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1.
Asian Journal of Andrology ; (6): 508-515, 2019.
Artigo em Inglês | WPRIM | ID: wpr-1009689

RESUMO

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.


Assuntos
Animais , Humanos , Masculino , Ratos , Apoptose , Dieta , Células Endoteliais , Disfunção Erétil/prevenção & controle , Fibrose , Hiper-Homocisteinemia/complicações , Metionina , Estresse Oxidativo , Pênis/patologia , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais/genética , Calicreínas Teciduais/genética
2.
Electron. j. biotechnol ; 13(5): 10-11, Sept. 2010. ilus, tab
Artigo em Inglês | LILACS | ID: lil-591892

RESUMO

Response surface methodology was undertaken to optimize the polyethylenimine-mediated transient transfection of suspension cultured HEK 293-F cells. A total of 15 combinations were designed according to Box-Behnken design to identify the effects of DNA concentration, polyethylenimine concentration and incubation time on transient transfection efficiency. The highest integral optic density of green fluorescent protein presenting r-protein yield was accessed using a DNA concentration of 1.75 ug/mL, a polyethylenimine concentration of 10.5 ug/mL, and an incubation time of 11.8 min. Analysis of variance demonstrated that the experimental values fit well with a quadratic model. The RSM-optimized transient transfection resulted in greater production of human tissue prokallikrein (TproK) than non-RSM-optimized conditions: protein yield was 32.0 mg/L and the maximum viable cell density reached 3.57 x 10(6) cells/mL in a 5 L stirred-tank bioreactor culture.


Assuntos
Humanos , Calicreínas Teciduais/genética , Expressão Gênica , Transfecção , Análise de Variância , Reatores Biológicos , Linhagem Celular , Polietilenoimina , Vetores Genéticos/genética
3.
Experimental & Molecular Medicine ; : 23-27, 2004.
Artigo em Inglês | WPRIM | ID: wpr-190978

RESUMO

To evaluate the feasibility of treating hypertension by human tissue kallikrein gene (KLK1) delivery and by enzyme (rK1) administration, two recombinant vectors expressing KLK1 cDNA were constructed for gene delivery (pcDNA-KLK1) and recombinant enzyme preparation (pOV-KLK1). Expression of the pcDNA-KLK1 vector in COS-1 cells was confirmed by immunofluorescence and in spontaneous hypertension rats (SHR) by enzymatic detection. Following intramuscular or intravenous injection with the pcDNA-KLK1 vector, systolic pressure of SHR was significantly decreased, which lasted for 20 d to two months depending on dose, route and/or time of injection. Egg white containing recombinant hK1 was prepared by injection of egg-laying hens with the oviduct-specific expression vector pOV-KLK1 and administered into SHR via oral gavage. Following administration, systolic pressure of the SHR was decreased to that of normal rats, which lasted for 3-5 d depending on the dosage used. These data suggest that both hKLK1 gene delivery and recombinant enzyme administration can be used as alternative strategies for treating human hypertension.


Assuntos
Animais , Feminino , Humanos , Ratos , Pressão Sanguínea/fisiologia , Células COS , Chlorocebus aethiops , Galinhas , Terapia Genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hipertensão/genética , Hipotensão/genética , Ratos Endogâmicos SHR , Proteínas Recombinantes/administração & dosagem , Calicreínas Teciduais/genética
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