WO2023236496A1 - 一种计数秀丽隐杆线虫子宫内发育胚胎数目的方法 - Google Patents

一种计数秀丽隐杆线虫子宫内发育胚胎数目的方法 Download PDF

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WO2023236496A1
WO2023236496A1 PCT/CN2022/140214 CN2022140214W WO2023236496A1 WO 2023236496 A1 WO2023236496 A1 WO 2023236496A1 CN 2022140214 W CN2022140214 W CN 2022140214W WO 2023236496 A1 WO2023236496 A1 WO 2023236496A1
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elegans
solution
coli
add
culture medium
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李辉
王晨
王晓丽
史崇丽
李叶勇
曾令君
彭怡
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上海大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
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  • the invention belongs to the field of biotechnology, and specifically relates to a method for counting the number of embryos developing in the uterus of Caenorhabditis elegans.
  • Caenorhabditis elegans is a non-parasitic nematode. It is an ideal model organism for research due to its easy culture, translucent body, short life cycle, and complete genome sequencing database. Most C. elegans are hermaphrodites and have a complete reproductive structure, including two gonadal arms, two seminal vesicles, and a uterus. They can reproduce through self-fertilization and are classic models for studying reproductive effects in biomedicine, environmental toxicology and other disciplines. . Usually, the physiological research indicators used to study the reproduction of C.
  • elegans include gonad apoptosis, genital morphology, number of embryos developing in the uterus, oocyte morphology and number, sperm morphology and number, number of eggs laid, egg hatching rate, etc. .
  • the normal growth cycle of C. elegans eggs after hatching is: L1 stage, L2 stage, L3 stage, L4 stage, youth stage, and adult stage.
  • the treatment of C. elegans exposure factors starts from the L1 stage, and the observation time of the number of embryos is the pregnancy period (that is, the adult period when embryos are observed in the uterus of C. elegans under a microscope). Therefore, based on the above conditions, a set of experimental procedures for counting the number of embryos developing in the uterus of C. elegans was designed.
  • the present invention provides a method for counting the number of embryos developing in the uterus of Caenorhabditis elegans.
  • the present invention mainly uses NaClO solution to pretreat C. elegans, and utilizes the strong oxidizing property of NaClO to dissolve protein macromolecules in the body of pregnant worms, making the body of C. elegans transparent or even completely dissolved, and the eggs can be released in a short time.
  • the structure and morphology will not be destroyed, thereby completely releasing and exposing the embryos developing in the uterus of the adult worm, making it easy to observe the embryonic morphology and count the number of embryos under a stereomicroscope.
  • the invention has a short detection cycle, simple and fast operation, and effectively improves the accuracy and reliability of the assessment of the number of embryos in the uterus among the reproductive indicators of C. elegans.
  • the technical solution of the present invention provides a method for counting the number of embryos developing in the uterus of Caenorhabditis elegans, which is characterized in that it specifically includes the following steps:
  • Step 1 Prepare E. coli bacterial liquid; use freshly prepared E. coli OP50 as C. elegans food. Divide E. coli into two types according to the purpose, one is used to coat NGM culture medium, and the other is used in TPHP For daily feeding of C. elegans during exposure;
  • the NGM culture medium preparation method is: add 3g NaCl, 17g agar, 2.5g peptone, 0.111g CaCl 2 , 0.24g MgSO 4 to 1L deionized water, stir to dissolve, and sterilize at 121°C for 25 minutes; after sterilization is completed , cool the culture medium to 55-60°C, then add 1 mL cholesterol solution and 25 mL potassium phosphate buffer solution, stir and mix, use a peristaltic pump to dispense 15 mL of culture medium into a 7cm petri dish, and wait for room temperature to cool and solidify to become NGM culture medium;
  • the cholesterol solution is prepared with absolute ethanol, with a concentration of 5 mg/mL;
  • the potassium phosphate buffer solution is prepared by adding 10.83g KH 2 PO 4 and 4.7g K 2 HPO 4 per 100 mL of ultrapure water, stirring to dissolve, and sterilizing at 121°C. Bacteria 25min;
  • Step 2 Coating E. coli; use a pipette to draw an appropriate amount of E. coli for coating onto the surface of the NGM culture medium, and then use a sterilized coating rod or the bottom of the test tube to spread the E. coli evenly in the center of the petri dish; After the E. coli is completely attached to the NGM culture medium and forms a thin film, place it upside down and place it in a light-proof biochemical incubator at 20°C for later use;
  • Step 3 Cultivation of C. elegans; inoculate an appropriate amount of C. elegans onto the surface of NGM culture medium with E. coli, and observe the growth status of C. elegans every day. If the E. coli is consumed, replace the medium in time. Until most of the C. elegans worms are in the gestational state;
  • Step 4 Lysis of C. elegans; wash the C. elegans cultured to the pregnancy stage from the petri dish into a 1.5 mL centrifuge tube with K solution, wash 3 times, and centrifuge to remove the supernatant; add 1 mL of lysis solution to the centrifuge tube solution and vortex vigorously for 1.5 minutes; then, centrifuge again to discard the supernatant, add lysis solution, and vortex vigorously until the pregnant worms are completely lysed and the eggs are fully exposed; collect the eggs, wash them 3-5 times, and remove the residual lysis solution ;
  • Step 5 Synchronization of Caenorhabditis elegans; add the eggs dropwise to the NGM medium without E. coli and culture them in the dark at 20°C for 16-20 hours. At this time, the C. elegans is in the L1 stage and rinse this stage with K solution. elegans into a 1.5mL centrifuge tube and wash 3 times;
  • Step 6 Prepare the exposure solution; accurately weigh 0.1000g of TPHP solid, dissolve it with dimethyl sulfoxide to 10g/L, and then use dimethyl sulfoxide to dilute TPHP again to concentrations of 10, 100, 1000, and 5000 mg/L.
  • the TPHP solution was used as the mother solution for later use; dimethyl sulfoxide was used as the blank control mother solution; the mother solution was further diluted 100 times with K solution, and then diluted 100 times with K+ solution, and the final TPHP concentrations were 0, 1, 10, 100, 500 ⁇ g/L Caenorhabditis elegans exposure solution;
  • Step 7 Expose C. elegans in the TPHP solution; add the prepared TPHP exposure solution to a sterile 6-well plate, and add 5 mL to each well; then add 6 ml of synchronized C. elegans in the L1 stage evenly Well plate, feed E. coli 100 ⁇ g/L every day, and culture for 72 hours;
  • Step 8 Selection of C. elegans exposure stage; after TPHP exposure, transfer C. elegans to NGM medium containing E.coli OP50 and culture until pregnancy, and then remove the C. elegans to 1.5mL. In the centrifuge tube, wash it 3 times with K solution;
  • Step 10 Transparent treatment of C. elegans; add the prepared NaClO solution to a sterile 24-well plate, and add 1 mL to each well; then add 10-20 C. elegans to each well, and let stand at room temperature for 5 minutes. , observe the status of C. elegans every 2 minutes until the nematode body becomes transparent;
  • Step 11 Count the number of C. elegans embryos in the uterus; place the 24-well plate containing the clearing C. elegans under an optical stereomicroscope, count the number of exposed embryos for each C. elegans, and count;
  • Step 12 Data analysis; data analysis uses SPSS software, sets the sample confidence interval to 95%, and calculates the intra-group average and average error of the number of embryos developed in nematodes in different treatment groups.
  • step 1
  • the first preparation method for coating NGM culture medium is to inoculate monoclonal E. coli into LB culture medium and culture it at 37°C and 150 rpm in the dark for 16-20 hours.
  • the K liquid culture medium preparation method is to add 2.386g KCl and 2.98g NaCl per 1L of ultrapure water, stir and mix, and sterilize at 121°C for 25 minutes.
  • step 1 the preparation method of LB culture medium is as follows: add 10g peptone, 5g yeast powder, and 5g NaCl per 1L of ultrapure water, stir to dissolve, and sterilize at 121°C for 25 minutes.
  • step 3 the method of replacing the culture medium is: wash the original C. elegans on the surface of the culture medium into a 1.5 mL centrifuge tube with K solution, wash it three times, centrifuge to remove the supernatant, and then remove the bottom of C. elegans was added dropwise to new culture medium with E. coli.
  • step 1 in step 4, the preparation method of the lysis solution is: add 2.5ml of NaClO solution with an effective chlorine content of 5.5-6.5%, 7.5ml of K solution, and 0.1g of NaOH to every 10ml of the lysis solution.
  • the K+ solution preparation method is: add 2.386g KCl, 2.98g NaCl, 0.333g CaCl 2 , 0.36g MgSO 4 per 1L of ultrapure water, stir and mix, sterilize at 121°C for 20 minutes, and cool Then add 1 mL of cholesterol solution and prepare freshly.
  • the present invention has the following advantages:
  • the exposure factor generally selects chemicals that are easily soluble in water, or are dissolved in water after dimethyl sulfoxide is used as a solubilizer.
  • the exposure treatment method of the invention is carried out in a liquid to make the exposure concentration of C. elegans uniform.
  • the present invention only requires the termination time of exposure, that is, the number of C. elegans embryos in the uterus to be counted is in the pregnancy period, and does not require the starting point of exposure.
  • the patent of this invention can be applied to the following experimental conditions:
  • Exposure factor treatment starts from the L1 period and stops during pregnancy.
  • the exposure factor treatment starts from the L1 period, and the exposure stops during the non-pregnancy period.
  • the C. elegans is transferred to the NGM medium coated with E. coli OP50 and continues to be cultured until the pregnancy period stops.
  • Exposure factor treatment starts from the L2-L4 period and stops during pregnancy.
  • Exposure factor treatment starts from the L2-L4 period, and the exposure stops during the non-pregnancy period.
  • the C. elegans is transferred to NGM medium coated with E. coli OP50 and continues to be cultured until the pregnancy period ends.
  • the NaClO solution used in the experiment is the concentration explored in the experiment of the present invention, and the preparation method is simple. This experimental concentration can quickly make pregnant worms transparent or even dissolve, so that the embryos can maintain their original shape for at least 1 hour.
  • This study chose a 24-well plate for experimental observation. On the one hand, the 24-well plate has high transparency. On the other hand, it can prevent the added NaClO solution from flowing out. Moreover, each well of the 24-well plate has just enough field of view under a stereomicroscope. Complete coverage to avoid double counting.
  • Figure 1 is a flow chart of the method of the present invention
  • Figure 2 is a schematic diagram of the number of C. elegans developing embryos treated by the method of the present invention
  • Figure 3 is a statistical diagram of the number of embryos in the uterus of C. elegans after exposure to TPHP after treatment using the method of the present invention.
  • This example provides a method for counting the number of embryos developing in the uterus of Caenorhabditis elegans after being exposed to the chemical triphenyl phosphate (TPHP) for 72 hours based on the method flow chart of the present invention ( Figure 1), which specifically includes the following steps:
  • Step 1 Prepare E. coli bacterial liquid. Freshly prepared Escherichia coli E.coli OP50 was selected as food for C. elegans in the experiment. E. coli can be divided into two types according to use, one is used to coat NGM culture medium, and the other is used to feed C. elegans daily when exposed to TPHP.
  • the first preparation method for coating NGM culture medium is to inoculate monoclonal E. coli into LB culture medium and culture it at 37°C and 150 rpm in the dark for 16-20 hours.
  • the NGM culture medium preparation method is: add 3g NaCl, 17g agar, 2.5g peptone, 0.111g CaCl 2 , 0.24g MgSO 4 to 1L deionized water, stir and dissolve, and sterilize at 121°C for 25 minutes. After sterilization, cool the culture medium to 55-60°C and add 1 mL of cholesterol solution and 25 mL of potassium phosphate buffer solution. Stir and mix evenly. Use a peristaltic pump to dispense 15 mL of the culture medium into a 7cm petri dish. Wait until it cools to room temperature and solidifies. NGM medium.
  • the cholesterol solution was prepared with absolute ethanol at a concentration of 5 mg/mL;
  • the potassium phosphate buffer solution was prepared by adding 10.83g KH2PO4 and 4.7g K2HPO4 per 100 mL of ultrapure water, stirring to dissolve, and sterilized at 121°C for 25 minutes.
  • the preparation method of K liquid culture medium is to add 2.386g KCl and 2.98g NaCl per 1L of ultrapure water, stir and mix, and sterilize at 121°C for 25 minutes.
  • step 1 the preparation method of LB culture medium is as follows: add 10g peptone, 5g yeast powder, and 5g NaCl per 1L of ultrapure water, stir to dissolve, and sterilize at 121°C for 25 minutes.
  • Step 2 E. coli coating.
  • Step 3 Cultivation of C. elegans. Inoculate an appropriate amount of C. elegans onto the surface of the NGM culture medium containing E. coli. Observe the growth status of C. elegans every day. If the E. coli is consumed, replace the medium in time until most C. elegans are in the pregnancy stage. status (nematode embryos in the uterus can be observed under a stereomicroscope).
  • the method of replacing the culture medium is: rinse the original C. elegans on the surface of the culture medium with K solution into a 1.5mL centrifuge tube, wash it three times, centrifuge to remove the supernatant, and then add the C. elegans at the bottom dropwise to in new culture medium with E. coli.
  • Step 4 Lysis of C. elegans.
  • C. elegans cultured to the pregnancy stage was washed from the petri dish into a 1.5 mL centrifuge tube with K solution, washed three times, and centrifuged to remove the supernatant. Add 1 mL of lysis solution to the centrifuge tube and vortex vigorously for 1.5 min. Subsequently, centrifuge again to discard the supernatant, add lysis solution, and vortex vigorously until the pregnant worms are completely lysed and the eggs are completely exposed. Collect the eggs and wash them 3-5 times to remove residual lysis solution.
  • the preparation method of the lysis solution is: add 2.5ml of NaClO solution with an effective chlorine content of 5.5-6.5%, 7.5ml of K solution, and 0.1g of NaOH to every 10ml of the lysis solution.
  • Step 5 Synchronization of C. elegans. Add the eggs dropwise to the NGM medium without E. coli and culture them in the dark at 20°C for 16-20 hours. At this time, C. elegans is in the L1 stage. Use K solution to rinse the C. elegans in this stage into a 1.5 mL centrifuge tube. Medium, wash 3 times.
  • Step 6 Prepare exposure solution. Accurately weigh 0.1000g of TPHP solid, dissolve it with dimethyl sulfoxide to 10g/L, and then use dimethyl sulfoxide to dilute the TPHP again into TPHP solutions with concentrations of 10, 100, 1000, and 5000 mg/L, which will be used as mother liquor for later use. . Dimethyl sulfoxide was used as the blank control stock solution. The mother solution was further diluted 100 times with K solution, and then diluted 100 times with K+ solution to finally obtain C. elegans exposure solutions with TPHP concentrations of 0 (control group), 1, 10, 100, and 500 ⁇ g/L.
  • step 6 the K+ solution preparation method is: add 2.386g KCl, 2.98g NaCl, 0.333g CaCl2, 0.36g MgSO4 per 1L of ultrapure water, stir and mix, sterilize at 121°C for 20 minutes, and add 1mL of cholesterol solution after cooling , ready for use.
  • Step 7 Expose C. elegans to TPHP solution. Add the prepared TPHP exposure solution to a sterile 6-well plate, adding 5 mL to each well. Subsequently, synchronized C. elegans in the L1 stage were evenly added to a 6-well plate, fed E. coli 100 ⁇ g/L daily, and cultured for 72 h.
  • Step 8 C. elegans exposure stage selection.
  • the method of the present invention is suitable for C. elegans in pregnancy, because C. elegans is not in pregnancy after being exposed to TPHP for 72 hours. Therefore, after TPHP exposure, the C. elegans was transferred to NGM medium containing E. coli OP50 and cultured until the pregnancy period. Then, the pregnant C. elegans were carefully removed into a 1.5 mL centrifuge tube and washed with K solution. 3 times for experimental research.
  • Step 9 Prepare NaClO solution.
  • Step 10 Transparency treatment of C. elegans. Add the prepared NaClO solution to a sterile 24-well plate, adding 1 mL to each well. Then add 10-20 C. elegans to each well. After leaving it at room temperature for 5 minutes, observe the status of C. elegans every 2 minutes until the nematode body becomes transparent and the embryos developing in the uterus are completely exposed, as shown in Figure 2 indicates that this state can be maintained for at least 1 hour. Therefore, this method recommends that the number of embryos in the uterus be counted within 1 hour.
  • Step 11 Count the number of C. elegans embryos in the uterus. Place the 24-well plate containing the clearing C. elegans under an optical stereomicroscope, count the number of exposed embryos for each C. elegans, and count at least 25 nematodes in each treatment group.
  • Step 12 Data analysis. SPSS software was used for data analysis. The sample confidence interval was set to 95%. The intra-group average and average error of the number of embryos developed in nematodes in different treatment groups were calculated. The data presentation format can be selected as average percentage or multiple according to experimental needs. The comparison method can also be used to compare the control group and the experimental group and between different experimental groups according to the experimental needs. Use Graphpad prism to draw, as shown in Figure 3.
  • the method of the present invention was used to expose Caenorhabditis elegans to TPHP, and then transferred to a petri dish and cultured until the pregnancy period.
  • Figure 2 after using NaClO to make the pregnant worms transparent, the intrauterine embryonic morphology and shape of the pregnant worms were obtained.
  • the study continued to count the number of embryos in the uterus of each pregnant worm under different concentrations of TPHP exposure conditions.
  • the TPHP exposure concentration in each group was no less than 25 pregnant worms.
  • SPSS was used for statistical analysis.

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Abstract

一种计数秀丽隐杆线虫子宫内发育胚胎数目的方法，以秀丽隐杆线虫作为模式生物，设计了一整套实验流程检测计数秀丽隐杆线虫子宫内的胚胎数目，能够使线虫虫体透明，并将孕虫体内不方便计数的胚胎完全暴露出来，从而便于在普通光学体视显微镜下计数胚胎数目，适用于从L1期开始暴露，最终状态处于孕期的秀丽隐杆线虫。

Description

一种计数秀丽隐杆线虫子宫内发育胚胎数目的方法
相关申请
本申请主张于2022年6月9日提交的、名称为“一种计数秀丽隐杆线虫子宫内发育胚胎数目的方法”的中国发明专利申请：202210650976.8的优先权。
技术领域
本发明属于生物技术领域，具体涉及一种秀丽隐杆线虫子宫内发育胚胎数目计数方法。
背景技术
秀丽隐杆线虫(Caenorhabditis elegans)是一种非寄生线虫，由于其易于培养，身体半透明，生命周期短，具有完整的基因组测序数据库，是一种理想的研究用模式生物。大部分秀丽隐杆线虫是雌雄同体，具有完整的生殖结构，包括两个性腺臂、两个精囊、一个子宫，可通过自体受精进行繁殖，是生物医药、环境毒理等学科中生殖效应研究经典模型。通常情况下，用于研究秀丽隐杆线虫生殖相关生理研究指标包括生殖腺凋亡、生殖器形态、子宫内发育胚胎数目、卵母细胞形态和数目、精子形态和数目、产卵数目、卵孵化率等。
由于秀丽隐杆线虫孕期结构有发育完整的生殖腺、子宫、肠道等，遮挡了线虫体内部分胚胎，导致直接观察孕虫体内胚胎数目不够准确。另外，由于秀丽隐杆线虫在显微镜下蠕动速度较快，导致线虫子宫内发育的胚胎形态和数目很难观察清楚，无法准确计数每条线虫子宫内的胚胎数目，从而导致生殖效应评估误差较大，结果不够准确。因此，需要考虑在短时间内不影响胚胎形态和质量的情况下，将秀丽隐杆线虫孕虫虫体进行进一步透明化后，记录胚胎形态或数目。
秀丽隐杆线虫卵孵化后的正常生长周期为：L1期，L2期，L3期，L4期，青年期，成年期。一般秀丽隐杆线虫暴露因素处理从L1时期开始，而胚胎数目观察时间为孕期(即显微镜下观察到秀丽隐杆线虫子宫内有胚胎的成年期)。因此基于以上条件，设计了一套用于计数秀丽隐杆线虫子宫内发育胚胎数目的实验流程。
发明内容
针对现有技术存在的问题，本发明提供了一种计数秀丽隐杆线虫子宫内发育胚胎 数目的方法。本发明主要采用NaClO溶液对秀丽隐杆线虫进行预处理，利用NaClO的强氧化性，溶解孕虫身体中蛋白质大分子，使秀丽隐杆线虫虫体透明化甚至完全溶解，且短时间内虫卵结构和形态不会被破坏，从而将成虫子宫内发育的胚胎完全释放和暴露出来，便于在体视显微镜下观察胚胎形态并计数胚胎数目。本发明检测周期短、操作简单快速，有效提高了秀丽隐杆线虫生殖指标中子宫内胚胎数目评估的准确性和可靠性。
本发明的技术方案提供了一种计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：具体包括以下步骤：
步骤1、制备大肠杆菌菌液；选用新鲜制备的大肠杆菌E.coli OP50作为秀丽隐杆线虫食物，根据用途将大肠杆菌分成两种，一种用于涂布NGM培养基，另一种在TPHP暴露时用于每日喂食秀丽隐杆线虫；
该步骤1中，NGM培养基配制方法为：1L去离子水中加入3g NaCl，17g琼脂，2.5g蛋白胨，0.111g CaCl 2，0.24g MgSO 4，搅拌溶解，121℃灭菌25min；灭菌结束后，将培养基冷却至55-60℃后加入1mL胆固醇溶液，25mL磷酸钾缓冲溶液，搅拌混匀，用蠕动泵分装15mL培养基到7cm培养皿中，待室温冷却凝固后为NGM培养基；
其中，胆固醇溶液用无水乙醇配制，浓度为5mg/mL；磷酸钾缓冲溶液配制方法为每100mL超纯水中加入10.83g KH 2PO 4，4.7g K 2HPO 4，搅拌溶解，121℃灭菌25min；
步骤2、大肠杆菌涂布；用移液枪吸取适量用于涂布的大肠杆菌于NGM培养基表面，随后，用灭菌后的涂布棒或试管底部将大肠杆菌在培养皿中央涂匀；待大肠杆菌完全附着在NGM培养基，形成一层薄膜后，将其倒置放入20℃避光生化培养箱中备用；
步骤3、秀丽隐杆线虫培养；将适量秀丽隐杆线虫接种到带有大肠杆菌的NGM培养基表面，每日观察秀丽隐杆线虫的生长状态，若大肠杆菌被消耗完毕，及时更换培养基，直至大部分秀丽隐杆线虫处于孕期状态；
步骤4、秀丽隐杆线虫裂解；将培养至孕期的秀丽隐杆线虫用K液从培养皿中冲洗至1.5mL离心管中，清洗3次，离心去掉上清液；在离心管中加入1mL裂解液，剧烈涡旋1.5min；随后，再次离心弃掉上清，加入裂解液，剧烈涡旋至孕虫完全裂 解、虫卵完全暴露为止；收集虫卵，清洗3-5遍，去除残余裂解液；
步骤5、秀丽隐杆线虫同期化；将虫卵滴加到不含大肠杆菌的NGM培养基上20℃避光培养16-20h，此时秀丽隐杆线虫处于L1时期，用K液冲洗该时期的秀丽隐杆线虫至1.5mL离心管中，清洗3次；
步骤6、配制暴露溶液；准确称取0.1000g TPHP固体，用二甲基亚砜溶解至10g/L，随后用二甲基亚砜再次将TPHP稀释成浓度为10、100、1000、5000mg/L的TPHP溶液，作为母液备用；二甲基亚砜作为空白对照组母液；将母液进一步用K液稀释100倍，再用K+溶液稀释100倍，最终得到TPHP浓度为0、1、10、100、500μg/L的秀丽隐杆线虫暴露溶液；
步骤7、秀丽隐杆线虫在TPHP溶液中暴露；将准备好的TPHP暴露液加到无菌6孔板中，每孔加5mL；随后将同期化后处于L1时期的秀丽隐杆线虫均匀加入6孔板，每日喂食大肠杆菌100μg/L，培养72h；
步骤8、秀丽隐杆线虫暴露阶段选择；TPHP暴露结束后，将秀丽隐杆线虫转移到含有大肠杆菌E.coli OP50的NGM培养基中培养至孕期，之后再取出孕期秀丽隐杆线虫至1.5mL离心管中，用K液清洗3次；
步骤9、配制NaClO溶液；采用有效含氯量为5.5-6.5％的NaClO，按照体积比NaClO：K液＝1:4的比例稀释成实验用NaClO溶液备用；
步骤10、秀丽隐杆线虫透明化处理；将配置好的NaClO溶液加入无菌24孔板中，每孔加入1mL；随后向每孔内加入10-20条秀丽隐杆线虫，室温静置5min后，每隔2min观察秀丽隐杆线虫状态，直至线虫虫体呈现透明；
步骤11、秀丽隐杆线虫子宫内胚胎数目计数；将含有透明化秀丽隐杆线虫的24孔板置于光学体视显微镜下，计数每条秀丽隐杆线虫暴露的胚胎数目并计数；
步骤12、数据分析；数据分析采用SPSS软件，设置样本置信区间为95％，计算不同处理组线虫体内发育胚胎数目的组内平均值、平均误差。
进一步地，该步骤1中：
第一种用于涂布NGM培养基的制备方法是将单克隆大肠杆菌接种于LB培养液中，37℃、150rpm避光培养16-20h。
第二种每日喂食秀丽隐杆线虫的大肠杆菌制备方法是将第一种大肠杆菌菌液12000rpm离心10min，此时大肠杆菌在离心管底部，弃掉上清液，并按照原有上清 液：K液(v:v)＝1:1进行替换。
进一步地，该步骤1中，K液培养基配制方法为每1L超纯水中加入2.386g KCl，2.98g NaCl，搅拌混匀，121℃灭菌25min。
该步骤1中，LB培养液配制方法为：每1L超纯水中加入10g蛋白胨，5g酵母粉，5g NaCl，搅拌溶解，121℃灭菌25min。
进一步地，该步骤3中：更换培养基的方法为：将培养基表面原有的秀丽隐杆线虫用K液冲洗至1.5mL离心管中，清洗3次，离心去掉上清液，随后将底部的秀丽隐杆线虫滴加到新的带有大肠杆菌的培养基中。
进一步地，该步骤1中：该步骤4中，裂解液的配制方法为：每10ml裂解液中加入2.5ml有效含氯量为5.5-6.5％的NaClO溶液，7.5ml K液，0.1g NaOH。
进一步地，该步骤6中，K+溶液配制方法为，每1L超纯水中加入2.386g KCl，2.98g NaCl，0.333g CaCl 2，0.36g MgSO 4，搅拌混匀，121℃灭菌20min，冷却后加入1mL胆固醇溶液，现用现配。
与现有技术相比，本发明具有以下优点：
第一、暴露因素一般选择易溶于水、或二甲基亚砜助溶后溶于水的化学药品，该发明方法暴露处理方式在液体中进行，使秀丽隐杆线虫暴露浓度均匀。
第二、实验条件适用性强。本发明只对暴露终止时间要求，即被计数子宫内胚胎数目的秀丽隐杆线虫处于孕期，对暴露的起始点不做要求。本发明专利可适用于以下实验条件：
(1)暴露因素处理从L1时期开始，孕期暴露停止。
(2)暴露因素处理从L1时期开始，非孕期暴露停止，将秀丽隐杆线虫转移至涂布大肠杆菌E.coli OP50的NGM培养基继续培养至孕期停止。
(3)暴露因素处理从L2-L4期间开始，孕期暴露停止。
(4)暴露因素处理从L2-L4期间开始，非孕期暴露停止，将秀丽隐杆线虫转移至涂布大肠杆菌E.coli OP50的NGM培养基继续培养至孕期停止。
第三、实验采用NaClO溶液为本发明实验探究的浓度，配制方法简单。该实验浓度下可使孕虫快速透明化甚至溶解，使胚胎保持原有形态至少维持1h；本研究选择先加NaClO溶液，后加秀丽隐杆线虫，可使线虫在加入时固定在中间位置，如果先加秀丽隐杆线虫，再加NaClO溶液，会使线虫被液体冲到孔板边缘，不利于观察 计数。本研究选用24孔板进行实验观察，一方面是24孔板透明度高，另一方面可以使加入的NaClO溶液不流出来，并且，24孔板的每个孔在体视显微镜下的视野刚好能完全覆盖，避免重复计数。
附图说明
图1为本发明方法流程图；
图2为利用本发明方法处理后的秀丽隐杆线虫发育胚胎数目示意图；
图3为利用本发明方法处理后TPHP暴露后秀丽隐杆线虫子宫内胚胎数目统计图。
具体实施方式
为了更清楚地表明本发明专利的技术方案及优点，下面将结合附图和具体实施方式，对本发明作出进一步地详细阐述。
该实施例基于本发明方法流程图(图1)提供了一种计数秀丽隐杆线虫经化学品磷酸三苯酯(TPHP)暴露72h后子宫内发育胚胎数目的方法，具体包括以下步骤：
步骤1、制备大肠杆菌菌液。实验选用新鲜制备的大肠杆菌E.coli OP50作为秀丽隐杆线虫食物。根据用途可以将大肠杆菌分成两种，一种用于涂布NGM培养基，另一种在TPHP暴露时用于每日喂食秀丽隐杆线虫。第一种用于涂布NGM培养基的制备方法是将单克隆大肠杆菌接种于LB培养液中，37℃、150rpm避光培养16-20h。第二种每日喂食秀丽隐杆线虫的大肠杆菌制备方法是将第一种大肠杆菌菌液12000rpm离心10min，此时大肠杆菌在离心管底部，弃掉上清液，并按照原有上清液：K液(v:v)＝1:1进行替换。
该步骤1中，NGM培养基配制方法为：1L去离子水中加入3g NaCl，17g琼脂，2.5g蛋白胨，0.111g CaCl 2，0.24g MgSO 4，搅拌溶解，121℃灭菌25min。灭菌结束后，将培养基冷却至55-60℃后加入1mL胆固醇溶液，25mL磷酸钾缓冲溶液，搅拌混匀，用蠕动泵分装15mL培养基到7cm培养皿中，待室温冷却凝固后为NGM培养基。其中，胆固醇溶液用无水乙醇配制，浓度为5mg/mL；磷酸钾缓冲溶液配制方法为每100mL超纯水中加入10.83g KH2PO4，4.7g K2HPO4，搅拌溶解，121℃灭菌25min。
该步骤1中，K液培养基配制方法为每1L超纯水中加入2.386g KCl，2.98g NaCl，搅拌混匀，121℃灭菌25min。
该步骤1中，LB培养液配制方法为：每1L超纯水中加入10g蛋白胨，5g酵母粉，5g NaCl，搅拌溶解，121℃灭菌25min。
步骤2、大肠杆菌涂布。用移液枪吸取适量用于涂布的大肠杆菌于NGM培养基表面，随后，用灭菌后的涂布棒或试管底部将大肠杆菌在培养皿中央涂匀，注意菌苔边缘距离培养皿壁约1cm，防止秀丽隐杆线虫爬到从培养皿边缘钻入底部或爬到侧壁。待大肠杆菌完全附着在NGM培养基，形成一层薄膜后，将其倒置放入20℃避光生化培养箱中备用。
步骤3、秀丽隐杆线虫培养。将适量秀丽隐杆线虫接种到带有大肠杆菌的NGM培养基表面，每日观察秀丽隐杆线虫的生长状态，若大肠杆菌被消耗完毕，及时更换培养基，直至大部分秀丽隐杆线虫处于孕期状态(体视显微镜下可观察到线虫子宫内胚胎)。更换培养基的方法为：将培养基表面原有的秀丽隐杆线虫用K液冲洗至1.5mL离心管中，清洗3次，离心去掉上清液，随后将底部的秀丽隐杆线虫滴加到新的带有大肠杆菌的培养基中。
步骤4、秀丽隐杆线虫裂解。将培养至孕期的秀丽隐杆线虫用K液从培养皿中冲洗至1.5mL离心管中，清洗3次，离心去掉上清液。在离心管中加入1mL裂解液，剧烈涡旋1.5min。随后，再次离心弃掉上清，加入裂解液，剧烈涡旋至孕虫完全裂解、虫卵完全暴露为止。收集虫卵，清洗3-5遍，去除残余裂解液。
该步骤4中，裂解液的配制方法为：每10ml裂解液中加入2.5ml有效含氯量为5.5-6.5％的NaClO溶液，7.5ml K液，0.1g NaOH。
步骤5、秀丽隐杆线虫同期化。将虫卵滴加到不含大肠杆菌的NGM培养基上20℃避光培养16-20h，此时秀丽隐杆线虫处于L1时期，用K液冲洗该时期的秀丽隐杆线虫至1.5mL离心管中，清洗3次。
步骤6、配制暴露溶液。准确称取0.1000g TPHP固体，用二甲基亚砜溶解至10g/L，随后用二甲基亚砜再次将TPHP稀释成浓度为10、100、1000、5000mg/L的TPHP溶液，作为母液备用。二甲基亚砜作为空白对照组母液。将母液进一步用K液稀释100倍，再用K+溶液稀释100倍，最终得到TPHP浓度为0(对照组)、1、10、100、500μg/L的秀丽隐杆线虫暴露溶液。
该步骤6中，K+溶液配制方法为，每1L超纯水中加入2.386g KCl，2.98g NaCl，0.333g CaCl2，0.36g MgSO4，搅拌混匀，121℃灭菌20min，冷却后加入1mL胆 固醇溶液，现用现配。
步骤7、秀丽隐杆线虫在TPHP溶液中暴露。将准备好的TPHP暴露液加到无菌6孔板中，每孔加5mL。随后将同期化后处于L1时期的秀丽隐杆线虫均匀加入6孔板，每日喂食大肠杆菌100μg/L，培养72h。
步骤8、秀丽隐杆线虫暴露阶段选择。本实发明方法适用于处于孕期的秀丽隐杆线虫，由于TPHP暴露72h后，秀丽隐杆线虫不处于孕期状态。因此，TPHP暴露结束后，将秀丽隐杆线虫转移到含有大肠杆菌E.coli OP50的NGM培养基中培养至孕期，之后再小心取出孕期秀丽隐杆线虫至1.5mL离心管中，用K液清洗3次，用于实验研究。
步骤9、配制NaClO溶液。本发明采用有效含氯量为5.5-6.5％的NaClO，按照体积比NaClO：K液＝1:4的比例稀释成实验用NaClO溶液备用。
步骤10、秀丽隐杆线虫透明化处理。将配置好的NaClO溶液加入无菌24孔板中，每孔加入1mL。随后向每孔内加入10-20条秀丽隐杆线虫，室温静置5min后，每隔2min观察秀丽隐杆线虫状态，直至线虫虫体呈现透明，子宫内发育的胚胎完全暴露，如图2所示，该状态可以维持至少1h。因此，该方法建议在1h内完成子宫内胚胎数目计数。
步骤11、秀丽隐杆线虫子宫内胚胎数目计数。将含有透明化秀丽隐杆线虫的24孔板置于光学体视显微镜下，计数每条秀丽隐杆线虫暴露的胚胎数目并计数，每个处理组至少计数25条线虫。
步骤12、数据分析。数据分析采用SPSS软件，设置样本置信区间为95％，计算不同处理组线虫体内发育胚胎数目的组内平均值、平均误差，数据呈现形式可以根据实验需求选择平均值百分比、或倍数。对比方式也可根据实验需求进行对照组与实验组及不同实验组组间比较。使用Graphpad prism绘图，具体如图3所示。
结果说明：利用本发明方法将秀丽隐杆线虫用TPHP暴露处理，之后转移至培养皿培养至孕期，如图2所示，利用NaClO使孕虫透明化后，得到了孕虫子宫内胚胎形态和数目示意图，该示意图共展示了22个胚胎，胚胎形态充分展示，胚胎数目粒粒可数。研究继续在不同浓度TPHP暴露条件下，统计每条孕虫子宫内胚胎数目，每组TPHP暴露浓度不少于25条孕虫，利用SPSS进行统计分析，采用单因素方差分析、Dunnett事后检验检测不同浓度组与对照组的组间比较，数据展示为平均值±标 准误差与对照组的百分比；**p<0.01表示TPHP暴露组和对照组之间具有显著统计学差异。如图3所示，当TPHP暴露浓度为1、10、100、500μg/L时，子宫内胚胎数目分别降低了14.2％、18.2％、18.1％、21.8％，且p<0.01，且均具有显著差异性，表明TPHP暴露能够显著降低线虫体内胚胎发育数目，具有潜在的生殖毒性风险，从而为该类化学品继续深入研究其生殖毒性效应和具体分子机制提供基础数据支撑。
尽管参考附图详地公开了本发明，但应理解的是，这些描述仅仅是示例性的，并非用来限制本发明的应用。本发明的保护范围由附加权利要求限定，并可包括在不脱离本发明保护范围和精神的情况下针对发明所作的各种变型、改型及等效方案。

Claims (7)

  1. 一种计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：具体包括以下步骤：
    步骤1、制备大肠杆菌菌液；选用新鲜制备的大肠杆菌E.coli OP50作为秀丽隐杆线虫食物，根据用途将大肠杆菌分成两种，一种用于涂布NGM培养基，另一种在TPHP暴露时用于每日喂食秀丽隐杆线虫；
    该步骤1中，NGM培养基配制方法为：1L去离子水中加入3g NaCl，17g琼脂，2.5g蛋白胨，0.111g CaCl 2，0.24g MgSO 4，搅拌溶解，121℃灭菌25min；灭菌结束后，将培养基冷却至55-60℃后加入1mL胆固醇溶液，25mL磷酸钾缓冲溶液，搅拌混匀，用蠕动泵分装15mL培养基到7cm培养皿中，待室温冷却凝固后为NGM培养基；
    其中，胆固醇溶液用无水乙醇配制，浓度为5mg/mL；磷酸钾缓冲溶液配制方法为每100mL超纯水中加入10.83g KH 2PO 4，4.7g K 2HPO 4，搅拌溶解，121℃灭菌25min。
    步骤2、大肠杆菌涂布；用移液枪吸取适量用于涂布的大肠杆菌于NGM培养基表面，随后，用灭菌后的涂布棒或试管底部将大肠杆菌在培养皿中央涂匀；待大肠杆菌完全附着在NGM培养基，形成一层薄膜后，将其倒置放入20℃避光生化培养箱中备用；
    步骤3、秀丽隐杆线虫培养；将适量秀丽隐杆线虫接种到带有大肠杆菌的NGM培养基表面，每日观察秀丽隐杆线虫的生长状态，若大肠杆菌被消耗完毕，及时更换培养基，直至大部分秀丽隐杆线虫处于孕期状态；
    步骤4、秀丽隐杆线虫裂解；将培养至孕期的秀丽隐杆线虫用K液从培养皿中冲洗至1.5mL离心管中，清洗3次，离心去掉上清液；在离心管中加入1mL裂解液，剧烈涡旋1.5min；随后，再次离心弃掉上清，加入裂解液，剧烈涡旋至孕虫完全裂解、虫卵完全暴露为止；收集虫卵，清洗3-5遍，去除残余裂解液。
    步骤5、秀丽隐杆线虫同期化；将虫卵滴加到不含大肠杆菌的NGM培养基上20℃避光培养16-20h，此时秀丽隐杆线虫处于L1时期，用K液冲洗该时期的秀丽隐杆线虫至1.5mL离心管中，清洗3次；
    步骤6、配制暴露溶液；准确称取0.1000g TPHP固体，用二甲基亚砜溶解至10g/L，随后用二甲基亚砜再次将TPHP稀释成浓度为10、100、1000、5000mg/L的TPHP溶液，作为母液备用；二甲基亚砜作为空白对照组母液；将母液进一步用K液稀释100倍，再用K+溶液稀释100倍，最终得到TPHP浓度为0、1、10、100、500μg/L的秀丽隐杆线虫暴露溶液；
    步骤7、秀丽隐杆线虫在TPHP溶液中暴露；将准备好的TPHP暴露液加到无菌6孔板中，每孔加5mL；随后将同期化后处于L1时期的秀丽隐杆线虫均匀加入6孔板，每日喂食大肠杆菌100μg/L，培养72h；
    步骤8、秀丽隐杆线虫暴露阶段选择；TPHP暴露结束后，将秀丽隐杆线虫转移到含有大肠杆菌E.coli OP50的NGM培养基中培养至孕期，之后再取出孕期秀丽隐杆线虫至1.5mL离心管中，用K液清洗3次；
    步骤9、配制NaClO溶液；采用有效含氯量为5.5-6.5％的NaClO，按照体积比NaClO：K液＝1:4的比例稀释成实验用NaClO溶液备用；
    步骤10、秀丽隐杆线虫透明化处理；将配置好的NaClO溶液加入无菌24孔板中，每孔加入1mL；随后向每孔内加入10-20条秀丽隐杆线虫，室温静置5min后，每隔2min观察秀丽隐杆线虫状态，直至线虫虫体呈现透明；
    步骤11、秀丽隐杆线虫子宫内胚胎数目计数；将含有透明化秀丽隐杆线虫的24孔板置于光学体视显微镜下，计数每条秀丽隐杆线虫暴露的胚胎数目并计数；
    步骤12、数据分析；数据分析采用SPSS软件，设置样本置信区间为95％，计算不同处理组线虫体内发育胚胎数目的组内平均值、平均误差。
  2. 根据权利要求1所述的计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：该步骤1中：
    第一种用于涂布NGM培养基的制备方法是将单克隆大肠杆菌接种于LB培养液中，37℃、150rpm避光培养16-20h。
    第二种每日喂食秀丽隐杆线虫的大肠杆菌制备方法是将第一种大肠杆菌菌液12000rpm离心10min，此时大肠杆菌在离心管底部，弃掉上清液，并按照原有上清液：K液(v:v)＝1:1进行替换。
  3. 根据权利要求1所述的计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：
    K液培养基配制方法为每1L超纯水中加入2.386g KCl，2.98g NaCl，搅拌混匀，121℃灭菌25min。
  4. 根据权利要求2所述的计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：
    LB培养液配制方法为：每1L超纯水中加入10g蛋白胨，5g酵母粉，5g NaCl，搅拌溶解，121℃灭菌25min。
  5. 根据权利要求1所述的计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：该步骤3中：更换培养基的方法为：将培养基表面原有的秀丽隐杆线虫用K液冲洗至1.5mL离心管中，清洗3次，离心去掉上清液，随后将底部的秀丽隐杆线虫滴加到新的带有大肠杆菌的培养基中。
  6. 根据权利要求1所述的计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：该步骤4中，裂解液的配制方法为：每10ml裂解液中加入2.5ml有效含氯量为5.5-6.5％的NaClO溶液，7.5ml K液，0.1g NaOH。
  7. 根据权利要求1所述的计数秀丽隐杆线虫子宫内发育胚胎数目的方法，其特征在于：该步骤6中，K+溶液配制方法为，每1L超纯水中加入2.386g KCl，2.98g NaCl，0.333g CaCl 2，0.36g MgSO 4，搅拌混匀，121℃灭菌20min，冷却后加入1mL胆固醇溶液，现用现配。
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