精氨酸加压素调控AQP4介导星形胶质细胞水通透性的信号转导机制的研究

发布时间：2018-05-25 19:36

本文选题：精氨酸加压素 + 水通道蛋白-4　；参考：《浙江大学》2010年博士论文

【摘要】： 目的构建绿色荧光蛋白(GFP)标识的水通道蛋白-4(AQP4)及其突变体示踪系统,利用激光共聚焦技术,建立表达AQP4及其突变体的大鼠星形胶质细胞水通透性的评价系统,并利用该系统探讨精氨酸加压素(AVP)对AQP4水通透性的调控及信号转导机制。方法依据大鼠AQP4-M23的cDNA序列和质粒pEGFP-C1多克隆位点(MCS)的特点设计引入合适的内切酶序列的引物,通过RT-PCR法获取AQP4-M23的DNA序列,凝胶电泳鉴定；利用pMD(?)20-T克隆载体通过T-A克隆扩增AQP4-M23,并进行基因测序鉴定；酶切T-A克隆所得AQP4-M23与pEGFP-C1质粒进行连接反应,完成pEGFP-C1-AQP4-M23的构建,鉴定正确后,扩增、提取纯化融合质粒。设计将AQP4-M23的第111位和180位的丝氨酸突变为丙氨酸的引物对,利用Muta-directTM定点突变试剂盒通过PCR技术使特定位点突变,构建pEGFP-C 1-S111A-AQP4-M23釉pEGFP-C 1-S180A-AQP4-M23两种含突变位点的融合质粒。水通透性测定：依据优化实验结果,将构建的融合质粒瞬时转染不表达AQP4的CTX-TNA2星形胶质细胞,37℃、5%CO2、完全培养基培养24h使目的蛋白表达。利用激光扫描共聚焦显微镜(LSCM)成像技术记录GFP荧光图像,以确定细胞表达与不表达目的蛋白,然后记录不同干预条件下,钙黄绿素荧光强度的变化,利用LSCM的脱机图像分析软件LAS AF Lite和免费图像处理软件Image J 1.42对脱机图像进行处理,分析GFP在细胞上的分布,观察V1aR激动剂Arg8-Vasoticin对AQP4定位的影响。选择表达目的蛋白的细胞及其周围不表达目的蛋白的细胞作为观察对象,分析换低渗环境前后不同时间点和不同干预条件下Calcein荧光强度的变化,取PBS由等渗液到换成低渗液即刻及其后10s内的荧光变化曲线作为分析对象,计算荧光强度随时间变化的速度,即时间导数τ,代表细胞水肿的速度,本实验中以-τ代表细胞膜对水的通透性的大小,以判断不同干预条件下AQP4-M23及其突变体对水的通透性。结果 1基于GFP标识的AQP4-M23及其突变体示踪系统的建立以SD大鼠脑组织总RNA为模板,利用自行设计的包含相应酶切位点序列的引物进行RT-PCR扩增AQP4-M23产物的凝胶电泳凝胶见大小正确的条带,T-A克隆扩增目的基因后测序,比对基因测序结果和基因库数据可见测序图中序号为24-929的碱基共计906个碱基和AQP4-M23序列完全一致,并在序列前后正确引入了内切酶HindⅢ和XbaⅠ酶切位点的序列,通过RT-PCR技术准确获取了AQP4-M23全长序列。利用基因重组技术连接真核表达质粒pEGFP-C1和所获得的含有合适酶切位点的AQP4-M23序列后,酶切证实目的基因(AQP4-M23)以正确方式插入质粒pEGFP-C1的MCS中。以融合质粒pEGFP-C1-AQP4-M23为模板,设计引物改变相应突变位点碱基通过PCR技术分别实现S111和S180。两个氨基酸残基对应碱基的突变,基因测序图谱分别示S111对应的碱基AGC突变为GCC、S180对应的碱基TCC突变为GCC。RT-PCR证实,CTX-TNA2星形胶质细胞系不表达内源性AQP4。LSCM扫描结果示,GFP标记于AQP4-M23的氨基端,不影响AQP4-M23定位在胞膜上,融合蛋白在细胞膜上表达,均匀分布,胞浆内仅痕量表达；S111和S180突变为丙氨酸后不影响突变体在细胞膜上的定位。下调仪器增益可消除GFP的荧光对钙黄绿素荧光强度测量的影响。表达了GFP标记的AQP4-M23的CTX-TNA2细胞,随蛋白表达量的增加,细胞膜对水的通透性增加,GFP荧光强度增加到一定程度后,进一步增加时,细胞膜对水的通透性未出现明显增加。 2 AVP对AQP4-M23水通透性的影响本实验中Arg8-vasotocin未改变转染了pEGFP-C1-AQP4-M23融合质粒的CTX-TNA2星形胶质细胞GFP荧光强度及其在细胞膜及胞浆内分布。表达AQP4-M23的CTX-TNA2细胞对水的通透性明显增加,达5倍左右(P0.01)；Arg8-vasotocin对不表达AQP4-M23的CTX-TNA2细胞对水的通透性无影响(P0.05); Arg8-vasotocin处理使表达AQP4-M23的CTX-TNA2细胞对的水通透性进一步增加(P0.01)；在Arg8-vasotocin处理前先加CaMKⅡ抑制剂KN-62预处理,可阻断Arg8-vasotocin的这种作用,在Arg8-vasotocin处理前先加PKC抑制剂BISI预处理,使表达AQP4-M23的CTX-TNA2细胞对的水通透性略有增加,但无统计学意义(P0.05)。 3 S111突变对AQP4-M23水通透性及AVP干预的影响和表达了AQP4-M23的CTX-TNA2细胞相比,AQP4-M23第111位的丝氨酸突变为丙氨酸后对水的通透性没有明显的影响；Arg8-vasotocin处理不能使表达S111A-AQP4-M23的CTX-TNA2细胞对的水通透性进一步增加,BISI预处理对表达S111A-AQP4-M23的CTX-TNA2细胞对水的通透性无明显影响(P0.05)。 4 S180突变对AQP4-M23水通透性及AVP干预的影响和表达了AQP4-M23的CTX-TNA2细胞相比,AQP4-M23第180位的丝氨酸突变为丙氨酸后对水的通透性没有明显的影响；Arg8-vasotocin处理仍可使表达S180A-AQP4-M23的CTX-TNA2细胞对的水通透性进一步增加(P0.01),KN-62预处理仍可阻断Arg8-vasotocin的这种作用(P0.05)。结论本实验成功构建GFP标识的AQP4及其突变体示踪系统,建立了表达AQP4及其突变体的大鼠星形胶质细胞水通透性的评价系统,AQP4可使星形胶质细胞对水的通透性明显增加,AVP可通过激活V1aR增加AQP4对水的通透性,直接或间接通过CaMKⅡ作用于AQP4的111位的丝氨酸是其可能的作用途径。
[Abstract]:Objective to construct the aquaporin -4 (AQP4) identified by green fluorescent protein (GFP) and its mutant tracer system. The water permeability evaluation system of rat astrocytes expressing AQP4 and its mutants was established by laser confocal technique. The system was used to study the regulation and signal transduction of arginine vasopressin (AVP) on the permeability of AQP4 water. Mechanism.
Methods based on the cDNA sequence of rat AQP4-M23 and the plasmid pEGFP-C1 polyclonal site (MCS), the primers were designed to introduce the appropriate endonuclease sequence. The DNA sequence of AQP4-M23 was obtained by RT-PCR method, gel electrophoresis was identified, and pMD (?) 20-T cloning vector was used to clone and expand AQP4-M23 by T-A, and the gene sequencing identification was carried out, and the enzyme cut T-A clone was cloned. AQP4-M23 and pEGFP-C1 plasmids were connected to complete the construction of pEGFP-C1-AQP4-M23. After identification, the fusion plasmid was amplified and purified. The primer pairs of AQP4-M23 111st and 180 serine were mutated to alanine, and the specific location point was mutated by PCR technology, and pEGF was constructed by Muta-directTM site mutation kit. P-C 1-S111A-AQP4-M23 enamel pEGFP-C 1-S180A-AQP4-M23 two fusion plasmid containing mutation site.
Water permeability measurement: Based on the optimized experimental results, the constructed fusion plasmid was transiently transfected into CTX-TNA2 astrocytes which did not express AQP4, 37 C, 5%CO2, and full medium culture 24h to express the target protein. The laser scanning confocal microscopy (LSCM) imaging technique was used to record the GFP fluorescence image, in order to determine the purpose of cell expression and non expression. Protein, and then record the changes in fluorescence intensity of calcein under different intervention conditions, processing the offline image by using LSCM's offline image analysis software LAS AF Lite and free image processing software Image J 1.42, analyze the distribution of GFP on the cell, observe the effect of V1aR agonist Arg8-Vasoticin on the positioning of AQP4, and choose the expression target egg. White cells and their surrounding cells that do not express the target protein are used as observation objects, and the changes of fluorescence intensity of Calcein under different time points and different intervention conditions before and after low permeability environment are analyzed. The fluorescence change curves of PBS from isosotic to hypotonic fluid immediately and after 10s are taken as the analysis object, and the change of fluorescence intensity with time is calculated. The velocity, the time derivative tau, represents the rate of cell edema. In this experiment, tau represents the permeability of the cell membrane to water, in order to determine the permeability of AQP4-M23 and its mutants to water under different intervention conditions.
Result
1 Establishment of AQP4-M23 and its mutant tracing system based on GFP identification
Taking the total RNA of the brain tissue of SD rats as a template, the gel electrophoresis gel of RT-PCR amplification of AQP4-M23 products with the primers containing the corresponding sequence of the corresponding enzyme cut site sequence was used to see the correct bands, and the T-A clones were amplified and sequenced after the amplification of the target gene. Compared with the gene sequencing results and the gene bank data, the sequence number of the sequence number is 24-929. The sequences of the endonuclease Hind III and Xba I sites were correctly introduced before and after the sequence of the 906 bases and AQP4-M23 sequences, and the whole sequence of AQP4-M23 was accurately obtained by RT-PCR technology. The enzyme of eukaryotic expression plasmid pEGFP-C1 and the AQP4-M23 sequence containing the appropriate enzyme cut site were obtained by gene recombination technology. It was confirmed that the target gene (AQP4-M23) was inserted into the MCS of plasmid pEGFP-C1 correctly. Using the fusion plasmid pEGFP-C1-AQP4-M23 as the template, the primers changed the base base of the corresponding mutation site to realize the mutation of the corresponding base of the two amino acid residues of S111 and S180. respectively, and the DNA sequencing Atlas showed the AGC mutation corresponding to the base of S111, respectively. For GCC, S180 corresponding base TCC mutation is confirmed to GCC.RT-PCR, CTX-TNA2 astrocyte line does not express endogenous AQP4.LSCM scanning results, GFP is labeled at the amino end of AQP4-M23, and does not affect the AQP4-M23 location on the cell membrane. The fusion protein is expressed on the cell membrane, uniform distribution, only trace expression in the cytoplasm; S111 and S180 mutation to alanine. It does not affect the location of the mutant on the cell membrane. Down regulation of the instrument gain can eliminate the effect of GFP's fluorescence on the fluorescence intensity measurement of calcein. The expression of the GFP labeled AQP4-M23 CTX-TNA2 cells, with the increase of protein expression, the permeability of the cell membrane to water, the increase of GFP fluorescence intensity to a certain extent, and further increase The permeability of the membrane to water did not increase significantly.
Effect of 2 AVP on water permeability of AQP4-M23
In this experiment, Arg8-vasotocin did not change the GFP fluorescence intensity of the CTX-TNA2 astrocytes transfected with the pEGFP-C1-AQP4-M23 fusion plasmid and its distribution in the cell membrane and cytoplasm. The permeability of the CTX-TNA2 cells expressing AQP4-M23 increased significantly, about 5 times (P0.01), and Arg8-vasotocin against the water of CTX-TNA2 cells that did not express AQP4-M23. There is no effect on permeability (P0.05); the water permeability of CTX-TNA2 cells expressing AQP4-M23 is further increased by Arg8-vasotocin treatment (P0.01), and CaMK II inhibitor KN-62 pretreatment before Arg8-vasotocin treatment can block the effect of Arg8-vasotocin, before Arg8-vasotocin treatment, PKC inhibitor is pretreated before Arg8-vasotocin treatment. 23 of CTX-TNA2 cells showed a slight increase in water permeability, but there was no statistical significance (P0.05).
Effect of 3 S111 mutation on AQP4-M23 water permeability and AVP intervention
Compared with the CTX-TNA2 cells expressing AQP4-M23, the serine mutation of AQP4-M23 111st bits to alanine has no obvious effect on the permeability of water; Arg8-vasotocin treatment can not further increase the water permeability of CTX-TNA2 cells expressing S111A-AQP4-M23, and BISI pretreatments for the CTX-TNA2 cells expressing S111A-AQP4-M23 to water. There was no obvious effect on permeability (P0.05).
Effect of 4 S180 mutation on AQP4-M23 water permeability and AVP intervention
Compared with CTX-TNA2 cells expressing AQP4-M23, the mutation of serine in AQP4-M23 180th bits to alanine had no significant effect on the permeability of water. Arg8-vasotocin treatment could further increase the water permeability of CTX-TNA2 cells expressing S180A-AQP4-M23 (P0.01), and KN-62 preconditioning could still block the effect of Arg8-vasotocin. P0.05). Conclusion the experiment successfully constructed the AQP4 of GFP identification and its mutant tracer system, and established the evaluation system of water permeability of astrocytes in rat astrocytes expressing AQP4 and its mutant. AQP4 can increase the permeability of astrocytes to water obviously. AVP can increase the permeability of AQP4 to water by activating V1aR, directly or indirectly through C. AMK 111 acting on the serine of AQP4 is the possible way of action.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

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