1.Experimental study of electrospun silk fibroin matrices seeded with urothelial cells for urethral reconstruction
Minkai XIE ; Lujie SONG ; Jihong WANG ; Suna FAN ; Yaopeng ZHANG ; Yuemin XU
Chinese Journal of Urology 2014;35(8):629-634
Objective To investigate the feasibility of urethral reconstruction by using stretched electrospun silk fibroin matrices.Methods Stretched electrospun silk fibroin matrix was prepared,and the structure of the material was assessed by electron microscopy.Canine urothelial cells were isolated,expanded and seeded onto the material for 1 week to obtain a tissue-engineered graft.The tissue-engineered graft was assessed using HE staining and electron microscopy scanning.A dorsal urethral mucosa defect was created in 9 female beagle dogs.In the experimental group,tissue-engineered mucosa was used to repair urethral mucosa defects in 6 dogs.No substitute was used in the 3 dogs of the control group.Retrograde urethrography was performed at 1,2 and 6 months after grafting.The urethral grafts were analyzed grossly and histologically.Results Electron microscopy scanning revealed that the material had a 3 dimensional porous structure.Urothelial cells grew on the material and showed good biocompatibility with the stretched silk fibroin matrices.Canines implanted with tissue-engineered mucosa voided without difficulty.Retrograde urethrography revealed no signs of stricture,and histological staining showed gradual epithelial cell development and stratified epithelial layers at 1,2 and 6 months.The canines in the control group showed difficulty in voiding.Retrograde urethrography showed urethral stricture,and histological staining showed that no or only one layer of epithelial cells developed.A severe inflammatory reaction was also observed in the control group.Conclusion Stretched electrospun silk fibroin matrices have good biocompatibility with urothelial cells,and could be a potential material for urethral reconstruction.
2.Study on proteomics of Hela cell apoptosis in bufalin-induced human cervical carcinoma.
Suna PAN ; Yuhong WANG ; Lixing FENG ; Cuiying FAN ; Dean GUO ; Xuan LIU ; Jinling FAN
China Journal of Chinese Materia Medica 2012;37(13):1998-2004
OBJECTIVETo seek possible effect targets of bufalin in HeLa cells by studying the impact of bufalin on cell protein expression profile after treatment on human cervical carcinoma cell lines HeLa.
METHODBufalin's ICs0was measured by MTr assay. The apoptosis of cells was observed by FCM (flow cytometry) and Hoechst 33342 staining assay. Differentiated expression protein spots were founded and identified using proteomic techniques, which could induce HeLa cell apoptosis.
RESULTBufalin showed remarkable cytotoxic effect on HeLa cells. IC50 (154 +/- 21.5) nmol X L(-1) indicated the possibility of inducing cell apoptosis. The protein expression profile showed 11 differentiated expression protein spots. Among the 11 proteins, nudix-type motif 5, vimentin, hnRNP C1/hnRNP C2 variant, HNRPK, HNRPK isoform a variant (two spots are the same protein), heat shock protein 27, macrophage-capping protein, SELENBP1 protein were down-regulated, while ribosomal protein, large, P0 and S-adenosylmethionine synthetase 2 were up-regulated by bufalin treatment. They may be effect targets of bufalin in HeLa cells. Western blotting showed consistent results in heat shock protein 27, vimentin and HNRPK between expression after treatment with bufalin and two-dimensional electrophoresis.
CONCLUSIONBufa-Lin can induce apoptosis in human cervical carcinoma cells HeLa and the effect of bufalin may be related to the joint intervention with multiple protein targets.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bufanolides ; pharmacology ; Cell Line, Tumor ; Female ; HeLa Cells ; Humans ; Neoplasm Proteins ; metabolism ; Proteomics ; methods ; Uterine Cervical Neoplasms ; drug therapy ; metabolism ; pathology
3.Expression, purification of proteasome subunit PSMB1 and application in screening of possible proteasome inhibitors.
Cuiying FAN ; Lixing FENG ; Dongmei ZHANG ; Suna PAN ; Xuan LIU ; De'an GUO ; Jinling FAN
Chinese Journal of Biotechnology 2012;28(2):233-242
Proteasome is a multi-subunit protease complex in eukaryocytes, and plays an important role in ubiquitin-proteosome pathway. Recombinant proteasome can be used to screen proteasome inhibitors. In this study, recombinant plasmid of pET28a-PSMB1 was constructed by inserting human proteasome catalytic subunit (PSMB1) cDNA (726 bp) into the prokaryotic expression vector pET28a(+), and transforming the plasmid into E. coli BL21(DE3) cells for expression. After overnight induction (1 mmol/L IPTG, 20 degrees C), an expected protein band with molecular weight of 27 kDa was observed on SDS-PAGE gel. The recombinant protein was then purified through affinity chromatography, and the purity is more than 95%. The amino acid sequence of the recombinant protein was validated by NanoLC-MS/MS. The data from in vitro BIAcore analysis showed that the recombinant PSMB1 could bind to celastrol. The binding affinity between PSMB1 and 10 micromol/L celastrol was more than 27RU.
Binding Sites
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Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Humans
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Proteasome Endopeptidase Complex
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biosynthesis
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genetics
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isolation & purification
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Proteasome Inhibitors
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isolation & purification
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Recombinant Proteins
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biosynthesis
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genetics
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metabolism
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Triterpenes
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metabolism
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Ubiquitin