ABSTRACT
Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.
Subject(s)
Urinary Bladder Neoplasms/metabolism , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/metabolism , Recombinant Fusion Proteins/genetics , Protamines/metabolism , Inclusion Bodies , Cloning, Molecular , Apoptosis , RNA, Small Interfering , Escherichia coli/metabolism , Receptor, Fibroblast Growth Factor, Type 3 , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/genetics , Flow CytometryABSTRACT
O processo de criopreservação causa estresse físico e químico aos espermatozóides, acarretando alterações bioquímicas, diminuição irreverssível da motilidade espermática, aumento da degeneração do DNA e liberação intracelular de enzimas e lipídeos. No presente estudo, foram estudadas a influência das estações não reprodutiva e reprodutiva, dos crioprotetores glicerol e etilenoglicol e do processo de congelação e descongelação sobre o complexo DNA-proteína de espermatozóides em garanhões. Foi comparados o sêmen puro, o sêmen puro e congelado sem crioprotetores, o sêmen diluído e exposto aos crioprotetores sem congelação e o sêmen diluído e congelado com crioprotetores. Foram utilizados seis garanhões, colhendo 12 ejaculados cada. A patologia do complexo DNA-Proteína foi avaliada em espermatozóides fixados com etanol-ácido-acético glacial 3: 1 (v/ v), tratados com HCL 4N a 25°C e corados com azul de toluidina a 0,025% em tampão McIlvaine, empregando microscopia óptica com aumento de 1000x. Os resultados mostraram que a anomalia do complexo DNA-Proteína dos espermatozóides diferem entre os grupos congelados e não congelados (P<0,05). O sêmen congelado sem crioprotetor não apresentou aumento significativo de patologia do complexo DNA-Proteína em relação ao sêmen congelado com crioprotetores, mas ambos mostraram aumento em relação ao sêmen puro ou diluído e exposto aos crioprotetores. A influência da estação reprodutiva mostrou diferença significativa (P<0,05) somente no sêmen puro e no sêmen puro e congelado sem crioprotetor. Conclui-se que o processo de congelação exerce influência negativa sobre o complexo DNA-Proteína de espermatozóides em garanhões.
The cryopreservation process cause stress physical and chemical to the spermatozoa, causing biochemistry alteration, irreversible reduction of the spermatic motility, increase of the DNA degeneration and intracellular enzyme and lipids release. The aim of this study was to evaluate the influence of non-breeding and breeding seasons, glycerol and ethylene glycol, cryopreservation and thawing processes on stallion spermatozoa DN A -protein complexo It was compared fresh semen, diluted semen frozen without cryoprotectants, diluted semen exposured to cryoprotectants but not frozen and d) diluted semen frozen with cryoprotectants. Six stallions had 12 semen collections each. DNA-protein complex pathology was assessed by optical microscopy (1000x) using spermatozoa treated with ethanol-acetic acid 3:1 (v/v), HCl 4N at room temperature and toluidin blue 0,025% in McIlavaine buffer. Results showed that DNA-protein complex were different between frozen and not frozen spermatozoa groups (P<0,05). Frozen semen without cryoprotectants had no increasing of DNA-protein complex pathology compared to semen cryopreserved with cryoprotectant, but both showed increasing in relation to fresh and diluted semen exposured to cryoprotectants. The influence of non breeding and breeding season showed significant difference (P<0,05) in the fresh semen and fresh semen frozen without cryoprotectants. Cryopreservation process had negative influence on spermatozoa DNA-protein complex.