| ObjectiveTo construct the GFP labeled aquaporin-4 tracing system, and establish the water permeability evaluation system for murine astrocytes that expressed AQP4 and its mutant by using of confocal microscope to investigate the signal transduction mechanism of the regulation of arginine vasopressin to AQP4-mediated water permeability.MethodsAppropriate primer sequences were designed and introduced by incision enzymes according to the characteristics of murine AQP4-M23 cDNA sequences and the pEGFP-C1 multiple cloning sites, then the AQP4-M23 sequences were acquired by RT-PCR authenticated by gel electrophoresis. The pMD(?)20-T cloning vector was used to expand AQP4-M23 by T-A clonal expansion and then authenticated by gene sequencing. Afterwards, the clonal AQP4-M23 and pEGFP-C1 plasmids were co-enzyme cut and, by the ligation reaction, to construct the pEGFP-C1-AQP4-M23. After the authentication, the ligated plasmids were expanded, extracted and purified.The pairs of primers of AQP4-M23 in which the serine was mutated to alanine in 111 and 180 sites were designed to construct the ligated plasmids that contained mutant sites of pEGFP-C1-S111A-AQP4-M23 and pEGFP-C1-S180A-AQP4-M23 by using of Muta-directTM site-directed mutagenesis kits.Measurement of water permeability:CTX-TNA2 astrocytes were transfected transiently with the ligated plasmids by using of TransFastTM transfection kit, cultured 24h to express protein.Laser scanning confocal microscope (LSCM) imaging technology was employed here to record the fluorescence image of GFP, and to measure the target protein in the astrocytes. The cells were then loaded with calcein. Variations of fluorescence intensity of the calcein were recorded by LAS AF Lite software and analyzed Image J 1.42 image processing software. Distribution of GFP on the membrane of astrocytes was elaborately measured to study the effects of Arg8-Vasoticin on the location of AQP4.The protein-expressed astrocytes were chosen for measurement as contrast to the non-protein-expressed astrocytes in their surroundings. The variations of the fluorescence intensity in different time points and different intervention conditions were analyzed after the culture medium was changed from isotonic to hypotonic state. The variation curve from the initiation of the change osmotic to 10s afterwards was chosen to observe the change rate of fluorescence intensity to time (derivative the fluorescence intensity to time,Ï„), which was related to the change rate of cellular edema, to determine the water permeability of the AQP4-M23 astrocytes and its mutant astrocytes in different intervention conditions.Results1. Construction of tracing system of GFP labeled AQP4-M23 and its mutant astrocytesRT-PCR primers that contain the restriction enzyme cutting sites were designed by using of total RNA in SD rat brain tissue as template, authenticated by electrophoresis and then T-A clonal expanded. The target gene was sequenced and matched with the Gene-Database. The sequencing diagram shown a total bases agreement with AQP4-M23, from the serial number 24-929, a total of 906 bases. The restriction enzyme cutting sites of Hindlll and Xba I were also correctly introduced. The total sequence of AQP4-M23 was acquired by the RT-PCR technique.The eukaryotic expression plasmids pEGFP-Cl and the sequences AQP4-M23, containing appropriate restriction Enzyme cutting sites, were ligated by the gene recombination technique. The sequences were enzyme cut and authenticated that the target genes were inserted into MCS.Use coupled pEGFP-C1-AQP4-M2 plasmid as template; the primers were designed to mutate the target bases. The two amino acid residues, S111 and S180 corresponding bases, were successfully mutated by PCR technique. Sequence diagram shown that the S111 corresponding site AGC was mutated to GCC, the S180 corresponding site TCC was mutated to GCC.CTX-TNA2 astrocytes do not express endogenous AQP4, further confirmed by our RT-PCR results. GFP was located at the amino terminal found by LSCM, which did not affect the location of AQP4-M23 on cell membrane. Fusion protein was found expressed on the cell membrane and no, if little, expression in the cytoplasm. The results demonstrated that the mutant of S111 or S180 to alanine has no effect on the transportation of the mutated gene to the cell membrane.Down-regulation of the gain of the instrument may eliminate the impact of GFP fluorescence on the measurement of calcein fluorescence.The permeability of astrocytes membranes for water increased with the increase in the amount of protein expression in the GFP labeled AQP4-M23 CTX-TNA2 astrocytes, but reached a plateau when the intensity fluorescence of GFP increased to a certain extent.2.The effect of AVP on the water permeability of AQP4-M23In our experiment, Arg8-vasotocin changed neither the GFP fluorescence intensity nor the distribution of it on the cell membrane or in the cytoplasm of pEGFP-C1-AQP4-M23 transfected astrocytes. The water permeability increased up to 5 folds in the AQP4-M23-expressed CTX-TNA2 astrocytes (P< 0.01). However, Arg8-vasotocin showed no influence on the water permeability in non-AQP4-M23-expressed CTX-TNA2 astrocytes (P> 0.05).The water permeability of was not affected by Arg8-vasotocin in non-AQP4-M23--expressed CTX-TNA2 astrocytes (P> 0.05) but further increased in AQP4-M23--expressed CTX-TNA2 astrocytes (P<0.01) when treated with Arg8-vasotocin, this effect could be blocked by pre-treatment with KN-62. Pre-treated with BISI increased little in the expression of AQP4-M23 in CTX-TNA2 astrocytes but had no statistical significance (P> 0.05).3. Influence of the S111 mutation on the water permeability and the treatment of AVPThe mutation of the 111st site of serine to alanine in AQP4-M23 demonstrated no influence on the water permeability contrast to the AQP4-M23-expressed CTX-TNA2 astrocytes. The water permeability did not increase in S111A-AQP4-M23-expressed CTX-TNA2 astrocytes when treated with Arg8-vasotocin. Pre-treated with BISI had no influence on the water permeability in S111 A-AQP4-M23-expressed CTX-TNA2 astrocytes (P>0.05).4. Influence of the S180 mutation on the water permeability and the treatment of AVPThe mutation of the 180th site of serine to alanine of AQP4-M23 demonstrated no influence on the water permeability contrast to the AQP4-M23-expressed CTX-TNA2 astrocytes. Water permeability further increased in the S180A-AQP4-M23-expressed astrocytes when pre-treated with Arg8-vasotocin (P<0.01) and could be blocked by pre-treatment with KN-62. ConclusionIn our experiment, tracing system of GFP labeled AQP4 and its mutants was successfully constructed; the water permeability evaluation system was established for murine astrocytes that expressed AQP4 and its mutants. The water permeability increased in the AQP4-M23-expressed astrocytes;AVP could increase the water permeability of AQP4 through activation of V1aR. The possible pathway maybe lies in that it acts on the 111th site of serine of AQP4, directly or indirectly through CaMKâ…¡.
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