细胞膜透性Δ电导率量化检测法研究初探

doi:10.3969/j.issn.1004-1524.20240719

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (6): 1344-1352.DOI: 10.3969/j.issn.1004-1524.20240719

细胞膜透性Δ电导率量化检测法研究初探

王忠(), 杨洪兵, 杨帆, 陈亦凡, 侯晓敏^*()

青岛农业大学生命科学学院,山东青岛 266109

收稿日期:2024-08-07 出版日期:2025-06-25 发布日期:2025-07-08
作者简介:王忠(1969—),男,山东荣成人,实验师,主要从事生物化学与分子生物学研究。E-mail:741770985@qq.com
通讯作者: *侯晓敏,E-mail:tsfhxm@hotmail.com
基金资助:
国家自然科学基金(32170172)

Preliminary exploration of the Δ conductivity quantification method for cell membrane permeability

WANG Zhong(), YANG Hongbing, YANG Fan, CHEN Yifan, HOU Xiaomin^*()

College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, Shandong, China

Received:2024-08-07 Online:2025-06-25 Published:2025-07-08

摘要/Abstract

摘要：

为提高细胞膜透性检测的可重复性和可比性,并在此基础上量化细胞膜透性,通过研究小麦叶片细胞膜透性,成功研发出能够量化研究细胞膜透性的新方法——细胞膜透性Δ电导率量化检测法(简称Δ电导率法)。Δ电导率法将细胞膜透性的单位定义为:1 g完整的细胞膜在特定压力下于1 L纯水中浸泡1 h,细胞外渗溶液的电导率值。计算公式:P=[(σ_e-σ_i)×V]/(m×R×t),其中,P代表细胞膜透性(单位为μS·cm^-1·L^-1·g^-1·h^-1),σ_e和σ_i分别代表选定的期末时点与初始时点的电导率值,V代表溶液体积,m代表样品干重,R代表细胞膜完整率,t代表所用时间。通过与现有的2种常用方法(抽气法和浸泡法)进行比较,结果表明,3种方法在定性研究上结论一致且可靠性相当,Δ电导率法的可重复性明显优于抽气法和浸泡法,证明Δ电导率法能够准确可靠地量化细胞膜透性。Δ电导率法为量化细胞膜透性奠定了基础,使细胞膜透性有可能成为标志性生物指标。

关键词: 细胞膜透性, Δ电导率法, 量化, 可重复性, 可靠性

Abstract:

To enhance the reproducibility and comparability of cell membrane permeability assays and further quantify cell membrane permeability based on these improvements, a novel method for quantifying cell membrane permeability was successfully developed through investigating wheat leaf cell membrane permeability: the Δ conductivity quantification method for cell membrane permeability (abbreviated as the Δ conductivity method). The Δ conductivity method defines the unit of cell membrane permeability as the conductivity value of the extracellular solution leaked from 1 g of biomass with intact cell membranes after immersion in 1 L of pure water for 1 h under specific pressure. The calculation formula is: P=[(σ_e-σ_i)×V]/(m×R×t), where, P represents cell membrane permeability (with the unit being μS·cm^-1·L^-1·g^-1·h^-1), σ_e and σ_i represent the electrical conductivity at the end time point and the initial time point, V represents solution volume, m represents dry weight of sample, R represents intact rate of cell membrane, t represents consumption time. By comparing with the two existing common methods(air extraction method and soaking method), the results demonstrated that the three methods showed consistent conclusions and comparable reliability in qualitative research. However, the Δ conductivity method exhibited significantly superior repeatability compared with the air extraction method and soaking methods, proving that the Δ conductivity method can accurately and reliably quantify cell membrane permeability. The Δ conductivity method establishes a foundation for quantifying cell membrane permeability, making it potentially feasible for cell membrane permeability to serve as a hallmark biological indicator.

Key words: cell membrane permeability, Δ, conductivity method, quantify, reproducibility, reliability

中图分类号:

Q945
S512.1

王忠, 杨洪兵, 杨帆, 陈亦凡, 侯晓敏. 细胞膜透性Δ电导率量化检测法研究初探[J]. 浙江农业学报, 2025, 37(6): 1344-1352.

WANG Zhong, YANG Hongbing, YANG Fan, CHEN Yifan, HOU Xiaomin. Preliminary exploration of the Δ conductivity quantification method for cell membrane permeability[J]. Acta Agriculturae Zhejiangensis, 2025, 37(6): 1344-1352.

图/表 7

图1 小麦叶片取样示意图 A,2叶1心期麦苗;B,叶片中段。

Fig.1 Schematic diagram of wheat leaf sampling A, Wheat seedlings at the two-leaf-with-one-heart stage; B, Mid-section of leaf.

图2 σi、σe选取示例图 A,取点前;B,取点后。

Fig.2 Example diagram for selecting points σi and σe A, Prior to selecting points; B, After selecting points.

图3 自制电导率检测系统 A,自制电导率检测系统实物图;B,自制电导率检测系统结构图。1,真空泵;2,KF转接气管接头;3,配电箱;4,电源;5,止回阀;6,放气阀;7,数字电接点压力表(控制真空泵运行);8,100 L真空罐;9,气管接头;10,多通路气管分接头;11,悬挂孔;12不锈钢管箍(固定真空管于木板上);13,尼龙扎带(固定真空管于木板上);14,样品管(5 mL离心管钻若干Φ 1 mm孔改装);15,Φ 1 mm镀锌铁丝(缠绕固定于样品管上);16,5 mL离心管盖(剪切钻孔改装);17,双气管球阀;18,加厚O型橡胶密封圈;19,真空管阀门;20,雷磁DDSJ-318电导率仪,21,雷磁DJS-1D电导电极;22,雷磁T-818-6温度电极;23,电脑;24,USB数据线(电导率仪连接电脑);25硅胶管(Φ 6 mm×12 mm);26,数字电接点压力表线缆;27,真空管(层析柱改装);28,磁铁。

Fig.3 Self-made conductivity detection system A, Physical image of a self-made conductivity detection system; B, Structural diagram of a self-made conductivity detection system. 1, Vacuum pump; 2, KF adapter for air tubing connection; 3, Distribution box; 4, Power supply; 5, Check valve; 6, Vent valve; 7, Digital electrical contact pressure gauge (for vacuum pump control); 8, 100 L Vacuum tank; 9, Air tube connector; 10, Multi-path air tube splitter; 11, Hanging hole; 12, Stainless steel clamp (for securing vacuum tubes to wooden boards); 13, Nylon cable tie (for securing vacuum tubes to wooden boards); 14, Modified sample tube (5 mL centrifuge tube with multiple Φ 1 mm holes drilled); 15, Φ 1 mm Zinc-coated steel wire (wound and secured around the sample tube); 16, Modified 5 mL centrifuge tube cap (sheared, drilled, and adapted); 17, Ball valve with dual gas tube connections; 18, Thickened O-ring rubber seal; 19, Vacuum tube valve; 20, Leici DDSJ-318 conductivity meter; 21, Leici DJS-1D conductivity electrode; 22, Leici T-818-6 temperature electrode; 23, Computer; 24, USB data cable (for connecting conductivity meter to computer); 25, Silicone tube (Φ 6 mm×12 mm); 26, Cable for digital electrical contact pressure gauge; 27, Vacuum tube (modified for chromatography column); 28, Magnet.

图4 细胞膜完整率统计过程示例 A,扫描图;B,ImageJ总面积图;C,ImageJ染色面积图。

Fig.4 Example of the statistical process for intact rate of cell membrane A, Scan image; B, ImageJ-generated total area graph; C,ImageJ-generated stained area graph.

表1 三种方法测定的细胞膜透性单因素ANOVA检测统计表

Table 1 The statistical table of single-factor ANOVA test for cell membrane permeability measured by three methods

方法 Method	分组名称 Group name	CMP	CMP_mean	P
抽气法	0.6gF	18.007 9±1.898 9	17.381 9±1.283 0	0.466 5
Air extraction method	0.5gF	17.514 6±0.993 4
	0.4gF	16.623 1±0.716 7
	0.6gS	13.146 1±0.323 0	13.546 1±0.925 8	0.001 0
	0.5gS	14.699 5±0.151 2
	0.4gS	12.792 8±0.463 1
浸泡法	0.6gF	15.078 2±0.999 3	16.701 5±1.659 0	0.001 5
Soaking method	0.5gF	18.640 5±0.418 3
	0.4gF	16.385 7±0.306 4
	0.6gS	10.522 8±0.233 9	13.936 0±2.788 0	0.000 1
	0.5gS	16.740 1±0.334 9
	0.4gS	14.545 3±1.048 4
Δ电导率法	0.6gF	2.610 1±0.249 1	2.615 7±0.149 8	0.566 8
Δ conductivity method	0.5gF	2.690 2±0.064 0
	0.4gF	2.546 8±0.090 5
	0.6gS	2.105 9±0.100 7	2.086 0±0.163 7	0.873 8
	0.5gS	2.111 7±0.300 3
	0.4gS	2.040 3±0.046 3

表2 三种方法生物重复层面的单因素ANOVA分析统计表

Table 2 Statistical table of one-way ANOVA analysis for biological replication levels using three different methods

方法 Method	个案数 Number of cases	F/S平均值 Average of F/S ratio	P
抽气法Air extraction method	27	1.287 5±0.114 0	0.235 3
浸泡法 Soaking method	27	1.225 8±0.162 8
Δ电导率法 Δ conductivity method	27	1.260 4±0.113 9

表3 三种方法技术重复层面单因素ANOVA分析统计表

Table 3 Statistical table of one-way ANOVA analysis for technique replication level using three methods

方法 Method	分组名称 Group name	个案数 Number of cases	F/S平均值 Average of F/S ratio	P
抽气法Air extraction method	0.6g	9	1.370 4±0.128 4	0.001 1
	0.5g	9	1.191 6±0.059 5
	0.4g	9	1.300 5±0.062 9
浸泡法Soaking method	0.6g	9	1.433 4±0.086 7	<0.000 1
	0.5g	9	1.113 8±0.029 1
	0.4g	9	1.130 3±0.070 2
Δ电导率法	0.6g	9	1.241 3±0.115 1	0.625 3
Δ conductivity method	0.5g	9	1.291 0±0.158 8
	0.4g	9	1.248 7±0.045 5

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细胞膜透性Δ电导率量化检测法研究初探

Preliminary exploration of the Δ conductivity quantification method for cell membrane permeability

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