Objective To investigate the expression of succinate dehydrogenase subunit A(SDHA)and subunit B (SDHB) in patients with renal cell carcinoma (RCC),and its relationship with the clinicopathologic characteristics. Methods The expression of SDHA and SDHB was detected in 179 cases of RCC by immunohisto-chemistry and the relationship between the SDHA ,SDHB expression and the clinicopathologic characteristics of RCC were analyzed. Results In 179 cases of RCC,18 cases(10.1%)were shown with negative or suspicious-negative expression of SDHA and SDHB,including 2 cases(1.1%)with suspicious-negative expression of SDHA and SDHB. 12 cases(66.7%)had tumor located in left kidney and 6 cases(33.3%)had tumor located in right kidney. The well-differentiation group contained 15 cases(83.3%),moderate-differentiation group contained 1 case (5.6%)and poor-differentiation group contained 2 cases(11.1%). Most of SDH negative and suspicious negative RCC had the following characteristics that the normal renal tubule could be seen within the tumor. The tumor was composed of polygonal cells arranged in nest ,and the polygonal cells were separated by a fine fibrovascular stroma. The nuclei were shown around with vesicular chromatin. The eosinophilic flocculet material ,vacuole and eosino-philic inclusion body could be seen in the cytoplasm of tumor cells. Conclusion The RCC cases with negative expression of SDHA and SDHB protein have unique histological features ,with significantly higher incidence in the left kidney than that in the right kidney.

ObjectiveIn order to compare the alteration of reelin-immunoreactive Cajal-Retzius cells (CR cells) in molecular layer of dentate gyrus of APPswe transgenic mice with wild type, the histochemical and developmental characteristics of CR cells were studied, therefore, the roles of CR cells in Alzheimer's disease would be revealed further.Methods The Thioflavine S staining, reelin immunofluorescence with or without reelin/glutamate and reelin/GABA immuno-double staining were carried out in the study. In the meantime, Western blotting was used to study the expression of reelin in hippocampi of the both wild type and transgenic mice. Results Reelin positive CR cells could be double-labeled with either glutamate or GABA immunostaining. Caspase-3 immunofluorescence demonstrated that some CR cells went through apoptosis during their development. Compared with wild type, CR cells in APPswe transgenic mice had significantly decreased in the molecular layer of the dentate gyrus. The result was supported with Western blotting analysis of reelin expression in hippocampus. Conclusion Reelin could be co-expressed with either glutamate or GABA, suggesting CR cells would be glutamatergic exciting neurons and GABAergic interneurons. The loss of CR cells during development probably was caused by the neuroapoptosis. Significant decrease of CR cells in hippocampus of APPswe transgenic mice indicated reelin may play an important role in AD pathological alterations.

Objective To observe the pathologic features on cardiac allograft and to test archived endomyocardial biopsy specimens for antibody-mediated rejection specific marker-C4d deposition and its characteristics by using immunoperoxidase (IP) techniques. Methods From January 2003 to December 2007,10 recipients underwent orthotopic cardiac transplantation and 17 specimens of endomyocardial biopsy were obtained either for a protocol basis (generally at 1 st month,3rd month,1st year and 2nd year post-transplant) and on immediate clinical indications.All specimens of endomyocardial biopsy were collected for histopathological examination and C4d immunohistochemical staining,simultaneously. All pathological diagnoses were done according to 2004 International Society for Heart and Lung Transplantation (ISHLT) recommendation working formulation and AMR Schema,and C4d staining intensity were graded and recorded as 0 to 3 +.Results Except 1 specimen unqualified,all 16 consecutive specimens of endomyocardial biopsy were qualified.There were 4 cases of acute T cell-mediated rejection (all graded 1 ),2 cases of Quilty lesion,and 7 cases of antibody-mediated rejection,who were documented according to ISHLT Schema and C4d deposition.Meanwhile,there were 6 cases showing evidence of antibody-mediated rejection without concurrent acute cellular rejection and only one case concordant with acute T cell-mediated rejection.One case of antibody-mediated rejection died 20 months posttransplantation due to combined transplant coronary artery disease (TCAD). The C4d in the cardiac allograft was deposited in microvasculature diffusively.Conclusion Antibody-mediated rejection is an important clinical entity following orthotopic heart transplantation and is difficult to diagnosis except to perform endomyoeardial biopsy.Immunoperoxidase staining for C4d is a sensitive and specific technique for detecting one marker of antibody-mediated rejection.

Objective To generate recombinant Pichia pastoris for high-copy expression of human tissue factor pathway inhibitor (TFPI). Methods The cDNA encoding human TFPI was inserted into the expression vector pPIC9K and the constructed expression vector rhTFPI-pPIC9K was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant plasmids were subsequently transformed into Pichia pastoris GS115 cells, and the transformants were confirmed by PCR amplification of the genomic DNA.The recombinant Pichia pastoris with high copies of TFPI cDNA was screened by G418 selection. Western blot and TFPI ELISA Kit were employed to analyzing. The temperature, time and concentration of methanol for the induction of recombinant protein were optimized. Results PCR analysis and DNA sequencing confirmed the successful construction of the expression vector rhTFPI-pPIC9K. Real time quantitative PCR and Western blot analysis demonstrated the positive correlation between TFPI expression level and the copy number of TFPI cDNA in Pichia pastoris cells. Optimization of the induction condition significantly elevated the expression level and activity of TFPI (9.95±0.78 mg/L and 3.91±1.37 U/mL). Conclusion The Pichia pastoris strain with high copy of TFPI expression was successfully constructed, which lays a solid foundation for the further investigation on the function of TFPI and its application in the prevention and therapy of diseases.

BACKGROUND:In recent years, many manufacturing techniques have been recently developed for soft tissue engineering scaffolds. Especialy additive manufacturing with a unique material accumulated forming principle can be feasible and reliable to manufacture the highly precise scaffolds with gradient structures and multi-materials for large soft tissue defect repairing.
OBJECTIVE:To summarize scaffolds manufacturing technologies in the soft tissue engineering applications developed in recent years and to predict the direction of development.
METHODS: A retrieval was performed for the literature about the manufacturing methods of soft tissue scaffolds using key words of “additive manufacturing, microfabrication, vascular tissue engineering, muscle tissue engineering, cartilage tissue engineering, stereolithography, 3D printing, biodegradable hydrogel” in English and Chinese, which were published between January 2010 and September 2013 in PubMed Database and China National Knowledge Infrastructure (CNKI) Database.
RESULTS AND CONCLUSION:For large soft tissue defects repairing, structure design of the scaffolds has been shifted from a simple planar structure to a more complex three-dimensional structure, and integration of scaffold structure, materials and cels, and growth factors during the manufacturing procedure can be used to obtain the resolution of vascularization. Additive manufacturings become one of the most promising approaches for the ideal soft tissue scaffolds with gradient and complex structure and multi-materials. In particular, the hydrogel/cellcomposite scaffolds fabrication, a hot but promising approach to develop the soft tissue engineering wil be made progress by the accurate principles and processes of the hydrogel additive manufacturing combined with the introduction of living cels and growth factors.

Objective To apply flow cytometry-panel reactive antibody (FLOW-PRA) and compare the application of traditional (enzyme-linked imrnunosorbent assay) ELISA-PRA in clinical organ transplantarion,so as to evaluate the concordance,sensitivity,accuracy and practicability of FLOW-PRA.Methods PRA was detected in 212 serum samples from 185 patients awaiting organ transplantation using FLOW-PRA and ELISA-PRA.Results It took 1.5 h and 3 h for FLOW-PRA vs ELISA-PRA.Concordance correlation coefficient for the results of the two methods was 94% (class Ⅰ)and 89% (class Ⅱ),respectively.Of all sera,24.5% (in comparison to ELISA-PRA,P<0.005)were class I positive,18.4% (P<0.05) class Ⅱ positive by flow cytometry,and 17.9% and 14.6% by ELISA,respectively.The positive incidence in Flow group was higher than in ELISA group.Low titer of antibodies was detected positively only by flow eytometry,furthermore,the antigen specificity of PRA could only be discriminated by FLOW-PRA.Conclusion Flow cytometry is more sensitive and more accurate than ELISA in PRA detection.FLOW-PRA is easy to operate and time-effective,and suitable for clinical application.

AIM:To investigate whether angiotensin-(1-7) [Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) acti-vation and necroptosis .METHODS:The expression levels of receptor-interacting protein 3 ( RIP3;an indicator of necrop-tosis) and TLR4 were determined by Western blot .Cell viability was measured by CCK-8 assay.The activity of lactate de-hydrogenase ( LDH) in the culture medium was measured with a commercial kit .The releases of interleukin-1β( IL-1β) and tumor necrosis factor-α( TNF-α) were measured by ELISA .The intracellular level of reactive oxygen species ( ROS) was analyzed by 2 ’ , 7 ’-dichlorfluorescein-diacetate ( DCFH-DA ) stating followed by photofluorography .Mitochondrial membrane potential ( MMP) was examined by rhodamine 123 staining followed by photofluorography .RESULTS:After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased . Co-treatment of the cells with 30μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG.Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100μmol/L necrostatin-1 ( Nec-1;a specific inhibitor of necroptosis ) and HG for 24 h attenua-ted the up-regulation of TLR4 expression induced by HG .Moreover, 1μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG.On the other hand, co-treatment of the cells with 1μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response , leading to the increase in the cell viability , and the decreases in the activity of LDH , ROS generation , MMP loss as well as the releases of IL-1βand TNF-α.CONCLUSION:Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR 4 activation and necroptosis .

[ ABSTRACT] AIM:To investigate the role of ATP-sensitive potassium ( KATP ) channels in the inhibitory effect of hydrogen sulfide ( H2 S) on high glucose ( HG)-induced inflammation mediated by necroptosis in H 9c2 cardiac cells. METHODS:The expression levels of receptor-interacting protein 3 ( RIP3; an indicator of necroptosis ) and cyclooxyge-nase-2 (COX-2) were determined by Western blot.The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α( TNF-α) were detected by ELISA .RESULTS:After H9c2 cardiac cells were treated with 35 mmol/L glucose ( HG) for 24 h, the expression of RIP3 was significantly increased .Pre-treatment of the cells with 100 μmol/L diazoxide ( DZ; a KATP channel opener) or 400 μmol/L NaHS (a donor of H2S) for 30 min considerably blocked the up-regulation of RIP3 induced by HG.Moreover, pre-treatment of the cells with 100 μmol/L 5-hydroxydecanoic acid (5-HD; a KATP channel
blocker) attenuated the inhibitory effect of NaHS on HG-induced up-regulation of RIP3.On the other hand, co-treatment of the cells with 100μmol/L necrostatin-1 ( a specific inhibitor of necroptosis ) or pre-treatment of the cells with 100 μmol/L DZ or 400 μmol/L NaHS attenuated HG-induced inflammatory responses , evidenced by decreases in the expression of COX-2 and secretion levels of IL-1βand TNF-α.However , pre-treatment of the cells with 100 μmol/L 5-HD significantly attenuated the above anti-inflammatory effects of NaHS.CONCLUSION:KATP channels play an important role in the inhib-itory effect of H2 S on HG-induced inflammation mediated by necroptosis in H 9c2 cardiac cells.

OBJECTIVE To investigate drug resistance status of the pathogens of nosocomial pulmonary infection in stroke patients and to provide the scientific reference for clinical prevention of nosocomial infections and reasonable use of antibiotics.METHODS By the methods combining prospective monitoring and retrospective review,patients′ clinical data were analyzed statistically.Referring to National Rules of Procedures in Clincal Laboratory,the strains were identified.The antibiotic susceptibility test was performed by K-B method and the results were read according to CLSI 2006.RESULTS The main pathogens of nosocomial pulmonary infection in stroke patients were Klebsiella Pneumoniae(22.0%),Pseudomonas aeruginosa(18.4%),Acinetobacter baumannii(12.7%),Staphylococcus aureus(12.3%) and Escherichia coli(11.4%).The detection rate of extensive-spectrum beta-lactamase(ESBLs) producing E.coli and K.pneumoniae was 43.2%.Meticillin-resistant S.aureus(MRSA) accounted for 39.0%.Pan-drug resistant strains were found in A.baumannii.CONCLUSIONS Drug resistance status of pathogens of nosocomial pulmonary infection in stroke patients is very serious.We should take intervention measures to prevent and control the onest and prevalence of resistant strains.