CN103424350B - High throughput analysis system and counting method for low-order-of-magnitude mutation-induced cells - Google Patents

High throughput analysis system and counting method for low-order-of-magnitude mutation-induced cells Download PDF

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CN103424350B
CN103424350B CN 201310344391 CN201310344391A CN103424350B CN 103424350 B CN103424350 B CN 103424350B CN 201310344391 CN201310344391 CN 201310344391 CN 201310344391 A CN201310344391 A CN 201310344391A CN 103424350 B CN103424350 B CN 103424350B
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cells
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潘天红
黄彪
邢讃
孙京京
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江苏大学
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Abstract

本发明公开了一种低数量级诱变细胞的高通量分析系统与计数方法。 The present invention discloses a low order of mutagenized cells and high throughput analysis systems counting method. 系统包括PC机/笔记本电脑、用于维持细胞生长环境的细胞培养箱、高通量的96x微电极板、正常细胞、诱变细胞和诱变剂。 The system includes a PC / laptop, cell growth environment for maintaining cell incubator, high-throughput microelectrode plate 96x normal cells, mutagens and mutagenized cells. 首先利用PC机/笔记本电脑中的实时细胞监控软件记录诱变细胞的生长曲线,并分析该诱变细胞响应曲线的动态特征,计算得到诱变细胞响应曲线的基准值和阈值,在此基础上,建立诱变细胞数目与生长时间之间的关系方程(亦即:估计模型),再利用非线性回归算法,辨识得到该估计模型的参数,利用辨识的估计模型实现对低数量级、待测诱变细胞数目的计数。 First, the cell using real-time monitoring software PC / laptop growth curves recorded in the mutagenized cells, and analyzing the mutagenized cells in response to dynamic characteristic curve, is calculated and the mutagenized cells in response to the reference value of the threshold curve, based on the establishing correlations among the number of cells mutagenized growth time (i.e.: estimation model), and then using non-linear regression algorithm, identification of the obtained parameters of the model estimation, the model identification using the estimated low magnitude of the test lure variable number of cells counted. 本发明最大的优势在于,采用非侵入式的检测手段,无需生物标记,且可以实现高通量的检测。 In that the greatest advantage of the present invention, using non-invasive detection methods, no biological marker, and detecting a high throughput can be realized.

Description

一种低数量级诱变细胞的高通量分析系统与计数方法 Mutagenized cells of a low magnitude high throughput analysis systems and counting method

技术领域 FIELD

[0001] 本发明涉及一种细胞统计技术与分析系统,特别是涉及一种低数量级的、诱变细胞的高通量统计方法与分析系统,属于细胞基因毒性分析领域。 [0001] The present invention relates to a technique and statistical analysis cell systems, particularly to a low magnitude, high throughput mutagenesis of the cell and method of statistical analysis system, cells belonging to genotoxic analysis.

背景技术 Background technique

[0002] 基因毒性(Genotoxicity)是指一些物质具有的特性,该特性能损害细胞内基因信息,破坏细胞内遗传物质的完整性。 [0002] genotoxicity (Genotoxicity) refers to a substance having a number of properties which can damage the genetic information in cells, disrupting the integrity of the genetic material of cells. 在大多数情况下,基因毒性可以使不同细胞和其它身体系统产生异变,从而引发生物体的各种疾病(如癌症,即:身体内的失去控制细胞生长)。 In most cases, it can make various genotoxic cells and other body systems generate mutation, organisms causing various diseases (such as cancer, namely: loss of control of cell growth in the body).

[0003] 在自然环境中,往往存在着大量的药物或污染物,这些物质具有一定基因毒性,可使正常细胞发生诱变,产生诱变细胞,导致生物体发生癌变。 [0003] In the natural environment, there is often a large amount of a drug or a contaminant, these substances have certain genotoxic mutagenesis allows the normal cells to produce mutagenized cells, resulting in cancerous organism. 因此,通过统计诱变细胞数目, 可以鉴定药物或污染物的危害等级,从而预测环境污染物对人体健康产生有害影响的可能性,即:实现人类健康风险评估。 Therefore, the number of statistical cell mutagenesis, can be identified hazard classes of drugs or contaminants, environmental pollutants to predict the possibility of harmful effects on human health, namely: achieving human health risk assessment. 但是,自然环境中的诱变剂浓度都非常低,低浓度的诱变剂产生诱变细胞数目也非常少,受检测灵敏度的限制,传统的细胞计数方法很难统计出低数量级的诱变细胞数目,例如:若培养皿中的诱变细胞数目不足1〇〇时,MTT比色法无法实现检测。 However, the concentration of mutagen natural environment are very low, low concentrations of mutagens to produce mutagenized cells are also very small number, the detection sensitivity is limited, the conventional method is difficult to count cell count low order of cells mutagenized number, for example: if the number is less than 1〇〇 mutagenesis of cell culture dish, the MTT colorimetric detection can not be achieved.

[0004] 专利"一种高精度细胞统计技术与分析装置"[申请号:201210062508. 5,公开号: CN102851208A]提出一种基于图像分析技术的细胞统计方法,通过调节显微成像装置,获取清晰的细胞显微图片,并对图片进行一系列的图像操作,获取识别结果,并对结果进行统计分析,得到所检测细胞的个数,尺寸与形态等。 [0004] Patent "statistical technique and a high-precision cell analyzer" [application number: 2012100625085, Publication Number: CN102851208A] proposed a statistical method based on image analysis of cells by microscopic imaging adjusting means, to obtain a clear the cell micro image, and a series of picture image operation, acquires the identification result, and the results were statistically analyzed, and the detected number, size and morphology of the cells. 此方法需要一套高精度的显微成像系统,常用于正常细胞计数,并且无法实现高通量检测。 This method requires a high precision microscopic imaging system, commonly used in normal cell counts, and can not achieve high-throughput assay.

[0005] 专利"用于对细胞和生物分子进行计数的系统和方法"[申请号: 200980121707. 5,公开号:CN102089418A]将细胞的样本与荧光标记试剂接触,并利用荧光光束,由检测设备成像技术,配以计算机软件,实现对待测细胞的计数。 "System and method for cell counting and biomolecules" [0005] patent [application number: 200980121707.5, Publication Number: CN102089418A] The sample cells labeled with a fluorescent reagent and a fluorescent light beam from the detection device imaging techniques, together with computer software, the counting of treated cells. 此方法是一种侵入式的检测方法,需要荧光标记试剂,细胞的活性受到荧光标记试剂的影响,实验员受感染的机会较大,且其测试步骤单一枯燥,实验员容易疲劳、出错,无法实现高通量检测。 This method is a method for detecting invasive, requires fluorescent labeling reagent, the activity of the cells affected by the fluorescent labeling reagent, the experimenter greater chance of infection, and a single boring step which tests, laboratory staff fatigue, error, not high throughput testing.

发明内容 SUMMARY

[0006] 针对上述现有技术的不足,本发明提出一种结合实时细胞分析仪(Real Time Cell Analyzer,RTCA)的诱变细胞的分析系统与计数方法,能实现低数量级诱变细胞的高通量检测。 [0006] The above-described deficiencies of the prior art, the present invention proposes a method of counting and analyzing system of one binding mutagenized cells in real time cell analyzer (Real Time Cell Analyzer, RTCA), to achieve the low magnitude of the high-pass mutagenized cells the amount of testing.

[0007] 依据本发明之目的,提出一种低数量级诱变细胞的高通量分析系统,该系统包括用于监测细胞生长和控制其生长环境参数的PC机/笔记本电脑、用于维持细胞生长环境的细胞培养箱(incubator ),一高通量的96x微电极板(E-Plate )、正常细胞、诱变细胞和诱变剂;其中,PC机/笔记本电脑安装有ACEA公司开发的实时细胞监控软件平台,该平台能控制细胞培养箱的环境参数,如:温度、湿度,以及二氧化碳浓度,从而保持细胞生长环境条件的均匀性;并能实时检测微电极板E-Plate的阻抗变化信号,并将该信号转化成细胞指数(Cell Index,CI)显示在屏幕上,同时也存储在PC机/笔记本电脑硬盘中;96x微电极板置于细胞培养箱中,细胞培养箱的环境参数(温湿度,二氧化碳浓度)由实时细胞监控软件控制,从而保持在整个诱变过程的环境条件不变;正常细胞接种在96x [0007] The object according to the present invention, provides a low magnitude mutagenized cells high throughput analysis systems, the system includes a monitoring control cell growth and the growth environment parameters PC / laptop, for maintaining cell growth incubator environment (incubator), a high throughput micro electrode plate 96x (E-plate), normal cells, mutagens and mutagenized cells; wherein, PC / laptop attached to real cells developed ACEA monitoring software platform to control the environmental parameters of the incubator, such as: temperature, humidity, carbon dioxide concentration, and to maintain the uniformity of the cell growth environment conditions; impedance changes and real-time detection signal E-plate micro-electrode plate, and and converting the signal to index cells (cell index, CI) displayed on the screen and also stored in the PC / laptop hard drive; 96X microelectrode plates were placed in the incubator, the incubator environmental parameters (temperature humidity, carbon dioxide concentration) controlled by a real-time monitoring software cell to maintain the ambient conditions constant throughout the mutagenesis procedure; normal cells were seeded at 96x 电极板中,诱变细胞和诱变剂加在96x微电极板的微孔中。 Electrode plate, mutagens and mutagenized cells applied to the microporous plate 96x microelectrodes.

[0008] 其中,正常细胞接种在96x微电极板(E-Plate)中,细胞会贴壁生长,其数量变化会导致E-Plate底部的金箔传感器的阻抗发生变化,微电极上的贴壁细胞越多,阻抗值变化就越大,实时细胞监控软件将这种变化转化成细胞指数(CI),显示在系统中。 [0008] wherein, normal cells were seeded at 96x micro electrode plate (E-Plate), the cells may adhere and grow its volume changes cause impedance foil sensor at the bottom of the E-Plate changes, adherent cells on the microelectrode the more, the greater the change in resistance value, the real-time monitoring software cell to convert this change to the cell index (CI), in the display system.

[0009] 利用上述分析系统的计数方法,包括如下步骤:正常细胞接种在96X微电极板中12小时后,在不同的微孔(we 11)中加入不同数量的诱变细胞与诱变剂,诱变剂将正常细胞全部杀死,让诱变细胞生长,利用实时细胞监控软件平台记录整个诱变细胞的生长曲线,并分析该诱变细胞响应曲线的动态特征,计算得到诱变细胞响应曲线的基准值和阈值;在此基础上,建立诱变细胞数目与生长时间之间的关系方程,即估计模型,再利用非线性回归算法,辨识得到该估计模型的参数,利用辨识的估计模型实现对低数量级、待测诱变细胞数目的计数。 [0009] With the above-described counting method analysis system, comprising the steps of: a normal cell inoculation After 12 h, cells were mutagenized with a different number of different microporous mutagen (we 11) in 96X microelectrode plate, all of mutagen will kill normal cells, so that growth, cell using real-time monitoring software platform mutagenized cells recording the growth curve of the whole cell mutagenesis, and the mutagenized cells in response to dynamic analysis of the characteristic curve, the calculated response curve mutagenized cells the reference and threshold values; on this basis, the relationship between the number of equations and mutagenized cell growth time, i.e. estimation model, and then using non-linear regression algorithm to identify parameters of the obtained estimation model, implemented by the estimated model identification low magnitude, the number of the test cell counts mutagenesis.

[0010] 本发明的细胞计数方法包括如下步骤: [0010] Cell counting method according to the present invention comprises the steps of:

[0011] (1)将正常细胞接种在96X的微电极板的微孔(well)中; [0011] (1) The normal cells were seeded in microwell plates 96X microelectrodes (Well); and

[0012] (2)等待正常细胞稳定12小时后,在微电极板的不同的微孔中加入不同数量的诱变细胞与诱变剂; After [0012] (2) waiting for the normal cells kept for 12 hours, the number of different cells with the mutagen mutagenic different microelectrodes microporous plate;

[0013] (3)诱变剂杀死正常细胞后,剩下的诱变细胞继续生长,实时细胞监控软件平台控制并记录整个诱变过程(200小时),记录并存储不同诱变细胞的生长曲线; After [0013] (3) mutagenic agents kill normal cells, the remaining cells continue to grow mutagenesis, real-time monitoring software platform to control cells and the entire recording mutagenesis procedure (200 hours), record and store different mutagenized cell growth curve;

[0014] (4)对所记录的诱变细胞生长曲线进行平滑处理,去除传感器的噪声信号; [0014] (4) recorded on the mutagenized cells growth curve smoothing, removal of noise signal of the sensor;

[0015] (5)分析诱变细胞响应曲线的动态特征,计算不同数量级诱变细胞生长曲线的基准值与阈值; [0015] (5) Mutation analysis of the dynamic response characteristic curve of the cell, calculate different magnitude mutagenized cell growth curve reference value and the threshold value;

[0016] (6)取所有生长曲线基准值的最大值为最终基准值,取所有生长曲线阈值的最小值为最终阈值;用最终基准值与最终阈值分别画一条水平直线,直线与每一条诱变细胞的生长曲线有两个交点,取交点之间的时间差值为诱变细胞的生长时间; The maximum value [0016] (6) to take all the growth curve of a reference value as the final reference value, taking all of the growth curve of the threshold minimum is the final threshold value; with the final reference value and the final threshold draw a straight horizontal line, a straight line with each attractant were variable cell growth curve has two intersection points, the time difference between the time taken for the growth of intersection mutagenized cells;

[0017] (7)建立诱变细胞数目与生长时间之间的幂函数方程(S卩:估计模型),并用非线性回归算法得到该幂函数方程的参数; [0017] (7) to establish the power function (S Jie: estimation model) between mutagenesis and the number of cells growth time, and to give the parameters of the power function by non-linear regression algorithm;

[0018] (8)利用幂函数方程(估计模型)估算待测诱变细胞样品的细胞数目。 [0018] (8) use of the power function (Estimation Model) to estimate the number of cells in the cell sample to be tested mutagenesis.

[0019] 本发明与现有技术比较的有益效果是: [0019] Advantageous effects of the present invention and comparative prior art are:

[0020] (1)采用非侵入式、无标记的检测方法,且细胞指数的读数是非损伤的; [0020] (1) non-invasive, label-free detection methods, and the reading of the index is non-damaged cells;

[0021] (2)采用96x微电极板进行细胞接种,可实现高通量诱变细胞评估; [0021] (2) The microelectrode 96x plate inoculated with cells, cells can achieve high throughput mutagenesis evaluation;

[0022] (3)采用实时细胞监控软件可以连续、实时的显示数据,记录整个诱变细胞的生长过程,可获得完整的细胞效益图谱,而不是假定细胞处于某种合适的处理阶段(终点法),进行细胞分析; [0022] (3) real-time continuous monitoring software cell, real-time data display, recording overall growth mutagenized cells, intact cells obtained spectrum efficiency, rather than assuming that the cells are in some suitable process stage (Endpoint ), analysis of cell;

[0023] (4)本计数方法可以实现低数量级细胞的估计。 [0023] (4) The present method may be implemented counting estimated magnitude lower cells.

附图说明 BRIEF DESCRIPTION

[0024] 图1为诱变细胞分析系统的结构原理图;其中,1-正常细胞,2-微电极板,3-诱变剂,4-诱变细胞,5-细胞培养箱,6-PC机; [0024] FIG. 1 is a block diagram Mutagenesis cell analysis system; wherein normal cells 1-, 2- microelectrode plate, mutagen 3-, 4- mutagenized cells, cell incubator 5-, 6-PC machine;

[0025] 图2为低数量级诱变细胞计数的流程示意图; [0025] FIG. 2 is a low magnitude mutagenized schematic flow cytometry;

[0026]图3为低数量级诱变细胞生长曲线,及其计算的基准值与阈值图; [0026] FIG. 3 is a low order of mutagenized cell growth curve, and calculating a reference value with a threshold value map;

[0027] 图4为诱变细胞估计模型曲线图。 [0027] FIG. 4 is a graph showing estimation model mutagenized cells.

[0028] 图5为低数量级诱变细胞估计结果图。 [0028] FIG. 5 is a low magnitude estimation result mutagenized cells FIG.

具体实施方式 detailed description

[0029] 请参阅第1图,其为本发明用于低数量级诱变细胞高通量计数的分析系统结构原理图,如图所示,本发明包括一用于监测细胞生长和控制其生长环境参数的PC机6, 一用于维持细胞生长环境的细胞培养箱5 (incubator),一高通量的96x微电极板2 (E-Plate), 正常细胞1,诱变细胞4,诱变剂3。 [0029] See Figure 1, which the present invention is a system configuration diagram for analyzing high-throughput counting of magnitude lower mutagenized cells, as illustrated, the present invention includes a control for monitoring cell growth and the growth environment parameters of a PC 6, a cell incubator for 5 (incubator) to maintain cell growth environment, a high-throughput 96x microelectrode plate 2 (E-plate), a normal cell, 4 cell mutagenesis, mutagens 3.

[0030] 其中,PC机6中安装有ACEA公司开发的实时细胞监控软件平台,该平台能控制细胞培养箱的环境参数,如:温度、湿度,以及二氧化碳浓度,从而保持细胞生长环境条件的均匀性;并能实时检测微电极板2的阻抗变化信号,并将该信号转化成细胞指数(Cell Index,CI)显示在屏幕上,同时也存储在PC机硬盘中。 [0030] wherein, PC machine 6 is installed in the cell in real time monitoring software platform developed by ACEA, the platform can be controlled incubator environmental parameters, such as: uniform temperature, humidity, and carbon dioxide concentration, to maintain environmental conditions of cell growth properties; impedance changes and real-time detection signal of the micro-electrode plate 2, and converting the signal into a cell index (cell index, CI) displayed on the screen and also stored on the PC hard drive.

[0031] 其中,96x微电极板2置于细胞培养箱5中,细胞培养箱5的环境参数(温湿度,二氧化碳浓度)由实时细胞监控软件控制,从而保持在整个诱变过程的环境条件不变。 [0031] wherein, 96X micro electrode plate 2 is placed in the cell culture incubator 5, cell incubator environmental parameters (temperature, humidity, carbon dioxide concentration) 5 controlled by the real-time monitoring software cells, thereby maintaining the ambient conditions in the whole process is not mutagenic change.

[0032] 其中,正常细胞1接种在96x微电极板2 (E-Plate)中,细胞会贴壁生长,其数量变化会导致E-Plate底部的金箔传感器的阻抗发生变化,微电极上的贴壁细胞越多,阻抗值变化就越大,实时细胞监控软件将这种变化转化成细胞指数(CI ),显示在系统中。 [0032] wherein normal cells 1 were seeded in (E-Plate) in 96x micro electrode plate 2, the cells adhered to the wall, the number of change causes the impedance foil sensor at the bottom of the E-Plate changes posted on the microelectrode the more adherent cells, the greater the change in resistance value, the real-time monitoring software cell to convert this change to the cell index (CI), in the display system.

[0033] 其中,在正常细胞1接种在微电极板2上12小时后,在不同的微孔(well)中加入不同数量的诱变细胞4与诱变剂3,该诱变剂3会将正常细胞全部杀死,让诱变细胞4生长, 实时细胞监控软件记录整个诱变细胞4的生长过程。 [0033] wherein, after the plate 2 microelectrode 12 hours, the number of different cells in different microporous mutagenesis (Well) in 14 normal cells were seeded mutagens 3, which will mutagen 3 all kill normal cells, so that the growth of 4, real-time monitoring software records the entire cell mutagenic cell growth 4 mutagenic cells.

[0034] 本发明的计数方法:首先利用实时细胞监控软件记录诱变细胞的生长曲线,并分析该诱变细胞响应曲线的动态特征,计算得到诱变细胞响应曲线的基准值(Basel ine )和阈值(Threshold),在此基础上,建立诱变细胞数目与生长时间之间的关系方程(亦即:估计模型),再利用非线性回归算法,辨识得到该估计模型的参数,利用辨识的估计模型实现对低数量级、待测诱变细胞数目的计数。 [0034] The counting method according to the present invention: Firstly, real-time monitoring software recording mutagenized cells cell growth curve, and analysis of the dynamic response characteristic curve of the mutagenized cells, mutagenized cell response curve calculated reference value (Basel ine) and estimated: (i.e. estimation model), and then using non-linear regression algorithm to identify parameters of the estimation model obtained by identification threshold (the threshold), on this basis, the relationship between the number of equations and mutagenized cell growth time model to achieve the low magnitude of the count, the number of test cells mutagenized.

[0035] 如图2所示,本发明的计数方法具体包含如下步骤: [0035] 2, the counting method of the present invention specifically comprises the following steps:

[0036] 第1步:将正常细胞1接种在96x的微电极板2中; [0036] Step 1: 1 in normal cells were seeded in micro plates 96x of the electrode 2;

[0037] 第2步:等待正常细胞1稳定12小时后,在微电极板2的不同的微孔中加入不同数量的诱变细胞4M』.(Mj.G {512, 256, 128, 64, 32, 16, 8, 4, 2, 1},j=l,2,…,10)与诱变剂3 ; [0037] Step 2: 1 stabilization waiting for the normal cells after 12 hours, different numbers of mutagenized cells was added 4M "at different microelectrodes microporous plate 2 (Mj.G {512, 256, 128, 64,. 32, 16, 8, 4, 2, 1}, j = l, 2, ..., 10) with a mutagen 3;

[0038] 第3步:诱变剂3杀死正常细胞后,剩下的诱变细胞4继续生长,实时细胞监控软件控制并记录整个诱变过程(200小时),记录并存储不同诱变细胞%在不同时刻的细胞指数CIji](i=l,2,…,200,亦即:实时细胞监控软件每1小时采样一个点),其结果如图3所示,其中各曲线代表的含义如下:a-无诱变细胞;b-1个诱变细胞;c-2个诱变细胞;d-4个诱变细胞;e-8个诱变细胞;f-16个诱变细胞;g-32个诱变细胞;h-64个诱变细胞;i-128 个诱变细胞;j-256个诱变细胞;k-512个诱变细胞;1-基准值CI b;m-阈值CI、 [0038] Step 3: After the mutagen 3 kill normal cells, the remaining 4 cells continue to grow mutagenesis, real-time control and monitoring software cell mutagenesis record the entire process (200 hours), record and store different cell mutagenesis % of cells at different time index CIji] (i = l, 2, ..., 200, namely: a real cell monitoring software sampling point per hour), and the results shown in Figure 3, wherein each curve represents the following meaning : a- no mutagenic cells; b-1 th mutagenized cells; c-2 th mutagenized cells; d-4 th mutagenized cells; e-8 th mutagenized cells; f-16 th mutagenized cells; G- 32 mutagenized cells; h-64 th mutagenized cells; i-128 th mutagenized cells; j-256 th mutagenized cells; k-512 th mutagenized cells; 1- reference value CI b; m- threshold CI,

[0039] 第4步:对所记录的不同数量级诱变细胞生长曲线进行平滑处理: [0039] Step 4: Mutation of magnitude different growth curve smoothing recorded:

Figure CN103424350BD00061

[0041] 其中,Num为移动平滑窗体的长度,这里取Num=6 ;k为采样时刻k=l,2,…,200 ; yj[k]为第%诱变细胞平滑处理后的细胞指数值。 [0041] wherein, Num is the length of the smooth movement of the form, where taking Num = 6; k is a sampling time k = l, 2, ..., 200; yj [k] is a cell index after the first smoothing processing cells mutagenized% value.

[0042] 第5步:计算不同数量级诱变细胞%生长曲线的基准值与阈值。 [0042] Step 5: Calculate the% cell growth mutagenesis different magnitude reference value and the threshold value curve. 为得到合理的基准值和阈值,首先要计算细胞指数的变化率Bjk]: To obtain a reasonable threshold value and the reference value, to calculate the first index of changes in cell Bjk]:

Figure CN103424350BD00062

[0044] 细胞指数的变化率h [k]直接反映诱变细胞在采样时刻k的生长速率,若为负值, 则表示细胞死亡,反之则表示细胞生长;绝对值越高表示细胞指数变化越大,细胞越有活力。 [0044] The rate of change in Cell Index H [k] directly reflect the mutagenized cell growth rate in the sampling instant k, if negative, it indicates cell death, cell growth indicates otherwise; the higher the absolute value of the index indicating changes in cell large, more vibrant cells.

[0045] 在此基础上,计算不同数量级诱变细胞的细胞指数变化率Bj [k]第一次大于0时的g值,并获取此时刻的细胞指数CI为该数量级诱变细胞的基准值(力: [0045] On this basis, the order of calculation of the different rates of change of mutagenized cells Cell Index Bj [k] is greater than the first value g 0 is, and acquires the cell index order of mutagenized cells for this time reference value CI (force:

Figure CN103424350BD00063

[0048] 其中,S为很小的数值,这里为避免系统噪声的影响,取S=1(T4(接近于〇)。 [0048] where, S is a small value, where in order to avoid system noise, taking S = 1 (T4 (close square).

[0049] 此外,计算不同数量级诱变细胞的细胞指数变化率Bjk]最大值时的$值,并获取此时刻的细胞指数CI为该数量级诱变细胞的阈值(夂: [0049] In addition, the index change in cell count distinct stages of mutagenized cells BJK] $ value of the maximum, and acquires the Cell Index CI this point for the magnitude of the threshold mutagenized cells (Wen:

Figure CN103424350BD00064

[0052] 第6步:取所有数量级诱变细胞的基准值(7;的最大值为最终基准值CIb: [0052] Step 6: Take all mutagenized cells magnitude reference value (7; final maximum value of the reference value CIb:

Figure CN103424350BD00065

[0054] 取所有数量级诱变细胞的阈值(7_:的最小值为最终阈值Cl% [0054] All taken mutagenized cells magnitude threshold (7_: minimum threshold value as the final Cl%

Figure CN103424350BD00066

[0056] 第7步:用最终基准值CIb画一条水平基准线,如图3所示,该基准线与每一条诱变细胞的生长曲线有一个交点h,取该交点的横坐标最小值为评估时间起点t (ks),即: [0056] Step 7: The final reference value CIb Videos with a horizontal reference line, shown in Figure 3, the reference line with a growth curve for each cell has a mutation intersection H, the abscissa takes the minimum value of the intersection point assess the starting point of time t (ks), namely:

Figure CN103424350BD00071

[0059] 用最终阈值CIt画一条水平阈值线,如图3所示,该阈值线与每一条诱变细胞的生长曲线有个交点h,取该交点的时间值t(kp与评估时间起点t(ks)的差值为该数量级诱变细胞%的生长时间tj: [0059] CIt draw a horizontal threshold line with the final threshold, as shown, the threshold line with the growth curve of each mutagenized cells are intersections h 3, to take the point of intersection of the time value t (KP and evaluation time origin t (KS) for the magnitude of the difference between the percent of cells mutagenized growth time tj:

Figure CN103424350BD00072

[0062] 第8步:建立诱变细胞数目M#生长时间之间的幂函数方程(即:估计模型): [0062] Step 8: the power function established between the number M # mutagenized cell growth time (i.e.: Estimation Model):

Figure CN103424350BD00073

[0064] 其中a。 [0064] wherein a. a2, a3为估计模型参数, a2, a3 to estimate model parameters,

[0065] 采用非线性回归算法得到该幂函数方程的参数,如图4所示,在此实施例中: 8^255. 1? a2=-〇. 99? a3=104. 8〇 [0065] The non-linear regression algorithm of the parameters of the power function, shown in Figure 4, in this embodiment:.?.? 8 ^ 255 1 a2 = 99 a3 = 104 8〇 -〇.

[0066] 第9步:用步骤1到步骤7的处理方法处理待测诱变细胞样品的生长曲线,得到待测诱变细胞的生长时间t,利用估计模型(9)即可估算出待测诱变细胞样品的细胞数目兪。 [0066] Step 9: Step 1 to Step processing processing method 7 cell growth curve mutagenesis sample to be tested, measured to obtain the growth time t mutagenized cells, using the estimated model (9) can be estimated to be tested mutagenesis cell sample cell number Yu.

Figure CN103424350BD00074

[0068] 为说明本发明的实施效果,将0. 2uL的诱变剂3加入待测试诱变细胞样品中,并将该待测样品接种到96x微电极板2中,总共需占据96x微电极板2的16个微孔,这16个微孔的生长曲线如图5所示。 [0068] To illustrate the effect of the present embodiment of the invention, the mutagen was added 0. 2uL 3 mutagenized cell sample to be tested, and the test sample was inoculated into the plate 2 microelectrode 96x, 96x occupy a total required microelectrode 16 microwell plate 2, this growth curve of pore 16 as shown in FIG. 由公式(10)即可算出,待测诱变细胞样品中诱变细胞数目为25。 (10) can be calculated from the formula, the number of the test cell sample mutagenesis mutagenized cells 25. 此结果与显微镜下人工计数的结果基本一致,说明本发明能够实现低数量级诱变细胞的计数,且具有较高的精度。 This result is consistent with the results of manual counting under a microscope, the description of the present invention enables a low count magnitude mutagenized cells, and has a high accuracy.

Claims (2)

  1. 1. 一种低数量级诱变细胞的高通量分析系统的计数方法,其特征在于,包括如下步骤: (1) 将正常细胞接种在96x的微电极板的微孔中; (2) 等待正常细胞稳定12小时后,在微电极板的不同的微孔中加入不同数量的诱变细胞与诱变剂; (3) 诱变剂杀死正常细胞后,剩下的诱变细胞继续生长,实时细胞监控软件平台控制并记录整个诱变过程,记录并存储不同诱变细胞的生长曲线; (4) 对所记录的诱变细胞生长曲线进行平滑处理,去除传感器的噪声信号; (5) 分析诱变细胞响应曲线的动态特征,计算不同数量级诱变细胞生长曲线的基准值与阈值; (6) 取所有生长曲线基准值的最大值为最终基准值,取所有生长曲线阈值的最小值为最终阈值;最终基准值与最终阈值分别所在的水平直线与每一条诱变细胞的生长曲线有两个交点,取交点之间的时间差值为诱变细胞 1. A method for counting the number of low-level high-throughput analysis of mutagenized cells system, characterized by comprising the steps of: (1) normal cells were seeded at 96x microwell plate microelectrodes; (2) waiting for the normal 12 hours after cell stabilizers, mutagenized cells were added different amounts of different mutagens in the microporous plate microelectrodes; (3) mutagenic agents kill normal cells, the remaining cells continue to grow mutagenesis, real-time cell control and monitoring software platform mutagenesis record the entire process, record and store different growth curve mutagenized cells; (4) mutagenic recorded cell growth curve smoothing, removal of noise signal of the sensor; (5) analysis of induced variant cells in response to dynamic characteristic curve, calculate different magnitude mutagenized cell growth curve of a reference value with a threshold value; maximum (6) to take all the growth curve of a reference value as the final reference value, taking all of the growth curve of the threshold minimum is the final threshold ; final horizontal straight reference value and the final threshold values ​​and the growth curve where the mutagenized cells each have two intersection points, the time difference between the intersection of the mutagenized cells taken 生长时间; (7) 建立诱变细胞数目与生长时间之间的幂函数方程,即估计模型,并用非线性回归算法得到该估计模型的参数;所述估计模型为:+〇3,其中ai,a2, a3为估计模型参数,为诱变细胞数目,b为诱变细胞的生长时间;j代表不同数量诱变细胞的序号,其取值范围为j = 1,2, 3, 4, 5, 6, 7, 8, 9, 10 ; (8) 利用估计模型估算待测诱变细胞样品的细胞数目。 Growth time; (7) mutagenesis established the power function between cell number and growth time, i.e. the estimated model, and the parameters obtained by nonlinear regression estimation model algorithm; the estimated model: + 〇3, wherein AI, a2, a3 is the estimated model parameters, the number of cells to mutagenesis, b is the growth time mutagenized cells; J represents a different serial number mutagenized cells, the range of j = 1,2, 3, 4, 5, 6, 7, 8, 9, 10; (8) the number of cells in the cell sample to be tested using the mutagenesis estimation model estimates.
  2. 2. 根据权利要求1所述的计数方法,其特征在于,所述步骤(3)的整个诱变过程为200 小时,实时细胞监控软件平台每1小时采样一个点。 2. A counting method according to claim 1, wherein said step (3) a process of mutagenesis of the entire 200 hours, real-time monitoring software platform cells per hour sample point.
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