by 5 grades according as the severities of their symptoms. There were 5 kinds of patterns such as deficiency of Qi, deficiency of Yin, deficiency of Yang, blood stasis and retention of phlegm and fluid by which the TCM syndrome differentiation was generalized.ResultsThe sequence of TCM patterns was deficiency of Yin, blood stasis, deficiency of Qi, deficiency of Yang and retention of phlegm and fluid, and the syndrome of the two formers were greater than 50%. The proportion of unity of deficient and excessive pattern was 80.5%. Three larger syndrome types were deficiency of both Yin and Yang combined with blood stasis (17.7%), Qi-Yin deficiency with blood stasis ( 16.9 %) and Yin deficiency with blood stasis (16.9%). There was a statistically significant difference in TCM syndromes which were divided into different groups by course of diseases (P<0.05). At onset of DM, the typical symptoms were less observed in the group whose course of disease was less than 5 years, and only 39.1% of patients had the typical symptoms. But at the same time, the prevalences of hypertension and hyperlipidemia were higher in this group than in the others, respectively 63.0% and 87.0%.ConclusionThe primary syndrome is unity of deficient and excessive pattern in chronic complications of type 2 DM and deficiency of Qi and blood stasis are the commonest patterns in course of DM.

BACKGROUND: A few mesenchymal stem cells (MSCs) can be found in peripheral blood.OBJECTIVE: To isolate rabbit peripheral blood MSCs and induce its differentiation into osteoblasts.DESIGN, TIME AND SE'I-I'ING: The cytological in vitro study was performed at the Central Laboratory of Shangdong Provincial Hospital from June to December 2008.MATERIALS: A total of 6 New Zealand rabbits were purchased from Shandong Academy of Agricultural Sciences. α-MEM and L-DiEM were bought from Hyclone.METHODS: Full-thickness skin (3 cm×3 cm) (dorsal muscular layer was left) was incised at various sites of rabbit back, every 3days. Incised skin was dressed following orthotopic transplantation. Each rabbit received four consecutive wounds. Peripheral blood was collected from femoral vein before injury and 1 week after injury. MSCs were harvested from peripheral blood by density gradient cantrifugation. MSCs were divided into 2 groups, which were respectively incubated in α-MEM supplemented with 10% fetal bovine serum and L-DiEM. Cells at passage 2 and 1 ×105/cm2 were incubated in a 12-well plate and induced with H-DiEM containing osteogenic inductor.MAIN OUTCOME MEASURES: The following parameters were measured: cell morphology before and after injury; colony forming efficiency of MSCs; outcome of osteogenic induction.RESULTS: Following primary medium change and before injury, obtained cells were normal. Twenty-four hours following incubation and after injury, MSCs were spindle or polygonal, and adhered to the wall. 5-6 days later, cell colonies appeared.Compared with the L-DiEM group, the number of primary culture colony formation in α-MEM group was significantly greater (P < 0.05). Peripheral blood MSCs were spindle, tdangle or polygonal, with the presence of processes, 2-3 nuclei and cell division phase, and slowly proliferated following osteogenic induction. At day 7 after differentiation of MSCs into osteoblasts, positive rate of alkaline phosphates was above 80%. At day 21, calcium deposition was detected by Alizarin Red S (positive staining).CONCLUSION: MSCs could be harvested from peripheral blood of wounded rabbits, with characteristics of osteogenic differentiation, α -MEM was more suitable than L-DiEM for peripheral blood MSCs to growin vitro.

Objective To explore the efficacy of unrelated cord blood transplantation treatment of mucopolysaccharidosis Ⅰ (MPS Ⅰ).Methods A 4-year-and-2-month-old boy with MPS Ⅰ who received treatment of human leucocyte antigen-mismatched unrelated cord blood stem cell transplantation after diagnosis was identified.The pre-treatment regimen was Busulfan + Cyclophosphamide + Fludarabine (Bu/Cy4 + Flud).Bu with the dosage of 1.2 mg/kg,once every 6 hours,4 days;Cy with the dosage of 50 mg/(kg · d) for 4 days and Flud with the dosage of 30 mg/(m2 · d) lasted for 4 days,respectively.The day that the graft was transplanted was defined as 0 day,days betore transplantation as negative days,days after transplantation as positive days.After pre-treatment,4.60 × 107/kg of cord blood nucleated cells and 3.05 × 105/kg CD34 positive cells were transplanted into the child.The combination of Antihuman thymocyte globulin,Cyclosporin A and Mycophenolate mofetil was administrated for prophylaxis of graft versus host disease(GVHD).After transplantation,the patient was given granulocyte colony stimulating factor to promote reconstitution of hematopoiesis.Results The myeloid and platelet engraftment time was respectively 15 days and 24 days after transplantation.Short tandem repeat (STR) DNA fingerprinting showed a full donor chimerism on day 21 after transplantation,and the full donor chimerism was stable afterwards.The peripheral-blood α-L-iduronidase (IDUA) activity returned to the normal value,and the IDUA gene sequencing did not demonstrate any mutation in 83 days after transplantation.On day 12 after transplantation,pulmonary infection with pulmonary hypertension occurred.Grade-Ⅱ acute intestinal GVHD occurred on day 15,Grade-Ⅱ acute cutaneous GVHD on day 51,and chronic GVHD (cutaneous,localized) on day 180.Otherwise,the patient complicated with hemorrhagic cystitis on day 35.These complications was cured favourably.In an 18-month-follow-up,the height of the boy increased by 3 cm,and his body weight had increased by 2.4 kg.His corneas regained clear,and his hepatosplenomegaly disappeared.The glycosaminoglycan of urine was negative.The neurocognitive performance of the boy had a little improvement.The abnormalities of fingers and other skeletons had no marked change.Conclusions Unrelated cord blood transplantation for MPS Ⅰ have definited effect.It is the first case report in China on treatment of MPS Ⅰ by unrelated cord blood transplantation.The researchers have accumulated some preliminary experience for future treatment of MPS Ⅰ by unrelated cord blood transplantation.

Objective To evaluate the clinical features and guanosine triphosphate cyclohydrolase 1 (GCH-1) gene mutation in a family with dopa-responsive dystonia (DRD).Methods The clinical features of this family were collected and their peripheral blood samples were screened for mutation in GCH-1 gene using PCR and DNA direct sequencing.Results The clinical features among each patient in this family were different.But all affected family members had quite a good response to levodopa treatment without significant adverse reactions.DNA test showed an AT deletion mutation at point of 631-632 in the 6th exon of GCH-1 gene in 5 affected members and 1 asymptomatic immediate family member.Conclusions Clinical heterogeneity is an important characteristic of DRD and clinical symptoms vary intra-families.Same gene type may cause different phenotype and not all carriers are patients.The deletion mutation at point of 631-632 in the 6th exon of GCH-1 gene should be considered as a pathogenic mutation for DRD.

OBJECTIVE:To determine organic solvent residual volume in fusidic acid by headspace gas chromatography.METHODS:The determination was performed on polyoxyl20000DM-WAX capillary,the column was subjected to tempera-ture programming,the detector temperature was250℃,the carrier gas was helium,the flow rate was7ml/min and the split ratio was1∶1.RESULTS:The linear detective concentration ranges of acetone,methanol and ethanol were2.04～102.50?g/ml(r=0.9992),2.00～99.98?g/ml(r=0.9995)and2.04～102.34?g/ml(r=0.9995),respectively,their average recovery rates were98.7%,99.2%and101.7%,respectively with RSD at2.11%,1.98%and2.01%,respectively.CONCLUSIONS:The method is simple,accurate,and suitable for the determination of organic solvent residual volume in fusidic acid.

Bone xenograft bone for the treatment of bone defect is one of the current research focus, which has advantages of extensive sources, low cost, simple preparation method. While the process of single bone xenograft bone in repairing bone defect is very long, and the clinical outcome is not satisfactory. The main problems focus on formation of bone and vascularization. Reconstituted bone xenograft combined with cells and xenogenic bone material could promote vascularization and bone fusion in vivo, thus achieve a clinical effect of autogenous bone in repairing bone defect.
Bone Transplantation ; methods ; Bone and Bones ; blood supply ; Humans ; Transplantation, Heterologous

Objective To report the development of clinical symptoms in a Chinese family with autosomal dominant progressive external ophthalmoplegia(adPEO).Methods Electromyologram and muscle biopsy were performed in the proband and 4 family members with the disease.Results The proband was a 57 year-old woman,who developed bilateral ptosis after the age of 30,external ophthalmoplegia after the age of 35 years old,weakness of extremities at the age of 37 years old and bulb palsy with palmus at the age of 47 years old.In the family there were 20 male and female members from five generations.All of them complained about bilateral ptosis between 26—33 years old,external ophthalmoplegia(12/15)and weakness of all extremities(14/15)between 35—45,facial and masticatory weakness(9/9)as well as dysphagia(8/9)between 44—60,accompanied with heart lesions(4/7)after 50 years old.Some patients died due to cardiac impairment.Electromyologram showed myopathic abnormalities in the examined patients. The main myopathological changes were ragged red fibers,cytochrome c oxidase negative fibers and ragged blue fibers in succinate dehydrogenase staining.Conclusions The adPEO started from extra-ocular muscles to limbs,finally facial and bulbar muscles.Heart lesions were presented in late stage and lead to death in some members.The developing process of symptoms suggested that we should pay more attention to cardiac manifestations in this disease.