技术领域technical field
本发明属于eGFP生物应用技术领域，具体涉及一种基于eGFP的GPR120基因表达的检测方法，本发明还涉及一种基于eGFP在GPR120基因表达检测中的应用。The invention belongs to the technical field of eGFP biological application, and in particular relates to an eGFP-based detection method for GPR120 gene expression, and also relates to an eGFP-based application in the detection of GPR120 gene expression.
背景技术Background technique
GPR120(G-protein coupled receptor 120；又名free fatty acid receptor 4，FFAR4)是脂肪酸受体家族一员，属于G蛋白偶联受体(GPCR)，可被长链脂肪酸激活，尤以n-3不饱和脂肪酸激活能力最强。GPR120在脑、垂体、肺、舌、胃肠、脂肪组织等许多组织表达，主要分布于胃肠多种内分泌细胞、脂肪细胞、巨噬细胞、成骨和破骨细胞以及味蕾细胞。GPR120激活可刺激GLP-1、CCK、GIP等胃肠激素分泌，影响机体的内分泌代谢活动，与肥胖等代谢异常密切相关。GPR120 (G-protein coupled receptor 120; also known as free fatty acid receptor 4, FFAR4) is a member of the fatty acid receptor family and belongs to G protein coupled receptors (GPCR), which can be activated by long-chain fatty acids, especially n-3 Unsaturated fatty acids have the strongest activation ability. GPR120 is expressed in many tissues such as the brain, pituitary, lung, tongue, gastrointestinal tract, and adipose tissue, and is mainly distributed in various endocrine cells, adipocytes, macrophages, osteoblasts and osteoclasts, and taste bud cells in the gastrointestinal tract. The activation of GPR120 can stimulate the secretion of gastrointestinal hormones such as GLP-1, CCK, and GIP, and affect the endocrine and metabolic activities of the body, which is closely related to metabolic abnormalities such as obesity.
GPR120表达水平高低显著影响其细胞调节作用，GPR120表达调控的研究将促进对GPR120功能的认识和发现可能的代谢调节靶分子，为GPR120靶点药物的研发提供一定的依据。基因表达研究所常用技术手段目前有反转录聚合酶链式反应(RT-PCR)和Northern印迹杂交(Northern Blot)，这两种方法虽然技术成熟，广泛应用于基因表达水平的检测，但是也存在一定的缺点：一是实验过程较为繁琐，二是不能在活细胞水平监测基因表达的变化。The expression level of GPR120 significantly affects its cell regulation. The study of GPR120 expression regulation will promote the understanding of GPR120 function and the discovery of possible metabolic regulation target molecules, and provide a certain basis for the development of GPR120 target drugs. Currently, the commonly used techniques in gene expression institutes include reverse transcription polymerase chain reaction (RT-PCR) and Northern blot hybridization (Northern Blot). Although these two methods are mature and widely used in the detection of gene expression levels, they are also There are certain disadvantages: first, the experimental process is relatively cumbersome, and second, it is not possible to monitor changes in gene expression at the level of living cells.
发明内容Contents of the invention
本发明的目的是提供一种基于eGFP的GPR120基因表达水平的检测方法及其应用，解决了现有GPR120基因表达水平的检测中实验步骤繁琐、不能在活细胞水平监测基因表达的变化的问题，为GPR120基因表达水平的检测提供了新思路。The purpose of the present invention is to provide a method for detecting the expression level of GPR120 gene based on eGFP and its application, which solves the problems that the existing GPR120 gene expression level detection has complicated experimental steps and cannot monitor the change of gene expression at the living cell level. It provides a new idea for the detection of GPR120 gene expression level.
本发明所采用的技术方案是，eGFP在GPR120基因表达水平的检测中的应用。The technical solution adopted in the present invention is the application of eGFP in the detection of the expression level of GPR120 gene.
本发明的特征在于，The present invention is characterized in that,
eGFP与GPR120基因表达水平的呈线性关系，即随着eGFP荧光平均强度值的增加，GPR120基因表达水平增加。There is a linear relationship between eGFP and GPR120 gene expression level, that is, as the average fluorescence intensity value of eGFP increases, the GPR120 gene expression level increases.
本发明所采用的另一个技术方案是，基于eGFP的GPR120基因表达水平的检测方法：Another technical solution adopted in the present invention is, the detection method of the expression level of GPR120 gene based on eGFP:
步骤1，首先通过CRISPR/Cas9技术将eGFP片段定点插入到GPR120基因的终止密码子TAA处，使eGFP随GPR120表达而表达，获得eGFP标记GPR120阳性细胞的转基因模型小鼠；Step 1, first insert the eGFP fragment into the stop codon TAA of the GPR120 gene at a fixed point by CRISPR/Cas9 technology, so that eGFP is expressed along with the expression of GPR120, and obtain a transgenic model mouse with eGFP-labeled GPR120-positive cells;
步骤2，随后应用荧光分析仪在488nm激光激发下对小鼠的阳性细胞荧光强度进行测定。Step 2, then use a fluorescence analyzer to measure the fluorescence intensity of positive cells in the mouse under the excitation of 488nm laser.
本发明的特征在于，The present invention is characterized in that,
步骤2的荧光强度通过单细胞平均荧光强度进行表达。The fluorescence intensity in step 2 is expressed by the mean fluorescence intensity of single cells.
步骤2的具体步骤：Specific steps for step 2:
步骤2.1，使用没有被eGFP标定的小鼠细胞进行荧光分析仪的激光强度指标校正；Step 2.1, use the mouse cells that have not been marked by eGFP to correct the laser intensity index of the fluorescence analyzer;
步骤2.2，收取步骤1中转基因模型小鼠的不同组织的eGFP阳性细胞，通过经步骤2.1校正后的荧光分析仪进行荧光强度测定，获取eGFP与GPR120基因表达水平之间的关系；同时通过RT-PCR方法检测eGFP阳性细胞中GPR120的基因表达水平，验证eGFP与GPR120基因表达水平之间的关系。Step 2.2: collect eGFP-positive cells from different tissues of the transgenic model mice in step 1, measure the fluorescence intensity with a fluorescence analyzer calibrated in step 2.1, and obtain the relationship between eGFP and GPR120 gene expression levels; at the same time, through RT- PCR method was used to detect the gene expression level of GPR120 in eGFP-positive cells, and the relationship between eGFP and GPR120 gene expression level was verified.
本发明有益效果是：The beneficial effects of the present invention are:
a)本发明通过实时监测GPR120基因表达，做到自身前后对照，控制组内误差，减少组间误差，保证测定结果更为可靠，从而弥补和克服了RT-PCR和Northern Blot等方法对不同组织细胞进行组间比较而产生的变异性、不能在活细胞水平监测基因表达的变化的问题；a) The present invention monitors the expression of the GPR120 gene in real time, achieves its own before and after comparison, controls the error within the group, reduces the error between the groups, and ensures that the measurement results are more reliable, thereby making up for and overcoming the effects of RT-PCR and Northern Blot on different tissues The variability caused by the comparison of cells between groups and the inability to monitor changes in gene expression at the living cell level;
b)本发明收取细胞后即刻确定GPR120基因表达水平，省去了RNA提取、RNA反转录和PCR等操作和反应过程，使GPR120基因表达检测更加简便快捷。b) The present invention determines the expression level of the GPR120 gene immediately after harvesting the cells, eliminating the need for operations and reaction processes such as RNA extraction, RNA reverse transcription, and PCR, and making the detection of the GPR120 gene expression easier and faster.
附图说明Description of drawings
图1是GPR120-ires-eGFP转基因小鼠的组织细胞中eGFP荧光阳性细胞，其中，图1A为GPR120-ires-eGFP转基因小鼠组织在488nm激光激发下eGFP荧光阳性细胞图，图1B为图1A的组织的普通光镜图，图1C为野生型小鼠组织在488nm激光激发下细胞图，图1D为图1C的组织的普通光镜图；Figure 1 is the eGFP fluorescence-positive cells in the tissue cells of GPR120-ires-eGFP transgenic mice, wherein Figure 1A is a picture of eGFP fluorescence-positive cells in GPR120-ires-eGFP transgenic mouse tissues under 488nm laser excitation, and Figure 1B is Figure 1A The ordinary light microscope image of the tissue, Figure 1C is the cell image of the wild-type mouse tissue under 488nm laser excitation, and Figure 1D is the ordinary light microscope image of the tissue in Figure 1C;
图2是小鼠组织中的扩增曲线图，其中，图2A为GPR120-ires-eGFP转基因小鼠的组织中GPR120的RT-PCR小扩增曲线，图 2B为GPR120-ires-eGFP转基因小鼠的组织中eGFP的RT-PCR小扩增曲线，图2C为野生型小鼠组织中GPR120的RT-PCR小扩增曲线，图2D为野生型小鼠组织中eGFP的RT-PCR小扩增曲线；Figure 2 is a graph of amplification curves in mouse tissues, wherein Figure 2A is a small RT-PCR amplification curve of GPR120 in tissues of GPR120-ires-eGFP transgenic mice, and Figure 2B is a small amplification curve of GPR120-ires-eGFP transgenic mice The RT-PCR small amplification curve of eGFP in the tissues of the wild-type mouse, Figure 2C is the RT-PCR small amplification curve of GPR120 in the wild-type mouse tissue, and Figure 2D is the RT-PCR small amplification curve of eGFP in the wild-type mouse tissue ;
图3是GPR120-ires-eGFP转基因小鼠不同组织细胞中eGFP荧光强度和GPR120基因表达水平之间的关系；Figure 3 is the relationship between eGFP fluorescence intensity and GPR120 gene expression level in different tissue cells of GPR120-ires-eGFP transgenic mice;
图4是GPR120-ires-eGFP转基因小鼠腹腔巨噬细胞在体外经脂多糖处理后eGFP荧光强度和GPR120基因表达水平之间的关系图，其中，图4A是GPR120基因表达水平在对照组和经脂多糖处理组的关系图，图4B是eGFP荧光强度均值在对照组和经脂多糖处理组的关系图，图4C是GPR120基因表达水平与细胞eGFP荧光强度均值的关系图。Figure 4 is a graph of the relationship between the eGFP fluorescence intensity and the expression level of the GPR120 gene after the peritoneal macrophages of GPR120-ires-eGFP transgenic mice were treated with lipopolysaccharide in vitro, wherein Figure 4A is the expression level of the GPR120 gene in the control group and the treated group The relationship diagram of the lipopolysaccharide treatment group, Figure 4B is the relationship diagram of the mean value of eGFP fluorescence intensity in the control group and the group treated with lipopolysaccharide, and Figure 4C is the relationship diagram of the expression level of the GPR120 gene and the mean value of the cell eGFP fluorescence intensity.
具体实施方式detailed description
下面结合附图和具体实施方式对本发明进行详细说明。The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.
本发明以eGFP标记GPR120阳性细胞的模型小鼠为对象，利用荧光显微镜技术测定小鼠eGFP阳性细胞的荧光强度，与eGFP阳性细胞的GPR120基因表达水平进行相关性分析，确定eGFP阳性细胞的荧光强度与GPR120基因表达水平之间的线性关系，使eGFP荧光强度作为GPR120基因表达水平的一个可靠的检测指标提供了依据。The present invention takes eGFP-labeled GPR120-positive cell model mice as objects, uses fluorescence microscopy to measure the fluorescence intensity of the mouse eGFP-positive cells, and performs correlation analysis with the GPR120 gene expression level of the eGFP-positive cells to determine the fluorescence intensity of the eGFP-positive cells The linear relationship with the expression level of the GPR120 gene provides a basis for the eGFP fluorescence intensity as a reliable detection index for the expression level of the GPR120 gene.
基于eGFP的GPR120基因表达水平的检测方法：Detection method of GPR120 gene expression level based on eGFP:
步骤1，首先通过CRISPR/Cas9技术将eGFP片段定点插入到GPR120基因的终止密码子TAA处，使eGFP随GPR120表达而表达，获得eGFP标记GPR120阳性细胞的转基因模型小鼠，这样eGFP随GPR120表达而表达，并且在mRNA水平，eGFP和GPR120相互分离，保证了GPR120蛋白的功能和代谢过程不受影响；Step 1, first insert the eGFP fragment into the stop codon TAA of the GPR120 gene by CRISPR/Cas9 technology, so that eGFP is expressed along with the expression of GPR120, and obtain a transgenic model mouse with eGFP-labeled GPR120 positive cells, so that eGFP grows along with the expression of GPR120 expression, and at the mRNA level, eGFP and GPR120 are separated from each other, ensuring that the function and metabolic process of GPR120 protein are not affected;
步骤2，随后应用荧光分析仪在488nm激光激发下对小鼠的阳性细胞荧光强度进行测定，并通过单细胞平均荧光强度表达，Step 2, then use a fluorescence analyzer to measure the fluorescence intensity of positive cells in the mouse under 488nm laser excitation, and express it through the average fluorescence intensity of single cells,
步骤2.1，使用没有被eGFP标定的小鼠细胞进行荧光分析仪的激光强度指标校正；Step 2.1, use the mouse cells that have not been marked by eGFP to correct the laser intensity index of the fluorescence analyzer;
步骤2.2，收取步骤1中转基因模型小鼠的不同组织的eGFP阳性细胞，通过经步骤2.1校正后的荧光分析仪进行荧光强度测定，获取eGFP与GPR120基因表达水平之间的关系；同时通过RT-PCR方法检测eGFP阳性细胞中GPR120的基因表达水平，验证eGFP与GPR120基因表达水平之间的关系。Step 2.2: collect eGFP-positive cells from different tissues of the transgenic model mice in step 1, measure the fluorescence intensity with a fluorescence analyzer calibrated in step 2.1, and obtain the relationship between eGFP and GPR120 gene expression levels; at the same time, through RT- PCR method was used to detect the gene expression level of GPR120 in eGFP-positive cells, and the relationship between eGFP and GPR120 gene expression level was verified.
1.实验对象1. Subjects
制备的eGFP标记GPR120的模型小鼠，细胞组织中能见eGFP阳性细胞，在488nm激光激发下发出绿色荧光，具体如图1所示：图1A为GPR120-ires-eGFP转基因小鼠组织的荧光阳性细胞图，可见阳性细胞，图1B为图1A的组织的普通光镜图，图1C为野生型小鼠组织的细胞图，未见荧光阳性细胞，图1D为图1C的组织的普通光镜图。In the prepared eGFP-labeled GPR120 model mouse, eGFP-positive cells can be seen in the cell tissue, which emit green fluorescence under 488nm laser excitation, as shown in Figure 1: Figure 1A shows the fluorescence-positive tissue of GPR120-ires-eGFP transgenic mice Cell map, positive cells can be seen, Figure 1B is the ordinary light microscope image of the tissue in Figure 1A, Figure 1C is the cell image of the wild-type mouse tissue, no fluorescent positive cells are seen, Figure 1D is the ordinary light microscope image of the tissue in Figure 1C .
2.细胞内RNA提取和反转录2. Intracellular RNA Extraction and Reverse Transcription
在eGFP标记GPR120的模型小鼠和野生型小鼠，分别提取小鼠的胃粘膜、十二指肠粘膜、空肠粘膜、结肠粘膜、脂肪组织、垂体、肺脏等组织器官的RNA提取并进行反转录，具体如下：取上述组织各1-2mg，每样加入350μL裂解液RL，使用组织破裂仪破裂组织，再将所有溶液转移至过滤柱CS中以12000rpm速度离心2min，收集滤液；再向滤液中加入350μL 70％的乙醇溶液，混匀后转入吸附柱CR3中以12000rpm速度离心1min，倒掉收集管中的废液，将吸附柱CR3放回收集管中；向吸附柱CR3中加入350μL去蛋白液RW1，以12000rpm速度离心1min，倒掉收集管中的废液，将吸附柱CR3放回收集管中；向吸附柱CR3中加入80μL DNaseI工作液，室温放置15min；向吸附柱CR3中加入350μL去蛋白液RW1，以12000rpm速度离心1min，倒掉收集管中的废液，将吸附柱CR3放回收集管中；向吸附柱CR3中加入500μL漂洗液RW，室温静置2min，以12000rpm速度离心1min，倒掉收集管中的废液，将吸附柱CR3放回收集管中；重复向吸附柱CR3中加入500μL漂洗液RW，室温静置2min，以12000rpm速度离心2min，倒掉收集管中的废液，将吸附柱CR3置于室温放置5min以彻底晾干残余的漂洗液；将吸附柱CR3转入RNase-Free离心管中，加入50μL Rnase-Free ddH2O，室温放置2min，以12000rpm速度离心2min，得到RNA溶液。In eGFP-labeled GPR120 model mice and wild-type mice, extract RNA from gastric mucosa, duodenal mucosa, jejunal mucosa, colonic mucosa, adipose tissue, pituitary gland, lung and other tissues and organs of the mice and reverse The details are as follows: Take 1-2 mg of each of the above tissues, add 350 μL of lysate RL to each sample, use a tissue disruptor to rupture the tissue, then transfer all the solutions to the filter column CS and centrifuge at 12,000 rpm for 2 minutes to collect the filtrate; Add 350μL of 70% ethanol solution to the solution, mix well, transfer to the adsorption column CR3 and centrifuge at 12000rpm for 1min, pour off the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube; add 350μL to the adsorption column CR3 Centrifuge the protein-removing solution RW1 at 12000rpm for 1min, pour off the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube; add 80μL DNaseI working solution to the adsorption column CR3, and place it at room temperature for 15min; Add 350 μL protein-removing solution RW1, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube; add 500 μL rinse solution RW to the adsorption column CR3, let stand at room temperature for 2 min, Centrifuge at a high speed for 1 min, discard the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube; add 500 μL of rinse solution RW to the adsorption column CR3 repeatedly, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and discard the collection tube For the waste liquid in the solution, place the adsorption column CR3 at room temperature for 5 minutes to completely dry the residual rinse solution; transfer the adsorption column CR3 to an RNase-Free centrifuge tube, add 50 μL RNase-Free ddH2O, and place it at room temperature for 2 minutes at a speed of 12000 rpm Centrifuge for 2 minutes to obtain RNA solution.
酶标仪检测RNA浓度与纯度，琼脂糖凝胶电泳检测RNA质量。在八连管中依次加入2μL 5x gDNA Eraser Buffer，再分别加入1μL gDNA Eraser，再分别加入1μg Total RNA，最后用RNase Free dH2O补足至10μL，混匀，室温反应5min。在各管中分别加入以下试剂，4μL5x PrimeScript Buffer2、4μLRNase Free dH2O、1μL PrimeScript RT Enzyme Mix I和1μL RT Primer Mix，总反应体系为20μL，混匀。反应条件为37℃、15min，85℃、5s。反应完成后将cDNA存于4℃备用，长期保存时转入-20℃。The RNA concentration and purity were detected by a microplate reader, and the RNA quality was detected by agarose gel electrophoresis. Add 2 μL of 5x gDNA Eraser Buffer to the eight-tube in sequence, then add 1 μL of gDNA Eraser, then add 1 μg of Total RNA, and finally make up to 10 μL with RNase Free dH2O, mix well, and react at room temperature for 5 minutes. Add the following reagents to each tube respectively, 4 μL 5x PrimeScript Buffer2, 4 μL RNase Free dH2O, 1 μL PrimeScript RT Enzyme Mix I and 1 μL RT Primer Mix, the total reaction system is 20 μL, and mix well. The reaction conditions are 37°C, 15min, 85°C, 5s. After the reaction was completed, the cDNA was stored at 4°C for later use, and transferred to -20°C for long-term storage.
3.GPR120和eGFP基因表达水平的检测3. Detection of GPR120 and eGFP gene expression levels
采用定量PCR方法对GPR120和eGFP的基因表达进行检测。在八连管中依次加入6μLdH2O，2μL模板cDNA，2μL FAS引物，10μL SYBR PremixEx TaqII(2x)，总反应体系为20μL，混匀。The gene expression of GPR120 and eGFP was detected by quantitative PCR method. Add 6 μL of dH2O, 2 μL of template cDNA, 2 μL of FAS primer, 10 μL of SYBR PremixEx TaqII (2x) to the eight-tube in sequence, the total reaction system is 20 μL, and mix well.
反应条件是：第一步是94℃、5min，第二步是94℃、30s，56℃、30s，72℃、1min循环进行40次，第三步是加温溶解扩增产物，获得溶解曲线。The reaction conditions are: the first step is 94°C, 5min, the second step is 94°C, 30s, 56°C, 30s, 72°C, 1min cycle for 40 times, the third step is to heat and dissolve the amplification product to obtain the melting curve .
其中，总反应体系中小鼠GPR120引物序列是：Wherein, the mouse GPR120 primer sequence in the total reaction system is:
上游引物：5’-GTG CCG GGA CTG GTC ATT GTG-3’；Upstream primer: 5'-GTG CCG GGA CTG GTC ATT GTG-3';
下游引物：5’-TTG TTG GGA CAC TCG GAT CTG G-3’。Downstream primer: 5'-TTG TTG GGA CAC TCG GAT CTG G-3'.
总反应体系中eGFP引物序列是：The eGFP primer sequence in the total reaction system is:
上游引物：5’-TCT TCT TCA AGG ACG ACG GCA ACT-3’；Upstream primer: 5'-TCT TCT TCA AGG ACG ACG GCA ACT-3';
下游引物：5’-CCT TGA TGC CGT TCT TCT GCT TGT-3’。Downstream primer: 5'-CCT TGA TGC CGT TCT TCT GCT TGT-3'.
以beta-actin基因表达为内对照，总反应体系中beta-actin引物序列是：Taking beta-actin gene expression as an internal control, the sequence of beta-actin primers in the total reaction system is:
上游引物：5’-CGT TGG CAT CCA CGA AAC TA-3’；Upstream primer: 5'-CGT TGG CAT CCA CGA AAC TA-3';
下游引物：5’-GGT GCT GGG AGG TAC AGG G-3’。Downstream primer: 5'-GGT GCT GGG AGG TAC AGG G-3'.
对实验结果表达水平分析得到如图2所示的扩增曲线。如图2A表示转基因小鼠的组织中GPR120的RT-PCR小扩增曲线，如图2B表示转基因小鼠的组织中eGFP的RT-PCR小扩增曲线，在一定阶段内两者均随着扩增循环次数的增大而升高。如图2C表示野生型小鼠组织中GPR120的RT-PCR小扩增曲线，在一定阶段内两者均随着扩增循环次数的增大而升高，如图2D表示野生型小鼠组织中eGFP的RT-PCR小扩增曲线，曲线结果无表达。The amplification curve shown in Figure 2 was obtained by analyzing the expression level of the experimental results. Figure 2A shows the RT-PCR small amplification curve of GPR120 in the tissue of transgenic mice, and Figure 2B shows the RT-PCR small amplification curve of eGFP in the tissue of transgenic mice. increased with increasing cycle times. Figure 2C shows the RT-PCR small amplification curve of GPR120 in wild-type mouse tissues, and both of them increase with the increase of the number of amplification cycles in a certain period, as shown in Figure 2D in wild-type mouse tissues The RT-PCR amplification curve of eGFP is small, and the curve result shows no expression.
以beta-actin基因表达为内对照，对各个不同组织中的GPR120基因表达水平进行分析，并应用荧光显微镜对各个组织中eGFP标记的细胞的绿色荧光强度进行测量。结果表明，组织中GPR120表达水平与组织中绿色荧光细胞的荧光强度之间呈显著正相关，结果如图3所示。Using beta-actin gene expression as an internal control, the expression level of GPR120 gene in different tissues was analyzed, and the green fluorescence intensity of eGFP-labeled cells in each tissue was measured by fluorescence microscope. The results showed that there was a significant positive correlation between the expression level of GPR120 in the tissue and the fluorescence intensity of the green fluorescent cells in the tissue, and the results are shown in FIG. 3 .
实施例Example
培养eGFP标记的GPR120的模型小鼠的肺泡巨噬细胞，具体方法如下：体外培养采用颈部脱臼法处死eGFP标记GPR120的模型小鼠，用75％的酒精对小鼠进行消毒处理；用10ml注射器吸取PBS缓冲液6ml左右，除去气泡备用；用眼科剪剪开颈部至胸腔外表皮，分离颈部气管穿线备用，剪开胸腔使肺部暴露，用镊子夹住气管前端，剪短气管将注射器针尖插入气管并用线扎紧；缓慢注入PBS缓冲液2-3ml使肺部充满，吸出肺内溶液再缓慢注入，循环3-5次，吸取肺内PBS缓冲液注入离心管，所得为含肺巨噬细胞的溶液；将离心管配平于低温水平离心机离心6min，转速为1200rpm；吸去上清液，底物即为肺巨噬细胞。The alveolar macrophages of eGFP-labeled GPR120 model mice were cultivated, and the specific method was as follows: in vitro culture, the eGFP-labeled GPR120 model mice were sacrificed by cervical dislocation, and the mice were sterilized with 75% alcohol; Draw about 6ml of PBS buffer solution, remove air bubbles for later use; use ophthalmic scissors to cut open the neck to the outer skin of the thoracic cavity, separate the trachea in the neck for threading, cut open the thorax to expose the lungs, clamp the front end of the trachea with tweezers, cut the trachea short and put the syringe Insert the needle tip into the trachea and tie it tightly with a thread; slowly inject 2-3ml of PBS buffer solution to fill the lungs, suck out the solution in the lungs and inject it slowly, cycle 3-5 times, absorb the PBS buffer solution in the lungs and inject it into the centrifuge tube, and the obtained lung giant Phage cell solution; balance the centrifuge tube and centrifuge in a low-temperature horizontal centrifuge for 6 minutes at a speed of 1200 rpm; suck off the supernatant, and the substrate is lung macrophages.
向肺巨噬细胞中加入细胞培养液，充分混匀，种于培养皿，使每组培养皿内细胞数均匀，标上日期及名称，置37℃的CO2培养箱中培养。培养8小时后，细胞分组，一组加入200ng/ml的脂多糖(LPS)处理24小时，另外一组为对照组。Add the cell culture solution to the lung macrophages, mix them well, plant them in a culture dish, make the number of cells in each group of culture dishes uniform, mark the date and name, and culture them in a CO2 incubator at 37°C. After culturing for 8 hours, the cells were divided into groups, one group was treated with 200 ng/ml lipopolysaccharide (LPS) for 24 hours, and the other group was the control group.
随后，应用荧光显微镜对各个组织中eGFP标记的细胞的荧光强度进行测量，测量荧光强度之后，收集细胞的RNA，反转录之后应用定量PCR方法观察GPR120基因表达水平。结果显示，如图4A所示，肺泡巨噬细胞的GPR120基因表达与LPS处理组比较，对照组显著升高；如图4B所示，同时肺泡巨噬细胞的eGFP荧光强度均值与LPS处理组比较，对照组也显著升高；如图4C所示，GPR120基因表达与细胞eGFP荧光强度均值之间存在显著正相关关系，随着eGFP荧光强度均值的增大，GPR120基因表达水平升高。Subsequently, the fluorescence intensity of eGFP-labeled cells in each tissue was measured using a fluorescence microscope. After measuring the fluorescence intensity, the RNA of the cells was collected, and after reverse transcription, quantitative PCR was used to observe the expression level of the GPR120 gene. The results showed that, as shown in Figure 4A, the GPR120 gene expression of alveolar macrophages was significantly higher than that of the LPS treatment group, and the control group was significantly increased; , the control group also significantly increased; as shown in Figure 4C, there was a significant positive correlation between the expression of GPR120 gene and the mean value of the fluorescence intensity of eGFP in cells. With the increase of the mean value of eGFP fluorescence intensity, the expression level of GPR120 gene increased.
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