实验报告
Experiment report

钙含量测定
Determination of calcium content

主要仪器及试剂
Main instruments and reagents

医用离心机 型号：TGL-16K （湖南湘仪实验室仪器开发有限公司，湖南)
Medical centrifuge Model: TGL-16K (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Hunan).

倒置显微镜 型号：SOPTOP OD630K （上海舜宇恒平科学仪器有限公司，上海）
Inverted microscope Model: SOPTOP OD630K (Shanghai Sunny Hengping Scientific Instrument Co., Ltd., Shanghai).

二氧化碳培养箱 型号：HF151（UV） （上海力申科学仪器有限公司，上海)
CO2 incubator Model: HF151(UV) (Shanghai Lishen Scientific Instrument Co., Ltd., Shanghai).

(4） 超净工作台 型号：SW-CJ-1F （苏州净化设备有限公司)
(4) Ultra-clean workbench Model: SW-CJ-1F (Suzhou Purification Equipment Co., Ltd.).

（5） 钙含量显色检测试剂盒 货号：S1063S （上海碧云天生物技术有限公司，上海)
(5) Calcium content chromogenic detection kit Item No.: S1063S (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai).

（6） Pluronic F-127 货号：P6791 （北京索莱宝科技有限公司，北京）
(6) Pluronic F-127 Catalog No.: P6791 (Beijing Solaibao Technology Co., Ltd., Beijing).

（7） HEPES 缓冲盐水 H1045 D-Hank's，不含钙镁，不含酚红（HBSS） 货号：H1070 （北京索莱宝科技有限公司，北京）
(7) HEPES Buffered Saline H1045 D-Hank's, Calcium Magnesium Free, Phenol Red Free (HBSS) Catalog No.: H1070 (Beijing Solaibao Technology Co., Ltd., Beijing).

二、 实验分组及步骤
2. Experimental grouping and procedures

（1）实验分组
(1) Experimental grouping

①空白对照组
(1) Blank control group

②号钙对照组；
(2) calcium control group;

③号样品组；
sample set (3);

④号样品组；
sample set (4);

（2）、（3）、（4）号样品分别配制成浓度为0.1、0.2、0.3、0.4、0.5mg/mL。
Samples (2), (3) and (4) were prepared at concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL, respectively.

（2）实验步骤
(2) Experimental procedures

（1） 检测缓冲液及显色液使用前应平衡至室温。
(1) The detection buffer and chromogenic solution should be balanced to room temperature before use.

（2） 标准品的制备
(2) Preparation of standards

取10μl 500mM的钙标准品，加入990μl去离子水，充分混匀后即可得到5mM的钙标准溶液。参考下表稀释钙标准品，制备0、0.1、0.2、0.4、0.6、0.8、1.0mM浓度标准品。
Take 10 μl of 500 mM calcium standard, add 990 μl of deionized water, and mix well to obtain a 5 mM calcium standard solution. Dilute calcium standards with reference to the table below to prepare standards at 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mM concentrations.

③ 检测工作液的配制：计算一次实验所需用量，把检测缓冲液与显色液以1:1混合配制成检测工作液待用。
(3) Preparation of detection working solution: calculate the amount required for an experiment, and mix the detection buffer and chromogenic solution at 1:1 to prepare the detection working solution for later use.

（4） 96孔板中每孔加入50μl标准品或样品。样品体积记录为V。
(4) Add 50 μl of standard or sample per well in a 96-well plate. The sample volume is recorded as V.

（5） 每孔加入150μl检测工作液，混匀。
(5) Add 150 μl of detection working solution to each well and mix well.

（6） 室温避光孵育5-10min。
(6) Incubate at room temperature in the dark for 5-10min.

（7） 酶标仪测量575nm处的吸光度，制作标准曲线。
(7) The microplate reader measures the absorbance at 575nm and makes a standard curve.

⑧ 计算标准品组中每个浓度组的平均吸光度，减去空白对照组的吸光度，即为各个浓度标准品的吸光度。以钙标准品的钙离子量为横坐标，吸光度为纵坐标，绘制出标准曲线。根据标准曲线计算测试孔中的钙含量。
(8) Calculate the average absorbance of each concentration group in the standard group, subtracting the absorbance of the blank control group, which is the absorbance of each concentration standard. The amount of calcium ions of the calcium standard was used as the abscissa and the absorbance was used as the ordinate to draw the standard curve. The calcium content in the test wells is calculated according to the standard curve.

根据如下计算公式，计算样品中钙浓度：
The calcium concentration in the sample is calculated according to the following formula:

C=A×n/V （μg/μl）或C=（A×n/V）/40（mol/L）
C=A×n/V (μg/μl) or C=(A×n/V)/40 (mol/L)

注：A为根据标准曲线确定的钙离子量（μg）
Note: A is the amount of calcium ions (μg) determined according to the standard curve.

n为样品稀释倍数；V为加入的样品体积；40为钙的原子量。
n is the dilution factor of the sample; V is the volume of the sample added; 40 is the atomic weight of calcium.

三、 实验结果
3. Experimental results

图 1 各组细胞钙含量情况
Figure 1 Calcium content in each group

结果讨论：
Discussion of results:

单层细胞模型有助于生物活性物质吸收转运。
The monolayer cell model facilitates the absorption and transport of bioactive substances.

Caco-2细胞是一种人类肠腺癌细胞，其特点是使用可变过滤支架的融合后长期培养中，能够自发地向“正常”肠道表型分化。Caco-2细胞被广泛用于体外研究，以深入了解矿物质吸收机制和肽代谢机制。在这项研究中，我们使用Caco-2细胞单层来评估三种样品对照合成的肽对钙转运的影响[1]。由图可知，1号空白对照组的钙浓度在0~0.010μg/μl之间。2号钙对照组配制成0.1~0.5mg/mL浓度后，钙浓度随着钙对照组样品浓度的增大而降低，细胞钙含量也随之降低，并且钙浓度低于0.010μg/μl，这说明体系中钙离子过多，部分钙离子未参与反应，影响钙螯合能力[2]。3号样品对照组呈现出随着样品浓度的增加，钙浓度也随之增大的现象，并且在0.5mg/mL浓度时，钙浓度最大，酪蛋白磷酸肽钙螯合物的钙结合量达到最大值，说明此浓度能使CPP与钙达到最大螯合效果，肽钙的利用率达到最大，螯合反应可以进行完全[3]。 4号样品对照组钙浓度又随样品浓度的增大而减小，钙含量呈现逐渐降低的趋势，与2号钙对照组出现了相同的情况，说明高浓度的钙溶液并没有增加螯合物的钙含量，此时钙的利用率低，反应进行不完全，使得钙螯合物在沉淀物中的比例相对减少，钙浓度下降，所以呈现下降趋势。总体而言，2、3、4号对照组在配制成0.1~0.5mg/mL浓度样品溶液后，发现钙离子浓度随着样品浓度的增大，钙离子含量出现相应的递增或递减规律，说明Caco-2单层细胞模型建立成功，具有转运药物的能力，单层细胞模型有助于生物活性物质吸收转运。
Caco-2 cells are human intestinal adenocarcinoma cells characterized by their ability to spontaneously differentiate into a "normal" intestinal phenotype in long-term culture after fusion using a variable filter scaffold. Caco-2 cells are widely used in vitro studies to gain insight into the mechanisms of mineral absorption and peptide metabolism. In this study, we use the Caco-2 cell monolayer to evaluate the effect of three sample control synthesized peptides on calcium transport[1]. As can be seen from the figure, the calcium concentration of the No. 1 blank control group is between 0~0.010μg/μl. After the No. 2 calcium control group was prepared to a concentration of 0.1~0.5 mg/mL, the calcium concentration decreased with the increase of the concentration of the calcium control group, and the cell calcium content also decreased, and the calcium concentration was lower than 0.010 μg/μl, which indicated that there were too many calcium ions in the system, and some calcium ions did not participate in the reaction, which affected the calcium chelation ability[2]. At the concentration of 0.5 mg/mL, the calcium concentration was the maximum, and the calcium binding amount of casein phosphopeptide calcium chelate reached the maximum, indicating that this concentration could maximize the chelation effect between CPP and calcium, and the utilization rate of peptide calcium reached the maximum, and the chelation reaction could be carried out completely[3]. The calcium concentration in the control group of sample No. 4 decreased with the increase of sample concentration, and the calcium content showed a gradual decreasing trend, which was the same as that of the No. 2 calcium control group, indicating that the high concentration of calcium solution did not increase the calcium content of chelate, and the utilization rate of calcium was low at this time, and the reaction was incomplete, so that the proportion of calcium chelate in the precipitate was relatively reduced, and the calcium concentration decreased, so it showed a downward trend. In general, the No. 2, 3 and 4 control groups found that the calcium ion concentration increased or decreased with the increase of the sample concentration with the increase of the sample concentration, indicating that the Caco-2 monolayer cell model was successfully established and had the ability to transport drugs, and the monolayer cell model was conducive to the absorption and transport of bioactive substances.

[1]廖伟，陈 H，金伟，等.从罗非鱼骨胶原水解物中新分离的三种钙螯合肽增强了肠道 Caco-2 细胞的钙吸收活性U]。农业与食品化学，2020,68（7）：2091-2098.
[1] Liao Wei, Chen H, Jin Wei, et al. Three newly isolated calcium chelating peptides from tilapia bone collagen hydrolysate enhanced calcium absorption activity in intestinal Caco-2 cellsU]. Agriculture and Food Chemistry,2020,68(7):2091-2098(in Chinese).

[2]李超楠.米蛋白肽-钙螯合物的制备及其性质研究[D].黑龙江八一农垦大学,2019.
[2] Li Chaonan. Preparation and properties of rice protein peptide-calcium chelate[D].Heilongjiang Bayi Agricultural University,2019.

[3]刘春燕.西瓜籽肽与钙螯合物的制备及其功能特性研究[D].江苏大学.2022.
[3] Liu Chunyan. Preparation and functional properties of watermelon seed peptide and calcium chelate[D].Jiangsu University.2022.