ApoE⁻⁻ 动脉粥样硬化模型

以预先生成的 cDNA 为输入的 SYBR Green 实时定量 PCR 检测。每孔加入 10 ng cDNA，技术三重复；使用 PowerUp SYBR Green Master Mix 与 5 µM 正反向引物；按源方案循环条件扩增并读取 CT，以看家基因和参考实验组进行 ΔΔCT 分析。具体基因、引物和生物学分组按各研究设计确定。

病变面积的复核由对实验分组不知情的第二观察者完成。

以 Image Pro Plus 经毫米空间标定后，按弓部、胸段、腹段分区测量内膜病变面积并导出至电子表格；分区内膜总面积与病变描记同步经显微镜核对，定量由第二观察者盲法复核。

各样品目标序列的 CT 值；以看家基因归一化并以参考实验组计算 ΔΔCT/相对表达量。本方案不包含具体基因或疾病特异结论，输出判读依赖所选的引物、看家基因和实验分组。

获得可用于主动脉根及头臂动脉病变面积和组成定量的固定或冷冻组织块及切片。

获得主动脉根和头臂动脉切片中油红 O 阳性病变面积，并按切片或血管截面面积归一化。

固定、清洁与剖开 1. 解剖所得主动脉每支置于 4% 多聚甲醛或 10% 中性缓冲福尔马林中固定 24–48 小时；固定后可存于生理盐水，室温或 2–8 EC 保存。 2. 去除外膜组织以清洁主动脉，注意不撕破或切伤主动脉及无名动脉、左颈总动脉、左锁骨下动脉等重要分支；清洁前先在生理盐水中浸泡数小时有助于清洁，操作全程以生理盐水保持组织湿润。 3. 无名动脉与左颈总动脉各保留 1 mm，左锁骨下动脉自根部完全切除；沿外侧弯经无名动脉、左颈总动脉剪开至左锁骨下动脉处，再沿升主动脉内侧弯向下剪开至腹段底部。 展平固定 1. 用探针（Fine Science Tools 货号 26002-20）将主动脉在黑色蜡盘上展平固定。 2. 涂生理盐水防止干燥。 3. 在蜡盘上标注小鼠编号，4 摄氏度保存；铺蜡后 3 天内必须完成成像与分析。 成像 用 Nikon 数码相机拍摄 en face 主动脉图像，画面内必须包含毫米标尺以校准测量；该 en face 方法设定三个区域，图像须分别包含：弓部区——心肌交界处至左锁骨下动脉远端 3 mm；胸段区——左锁骨下动脉远端 3 mm 至最后一支肋间动脉；腹段区——最后一支肋间动脉至髂动脉分叉。图像存为 JPEG 或 TIFF。 定量与盲法复核 1. 使用 Image Pro Plus 5.0 或 7.0 软件进行定量。 2. 以图像为底标注病变的同时，通过显微镜同步核对主动脉实况。 3. 定量结果由不知实验分组的第二观察者复核。复核完毕后，主动脉去针，转入正确标记的加样管，用含 0.02% 叠氮钠的生理盐水于室温保存。 软件空间标定 1. 打开 Image Pro（5.0 或 7.0）及待测图像。 2. 进入 Measure ▸ Calibrate ▸ Spatial Calibration。 3. 选择 Spatial Calibration Wizard 按提示操作，或点击 New 命名本次标定。 4. 选择 Image，将定标线对准标尺并把参考单位改为 mm，点击 OK 后 Apply。 图像标注 1. 点击标注按钮（Annotate），输入文字（研究名、小鼠编号、日期）后确定，将标签烧录进图像（双箭头按钮）。 2. 保存图片，并对其余图像重复标注。 分区描记与病变面积测量 1. 为每张打开的图像选择正确标定：Measure ▸ Calibration ▸ Set System ▸ Apply。 2. 打开 Measurements 测量面板，选择直线工具在标尺上量取 3 mm。 3. 用直线工具定义弓部区边界：使线的一端落在左锁骨下动脉根部，作为弓部与胸段的分隔标记。 4. 选择多边形工具描记弓部区内膜总面积，再在图像上描记弓部区病变，同时通过显微镜核对主动脉实况。 5. 自弓部区末端量至最后一支肋间动脉（约 17 mm），勾出胸段；通过显微镜核对并在图像上描记胸段病变。 6. 勾出腹段：自胸段末端至髂动脉分叉（可选：定量可包含双侧髂动脉各 1 mm），描记腹段病变并经显微镜核对。 数据导出 1. 在 Input/Output 选项卡选择测量项并点击 Export Now，粘贴入电子表格。 2. 保存测量图像：Input/Output 选项卡另存为“小鼠编号….msr”，此时可打印带叠加层的图像。 3. 在图像打印、保存且测量值已录入电子表格之前，不得关闭图像。

qPCR 反应体系 **Option 2****: SYBR qPCR** qPCR 反应体系 Prepare SYBR Master mix (ul per well): 1. 5ul PowerUp SYBR Green Master Mix (#A25776, Applied Biosystem-ThermoFisher) 1. 1ul Primers (Forward and Reverse, diluted in Nuclease-free water at 5µM) qPCR 反应体系 Add 6ul of SYBR Master mix to each well with an automatic pipette (Multipette E3, Eppendorf) qPCR 反应体系 Add 4ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics) 热循环 Perform PCR using the following cycling conditions in a LightCycler® 480 System (Roche): 1. 50˚C for 2min 1. 95˚C for 2min 1. 95˚C for 15s + 60˚C for 1min (x40cycles) 数据分析 Using the LightCycler Software obtain the threshold cycles (CT) signals from each samples. 数据分析 Perform ΔΔCT-method to analyze the expression data using the endogenous control genes and the reference experimental group.

Prepare a 1 ml syringe by filling with 15µl 0.5 M EDTA ph 8.0 and then capping with a 23g needle. The syringe will be us Prepare a 1 ml syringe by filling with 15µl 0.5 M EDTA ph 8.0 and then capping with a 23g needle. The syringe will be used for drawing blood from the heart. Prepare a 10 ml syringe with 50-100 µl of heparin (1000 units per ml), then fill the syringe to 10 ml with PBS. Remove a Prepare a 10 ml syringe with 50-100 µl of heparin (1000 units per ml), then fill the syringe to 10 ml with PBS. Remove all the air from the syringe. Cap with a 30g needle. The syringe will be used to flush the heart. Anesthetize the mouse. Anesthetize the mouse. Determine weight and length of the mouse. Determine weight and length of the mouse. Tape the mouse’s arms to 3-4 layers of paper towels and place under a light source. Tape the mouse’s arms to 3-4 layers of paper towels and place under a light source. Cut the skin of the mouse from the abdomen to the top of the thorax. Cut the skin of the mouse from the abdomen to the top of the thorax. Open the abdominal wall below the ribcage. Open the abdominal wall below the ribcage. Lift the sternum with tweezers and cut the diaphragm. Then cut away the lower part of the ribcage to partially expose th Lift the sternum with tweezers and cut the diaphragm. Then cut away the lower part of the ribcage to partially expose the heart. Draw blood from the heart by sticking the needle of the 1 ml syringe (see 1) into the apex of the left ventricle. Draw b Draw blood from the heart by sticking the needle of the 1 ml syringe (see 1) into the apex of the left ventricle. Draw blood by slowly pulling the plunger, twisting the needle. Do not draw for over a minute to avoid blood clots. Transfer the blood into an Eppendorf tube. Mix the tube by inverting it a few times. Centrifuge the tube of blood and transfer the plasma into a fresh tube. Make a small incision in the right atrium for drainage. Make a small incision in the right atrium for drainage. Stick the 10 ml syringe into the apex of the left ventricle and flush the blood from the mouse with 10 ml of PBS. Stick the 10 ml syringe into the apex of the left ventricle and flush the blood from the mouse with 10 ml of PBS. If the liver is to be saved take several sections and immediately freeze at –80ºC. If the liver is to be saved take several sections and immediately freeze at –80ºC. Dry the mouse by turning it over and pressing it against paper towels. Transfer to clean, dry paper towels and re-tape t Dry the mouse by turning it over and pressing it against paper towels. Transfer to clean, dry paper towels and re-tape the arms. Place the mouse under a dissecting microscope. Place the mouse under a dissecting microscope. Remove the remaining ventral part of the ribcage, carefully cut the right clavicle leaving the brachiocephalc artery and Remove the remaining ventral part of the ribcage, carefully cut the right clavicle leaving the brachiocephalc artery and its branches intact. Using microdissection scissors, cut out all of the fat around the ascending aorta and brachiocephalic artery. Cut all the fat and tissue surrounding the heart, including the pulmonary artery, and veins. Cut all the fat and tissue surrounding the heart, including the pulmonary artery, and veins. Flush the heart again through the right ventricle, left atrium, and left ventricle using a 10 ml syringe containing a to Flush the heart again through the right ventricle, left atrium, and left ventricle using a 10 ml syringe containing a total of 3 ml of PBS to clean out residual blood. Cut the brachiocephalic artery distal to where it branches into the right subclavian and right carotid and then cut it a Cut the brachiocephalic artery distal to where it branches into the right subclavian and right carotid and then cut it at its branching site from the aorta so you end up with a Yshaped piece. Put it in a base mold (Fisher cat no 22-038217) and using a 10 ml syringe perfuse it with 1 ml of PBS to get it clean. It is important while handling the brachiocephalic artery to touch it only at its ends and not in the middle. Cut the aorta proximal to the branching site of the brachiocephalic artery. Cut the aorta proximal to the branching site of the brachiocephalic artery. Take out the heart with the aorta. The atria must remain intact as they serve as landmarks when cutting the heart in the Take out the heart with the aorta. The atria must remain intact as they serve as landmarks when cutting the heart in the atherosclerosis assay. Flush the heart-aorta preparation through the aorta with a 10 ml syringe containing 2 ml of PBS and place the heart in a Flush the heart-aorta preparation through the aorta with a 10 ml syringe containing 2 ml of PBS and place the heart in a 20 ml scintillation vial containing 10 ml of formaldehyde solution (10% v/v in aqueous phosphate buffer, Mallinckrodt). Take the cleaned piece of the brachiocephalic artery and place it in a new base mold, fill it up to the first lip of the Take the cleaned piece of the brachiocephalic artery and place it in a new base mold, fill it up to the first lip of the base mold with Tissue Tek O.C.T. compound. Move the brachiocephalic artery around carefully to make sure it fills with OCT. Turn the Y-shaped piece of brachiocephaic artery piece so the prongs of the fork face the bottom of the base mold and fr Turn the Y-shaped piece of brachiocephaic artery piece so the prongs of the fork face the bottom of the base mold and freeze it in that position on a block of dry ice.

**Preparation of Aortic Root and Brachiocephalic Artery for Lesion Quantitation:** **Preparation of Aortic Root and Brachiocephalic Artery for Lesion Quantitation:** 1. Prepare a 1 ml syringe by filling with 15µl 0.5 M EDTA ph 8.0 and then capping with a 23g needle. The syringe will be used for drawing blood from the heart. 2. Prepare a 10 ml syringe with 50-100 µl of heparin (1000 units per ml), then fill the syringe to 10 ml with PBS. Remove all the air from the syringe. Cap with a 30g needle. The syringe will be used to flush the heart. 3. Anesthetize the mouse. 4. Determine weight and length of the mouse. 5. Tape the mouse’s arms to 3-4 layers of paper towels and place under a light source. 6. Cut the skin of the mouse from the abdomen to the top of the thorax. 7. Open the abdominal wall below the ribcage. 8. Lift the sternum with tweezers and cut the diaphragm. Then cut away the lower part of the ribcage to partially expose the heart 9. Draw blood from the heart by sticking the needle of the 1 ml syringe (see 1) into the apex of the left ventricle. Draw blood by slowly pulling the plunger, twisting the needle. Do not draw for over a minute to avoid blood clots. Transfer the blood into an Eppendorf tube. Mix the tube by inverting it a few times. Centrifuge the tube of blood and transfer the plasma into a fresh tube. 10. Make a small incision in the right atrium for drainage. 11. Stick the 10 ml syringe into the apex of the left ventricle and flush the blood from the mouse with 10 ml of PBS. 12. If the liver is to be saved take several sections and immediately freeze at –80°C. 13. Dry the mouse by turning it over and pressing it against paper towels. Transfer to clean, dry paper towels and re-tape the arms. 14. Place the mouse under a dissecting microscope. 15. Remove the remaining ventral part of the ribcage, carefully cut the right clavicle leaving the brachiocephalc artery and its branches intact. Using microdissection scissors, cut out all of the fat around the ascending aorta and brachiocephalic artery. 16. Cut all the fat and tissue surrounding the heart, including the pulmonary artery, and veins. 17. Flush the heart again through the right ventricle, left atrium, and left ventricle using a 10 ml syringe containing a total of 3 ml of PBS to clean out residual blood. 18. Cut the brachiocephalic artery distal to where it branches into the right subclavian and right carotid and then cut it at its branching site from the aorta so you end up with a Y-shaped piece. Put it in a base mold (Fisher cat no 22-038217) and using a 10 ml syringe perfuse it with 1 ml of PBS to get it clean. It is important while handling the brachiocephalic artery to touch it only at its ends and not in the middle. 19. Cut the aorta proximal to the branching site of the brachiocephalic artery. 20. Take out the heart with the aorta. The atria must remain intact as they serve as landmarks when cutting the heart in the atherosclerosis assay. 21. Flush the heart-aorta preparation through the aorta with a 10 ml syringe containing 2 ml of PBS and place the heart in a 20 ml scintillation vial containing 10 ml of formaldehyde solution (10% v/v in aqueous phosphate buffer, Mallinckrodt). 22. Take the cleaned piece of the brachiocephalic artery and place it in a new base mold, fill it up to the first lip of the base mold with Tissue Tek O.C.T. compound. Move the brachiocephalic artery around carefully to make sure it fills with OCT. 23. Turn the Y-shaped piece of brachiocephaic artery piece so the prongs of the fork face the bottom of the base mold and freeze it in that position on a block of dry ice. **Embedding Hearts in Gelatin for Aortic Root Analysis:** **Embedding Hearts in Gelatin for Aortic Root Analysis:** 1. Fix hearts in buffered formalin for 2 weeks at 4°C. 2. Place each heart in an embedding capsule; and place up to 40 capsules in a 1000 ml beaker. Wash under cold running tap water for 1 hour. 3. Make gelatin solutions containing 5%, 10% and 25% gelatin in H²0 and dissolve at 50°C in a water bath. 4. Place washed heart capsules in a shallow plastic container, float container in a 42°C water bath and cover heart capsules with 5% gelatin for 2 hours. 5. Pour back gelatin into a bottle (can be stored at 4°C for future use) and cover hearts with 10% gelatin for 2 hours. 6. Pour back gelatin into a bottle (can be stored at 4°C for future use) and cover hearts with 25% gelatin. Incubate in water bath over night. 7. Place container with heart capsules in refrigerator for 3 hours. 8. Remove heart capsules from solidified gelatin, discard gelatin. 9. Remove embedded hearts from capsules, trim gelatin around the heart to a cube and place trimmed hearts back into buffered formalin. 10. Store at 4°C for a minimum of 24 hours before sectioning the hearts. **Sectioning Hearts for Aortic Root Analysis:** **Sectioning Hearts for Aortic Root Analysis:** 1. Locate the left and right atria. Using a sharp razor-blade cut the bottom half of the heart off in a plane parallel to the atria. **NOTE:** It is essential that the gross cut is parallel to the atria so that a cross section of all three aortic valves is in the same geometric plane. Discard lower half of the heart. 2. Make a second cut above the top of the heart parallel to the first cut on the bottom. 3. Store the heart at 4°C in buffered formalin until ready to section. 4. Mount the trimmed heart on a sample pedestal in the cryostat using OCT compound, the aortic side of the block facing the pedestal. 5. Cover the rest of the gelatin block with OCT and let it freeze. 6. Set section thickness to 30µm and start sectioning until the part of the block that contains the heart tissue is visible. If a complete heart section is not visible, reposition the pedestal. 7. Once a complete heart section is visible, place it on a slide and to check the anatomic location of the section (initially you will see just a thick muscular wall). (**note**: to flatten the section for better viewing under the microscope dip the slide in water). 8. Cut approx. 20 of these 30µm sections and check under the microscope to see if the aortic sinus approaches. The presence of the left and right atria suggests that the sinus is close. The aortic sinus will usually appear rounded, the aortic wall won’t be thick yet and the valves won’t be distinct. Start saving the sections when the valve leaflets become clearly visible. (Reposition the pedestal if necessary to have all 3 leaflets of the valve in one plane) 9. Change thickness of sections to 12 µm and save every other section. Put 4 sections on one slide in this order, and label mouse and slide no: [ms.JPG]]() 10. Cut and save sections until the aorta is rounded and all of the valve leaflets are gone. Usually, this is after 20 to 24 sections (5 to 6 slides). 11. Place slides in a covered glass dish with formalin vapor (put some formalin on bottom of dish) and store slides at 4°C for at least 12 hours prior to staining. **Staining Heart Sections for Aortic Root Analysis:** **Staining Heart Sections for Aortic Root Analysis:** 1. 1 min. H²O 2. 1 min. H²O 3. 30 sec. 60% isopropanol 4. 18 min filtered oil red-O (0.24% in 62.5% 2-propanol) 5. 30 sec. 60% isopropanol rinse 6. 1 min. H²O 7. 1 min. H²O 8. 2 min Harris Hematoxylin 9. 1 min Bluing Solution (H²O with a few drops of ammonia) 10. 1 min. H²O 11. 30 sec. Light Green 12. 1 dip H²O 13. Keep in H²O until ready to put cover slip on the slide 14. Fix cover slips with glycerol gelatin 15. Wash slides to remove excess glycerol gelatin **Sectioning Brachiocephalic Artery for Quantitation of Cross Sectional Lesional Area:** **Sectioning Brachiocephalic Artery for Quantitation of Cross Sectional Lesional Area:** 1. Set cutting temperature of microtome to –30°C. Mount specimens embedded in OCT on cutting pedestal, the opening of the Y shaped vessel should face towards the microtome cutting blade. 2. Set section thickness to 10µm. Start sectioning the right carotid and right subclavian arteries up to the point where they first combine into the brachiocephalic. A section of this point will not have the separation between the two vessels and will be considered the starting point for the following distances. 3. Once this point has been defined, cut 200µm discarding the sections. 4. Use Fisher Colorfrost plus slides to save sections 5. Save the following 2 sections (10µm each) on a the first slide (slide 1) in the following order: [ae.JPG]]() 6. Proceed sectioning and discard the following 80µm (8 sections) 7. Save the next 2 sections (10µm, each) → slide 2 8. Proceed sectioning and discard the following 80µm (8 sections) 9. Save the next 2 sections (10µm, each) → slide 3 10. Proceed sectioning and discard the following 80µm (8 sections) 11. Save the next 2 sections (10µm, each) → slide 4 12. Save 4 additional sections (10µm each), one per slide → slides a,b,c,d The purpose is to have consecutive sections available for immunostaining. These sections are not relevant for quantification and can be discarded if immunostaining will not be done. 13. Proceed sectioning and discard the following 40µm (4 sections) 14. Save the next 2 sections (10µm, each) → slide 5 15. Proceed sectioning and discard the following 80µm (8 sections) 16. Save the next 2 sections (10µm, each) →slide 6 17. Proceed sectioning and discard the following 80µm (8 sections) 18. Save the next 2 sections (10µm, each) →slide 7 [d.JPG]]() **Staining Brachiocephalic Artery Sections for Lesion Quantitation:** **Staining Brachiocephalic Artery Sections for Lesion Quantitation:** a. 1 min. H²O b. 1 min. H²O c. 30 sec. 60% isopropanol d. 18 min filtered oil red-O (0.24% in 62.5% 2-propanol) e. 30 sec. 60% isopropanol rinse f. 1 min. H²O g. 1 min. H²O h. 2 min Harris Hematoxylin i. 1 min Bluing Solution (H²O with a few drops of ammonia) j. 1 min. H²O k. 30 sec. Light Green l. 1 dip H²O m. Keep in H²O until ready to put cover slip on the slide n. Fix cover slips with glycerol gelatin o. Wash slides to remove excess glycerol gelatin **Quantification of Aortic Root and Brachiocephalic Artery Lesion Area by Image Pro Software:** **Quantification of Aortic Root and Brachiocephalic Artery Lesion Area by Image Pro Software:** 1. Use a microscope equipped with a video imaging system attached to a computer with Image Pro software 2. Quantify all 5 slides obtained from one aortic root and seven slides obtained from one brachiocephalic artery, preferentially use section number 1 on each slide. If section number 1 is not readable, go to the second section of the slide. 3. Measure only the area of oil red-O stained lesions on top of the internal elastic lamina, do not quantify any oil red-O stained material underneath the internal elastic lamina. In some cases, lesions may be sheared off the internal elastic lamina and are found in the lumen. Those lesions should also be counted. 4. The atherosclerotic lesion area is expressed as the mean of the lesion areas of one section per slide from the 5 aortic root slides or the 7 brachiocephalic artery slides.