BACKGROUND:Previous studies have mainly focused on costal cartilage, articular cartilage, nasal septal cartilage, and auricular cartilage, but in vitro culture of human vertebral endplate cartilage is stil rarely reported. OBJECTIVE: To discuss the feasibility of culture of vertebral endplate chondrocytes from type I neurofibromatosis associated with scoliosis patientsin vitro and to study the biological characters of the chondrocytes. METHODS: Through two-step enzymatic digestion and tissue culture, the chondrocytes from the vertebral endplate of seven type I neurofibromatosis patients isolated and cultured in monolayer and passaged to observe the changes of cel morphology under inverted phase contrast microscope. Colagen type II expression was detected by immunocytochemistry to identify whether the cels had chondrocyte characters. The growth kinetics was detected by using MTT colorimetric assay to draw the growth curve of passage 2 chondrocytes. RESULTS AND CONCLUSION:A few chondrocytes crawled from the cartilage after 2 weeks culture and cels were passaged at 3 weeks. Along with passage going on, the phenotype of chondrocytes was changed from polygonal, round, triangle, and irregular shapes to fusiform. The colagen type II expression in passage 2 cels was positive by immunohistochemical staining. MTT test showed the growth curve of the passage 2 chondrocytes presented a transverse “S”. Cels were found logarithmic growth at days 4-7, reached platform stage at days 8-13, and decreased at day 14. It is an effective and simple procedure by two-step enzymatic digestion and tissue explant method to culture vertebral endplate chondrocytes with high purity and good viability from type I neurofibromatosis patients associated with scoliosisin vitro. Passage 2 chondrocytes from the vertebral endplate exhibit the best viability at days 4-7, which can be used as targets for research of pathogenesis of type I neurofibromatosis with atrophic scoliosis.

Objective To investigate the regulatory effect of adrenomedullin (AM) on the cell-cell contact formation of podocytes and the possible mechanism.Methods Podocytes were treated with AM (10-7 mol/L),AM combined with a PKA inhibitor H89 (10-4 mol/L),and forskolin (10-5 mol/L) as positive control respectively for 12 hours.Immunofluorescent staining was applied to observe the distribution of cell adhesion molecules and actin-associated proteins.Western blotting assay was used to assess their protein levels.Rho GTPases activity was analyzed by GST-pull down assay and their protein levels were tested by Western blotting.Results AM induced the redistribution of adhesion molecules,actin-associated proteins as well as the F-actin at cell-cell contacts between podocytes.This effect was similar to that of forskolin and could be blocked by H89.The levels of those proteins did not change significantly (P ＞ 0.05).AM up-regulated the activities of RhoA,Rac1 and Cdc42 (P ＜ 0.05),which were partially blocked by H89.The protein levels of Rho GTPases showed no difference compared with the control (P ＞ 0.05).Conclusions AM may promote cell-cell contact formation of podocytes,probably through enhancing the activity of Rho GTPases and then resulting in the redistribution of adhesion molecules,actin-associated proteins and F-actin,which is partially mediated through cAMP-PKA signaling pathway.

Objective:To accumulate dendritic cells(DC) into the peripheral blood and induce the specific cytotoxic T lymphocyte against tumor.Methods:B220~-CD11c~+ cells were sorted from the peripheral blood of C57BL/6 mice injected i.v.with P.acnes,and cultured for about six days in the presence of GM-CSF,IL-4 and mTNF-?.Phenotype and mixed leukocyte reaction(MLR) of these cells were assayed.CTL to anti-tumor,which was developed by cultured B220~CD11c~+ cells loading tumor antigen,was detected.Results:B220~-CD11c~+ cells were rapidly accumulated in the circulation of the mice after P.acnes injection.These cells could differentiate into mature DC when cultured in the presence of cytokines for several days.Moreover,cultured B220~-CD11c~+ cells loading tumor antigen could stimulate naive splenic CD3~+T cells generate high level of IFN-? and tumor specific cytolytic reactivity in vitro.Conclusion:B220~-CD11c~+ DC precursors rapidly accumulated in the peripheral blood after injection of(P.acnes) in mice.T cells could develop specific CTL to anti-target tumor cells in vitro when stimulated with P.acnes-mobilized DC loading tumor antigen.

BACKGROUND:With the extensive application of anterior titanium plate, postoperative complications such as dysphagia, titanium loose, screw exit and disc degeneration of neighboring segments induced more and more attention of researchers. However, the application of anterior cervical cage is expected to avoid these complications. ＜br> OBJECTIVE:To observe primary curative effect of anterior cervical cage ROI-C in anterior cervical spine surgery. ＜br> METHODS:A total of 32 patients with cervical spondylosis were treated with anterior cervical cage ROI-C in the Wuxi Ninth Hospital Affiliated to Soochow University from April to December 2013. The cage was implanted to promote interbody fusion. Of 32 cases, 23 cases affected cervical spondylotic myelopathy, 2 cases affected nerve root type cervical spondylosis, 3 cases affected cervical hyperextension injury, 1 case affected cervical disc herniation, 2 cases affected cervical instability and 1 case affected segmental cervical ossification of the posterior longitudinal ligament. Japanese Orthopaedic Association and NDI scores were determined to assess neurological symptoms and functional improvement before internal fixation and during final fol ow-up. Simultaneously, adverse reactions were recorded. ＜br> RESULTS AND CONCLUSION:A total of 32 patients finished the regular fol ow-up for 4 to 8 months. Clinical symptoms and spinal cord function of al patients were obviously improved. No ROI-C loosing or displacement or secondary surgery was found. The average fusion time was 4.2 months (3 to 5 months). Mean score of Japanese Orthopaedic Association was increased from 9.2 points pre-surgery to 13.8 points post-surgery. Japanese Orthopaedic Association and NDI scores were higher during final fol ow-up than before fixation (P＜0.05). These data indicated that ROI-C effectively restored intervertebral height in anterior cervical spine surgery, stably reconstructed cervical vertebra, obtained interbody fusion, effectively avoided related surgical complications induced by plate implantation, improved neurological symptoms and function, and showed good short-term effects.

OBJECTIVETo analyze the expression of the CD(40) and its ligand (CD(40)L) on bone marrow stromal cells (BMSC), and investigate interleukin-3 (IL-3), IL-6, Flt3 ligand (FL) and stem cell factor (SCF) production of BMSC after stimulated with CD(40) agonistic monoclonal antibody (5C11) and its role in the regulation of hematopoiesis.

METHODSBMSC were freshly isolated from adult bone marrow. The expression of CD(40) on these cells was determined with flow cytometry, the concentrations of IL-3, IL-6, FL and SCF in the supernatant at 24, 48 and 72 hours after BMSC cultured with 5C11 at a dose of 20 micro g/ml were determined by the ELISA assay. After BMSC incubated with 5C11 for 24 hours, the supernatant was collected and cultured with purified cord blood CD(34)(+) cells for 7 days under 37 degrees C in a fully humidified atmosphere supplement with 5% CO(2). Colony formation assay and Annexin V assay were employed to determine the proliferation and apoptosis of the CD(34)(+) cells.

RESULTSBMSC expressed CD(40) and the production of IL-6 and FL increased after stimulated with 5C11 while IL-3 and SCF had no change. The supernatant collected from the stimulated BMSC promoted proliferation of CD(34)(+) cells, increased the CFU-GM yields and had anti-apoptosis effects.

CONCLUSIONCD(40) ligandization on BMSC increased the production of IL-6 and FL and promoted the proliferation of CD(34)(+) cells. The couple CD(40)/CD(40)L may be involved in the control of hematopoiesis via modulation of the cytokine network in the bone marrow.

Adult ; Antibodies, Monoclonal ; pharmacology ; Antigens, CD34 ; analysis ; Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; metabolism ; CD40 Antigens ; immunology ; metabolism ; CD40 Ligand ; metabolism ; Cell Division ; drug effects ; Culture Media, Conditioned ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; immunology ; Flow Cytometry ; Humans ; Interleukin-6 ; biosynthesis ; Membrane Proteins ; biosynthesis ; Stromal Cells ; cytology ; metabolism

Objective To examine the expression of response gene to complement 32 (RGC-32) in renal tissue of children with IgA nephropathy (IgAN), and to explore its significance. Methods The subjects were 45 children diagnosed as IgAN by renal biopsy. The expression of RGC-32, α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1) was examined by immunohistochemistry staining. The correlation of RGC-32 expression with α-SMA,TGF-β1, degree of renal pathological lesions and clinical index in IgAN was assessed by Spearman correlation analysis. Results RGC-32 protein located in renal tubular epithelial cells in normal and IgAN renal tissues. The positive expression index of RGC-32 in nomal group, IgAN mild group, moderate group and severe group was (18.29±6.22)%, (23.90±9.65)%, (31.23±9.86)%,and (34.52±10.63)% respectively. With more severity of renal pathological lesions, the expression of RGC-32 in IgAN was enhanced. The RGC-32 expression was positively correlated with the score of glomerulus and renal interstitium in children with IgAN (r=0.385, 0.347, P＜0.05), as well as α-SMA, TGF-β1 (r=0.594, 0.521, P＜0.01), but was not correlated with Scr, urinary NAG/Cr,Alb/Cr, IgG/Cr, and α1-M/Cr (r =0.117, -0.115, -0.138, -0.176, -0.028, all P ＞0.05).Conclusions RGC-32 protein locates in renal tubular epithelial cells in normal and IgAN renal tissues. RGC-32 may participate in the course of renal tubulointerstitial lesions in children with IgAN, especially in the course of epithelial-mesenchymal transition (EMT) induced by TGF-β1.

Object: To investigate the effects of Huaiqihuang Granule, a compound Chinese herbal medicine, on expressions of nephrin and podocin of slit diaphragm of glomerular podocytes in rats with adriamycin-induced nephrosis and to explore the mechanism in reducing the proteinuria. Methods: Twenty SD rats were randomly divided into five groups: control group, model group, glucocorticoid group, Huaiqihuang Granule group and Huaiqihuang Granule plus glucocorticoid group. The 24-hour urine was collected 7, 14, 21 and 28 days after adriamycin injection respectively to measure 24-hour urinary protein, and all rats were sacrificed after 28-day treatment. Pathological changes in renal tissues were observed under a light microscope and an electron microscope. Expressions of nephrin and podocin mRNAs in renal cortex were determined by real-time polymerase chain reaction, and protein levels of nephrin and podocin were detected by Western blotting. Results: (1) In the model group and the treatment groups, the level of urinary protein increased significantly from the 14th day. (2) Under the light microscope, inflammatory cells and slight fibroplasia were found in renal interstitium of the model group, but there were less inflammatory cells in renal interstitium in the intervention groups than in the model group. Under the electron microscope, 29 days after adriamycin injection, extensive fusion of foot processes was observed. (3) The expressions of nephrin and podocin were higher in treatment groups than in the model group. (4) Proteinuria level was negatively correlated with the expressions of nephrin mRNA and nephrin and podocin proteins. Conclusion: The above results indicate that Huaiqihuang Granule can maintain the integrity of the slid diaphragram in podocyte, alleviate the lesion of glomerular filtration membrane, and decrease the proteinuria by up-regulating the expressions of nephrin and podocin. Huaiqihuang Granule plus glucocorticoid maybe has better effects than glucocorticoid alone.

Objective:To investigate the expression of inducible costimulatory ( ICOS) and inducible costimulatory ligand ( ICOSL) on peripheral blood mononuclear cells ( PBMCs ) and their clinical relationship with rheumatoid arthritis ( RA ) patients.Methods:Peripheral blood samples were collected from 85 RA patients and 50 HC in this study.Expression of ICOS and ICOSL on PBMC from the subjects were detected by flow cytometry and real-time polymerase chain reaction( RT-PCR).The alteration of ICOS and ICOSL were observed after hormone therapy in 15 patients with RA and the relationship between their expression level and patients′clinical manifestations were analysed.Results:The ICOS and ICOSL mRNA level of RA patients′PBMCs were significantly higher than that in HC.The expression level of ICOS on CD4+T cells was higher than than that in HC[(7.08±4.72)% vs (3.01+1.39)%,P<0.0001].The expression of ICOSL on monocytes[(5.77±3.45)%vs (3.64±1.43)%,P<0.05] and B cells [(5.78± 4.52)%vs (3.97±1.63)%,P<0.05] were significantly elevated in RA patients.In RA patients with active disease,however,ICOSL expression on monocytes and B cells were increased as compared with those in inactive RA patients [ ( 5.45 ±3.50 )% vs ( 4.04 ± 1.55)%,P=0.036],[(6.59 ±5.74)%vs (5.63±4.30)%,P=0.016].Furthermore,after receiving immunosuppressive therapy, the expressions of ICOS and ICOSL were notably reduced as compared with pre-therapy levels on PBMCs from patients [ ( 5.45 ±3.50)%vs (4.04±1.55)%,P=0.036],[(6.59 ±5.74)%vs (5.63±4.30)%,P=0.016].Conclusion:The high levels of ICOS and ICOSL expression were closely correlated with the degree of disease and therapeutic response,suggesting that ICOS/ICOSL pathway may play a critical role in pathogenesis of RA.

ObjectiveTo explore the effects of Jinyaodai on neurological behavior and Akt expression in the cortex of rats following spinal cord contusion injury.MethodsRats were randomly divided into control group,spinal cord contusion group and Jinyaodai group.The weight-drop device was employed to prepare the spinal cord injury(SCI) model.Jinyaodai was administrated every day by using a stomach tube.Rats were performed the BBB assessment,and the detection of Akt expression and count of Neun positive neurons in cortex following SCI.ResultsCompared with control group,deficit of motor function in hindlimbs was seen at 3 dpo following cord contusion,and partial functional recovery could be seen from 7 dpo to 1 m.Treatment of Jinyaodai greatly increased the BBB scores ( 14.1 ± 1.4 ) more than SCI group ( 7.8 ± 1.3 ) at 1 month (P ＜ 0.05 ) ; Simultaneously,compared with SCI rats,treatment of Jinyaodai significantly increased the expression of Akt (0.53 ± 0.05,0.68 ± 0.07,P ＜0.05 ) and the number of neurons ( 11 ± 2, 15 ± 1 ; P ＜ 0.05 ) in the lesion-induced cortex of rats.Conclusion Jinyaodai may play an essential roles in functional recovery after spinal cord injury,in which the underlying mechanism may be involved in the expression of Akt in cortex.

AIM: To investigate the effects of aging on the angiotensinⅡ receptors (ATR) in different parts of the kidney. METHODS: Expression of AT 1 receptor in renal cortex was determined by Western and Northern blotting. Five micrometer thick cryostat sections of 3 or 24-month Wistar rats were mounted onto a 1.35?m thin polyethylene membrane. Glomeruli, tubules and arteries of rat kidney were isolated by laser microdissection and pressure catapulting system, AT 1aR mRNA, AT 1bR mRNA AT 2R mRNA were measured with reverse-transcription PCR(RT-PCR). RESULTS: Western and Northern blotting showed that protein and gene expression of AT 1 receptor in kidney cortex decreased in 24 month Wistar rats compared to those in 3 month Wistar rats. Glomeruli, tubules and arteries were quickly isolated by laser microdissection and pressure catapulting system without contamination. Compared to young group, AT 1aR mRNA expression was decreased in the tubules of aged rats, no changes was found in isolated glomeruli. AT 1bR mRNA was decreased in glomeruli and tubules. Both AT 1aR mRNA, AT 1bR mRNA were increased in the arteries. AT 2R mRNA was only increased in the tubules, whereas there were no significant changes in the glomeruli or arteries. CONCLUSION: Angiotensin Ⅱ receptors are regulated selectively in different portions of aged kidney, which might play an important role in aging related changes of the kidney.