【作者】 贾彩霞；

【导师】 张海林；

【作者基本信息】 河北医科大学 ， 药理学， 2007， 硕士

【摘要】 磷脂是细胞膜特有组分,主要包括卵磷脂、脑磷脂、肌醇磷脂,其中肌醇磷脂尤其是磷脂酰肌醇二磷酸(phosphatidylinositol-4,5-bisphosphate, PIP2)在信号转导中具有重要作用。PIP2占细胞膜总磷脂的含量很少(不到1%),但却在胞内第二信使的产生、胞吐与胞饮调控、细胞骨架与胞膜贴附等一系列细胞的生理过程及生命活动中发挥着重要作用。此外,PIP2还可以调控很多离子转运体和离子通道的功能,如Na/Ca交换体,Na/H交换体,电压门控性钙离子通道,TRP通道,内向整流性钾离子通道(Kir通道)以及KCNQ通道等。PIP2的代谢在对离子通道的功能调节中发挥重要作用。近年研究表明,神经递质、蛋白激酶、pH值、带电离子、药物、脂类代谢物等均可作为调节因素调节离子通道的功能,而上述调节因素可能通过影响细胞膜磷脂PIP2与离子通道的相互作用而影响通道功能,PIP2可能是决定离子通道功能调节的最终的关键因素。因此对PIP2的定量研究对于探讨其在离子通道的调控机制方面具有重要意义,可为研究PIP2代谢在离子通道功能调节中的作用提供直接的证据。对于膜磷脂PIP2的定量,以往大部分都采用传统的放射性同位素标记的方法,或者采用衍生化的方法对PIP2的结构进行再修饰,以提高其检测灵敏度,然后采用离子抑制电导检测或紫外检测的方法进行液相色谱分析。本实验建立了一种直接有效分离并检测细胞膜磷脂PIP2的新方法—反相高效液相色谱法,并结合药理学方法、液-质联用技术和激光共聚焦扫描技术对该方法的准确性和可靠性进行了进一步验证。目的:建立并验证一种直接有效测定爪蟾卵母细胞膜磷脂PIP2含量变化的反相高效液相色谱方法。方法:(1)高效液相色谱方法的建立:主要色谱条件:色谱柱为Diamonsil C18柱(250mm×4.6mm, 5μm),保护柱为Diamonsil C18柱(4mm×4.6mm, 5μm),流动相为甲醇-乙腈-水(40:40:20, v/v/v),以1.0ml/min进行等梯度洗脱,紫外检测波长205nm,柱温25℃。(2)高效液相色谱方法对爪蟾卵母细胞膜磷脂PIP2含量变化的测定,即药理学方法对PIP2色谱峰的定性确证:用wortmannin(μM水平可阻断PI4K激酶,抑制PIP2的合成)孵育爪蟾卵母细胞两小时后,加入1ml裂解缓冲液匀浆,以5500rpm低速离心5min,取上清液,再以17500rpm高速离心2h,弃上清液,沉淀加入50μl裂解缓冲液制成悬浮液。悬浮液加入900μl氯仿-甲醇(9:1, v/v)提取细胞膜磷脂,以反相高效液相色谱技术分析wortmannin处理组和空白对照组(未用wortmannin孵育)膜磷脂PIP2的含量变化。样品和标准对照品均用甲醇-乙腈-水(40:40:20, v/v/v),即流动相溶解,进样量:20μl。(3)应用液-质联用技术对标准对照品PIP2和细胞膜磷脂PIP2的色谱峰进行进一步定性确证。标准品溶液和样品溶液配制方法同前,液相色谱条件同前,主要质谱条件:离子源类型:Turbo Spray电喷雾式;电离方式:负离子模式;扫描类型:Q1全扫描模式。(4)应用激光共聚焦扫描方法对所建立色谱方法的进一步验证:将克隆于pOMU质粒载体中的PLCδ1PH-GFP质粒DNA用XbaⅠ限制性内切酶线性化,用RibomaxTM Large Scale RNA Production Systems T7 Kit体外转录成相应的cRNA,用微注射的方法按每个卵母细胞50nl注射,注射后的细胞在含2.5mM丙酮酸钠的ND96液中19-20℃培养,表达24小时后用wortmannin孵育细胞两小时,激光共聚焦显微镜观察细胞膜PIP2水解情况,并与空白对照(未用wortmannin孵育)进行比较,观察细胞膜磷脂PIP2的含量变化。结果:(1)应用建立的RP-HPLC法能够有效分离出细胞膜磷脂PIP2并检测出其含量变化。PIP2的保留时间为2.1min,在0.025~0.3μg/μl浓度范围内线性关系良好,回归方程为y=100.09x+0.7483(r=0.9979),最小检测限为6.25×10-3μg/μl。精密度试验测得色谱峰高的RSD为6.0%,重复性试验测得色谱峰高的RSD为5.7%,均符合生物样品分析要求。(2)本实验每组均从50个爪蟾卵母细胞中提取膜磷脂,最后以100μl溶剂溶解进样分析,每次进样20μl,测得未用wortmannin预孵育的爪蟾卵母细胞,即空白对照组, PIP2的色谱峰高为11.16±0.28(n=5),而以10μM wortmannin预孵育爪蟾卵母细胞两小时的实验组,PIP2的峰高值明显下降,其色谱峰高为5.30±0.13(n=5),两组存在显著差异(P<0.01),峰高下降率达52.5%。(3)应用液-质联用技术在相同的出峰时间分别测定标准品PIP2和爪蟾卵母细胞膜磷脂样品中PIP2的质谱图,所得的质谱图吻合较好,其中m/z181, m/z233, m/z249, m/z317, m/z369, m/z385, m/z453, m/z589, m/z=657, m/z673, m/z725, m/z809等质谱峰在两个来源的受试品种完全吻合,且在二者的质谱图中均得到了PIP2的特征质谱峰。在标准品质谱图中,主要的特征质谱峰有:m/z1133([M-5H+4Na]-),m/z 929([M-1-H3PO4-H2O]-),m/z 521([M-2H]2-),m/z 437,m/z 369([M-2H-R2COOH]2-);在样品质谱图中,主要的特征质谱峰有: m/z 741([M-2H-R2COO-]-),m/z 401,m/z 369( [M-2H-R2COOH]2-),m/z 348([M-3H]3-)。(4)向卵母细胞注射cRNA表达24小时后,再以10μM wortmannin孵育两小时,LSCM观察爪蟾卵母细胞膜磷脂PIP2的含量变化:未用wortmannin预孵育但表达有PLCδ1PH-GFP的空白对照组细胞膜处的绿色荧光强度均值为127.90±3.6(n=7);而以10μM wortmannin预孵育表达有PLCδ1PH-GFP的爪蟾卵母细胞两小时的实验组,细胞膜处的绿色荧光强度明显减弱,荧光强度均值为63.74±4.3(n=6),两组存在显著差异(P<0.01),荧光强度下降率达到50.2%。结论:本实验中建立的反相高效液相色谱方法能够有效分离爪蟾卵母细胞膜磷脂PIP2并检测其含量变化,具有较好的准确性和可靠性。更多还原

【Abstract】 Phospholipids are characteristic components of cell membrane, mainly including phosphatidylcholine, phosphatidylserine and phosphatidylinositol. Phosphatidylinositol-4,5-bisphosphate (PIP2) is one type of phosphatidylinositol and plays an important role in cell signaling. PIP2, although comprises less than 1% of cell membrane phospholipids, plays multiple important roles in a wide variety of cellular processes, such as generation of second messengers, regulation of both endocytosis and exocytosis, membrane attachment to the cytoskeleton, etc. In addition, PIP2 can also modulate the function of many ion transporters and channels, such as Na/Ca exchanger, Na/H exchanger, voltage-dependent Ca channels, TRP channels, Kir channels and KCNQ channels, etc. The metabolism of PIP2 is involved in modulation of ion channel currents. In recent years, it has been found that neurotransmitters, protein kinases, pH , charged ions, drugs and metabolites of lipids can be involved in the regulation of ion channels by affecting the interaction of membrane PIP2 and ion channels. PIP2 may be the ultimate and crucial mediator in the current modulation of ion channels. Therefore, to develop the method of quantification of membrane PIP2, which will provide direct evidence for the involvement of PIP2, is greatly important for the study of mechanism for the modulation of ion channels.There are generally two ways of measuring PIP2. The classical way is to use radioisotope, which requires high specific activities and long labeling times, and the other way is to measure derivatives of PIP2 with suppressed conductivity detection or UV detection, which requires modification of the PIP2 structure. In our work, we set up a nonradioactive, fast and convenient method by reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection, which separated and quantified the membrane PIP2 in Xenopus oocytes effectively and directly. We also used the pharmacological agent wortmannin, high performance liquid chromatography/mass spectrometry (HPLC/MS) technique and laser scanning confocal microscopy (LSCM) to verify the accuracy and reliability of the established RP-HPLC method.Objective: To establish and test the reliability of a method of reversed-phase high performance liquid chromatography (RP-HPLC) to measure membrane PIP2 in Xenopus oocytes.Methods: (1) The establishment of RP-HPLC method: Chromatography was carried out on a Diamonsil C18 column (250mm×4.6mm,5μm) with a Diamonsil C18 guard column(4mm×4.6mm,5μm). The mobile phase contained methanol-acetonitrile-water (40:40:20, v/v/v) and the flow rate was 1.0ml/min using isocratic elution. The detection wavelength was at 205nm and column temperature at 25℃. (2) The analysis of membrane PIP2 in Xenopus oocytes by RP-HPLC: For wortmannin-treated group, oocytes were first incubated with wortmannin for 2 h, and then were manually lysed in 1ml cold lysis buffer and homogenized in the crushed ice. The homogenate was transferred to a 2-mL microfugetube and centrifuged for 5min at 5500rpm at 4℃. The supernatant was then centrifuged for 2h at 17500rpm at 4℃. The pellets were resuspended with 50μl lysis buffer (1μl per oocyte). 900μl methanol-chloroform (1:9, v/v) solution was then added to the suspension and phospholipids were extracted. Membrane PIP2 from wortmannin-treated group and control group (without wortmannin) were determined by RP-HPLC. Both the standard PIP2 and sample membrane PIP2 were dissolved with mobile phase solution containing methanol-acetonitrile-water (40:40:20, v/v/v), 20μl of each solution was injected for analysis. (3) The qualitative verification of standard PIP2 and sample membrane PIP2 by HPLC/MS: The preparation of standard PIP2 and sample membrane PIP2 and chromatographic conditions were the same as described above. The main MS parameters were as follows: source type: turbo spray (ESI); polarity: negative; scan type: Q1 MS. (4) Further verification of the established chromatographic method employed laser scanning confocal microscopic (LSCM) technique: PLCδ1PH-GFP cDNA subcloned into the pOMU plasmid vector was linearized with XbaⅠrestriction endonuclease. PLCδ1PH-GFP cRNA was transcribed using RibomaxTM Large Scale RNA Production Systems T7 Kit in vitro. Each oocyte was injected with 50nl of water containing the desired cRNA. The injected oocytes were cultivated in ND96 solution containing 2.5mM sodium pyruvate at 19-20℃. After expressing for 24h, oocytes were incubated with wortmannin for 2h and PIP2 hydrolysis from wortmannin-treated group and control group were monitored using LSCM.Results: (1) Membrane PIP2 in Xenopus oocytes was separated and quantified effectively and directly by RP-HPLC with the retention time of 2.1min. The calibration curve of PIP2 was linear in the range of 0.025~0.3μg/μl. The regression equation was y=100.09x+0.7483 (r=0.9979). The lowest detectable limit was 6.25×10-3μg/μl. The precision was determined by analyzing the same standard PIP2 solution for five times, the relative standard deviation (RSD) value of peak height was calculated as 6.0%. The repeatability was determined by analyzing five sample solutions, the RSD value of peak height was calculated as 5.7%. (2) Phospholipids were extracted from 50 Xenopus oocytes for each group and dissolved with 100μl mobile phase solution. The peak height of PIP2 for control group and wortmannin-treated group was 11.16±0.28 (n=5) and 5.30±0.13 (n=5) respectively. The difference was significant between these two groups (P<0.01). The peak height of PIP2 from wortmannin-treated group decreased by 52.5% compared with control group. (3) The mass spectrum was obtained at the same retention time and there was good agreement between standard PIP2 and sample membrane PIP2. The values of m/z181, m/z233, m/z249, m/z317, m/z369, m/z385, m/z453, m/z589, m/z657, m/z673, m/z725, m/z809 were all the same in both sample groups. The characteristic MS peak of PIP2 including m/z1133 ([M-5H+4Na]-),m/z 929 ([M-1-H3PO4-H2O]-),m/z 521 ([M-2H]2-),m/z 437 and m/z 369 ([M-2H-R2COOH]2-) were found in the spectrum of standard PIP2 and m/z 741 ([M-2H-R2COO-]-), m/z 401,m/z 369 ([M-2H-R2COOH]2-),m/z 348 ([M-3H]3-) in the spectrum of oocyte membrane PIP2. (4) Membrane PIP2 hydrolysis in oocytes monitored by LSCM: The fluorescence intensity of membrane for control group and wortmannin-treated group was 127.90±3.6 (n=7) and 63.74±4.3 (n=6) respectively. There was significant difference between these two groups (P<0.01). The fluorescence intensity of oocyte membrane from wortmannin-treated group decreased by 50.2% compared with control group.Conclusion: The established RP-HPLC method can be applied in detecting the content of membrane PIP2 in Xenopus oocytes with high efficiency, accuracy and reliability.更多还原