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PKDL通道的酸反应特性的电生理研究

Electrophysiological Research on the Offset Response of PKDL Channels to Acid Stimulation

【作者】 赵杰

【导师】 罗建红; 杨巍;

【作者基本信息】 浙江大学 , 神经生物学, 2011, 硕士

【摘要】 背景和目的:TRPP是TRP家族中重要的一个亚家族。人们发现PKD2突变后会导致常染色体多囊肾疾病(ADPKD, autosomal dominant polycystic kidney disease)后,因此命名了TRPP。TRPP亚家族包括PKD1、PKD2、PKD2L1、PKD1L3、PKD2L2等等。其中的PKD1/PKD2形成的受体-通道复合物,是与细胞的机械敏感性紧密相联的。而TRPP的另外两个成员PKD1L3和PKD2L1组成的通道复合物与PKD1和PKD2组成的通道复合物非常相似,2006年有三个研究小组均在国际知名杂志发表论文,认为PKD1L3和PKD2L1是对酸味敏感的感受体。此文中的PDKL通道就指的是PKD1L3和PKD2L1组成的通道。在发现PKDL通道的对酸反应后,杨巍和陈培华首先验证了共同表达PKD1L3和PKD2L1后产生的内向电流不是一个延迟反应,而是一个off响应。然后研究off响应与给酸和撤酸溶液的pH的关系,最后还研究了off响应的Ca2+依赖性。尽管酸通道的具体编码酸味觉的机制还不是很清楚,但本文尝试着用电生理和工程学的一些方法对此进行一些解读。实验方法:HEK293细胞培养于含10%胎牛血清和1%双抗的DMEM培养基中,3-4天传代一次,传代后24小时左右转染。转染时使用脂质体Lipofectamine2000 (Invitrogen),一般每35 mm培养皿,加入3-4μg质粒。将GFP. PKD1L3和PKD2L1共同转染到HEK293细胞中,室温下(22-25℃)对转染后24-48小时的HEK293细胞在电压钳模式下进行全细胞膜片钳记录。用Multiclamp700A放大器(Axon, USA)和pCLAMP系统软件(Version 9.0, Axon, USA)进行电信号的放大以及数据的记录和分析。数据采集接口为Digidata 1322A (Axon, USA),采样频率为10 kHz,1 kHz滤波。将种在小玻片的细胞放置在持续灌流细胞外液(ECS)的记录槽中进行实验。HEK293细胞的钳制电压为-60 mV。不同外液进行切换时,采用快速溶液切换系统RSC-160 (Biologic Science Instruments,France)完成。结果:1.撤酸后的不同pH对off电流的衰减时间的影响没有明显差异。2.胞外Ca2+浓度对off电流的衰减时间有较大的影响。胞外Ca2+浓度0mM时,衰减时间可以近二十秒,而当Ca2+浓度达到10 mM以及以上时,衰减时间则小于5秒。3.胞内Ca2+浓度也对off电流的衰减时间有较大的影响。含有0.5 mM EGTA的内液产生的off电流从峰值返回基线最快,均小于1秒,而含有10 mM BAPTA的内液产生的电流衰减时间很长,可达到6秒左右,含有5 mM EGTA的内液的电流的衰减时间居中。它们三者之间有统计学显著差异,*p<0.05。4.单独转染PKD2L1、PKD2均在给酸后有ofp向应。结论:PKDL通道的off电流不仅与所给酸的pH值有关,而且与撤酸后的pH值有关。PKDL通道受胞内外Ca2+浓度调节。单独转染PKD2L1、PKD2均在给酸后有off响应。

【Abstract】 Background and Objective:TRPP is a very important subfamily in TRP. Since PKD2 was found to be related to Autosomal Dominant Polycystic Kidney Disease, it was named as TRPP. This subfamily includes PKD1、PKD2、PKD2L1、PKD1L3、PKD2L2 and so forth. The complex formed by PKD1L3 and PKD2L1 is tightly related to cell sensitivity to mechanical stimulation. The complex formed by PKD1 and PKD2 closely resembles PKD1L3/2L1 complex. In 2006, according to the papers published by three research groups, it was suggested that PKDL channel was the sought-after candidate for proton sensing in taste cells. In this passage, we firstly made sure that the inward current is an offset response rather than a delayed response. Then, we studied the relationship between off-response and pH. At last, we examined the Ca2+ dependency of off current. Although the mechanism of the acid code is still unclear, we try to give some explanation in this passage using the electrophysiology method.Methods:HEK293 cells are raised in DMEM culture medium (also contain fetal bovine serum, Kanamycin and Ampicillin). We used Lipofectamine 2000 (Invitrogen) to do the transfection with GFP、PKD1L3、PKD2L1.24-48 hours after the transfection, we did the whole-cell patch clamp experiment on the HEK293 cells. We used Multiclamp 700A amplifier and pCLAMP software to amplify electronic signals and analyzed signals. The data collection interface is Digidata 1322A. The sampling frequency was 10 kHz, the filter 1 kHz. Holding potential was-60 mV. We used the Rapid Solution Changer to change different ESCs.Results:1. The pH of the solution after giving acid does not have obvious effect on the off-response.2. The concentration of Ca2+ in ECS has great effect on the attenuation time of the off current. When Ca2+ free ECS was used, it took nearly 20 seconds for the off current to return down to baseline, while when Ca2+ was 10 mM, it took only less than 5 seconds.3. The concentration of Ca2+ in ICS also has a great effect on the attenuation time of the off current. When there was 0.5 mM EGTA in ICS, the off current returned down to baseline so fast within less than 1 second. The attenuation time differed significantly among three the ICSs.4. The cells transfected with PKD2L1 or PKD2 alone also exhibit the off current.Conclusion:Not only the pH of acid but also the pH of the solution after giving acid can have some kind of effect on the off response. PKDL channels seem regulated by different Ca2+ concentration in ECS and ICS.

【关键词】 PKD2L1PKD1L3酸通道膜片钳全细胞记录
【Key words】 acid sensationoff-responsePKD1L3PKD2L1whole-cell recordingpatch-clamp
  • 【网络出版投稿人】 浙江大学
  • 【网络出版年期】2011年 07期
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