ABSTRACT
<p><b>OBJECTIVE</b>To study the membrane mobility of aquaporin 7 (AQP7) by cloning stably transfected CHO cells with expression of pEGFP-C1-AQP7, in which AQP7 cDNA was fused downstream and in frame to pEGFP-C1 gene.</p><p><b>METHODS</b>The full sequence of AQP7 was amplified by RT-PCR and then recombined in the downstream of the green fluorescent protein gene in the pEGFP-C1 vector. The recombinant vector pEGFP-C1-AQP7 was stably transfected into CHO cells. With fluorescent microscopy, immunocytochemical stain and Western blot, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization.</p><p><b>RESULTS</b>(1) The sequence of AQP7 cDNA of the Wistar rat was logged into the GenBank (access number: AY157737). (2) Identification demonstrated that pEGFP-C1-AQP7 fusion protein stably expressed in CHO cells. (3) With fluorescence microscopy, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization.</p><p><b>CONCLUSION</b>The CHO cell line with stable pEGFP-C1-AQP7 expression was set up successfully for advanced research.</p>
Subject(s)
Animals , Cricetinae , Male , Rats , Aquaporins , Genetics , CHO Cells , Cricetulus , DNA, Complementary , Genetics , Green Fluorescent Proteins , Genetics , Molecular Sequence Data , Rats, Wistar , Recombinant Fusion Proteins , Reverse Transcriptase Polymerase Chain Reaction , Testis , Metabolism , TransfectionABSTRACT
AIM: Aquaporins(AQP) are very important for the water transport across cell membrane. Aquaporin 7(AQP 7) and aquaporin 8(AQP8) expressed in the rat testis, but the biophysical functions of these two aquporins in testis and the regulatory mechanism of their expression remain unclear. The aim of this study was to find out if the expression of these two aquporins was correlative with the function of testis, and if panaxadiol saponins (PDS) affected their expression. METHODS: 4 weeks, 8 weeks and 12 weeks old rats were divided into saline and PDS injection groups. Semi-quantified RT-PCR method was employed to detect aquaporin 7 and 8 gene expression: using total RNA extracted from rat testis as PCR template, then using optical scanner to measure the OD value of bands on agrose electrophoresis. OD value of target gene products was divided by which of contol gene. The ratio was identified as the quantity of target gene expression. Western blot was also employed to detect expression of these two proteins in the testis. RESULTS: ①OD value of AQP 7 mRNA products in testis of 4 weeks, 8 weeks, 12 weeks rats were 48 227, 51 536, 59 567 respectively and the ratio divided by OD value of control gene was 0.82, 0.85, 0.99; OD value of AQP 8 mRNA products in testis of 4 weeks, 8 weeks, 12 weeks rats were 23 092, 39 302, 43 316 respectively, the ratio divided by OD value of control gene was 0.39, 0.65, 0.72. Thus, AQP 7 and 8 mRNA expression increased with age. ②After PDS injection for 2 weeks. AQP 7 and 8 mRNA expression in 4 weeks old rats increased to 0.97 and 0. 72,which in control group were 0. 82 and 0. 39; the ratio decreased to 0.77 and 0.55 in 8 weeks old rats, which in control group were 0.85 and 0.65. There was no PCR product of neither aquaporins in 12 weeks old PDS injected rats, except products of control gene. ③ Western blot of AQP 7 and 8 protein showed little difference between PDS and saline injected 12 weeks old rats. CONCLUSION: ①Quantity of AQP 7 and AQP 8 expression was related to testis function of rats. ②PDS influenced the expression of AQP 7 and AQP 8 mRNA, perhaps by promoting the secretion of LH in pituitary.
ABSTRACT
AIM:Aquaporins (AQP) are very important for the water transport across cell membrane. There are at least 10 mammalian AQPs( aquaporins 0-9) distributed in various organs and different kinds of cells. Each AQP has a distinct organ distribution, and this distribution could be useful in presuming the biological function of the aquaporin. The aim of this study was to figure out the distribution of aquaporin 7 (AQP7) and aquaporin 8(AQP8).METHODS：Semi-quantified RT-PCR was employed in this research. The ratio of OD value of target gene products divided by which of control gene products was calculated. Among 35 organs, testis, epididymis, skin, muscle, rectum, lung, bronchus, lymph node, stomach, duodenum, jejunum, ileum, colon, pancreas, liver, gall bladder, spleen, mammary gland, uterus, placenta, tonsil, urinary bladder, thyroid came from normal area of removed samples during operation. cDNA library of Prostate, thymus, salivary gland, penis, carotiol artery, adrenal gland, occipital lobe of brain, temporal lobe of brain, frontal lobe of brain, parietal lobe of brain, mid brain, choroid plexus are purchased from OriGene biotechnique company.RESULTS：①AQP 7 mRNA was found in testis, muscle, gall bladder, carotiol artery, lymph node and adrenal gland, and maximum expression of AQP 7 was in testis.②AQP 8 mRNA was found in pancreas, testis, skin and colon. and maximum expression of AQP 8 was in pancreas.CONCLUSION:Coexistence of AQP 7 and 8 in testis was confirmed, which suggested that both of these two aquaporins were involved in the regulation of testis function.
ABSTRACT
AIM: To find out whether different dosage of rare earth element-lanthanum can influence the expression of aquaporin 7(AQP 7) in the testis of rats. METHODS:Rats were fed with lanthanum nitrate ［La(NO3)3］ and killed 6 months later. Testes were then removed immediately to extract total RNA. Northern blot analysis is performed finally. RESULTS:0.1 mg/kg La(NO3)3 depressed the expression of AQP 7 in rat testis, while 20 mg/kg La(NO3)3 had no significant effect on it. CONCLUSION: AQP 7 expession is found in the rat testis; La(NO3)3 can depress the expression of AQP 7 in the rat testis.
ABSTRACT
AIM: To find out whether different dosage of rare earth element-lanthanum can influence the expression of aquaporin 7(AQP 7) in the testis of rats. METHODS:Rats were fed with lanthanum nitrate [La(NO 3) 3] and killed 6 months later. Testes were then removed immediately to extract total RNA. Northern blot analysis is performed finally. RESULTS:0.1 mg/kg La(NO 3) 3 depressed the expression of AQP 7 in rat testis, while 20 mg/kg La(NO 3) 3 had no significant effect on it. CONCLUSION: AQP 7 expession is found in the rat testis; La(NO 3) 3 can depress the expression of AQP 7 in the rat testis.