本文選題：精氨酸加壓素 + 水通道蛋白-4��； 參考：《浙江大學(xué)》2010年博士論文

【摘要】： 目的構(gòu)建綠色熒光蛋白(GFP)標(biāo)識的水通道蛋白-4(AQP4)及其突變體示蹤系統(tǒng),利用激光共聚焦技術(shù),建立表達(dá)AQP4及其突變體的大鼠星形膠質(zhì)細(xì)胞水通透性的評價(jià)系統(tǒng),并利用該系統(tǒng)探討精氨酸加壓素(AVP)對AQP4水通透性的調(diào)控及信號轉(zhuǎn)導(dǎo)機(jī)制。 方法依據(jù)大鼠AQP4-M23的cDNA序列和質(zhì)粒pEGFP-C1多克隆位點(diǎn)(MCS)的特點(diǎn)設(shè)計(jì)引入合適的內(nèi)切酶序列的引物,通過RT-PCR法獲取AQP4-M23的DNA序列,凝膠電泳鑒定；利用pMD(?)20-T克隆載體通過T-A克隆擴(kuò)增AQP4-M23,并進(jìn)行基因測序鑒定；酶切T-A克隆所得AQP4-M23與pEGFP-C1質(zhì)粒進(jìn)行連接反應(yīng),完成pEGFP-C1-AQP4-M23的構(gòu)建,鑒定正確后,擴(kuò)增、提取純化融合質(zhì)粒。設(shè)計(jì)將AQP4-M23的第111位和180位的絲氨酸突變?yōu)楸彼岬囊飳?利用Muta-directTM定點(diǎn)突變試劑盒通過PCR技術(shù)使特定位點(diǎn)突變,構(gòu)建pEGFP-C 1-S111A-AQP4-M23釉pEGFP-C 1-S180A-AQP4-M23兩種含突變位點(diǎn)的融合質(zhì)粒。 水通透性測定：依據(jù)優(yōu)化實(shí)驗(yàn)結(jié)果,將構(gòu)建的融合質(zhì)粒瞬時(shí)轉(zhuǎn)染不表達(dá)AQP4的CTX-TNA2星形膠質(zhì)細(xì)胞,37℃、5%CO2、完全培養(yǎng)基培養(yǎng)24h使目的蛋白表達(dá)。利用激光掃描共聚焦顯微鏡(LSCM)成像技術(shù)記錄GFP熒光圖像,以確定細(xì)胞表達(dá)與不表達(dá)目的蛋白,然后記錄不同干預(yù)條件下,鈣黃綠素?zé)晒鈴?qiáng)度的變化,利用LSCM的脫機(jī)圖像分析軟件LAS AF Lite和免費(fèi)圖像處理軟件Image J 1.42對脫機(jī)圖像進(jìn)行處理,分析GFP在細(xì)胞上的分布,觀察V1aR激動劑Arg8-Vasoticin對AQP4定位的影響。選擇表達(dá)目的蛋白的細(xì)胞及其周圍不表達(dá)目的蛋白的細(xì)胞作為觀察對象,分析換低滲環(huán)境前后不同時(shí)間點(diǎn)和不同干預(yù)條件下Calcein熒光強(qiáng)度的變化,取PBS由等滲液到換成低滲液即刻及其后10s內(nèi)的熒光變化曲線作為分析對象,計(jì)算熒光強(qiáng)度隨時(shí)間變化的速度,即時(shí)間導(dǎo)數(shù)τ,代表細(xì)胞水腫的速度,本實(shí)驗(yàn)中以-τ代表細(xì)胞膜對水的通透性的大小,以判斷不同干預(yù)條件下AQP4-M23及其突變體對水的通透性。 結(jié)果 1基于GFP標(biāo)識的AQP4-M23及其突變體示蹤系統(tǒng)的建立 以SD大鼠腦組織總RNA為模板,利用自行設(shè)計(jì)的包含相應(yīng)酶切位點(diǎn)序列的引物進(jìn)行RT-PCR擴(kuò)增AQP4-M23產(chǎn)物的凝膠電泳凝膠見大小正確的條帶,T-A克隆擴(kuò)增目的基因后測序,比對基因測序結(jié)果和基因庫數(shù)據(jù)可見測序圖中序號為24-929的堿基共計(jì)906個(gè)堿基和AQP4-M23序列完全一致,并在序列前后正確引入了內(nèi)切酶HindⅢ和XbaⅠ酶切位點(diǎn)的序列,通過RT-PCR技術(shù)準(zhǔn)確獲取了AQP4-M23全長序列。利用基因重組技術(shù)連接真核表達(dá)質(zhì)粒pEGFP-C1和所獲得的含有合適酶切位點(diǎn)的AQP4-M23序列后,酶切證實(shí)目的基因(AQP4-M23)以正確方式插入質(zhì)粒pEGFP-C1的MCS中。以融合質(zhì)粒pEGFP-C1-AQP4-M23為模板,設(shè)計(jì)引物改變相應(yīng)突變位點(diǎn)堿基通過PCR技術(shù)分別實(shí)現(xiàn)S111和S180。兩個(gè)氨基酸殘基對應(yīng)堿基的突變,基因測序圖譜分別示S111對應(yīng)的堿基AGC突變?yōu)镚CC、S180對應(yīng)的堿基TCC突變?yōu)镚CC。RT-PCR證實(shí),CTX-TNA2星形膠質(zhì)細(xì)胞系不表達(dá)內(nèi)源性AQP4。LSCM掃描結(jié)果示,GFP標(biāo)記于AQP4-M23的氨基端,不影響AQP4-M23定位在胞膜上,融合蛋白在細(xì)胞膜上表達(dá),均勻分布,胞漿內(nèi)僅痕量表達(dá)；S111和S180突變?yōu)楸彼岷蟛挥绊懲蛔凅w在細(xì)胞膜上的定位。下調(diào)儀器增益可消除GFP的熒光對鈣黃綠素?zé)晒鈴?qiáng)度測量的影響。表達(dá)了GFP標(biāo)記的AQP4-M23的CTX-TNA2細(xì)胞,隨蛋白表達(dá)量的增加,細(xì)胞膜對水的通透性增加,GFP熒光強(qiáng)度增加到一定程度后,進(jìn)一步增加時(shí),細(xì)胞膜對水的通透性未出現(xiàn)明顯增加。 2 AVP對AQP4-M23水通透性的影響 本實(shí)驗(yàn)中Arg8-vasotocin未改變轉(zhuǎn)染了pEGFP-C1-AQP4-M23融合質(zhì)粒的CTX-TNA2星形膠質(zhì)細(xì)胞GFP熒光強(qiáng)度及其在細(xì)胞膜及胞漿內(nèi)分布。表達(dá)AQP4-M23的CTX-TNA2細(xì)胞對水的通透性明顯增加,達(dá)5倍左右(P0.01)；Arg8-vasotocin對不表達(dá)AQP4-M23的CTX-TNA2細(xì)胞對水的通透性無影響(P0.05); Arg8-vasotocin處理使表達(dá)AQP4-M23的CTX-TNA2細(xì)胞對的水通透性進(jìn)一步增加(P0.01)；在Arg8-vasotocin處理前先加CaMKⅡ抑制劑KN-62預(yù)處理,可阻斷Arg8-vasotocin的這種作用,在Arg8-vasotocin處理前先加PKC抑制劑BISI預(yù)處理,使表達(dá)AQP4-M23的CTX-TNA2細(xì)胞對的水通透性略有增加,但無統(tǒng)計(jì)學(xué)意義(P0.05)。 3 S111突變對AQP4-M23水通透性及AVP干預(yù)的影響 和表達(dá)了AQP4-M23的CTX-TNA2細(xì)胞相比,AQP4-M23第111位的絲氨酸突變?yōu)楸彼岷髮λ耐ㄍ感詻]有明顯的影響；Arg8-vasotocin處理不能使表達(dá)S111A-AQP4-M23的CTX-TNA2細(xì)胞對的水通透性進(jìn)一步增加,BISI預(yù)處理對表達(dá)S111A-AQP4-M23的CTX-TNA2細(xì)胞對水的通透性無明顯影響(P0.05)。 4 S180突變對AQP4-M23水通透性及AVP干預(yù)的影響 和表達(dá)了AQP4-M23的CTX-TNA2細(xì)胞相比,AQP4-M23第180位的絲氨酸突變?yōu)楸彼岷髮λ耐ㄍ感詻]有明顯的影響；Arg8-vasotocin處理仍可使表達(dá)S180A-AQP4-M23的CTX-TNA2細(xì)胞對的水通透性進(jìn)一步增加(P0.01),KN-62預(yù)處理仍可阻斷Arg8-vasotocin的這種作用(P0.05)。結(jié)論本實(shí)驗(yàn)成功構(gòu)建GFP標(biāo)識的AQP4及其突變體示蹤系統(tǒng),建立了表達(dá)AQP4及其突變體的大鼠星形膠質(zhì)細(xì)胞水通透性的評價(jià)系統(tǒng),AQP4可使星形膠質(zhì)細(xì)胞對水的通透性明顯增加,AVP可通過激活V1aR增加AQP4對水的通透性,直接或間接通過CaMKⅡ作用于AQP4的111位的絲氨酸是其可能的作用途徑。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346

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