Objective To establish the methods of efficiently isolating chondrocytes from epiphyseal cartilage and observe the basic biological characteristics of the cultured ones. Methods The improved isolation method through three-step enzymatic digestion using 1 g/L trypsase in 1 g/L ethylenediaminetetraacetic acid, 1 g/L hyaluronidase and 2 g/L type Ⅰ collagenase was established to isolate the primary passage chondrocytes from epiphyseal cartilage of young rabbits and its efficiency evaluated. The cellular biological characteristics of the chondrocytes were observed under the inverted and light microscopes. Results (1) All chondrocytes were isolated from the matrix after it was completely digested by three above-mentioned enzymes. The harvested chondrocytes significantly and positively correlated with the wet weight of cartilage. The number of chondrocytes from 1 g of wet cartilage was 32.3?106 with a viability of 97.6% on average. (2) The chondrocytes of the primary and the first passages showed triangle or multi-angle shapes (oval during growth and fusion) with the positive immunohistochemical stain of collagen type Ⅱ and the strong heterochromia to toluidine blue. The chondrocytes after the third passage became spindle-shaped with the negative stain of collagen type Ⅱ and the weak heterochromia to toluidine blue. Conclusions (1) The chondrocyte-isolating method through three-step enzymatic digestion has advantages of high efficiency in harvesting primary chondrocytes, high cellular viability and simple manipulation. (2) The chondrocytes differentiate and lose their special phenotypes gradually with passage.

Objective To analyze pathologic features, therapeutic strategies and prognosis of patients with metastatic solid pseudopapillary tumor of the pancreas (m-SPTP). Methods Twenty five patients with m-SPTP undergoing radical resection between June 1985 and Dec 2005 were retrospectively analyzed. Kaplan-Meier analysis was used to screen out risk factors of the prognosis of m-SPTP patients.Results Twenty-three postoperative patients were followed up until Dec 2007. The follow-up rate was 92% and the median follow up time was 78 months. The 5-year overall survival rate was 82%. Kaplan-Meier analysis showed that five variables including age, tumor size, pathologic features, number of metastatic lesions and lymphatic metastasis were related to overall survival. Conclusions Solid pseudopapillary tumor of the pancreas is low-grade malignant potential with a favorable prognosis, but not for the m-SPTP.Patients of ≥40 years old, tumor size (≥6 cm), pathologic features (including presence of areas with diffuse growth pattern and tumor necrosis), multiple metastatic lesions and lymphatic metastasis have poor prognosis.

To summarize the status of chinese medicine syndrome study in chronic prostatitis, emphatically analyze the key problems. In the national standard and textbook, the names of four syndrome types are not unifi ed, difference of symptoms description still exist, and diagnostic criteria of basic syndrome type needs to be clarified. Analysis of literature showed that syndrome differentiation was commonly based on the experience of physician in clinical practice. In addition to the four syndrome types, occurrence frequency of spleen qi deficiency and liver qi stagnation syndrome types are relatively high. Research method of syndrome should be improved urgently. This paper proposes counter measures: Clarify the commonly basic syndrome types and syndrome differentiation standards. Formulate the quantitative diagnostic criteria of basic syndrome types.conduct the prospectively multicenter clinical survey.

Objective To establish the measurement for evaluating the efficiency of isolating and harvesting marrow mesenchymal stem cells (MSCs) and observe the main biological characteristic of MSCs in vitro during the passage cultivation. Methods ①The marrow nucleated cells in young rabbit marrow were isolated by the gradient centrifugation and cultivated to isolate and harvest MSCs. The efficiency of isolating and harvesting MSCs was measured by the number of the first passage MSCs from every 10~(6) of the marrow nucleated cells. ②The changes of MSCs morphology, growth and proliferation, synthesis of collagen typeⅠand activity of alkaline phosphatase (ALP) were observed during the passage cultivation. Results ① After cultivated at 2.5?10~(5)/cm~(2) marrow nucleated cells for 9-10 d, the first passage MSCs were harvested. The number of the first passage MSCs had a remarkable statistical positive correlation with the number of the marrow nucleated cells from one rabbit(r=0.932, P

Objective To evaluate the impact of influenza virus hemagglutitin (HA) 306-318 peptide on T cell activation and to investigate the inhibitory effect of the altered HA306-318 peptide on T cell proliferation. Methods HA306-318 peptide and its mutant containing amino acid substitutions were synthesized. They were used in T cell activation assay using HLA-DR1 transfected cells. Responses were determined by MTT proliferation assays. The HA mutant without stimulating effect on T cells was then examined by inhibiting HLA-DR1-restricted T cell activation. Results It was demonstrated in this study that the altered HA306-318 peptide bound to HLA-DR1 molecule on L57.23 cells transfected with HLA-DR1 cDNA, but not on the control cells. The altered HA306-318 peptide had no stimulating effect on T cells compared with the wild type HA306-318 which induced T cell proliferation. It was shown that the altered HA peptide inhibited T cell activation mediated by wild type HA306-318 as well as by CⅡ263-272 which was the specific T cell antigen. Conclusions This study suggests that HA306-318 peptide is antigenic and can induce responses in HLA-DR1 specific T cells. Altered HA306-318 peptide is hyporesponsive in T cell activation with inhibitory effect on antigen-driven T cell responses, and it is potentially a therapeutic agent in rheumatoid arthritis.

0.05). The positive rate of the SE in rats experiencing longer arthritis duration (≥4 weeks) was significantly higher than that with shorter arthritis duration (

Objective To establish the technique of biphasic seeding of articular chondrocyte s in vitro for cartilage engineering in u sing a self designed biological gel a nd the three-dimensional scaffold a nd observe the efficiency of cartilage regeneration in vitro-tissue engin eered articular cartilage from cell scaffold complex.Methods The articular chondrocytes were iso lated enzymatically from the epiphyseal cartilage of young rabbits,and were then plated i nto the tissue culture flasks and were cultivated.The first passage ar-ticular chondrocytes were collecte d and mixed fully with the self-made l iquid biological gel-matrix at appr oxi-mately 2.5?10 7 cells /ml to form cell-gel fluid.The cell-gel fluid was dropped into the p orous CPPf /PLLA(calcium polyphosphate fiber /poly -L -lactic acid)scaffold,and a cell-gel-scaffold c omplex was formed after being solidified.The complexes were cultivated for 4weeks.The changes of complexes in morphology and synthesis of collagen typeⅡandⅠand aggregates were investigated under the gross and the phase and light microscope.The GAG sulfate content in complexes was quantitatively mea sured by the modified dimethyl-methylene blue method.Results1)After feeded,the porous CPPf /PLLA s caffolds were completely filled with the liquid chondrocyte-gel and the chondrocyte-gel-scaffold comp lexes were well formed by solidification.During the cultivation,the complex es could keep its original shape and m aintain the stable homogeneous three-dimensional distribution of chondrocytes in themselves without cell falling.In the same time,the com-plexes were gradually increasing th e consistency with the elasticity an d lubrication surface.2)The chondro-genesis began in the periphery area a nd extended to the central area of the complexes with the passage of cultivation period.After the 2nd we ek,the complexes were gradually reorganized into the mature engineered cartilage with typical cartilaginous histological structure,with ric h typeⅡcollagen and the strong typical het-erochromia to toluidine blue,but wi th gradually fading negative immunological stain of collagen typeⅠ.Meantime,the scaffold was graduall y de-graded in the complexes.The average content of GAG sulfate in the enginee red cartilages at 4th week was(15.70?2.00)mg /g(wet weight ),and was over 30%of the natural articular cartila ge.Conclusion The technique of chondrocytes biphasic seeding for three-dimensional scaffold has advantages of simple manipulation,fix ing cell stably in the scaffold,main-taining the original shape of the com plexes and the stable homogeneous th ree-dimensional distribution of chondrocytes in the scaffold,and ad vancing the regeneration and matura tion of tissue-engineered articula r cartilage.[

Objective:To evaluate the impact of altered collagen Ⅱ(CⅡ) peptide(S268-270) on T cell activation.Methods:Altered CⅡ263-272 with G268,P269 and K270 consecutively substitution with A or G were synthesized using solid phase various concentrations were added to HLADR1 transfected APC.Expression of FITC stainning was analyzed by fluorescence-activated cell sorting,and the binding of altered CⅡ peptide to HLA-DR1 molecule on cell membrane was evaluated.Irridiated HLA-DR1 expressing APC was incubated with CⅡ263-272 or hemagglutinin(HA)306-318 and altered CⅡ peptides at various concentrations for 2 hours before T cells(3.19) were added.Supernatant was harvested and then added to IL-2 dependent CTLL cell.The cell proliferation was examined by methotetrazolium(MTT) method.Results:The altered CⅡ peptide and wild type of CⅡ263-272 were able to competitively bind to HLA-DR1 molecule expressed on cell membrane of transfected APC.CⅡ or HA induced T cell activation was significantly inhibited by the altered peptide S268-270.Conclusion:Altered CⅡ263-272 with G268,P269 and K270 consecutively substitutions with A or G inhibited CⅡ263-272 and HA306-318 induced T cell activation through competitively binding to HLA-DR1,suggesting a new approach in inhibition of T cell activation in RA.

Objective:To evaluate the inhibitory effect of altered HA308-317 peptides on HLA-DR4 restricted CII specific T cell response.Methods:Three altered HA308-317 peptides and CII263-272 were synthesized using solid-phase techniques. The binding of altered HA308-317 peptides for HLA-DR4 molecules was assayed using flow cytometry. The suppressive effect of altered HA308-317 peptides on CII-mediated T cell proliferation was determined using 3H incorporation assay. The level of IL-2 in the supernatants was identified by ELISA. The expression of CD25 and CD69 on T cell surface were studied using flow cytometry.Results:The altered HA308-317 peptides were able to bind to HLA-DR4 molecules and competed with CII263-272. Altered HA308-317 peptides inhibited T cell proliferation and IL-2 production induced by CII263-272( P

OBJECTIVE:To establish a method for the determination of weight average molecular weight(Mw)and molecular weight distribution (D) of ferric carboxymaltose. METHODS:HPGPC method was adopted to detect the Mw and D of 3 batches of Ferric carboxymaltose injection (imported) and its raw material (self-made). The determination was performed on TSK G4000 PWXL column with 0.1% sodium azide solution with the flow rate of 0.5 ml/min. The detector was refractive index detector;the column temperature was set at 35 ℃,and sample size was 20 μl. The results were calculated with GPC software. RESULTS:RSDs of precision,stability and reproducibility tests were all lower than 3.0%(n=6);Mw and D of 3 imported samples were 157 667 and 1.30;those of self-made samples were 162 000 and 1.42. CONCLUSIONS:The method has high precision,good stability,repeat-ability and durability. It can be used for the determination of Mw and D of ferric carboxymaltose.