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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Abstract

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17β-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17β-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17β-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Introduction

Epidemiological and clinical studies have shown that postmenopausal women are at considerably greater risk of cardiovascular disease than premenopausal women1,2. Hormone replacement therapy (HRT) may reduce the relative risk of cardiovascular disease to 0.37-0.793. Among other complications, atherosclerosis caused by cardiovascular diseases is the leading cause of death worldwide4. However, laboratory models involving estrogen-deficient mice presenting atherosclerosis prone status are lacking. This protocol provides an in vivo estrogen deficiency mouse model for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Previous studies show that the application of OVX in atherosclerotic rodents fed a high-cholesterol diet can mimic postmenopausal women suffering from atherosclerosis5,6,7,8. A reproducible and convenient animal model resembling the atherosclerotic state in menopausal women is the basis of exogenous estrogen research. Here, a double dorsal-lateral incision of bilateral ovariectomy was applied in atherosclerosis-prone apolipoprotein E knockout (apoE-/-) mice9,10. Compared with middle abdominal or dorsal incision, double dorsal-lateral incision is an easier, less time-consuming method that can avoid severe abdominal cavity adhesion and inflammation. Peroral administration via hazelnut spread (see Table of Materials) is noninvasive and convenient, rendering it widely applicable as a long-term administration mode11. Slow-release pellet implantation is also popular6. However, implants mayaggravate the incidence of infection especially in mice subjected to OVX. Other noninvasive administration modes, such as oral gavage and water administration, also have many drawbacks. Oral gavage typically stress mice and may cause esophageal injury. Administering the hormone via drinking water is highly beneficial; however, the adding of DMSO as an emulsifier is inevitable as exogenous estrogens are insoluble in water. Here, we chose peroral 17β-estradiol or phytoestrogen hormone replacement via hazelnut spread for long-term administration.

Recently, the beneficial effect of HRT on the cardiovascular system of postmenopausal women has been contested in women's health initiative (WHI) trials12. On the one hand, exogenous estrogen alone exerts a beneficial effect on the cardiovascular system; on the other hand, it can combine with metohydroxyprogesterone acetate to increase the risk of cardiovascular events. More seriously, HRT may lead to breast and uterine tumor progression, and this effect has markedly limited its use13,14. More interest has been focused on the cardiovascular-protective effects of exogenous estrogens lacking mitotic activity in tumor cells15,16,17. Multiple studies in humans and animals suggest that phytoestrogens with structures similar to that of estrogens can play a beneficial role in cardiovascular protection15,18.

Thus, the aims of the present work are (i) to build an in vivo estrogen deficiency mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice and (ii) to compare the cardiovascular protective effects of perorally administered 17β-estradiol and pseudoprotodioscin (PPD), via hazelnut spread. 17β-estradiol is one kind of exogenous estrogen that belongs to female sexual hormones6,11,19. PPD, a steroid saponin and phytoestrogen from Dioscorea plants, has been previously reported to exert anti-tumor properties20.

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Protocol

All animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College (Permission No.: SYXK (Beijing) 2013-0023). The origin of apoE-/- mice is C57BL/6J9,10.

1. Bilateral Ovariectomy via a Double Dorsal-lateral Incision in apoE-/- Mice

  1. At weaning (age 28 days), anesthetize female apoE-/- C57BL/6J mice with avertin (tribromoethanol; 200 mg/kg; intraperitoneally).
    NOTE: 32 apoE-/- mice were randomly divided into 4 groups: SHAM, OVX, OVX/E2, and OVX/PPD group (n = 8 per group).
  2. Place the animal in prone position on a heating pad. Apply eye lubricant for eye protection during anesthesia.
  3. Maintain body temperature within 36 ± 0.5 °C. Administer 5 mg/kg body weight of the analgesic carprofen subcutaneously to the lateral aspect of the mouse’s neck.
  4. Shave a 3 x 5 cm2 mouse area cephalic from the iliac crest. Prior to covering the animal with a 3 x 5 cm2 aperture surgical sheet, clean the shaved area thoroughly with iodine and then 70% ethanol. Use sterile instruments and gloves during the experiment. 
  5. Use scissors and forceps to make an incision 1 cm lateral to the midline and 1 cm lateral to the costal ribs.
  6. Bluntly dissect the subcutaneous tissue using forceps.
  7. Use dissecting goggles (see Table of Materials) to identify the white adipose tissue in the abdominal cavity.
  8. Use microscissors and microforceps to make a 0.5-1 cm incision through the fascia until the abdominal cavity is reached.
    NOTE: For the sham-operated group, close the wounds directly. Suture the muscle layer and skin separately using a monofilament suture.
  9. When the white adipose tissue in the abdominal cavity can be seen, grab the adipose tissue using microforceps and gently pull it out. A pink mulberry-shaped ovary wrapped by adipose tissue in the abdominal cavity can be seen.
  10. Ligate the 0.5-1 cm proximal vessel and the uterine horn using a monofilament suture. Remove the ovary using microscissors and place the remaining tissue back into the abdominal cavity.
    NOTE: The main adverse symptoms for the OVX operation is ureteral ligation which leads to high mortality in OVX-operated mice. This can be avoided by identifying the tissues using a dissecting goggle.
  11. Close the wounds. Suture the muscle layer and skin separately using a monofilament suture.
  12. Use scissors and forceps to make another incision 1 cm lateral to the midline and 1 cm lateral to the costal ribs on the other side. Repeat the above procedure (1.5 to 1.11).
  13. Let the animal wake from anesthesia. Separately keep the mouse on the first day after surgery.
  14. Clean or replace the cage frequently during the recovery phase.
  15. Approximately 24 h after surgery, administer another 5 mg/kg body weight of the analgesic carprofen subcutaneously.

2. Peroral Administration of 17β-estradiol or PPD via Hazelnut Spread

  1. Thoroughly dissolve 17β-estradiol or PPD in sesame oil, and then mix the sesame oil with hazelnut spread (see Table of Materials). A daily portion for each 30 g mouse contains 3 μg of 17β-estradiol or 15 μg of PPD, 4 μL of sesame oil, and 60 mg of hazelnut spread. Prepare a placebo for each 30 g mouse contains 4 μL of sesame oil and 60 mg of hazelnut spread.
    NOTE: The daily administration portion of 17β-estradiol or PPD was based on previous studies6,11 and preliminary experiments.
  2. One week after OVX, feed the mice with a high-cholesterol diet (1.25% cholesterol, 0% cholate) for 12 weeks. A typical experimental treatment scheme, as used in this study, is illustrated in Figure 1.
  3. 5 days before the peroral administration of hazelnut spread at week 4, train the mice to eat the placebo containing approximately 30 mg hazelnut spread for 2-5 mice for 5 days. Train the mice in groups in their home cages during the first 3 days. Place the mice in separate cages on the fourth and fifth days of training and serve the daily portion to resemble the experimental situation.
  4. During the last 9 weeks, place the mice in separate cages and then serve a daily portion the hazelnut spread portion for every feeding occasion.
    NOTE: Serve a daily portion containing 17β-estradiol (0.1 mg·kg-1) or PPD (0.5 mg·kg-1) via hazelnut spread in OVX/E2 and OVX/PPD group respectively; serve a daily portion containing hormone-free hazelnut spread in SHAM and OVX group.

3. Determination of Intima-media Thickness and Cardiac Dysfunction Using a Microultrasound System

  1. Ultrasonographic biomicroscopy
    1. One day before termination, examine intima-media thickness and cardiac dysfunction using a microultrasound system (see Table of Materials) as previously described21.
    2. Before examination, give each mouse a 200 mg/kg intraperitoneal injection of avertin (tribromoethanol) as anesthesia (n = 8 mice per group).
    3. Shave the neck hair of each mouse carefully. Apply warm ultrasound transmission gel liberally to ensure optimal image quality.
    4. Obtain baseline ultrasonographic images of the aortic root and ascending aorta with the 30 MHz scan head at a 12.7 mm focus and a resolution of 40 μm.
    5. Use electrocardiography with a lead II configuration for monitoring.
    6. Capture right parasternal long-axis images of the ascending aorta, aortic arch, and brachiocephalic artery branch in one plane in systole (Figure 3).
  2. Measurements of intima-media and maximal plaque thickness
    1. Adjust the distance between the transducer and the arterial site readily to obtain clear images.
    2. Store a 10 s cine loop digitally for offline examination on an image analysis system.
    3. Choose an optimal freeze-frame ultrasonographic image manually for further measurements. Check the images in the minor curvature of the ascending aorta. If plaque in the ascending aorta can be seen, measure the maximal plaque thickness. If plaque in the ascending aorta cannot be seen, measure the maximal IMT.
    4. Measure the IMT (distance between the vascular luminal-intimal interface and the medial-adventitial interface). Measure the maximal plaque thickness (the thickest distance between the border of the vascular lumen and adventitial layer).
    5. Average data from three lesion sites (Figure 3).
  3. Determination of cardiac dysfunction using echocardiography
    NOTE:     Examine cardiac function through echocardiography using a microultrasound system, as previously described22.
    1. Direct an ultrasound beam toward the heart, near the papillary muscles.
    2. Achieve two-dimensional electrocardiogram-based kilohertz visualization.
    3. Perform in vivo transthoracic echocardiography of the left ventricle using a 30 MHz scan head.
    4. Measure parameters associated with cardiac function digitally from M-mode tracings.
    5. Average the data from three to five cardiac cycles (Table 1).
  4. Intra- and interobserver variability
    1. For validation of intraobserver variability, analyze the data by one operator on two different occasions.
    2. For evaluation of interobserver variability, analyze the data by a different operator.

4. Weekly Body Weight Measurement and Plasma Total Cholesterol (TC) and Triglyceride (TG) Determination

  1. Weekly body weight measurement
    1. Measure body weights once a week from week -1 to week 12.
      NOTE: n = 8 mice per group.
  2. Plasma preparation
    1. Before collecting blood samples through intracardiac puncture, prepare syringes and tubes. Use EDTA as an anticoagulant. Add 10 µL of 0.5 M EDTA to each 2 mL syringe, and add 8 µL of 0.5 M EDTA to each 1.5 mL tube.
    2. At week 12, after an overnight fast, anesthetize the mice with avertin (tribromoethanol; 200 mg/kg; intraperitoneally).
      NOTE: n = 3 mice per group.
    3. Prepare the ventral chest area with 70% ethanol.
    4. Use scissors and forceps to open the thoracic cavity and cut the ribs until the beating heart is exposed.
    5. Insert the 25 G needle into the right ventricle. Aspirate slowly until blood starts to flow into the syringe.
      NOTE: We use disposable syringes in sterile condition with 25 G needles (see Table of Materials).
    6. Continue to aspirate with steady, even pressure. If no blood is seen, reposition the needle and repeat aspiration.
    7. Keep mice deeply anesthetized before collecting the required blood volume. Normally, up to 1 mL of blood can be collected. Euthanize the mice by cervical dislocation under this deep anesthetic condition.
    8. Pipette blood samples into 1.5 mL tubes and invert the blood thoroughly to ensure mixing EDTA into the blood. Then place blood samples on ice immediately.
    9. Centrifuge samples for 20 min at 400 x g at 4 °C within 30 min of collection.
    10. Collect the supernatant carefully. Aliquot and store plasma samples at -80 °C.
  3. Construct standard curves for TC or TG content measurement
    1. For the TC standard curve, prepare various concentrations of cholesterol standards: 0 mmol/L, 0.52 mmol/L, 1.03 mmol/L, 2.07 mmol/L, 4.14 mmol/L, 6.20 mmol/L, 8.27 mmol/L and 10.34 mmol/L. Measure O.D. for each cholesterol standard. Set average O.D. for each cholesterol standard. As the vertical (Y) axis value, set concentration as the horizontal (X) axis value. Create a standard curve by using a statistical software.
    2. For the TG standard curve, prepare various concentrations of TG standards: 0 mmol/L, 0.45 mmol/L, 0.90 mmol/L, 1.81 mmol/L, 3.62 mmol/L, 5.42 mmol/L, 7.23 mmol/L and 9.04 mmol/L. Measure O.D. for each TG standard. Set average O.D. for each TG standard as the vertical (Y) axis value; set concentration as the horizontal (X) axis value. Create a standard curve by using a statistical software.
      NOTE: A four-parameter Logistic curve fitting (4-pl) was used for the standard curve construction in the present study. Check the standard curve before measuring the plasma samples and ensure that r2 is greater than 0.995.
  4. TC content measurement
    1. Label the bottle of color reagent (25 mL) from TC assay kit as “TC Working Solution”.
    2. Vortex the refrigerated specimens briefly. Prepare dilutions: 20 μL of plasma in 80 μL of distilled water. Briefly vortex dilutions.
    3. Add 2.5 μL of cholesterol standards (5.17 mmol/L) or 2.5 μL of diluted plasma or 2.5 μL of distilled water (blank) to the appropriate wells of a 96-well plate. Triplication is recommended.
    4. To all wells, add 250 μL of the color reagent.
    5. Incubate at 37 °C for 10 min.
    6. Turn the microplate reader on and allow a 10 min warm up.
    7. Remove the plate(s) from the incubator and read the microplate reader at 510 nm. Ensure that no bubbles or dust are present in the microtiter wells or at the bottom of the plate, respectively.
    8. Calculate the TC concentration as follows:
      ​TC cont. = cholesterol standards cont. × (plasma sample O.D.-blank O.D)/(cholesterol standards O.D.-blank O.D)
  5. TG content measurement
    1. Label the bottle of color reagent (25 mL) from TG assay kit as “TG Working Solution”.
    2. Vortex the refrigerated specimens briefly.
    3. Add 2.5 μL of TG standards (2.26 mmol/L) to or 2.5 μL of diluted plasma or 2.5 μL of distilled water (blank) to the appropriate wells of a 96-well plate. Triplication is recommended.
    4. To all wells, add 250 μL of the color reagent.
    5. Incubate at 37 °C for 10 min.
    6. Turn the microplate reader on and allow a 10 min warm up.
    7. Remove the plate(s) from the incubator and read the microplate reader at 510 nm.
      NOTE: Ensure that are no bubbles or dust are present in the microtiter wells or on the bottom of plate.
    8. Calculate the TG concentration as follows:
      TG cont. = TG standards cont. × (plasma sample O.D.-blank O.D)/(TG standards O.D.-blank O.D)

5. En Face Analysis of Aortic Atherosclerotic Lesions

  1. Aorta isolation and excision
    1. At week 12, after an overnight fast, anesthetize the mice with avertin (tribromoethanol; 200 mg/kg; intraperitoneally). Euthanize the mice by cervical dislocation under this deep anesthetic condition.
      NOTE: We used 3 mice per group.
    2. Prepare the ventral chest area with 70% ethanol. Use scissors and forceps to open the thoracic cavity and cut the ribs until the beating heart is exposed.
    3. Fill a 50 mL syringe with phosphate buffered saline at pH 7.4 (see Table of Materials). Insert a 25 G of needle into the left ventricle and cut the right atrium to avoid high pressure from perfusion.
    4. Perform in situ perfusion at a flow rate of 0.05-0.08 mL/min. Absorb perfusion fluid with tissues.
    5. Remove ribs and lungs in thoracic cavity with scissors and forceps. Then, open the abdominal cavity and remove the organs inside for a better view of the aorta.
    6. Remove the aorta by holding the heart with the microforceps and separating the aorta from spine dorsally with microscisssors until the iliac bifurcation.
      NOTE: When dissecting near the renal atrial branches, cut deeply using microscissors to avoid aorta damage.
    7. Fix the heart and aorta for 48 h in 4% paraformaldehyde. Store the aortas in saline at room temperature or at 2-8 °C for a few hours.
      NOTE: This procedure will facilitate cleaning.
  2. Preparation of aorta
    1. Remove the heart. Carefully remove the adventitial tissues from the aortas using microforceps and microscisssors under a stereomicroscope. Use saline to keep the tissue moist during cleaning.
      NOTE: Be careful to not tear or nick the aorta and some important branches, such as the innominate artery, left common carotid artery, and left subclavian artery.
    2. Leave 1 mm of the innominate and left common carotid arteries and cut off the entire left subclavian artery.
    3. Cut open the outer curvature through the innominate artery, then to the left common carotid artery, and then to the left subclavian artery.
    4. Cut open along the inner curvature of the ascending portion to the bottom of the abdominal portion.
    5. Pin the aorta flat onto a black plastic sheet and apply saline to keep aortas from drying.
  3. Image of the intimal region of aorta
    1. Take pictures of en face aortas with a stereo microscope. Include a millimeter scale ruler in the images to calibrate measurements.
    2. Include the arch and thoracic regions in the same image and the abdominal region in another. Save images as JPEG or TIFF.
      NOTE: The arch region is from the junction of the myocardium to 3 mm distal from the left subclavian artery, the thoracic region is 3 mm distal to the left subclavian artery to the last intercostal artery, and the abdominal region is the last intercostal artery to the iliac bifurcation.
  4. Quantification of atherosclerotic lesion-en face method
    1. Calibration
      1. Open the image with image analysis software (see Table of Materials), go to Spatial Calibration and follow the instructions.
      2. Change reference units to mm by positioning the ruler over the line.
    2. Measurement
      1. Set correct calibration for each image.
      2. Measure 3 mm on the ruler.
      3. Arch and thoracic region measurement: Outline the arch region from the junction of the myocardium to 3 mm distal from the left subclavian artery and the thoracic region from 3 mm distal to the left subclavian artery to the last intercostal artery. Trace lesions in the arch and thoracic region and look at the aorta through the microscope.
      4. Abdominal region measurement: Outline the abdominal region from the end of thoracic region to the iliac bifurcation. Trace lesions in the abdominal region and look at the aorta through the microscope.
      5. Calculate the lesion area relative to the inner surface of aorta.
      6. Verify quantification through a second observer who is blind to the study groups.

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Results

A typical experimental treatment scheme, as used in this study, is illustrated in Figure 1. At weaning (age 28 days), female apoE-/- C57BL/6J mice were anesthetized withavertin (tribromoethanol; 200 mg/kg; intraperitoneally). Mice were bilaterally OVX or sham operated through a 1 cm dorsal incision. One week after bilateral OVX, the mice were fed a high-cholesterol diet (1.25% cholesterol, 0% cholate) for 12 weeks. 17β-Estradiol (0.1 mg·k...

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Discussion

The methodology described here is a mouse model resembling lipid disruption and atherosclerosis seen in menopausal women. It is well-documented that estrogen deficiency in postmenopausal women can aggravate the incidence of pre-existing or ongoing hypercholesterolemia with progressively complex and widespread atherosclerostic lesions1. To mimic the atherosclerosis-prone status in clinic, apoE-deficient mice, a reproducible and convenient source of animals with which to study atherogenesis

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Disclosures

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81202526 to J.X.), the National Natural Science Foundation of China (81302769 to B.S.), the Beijing Municipal Natural Science Foundation (47144226 to B.S.), the Chinese Postdoctoral Science Foundation (20110490325 to J.X.), and the Ph.D. Programs Foundation of Ministry of Education of China (20121106120031 to B.S.).

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Materials

NameCompanyCatalog NumberComments
17β-estradiol, >98%Sigma-AldrichE8875-250MGEstrogen
Disposable syringes (with 25 G needles)Hunan Luzhou Huikang Development Co., Ltd0.5*19TWLBCardiac bleeding
High-cholesterol mouse dietHuafukang Bio-TechnologyN/A1.25% cholesterol, 0% cholate
High-Resolution In Vivo Micro-Imaging SystemVisualSonicsVevo®770Measurements of intima-media thickness and cardiac dysfunction
2-Methyl-2-butanolSigma-Aldrich152463-250MLPreparation of avertin
Micro Dissecting forceps, Curved 8mmKanghua Medical Equipment Co., LtdSurgical tools
Micro Dissecting forceps, Straight 8 mmKanghua Medical Equipment Co., LtdSurgical tools
Micro Dissecting Scissors, Curved/Sharp 8 mmKanghua Medical Equipment Co., LtdSurgical tools
Micro Dissecting Scissors, Straight/Sharp 8 mmKanghua Medical Equipment Co., LtdSurgical tools
Monofilament suture 4-0 1/2 5 x 12 19 mmShanghai Pudong Jinhuan Medical Supplies Co., LtdR413Suture and ligation of the tissues
Nut cream (Nutella)FerreroN/AMedium for peroral 17β-estradiol or PPD
OptiVisor optical glass binocular magnifierDohegan Optical Company Inc.N/AAssistant of identifying the tissues during ovariectomy
Phosphate-buffered saline at pH 7.4SIGMAP3813Preparing 1 L saline
Pro MultiLabel Microplate ReaderTecanInfinite M1000Plasma TC and TG determination
PseudoprotodioscinShanghai Winherb Medical S & T DevelopmentW-0427CAS registry no. 102115-79-7
Rimadyl, 50 mg/mLPfizer Pharma GmbH462986Postoperative analgesia after ovariectomy
Sesame oilSigma-AldrichS3547-1LDissolving the 17β-estradiol or PPD
Solcoseryl Eye-GelMenarini, Solco Basle Ltd.Eye protection during anesthesia
Stereo microscopeMCALONMCL-6STVImage of the intimal region of aorta
Table model high speed centrifugeSIGMA1-14KPreparation of plasma
Scissors, slight Curve (14 cm)Kanghua Medical Equipment Co., LtdSurgical tools
Scissors, straight Flat (14 cm)Kanghua Medical Equipment Co., LtdSurgical tools
Tissue forceps, serrated, slight Curve (14 cm)Kanghua Medical Equipment Co., LtdSurgical tools
Tissue forceps, serrated, straight Flat (14 cm)Kanghua Medical Equipment Co., LtdSurgical tools
TribromoethanolSigma-AldrichT48402-5GPreparation of avertin
Triglycerides (TG) assay kitInstitute of Nanjing Jiancheng Biology EngineeringA110-1Plasma TG determination
Total cholesterols (TC) assay kitInstitute of Nanjing Jiancheng Biology EngineeringA111-1Plasma TC determination

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