WO2024109771A1 - 一种基于有机荧光探针评估活生殖细胞及其早期胚胎线粒体状态的应用方法 - Google Patents

一种基于有机荧光探针评估活生殖细胞及其早期胚胎线粒体状态的应用方法 Download PDF

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WO2024109771A1
WO2024109771A1 PCT/CN2023/133068 CN2023133068W WO2024109771A1 WO 2024109771 A1 WO2024109771 A1 WO 2024109771A1 CN 2023133068 W CN2023133068 W CN 2023133068W WO 2024109771 A1 WO2024109771 A1 WO 2024109771A1
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embryos
mitochondrial
fluorescent probe
mitochondria
embryo
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French (fr)
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雷晓华
李秦
张鹏飞
罗媛
赵华山
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中国科学院深圳先进技术研究院
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    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the invention belongs to the field of biotechnology and relates to an application method of a novel fluorescent probe for evaluating the mitochondrial status of germ cells and early embryos.
  • Photodynamic therapy (PDT) and sonodynamic therapy (SDT) are promising cancer treatments. They use photosensitizers or sonodynamic agents to react with water molecules or oxygen molecules in the environment under light or ultrasound excitation to produce reactive oxygen species (ROS) such as singlet oxygen and free radicals, thereby killing tumor cells.
  • ROS reactive oxygen species
  • PDT has the advantages of high selectivity for target tissues, low toxicity and side effects, and no damage to internal organs, but poor penetration; compared with PDT, SDT also has the advantages of strong penetration and non-invasiveness, but low selectivity for target tissues.
  • Sonodynamic therapy uses the strong tissue penetration ability of ultrasound to cause sonodynamic agents accumulated in deep tumors to produce ROS or cavitation effects to treat tumors, which has successfully attracted widespread attention.
  • tumor sonodynamic therapy has been used in clinical practice, and combined with immunotherapy and endocrine therapy, it has successfully cured three patients with advanced breast cancer.
  • sonosensitizers are: porphyrin, porphyrin derivatives, chemotherapy drugs DOX, acid yellow, methylene Assisted reproductive technology is one of the important means to solve human infertility, livestock breeding and artificial breeding of endangered wild animal species, and the screening of high-quality live germ cells and early embryos is crucial to the success rate of assisted reproductive technology.
  • Mitochondria are important and unique organelles in the cytoplasm. During the development of germ cells and early embryos, mitochondria undergo a series of changes in morphology and quantity. Their structure, distribution and number in cells are closely related to the function of mitochondria. In mitochondria, ATP (adenosine triphosphate) is produced through oxidative phosphorylation to provide energy for processes such as germ cell meiosis, cytoplasmic maturation, preimplantation embryo cleavage, and lineage differentiation. Animal models have confirmed that the activity state and function of germ cell or embryonic mitochondria are closely related to embryo quality and developmental potential. Mitochondrial abnormalities can lead to reduced blastocyst development rate, impaired implantation potential, changes in placental development and abnormal fetal growth rate. Therefore, the detection of abnormalities in the structure, distribution and number of mitochondria can be used as an important indicator to judge the quality and developmental potential of germ cells and early embryos.
  • ATP adenosine triphosphate
  • MitoTracker (Red) fluorescent probe when used to analyze the distribution of mitochondria in MII stage oocytes of aged mice, 500 nM MitoTracker dye needs to be added to the M2 culture medium and incubated in an incubator at 37 °C and 5% CO 2 for 30 min. After the incubation, the M2 culture medium needs to be washed three times again, and finally the oocytes are fixed on a glass slide and imaged under a laser confocal microscope.
  • the use of the above probes is cumbersome, and the quality of the samples after the mitochondrial probe detection is reduced, which is not conducive to continuous tracking and observation of subsequent mitochondrial changes in the same sample and the relationship between mitochondrial status and subsequent developmental potential.
  • the purpose of the present invention is to provide an application of a mitochondrial targeted fluorescent probe in detecting and monitoring the mitochondrial distribution status of living germ cells and early embryos of humans and animals, thereby providing an effective means for obtaining high-quality germ cells and early embryos in human assisted reproductive treatment and animal breeding-related technologies and scientific research.
  • One aspect of the present invention provides a mitochondrial fluorescent probe for use in preparing a reagent for detecting the distribution state of mitochondria in the development of reproductive cells and embryos; or in preparing a reagent for evaluating the development process and quality of reproductive cells and embryos.
  • the mitochondrial fluorescent probe is a fluorescent probe that targets mitochondria and displays the distribution status of mitochondria.
  • the mitochondrial fluorescent probe is selected from MG-Rho-2, MitoTracker® Deep Red FM, MitoTracker Green FM, MitoTracker Orange CMTMRos1, MitoTracker Orange CMTMRos 2, TPP-KIz, TPP-KIzTZ, MCY-BF2, SiR-Mito 8, and SiR-Mito 11.
  • the mitochondrial fluorescent probe is a composition comprising the mitochondrial fluorescent probe, and the composition comprising the mitochondrial fluorescent probe comprises the mitochondrial fluorescent probe and a culture medium for culturing germ cells and embryos.
  • the germ cells and embryos are selected from humans or mammals, wherein the mammals are common mammals or mammals on the list of national key protected wild animals; the common mammals are preferably mice, rats, humans, rabbits, cattle, sheep, pigs, horses, monkeys, dogs, and pandas. Mammals on the list of national key protected wild animals are, for example, Vietnamese antelopes, wild donkeys, yaks, Przewalski's gazelles, snow leopards, giant pandas, and Siberian tigers.
  • the reproductive cell is selected from oocytes and sperms; further, the oocyte is selected from immature oocytes or mature oocytes.
  • the embryo is selected from the group consisting of a fertilized egg, a 2-cell embryo, a 4-cell embryo, an 8-cell embryo, a morula and a blastocyst.
  • the embryo development process is selected from any one of in vivo naturally fertilized embryos, parthenogenetic activated embryos, in vitro fertilized embryos, intracytoplasmic sperm injection embryos, and somatic cell nuclear transfer embryos.
  • embryos can be used for other experiments or embryo transfer to further evaluate reproductive outcomes.
  • the detection of mitochondrial status is selected from the detection and monitoring of the mitochondrial distribution status of mature oocytes, the mitochondrial distribution status of various stages of preimplantation embryo development, and the mitochondrial distribution status of preimplantation embryos in the blastocyst stage.
  • the method for detecting the state of mitochondria is to observe the distribution state of mitochondria using a laser confocal imaging system.
  • the method for detecting the mitochondrial state also includes the step of further analyzing the mitochondrial distribution state after obtaining the mitochondrial distribution state.
  • the analysis is to analyze the fluorescence intensity and aggregation state of the mitochondrial probe.
  • Another aspect of the present invention provides a use of a mitochondrial fluorescent probe in preparing a reagent for screening embryos in assisted reproductive therapy.
  • the mitochondrial fluorescent probe is selected from MG-Rho-2, MitoTracker® Deep Red FM, MitoTracker Green FM, MitoTracker Orange CMTMRos1, MitoTracker Orange CMTMRos 2, TPP-KIz, TPP-KIzTZ, MCY-BF2, SiR-Mito 8, and SiR-Mito 11.
  • the present invention provides a detection composition comprising a mitochondrial fluorescent probe, wherein the detection composition contains the mitochondrial fluorescent probe and a culture medium for culturing germ cells or embryos.
  • the concentration of the mitochondrial fluorescent probe in the detection composition is 0.01 ⁇ M-2 ⁇ M, for example, 1 ⁇ M, 0.5 ⁇ M, 0.25 ⁇ M, 0.1 ⁇ M, 0.05 ⁇ M, 0.025 ⁇ M, 0.01 ⁇ M.
  • the mitochondrial fluorescent probe is selected from MG-Rho-2, MitoTracker® Deep Red FM, MitoTracker Green FM, MitoTracker Orange CMTMRos1, MitoTracker Orange CMTMRos 2, TPP-KIz, TPP-KIzTZ, MCY-BF2, SiR-Mito 8, and SiR-Mito 11.
  • the present invention provides a method for screening germ cells, preimplantation embryos and embryonic blastocysts using a mitochondrial fluorescent probe, comprising the following steps:
  • step S2) placing the germ cells, preimplantation embryos and embryonic blastocysts in the detection composition obtained in step S1) for incubation or culture;
  • step S4 analyzing the distribution status of mitochondria obtained in step S3) to evaluate or screen the quality and vitality of corresponding germ cells, preimplantation embryos, and embryonic blastocysts.
  • the pre-implantation embryo is a fertilized egg, a 2-cell embryo, a 4-cell embryo, an 8-cell embryo, a morula and a blastocyst.
  • the reproductive cells are selected from oocytes and sperms.
  • step S2 Furthermore, the incubation time in step S2) is 0.1-12 hours.
  • the culture time is 12-72 hours.
  • the pre-implantation embryos are cultured to embryos of any stage. Further, the embryos of any stage are selected from 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae or blastocysts.
  • step S3) the observation is performed once or twice or more following the culture or incubation in step S2).
  • step S3 the method for observing the distribution state of mitochondria is to use a laser confocal imaging system for imaging observation.
  • step S4) the embryonic blastocyst can be transplanted into a subject after being evaluated in step S4).
  • the evaluation or screening method is to evaluate the distribution status of mitochondria obtained in step S3), and the quality and vitality of germ cells, preimplantation embryos and embryo blastocysts with uniform mitochondrial distribution are higher than those of germ cells, preimplantation embryos and embryo blastocysts with uneven mitochondrial distribution.
  • the means of analysis and evaluation is to use Image J software to detect the fluorescence intensity in the specified fluorescence area of the image, obtain the fluorescence intensity parameter results, and import the parameter results into Graphpad to generate a result schematic diagram.
  • the present invention provides a method for screening embryonic blastocysts for transplantation, the screening method comprising the following steps:
  • step S02 placing the embryonic blastocyst in the detection composition obtained in step S01) for incubation or culture;
  • step S04 analyzing the distribution status of mitochondria obtained in step S02) to evaluate the quality and vitality of the corresponding embryonic blastocyst;
  • the pre-implantation embryo is a fertilized egg, a 2-cell embryo, a 4-cell embryo, an 8-cell embryo, a morula and a blastocyst.
  • the reproductive cells are selected from oocytes and sperms.
  • step S02 the incubation time is 0.1-12 hours.
  • the culture time is 12-72 hours, further, the pre-implantation embryo is cultured to an embryo of any stage, further, the embryo of any stage is selected from a 2-cell embryo, a 4-cell embryo, an 8-cell embryo, a morula or a blastocyst.
  • step S03 the observation is performed once or twice or more following the culture or incubation in step S02).
  • step S03 the method for observing the distribution state of mitochondria is to use a laser confocal imaging system for imaging observation.
  • step S04 the embryonic blastocyst can be transplanted into a subject after being evaluated in step S04).
  • the evaluation or screening method is to evaluate the distribution status of mitochondria obtained in step S03), and the quality and vitality of germ cells, preimplantation embryos and embryo blastocysts with uniform mitochondrial distribution are higher than those of germ cells, preimplantation embryos and embryo blastocysts with uneven mitochondrial distribution.
  • the means of analysis and evaluation is to use Image J software to detect the fluorescence intensity in the designated fluorescence area of the image, obtain the fluorescence intensity parameter results, and import the parameter results into Graphpad to generate a result schematic diagram.
  • the inventors unexpectedly discovered that the probe used in the present invention can achieve rapid observation and imaging under laser confocal, with strong fluorescence intensity. More importantly, the mitochondrial fluorescent probe detection reagent used in the present invention overcomes the prejudice of the prior art, can judge the quality of oocytes, embryos, and blastocysts without affecting their further growth and differentiation, and has extremely low side effects on cells, so that embryos and germ cells can survive further in subsequent experiments, and the probe can maintain similar effects in vitro in further in vivo experiments. This overcomes the problem that the detection reagent in the prior art can only evaluate the quality of germ cells or embryos, but cannot be applied to the screening process, and has a pioneering impact on assisted reproductive technology.
  • Figure 1 is a representative image of the mitochondrial distribution status of mature yak oocytes detected by Mito Tracker mitochondrial fluorescent probe.
  • Figure 2 is a representative image of the mitochondrial distribution status of mouse mature oocytes detected by MG-Rho-2 mitochondrial fluorescent probe.
  • Figure 3 shows representative images of normal and abnormal mitochondrial distribution in mature oocytes assessed by MG-Rho-2 probe.
  • Figure 4 shows representative images of the continuous monitoring of mitochondrial distribution status during preimplantation embryo development using the MG-Rho-2 probe.
  • Figure 5 shows representative images of the Mito Tracker probe continuously monitoring the distribution status of mitochondria during preimplantation embryo development.
  • Figure 6 shows representative images of normal and abnormal mitochondrial distribution in blastocysts assessed by MG-Rho-2 probe.
  • Figure 7 is a representative image of the results of transplantation of blastocysts with normal and abnormal mitochondria screened using the MG-Rho-2 probe.
  • a detection composition comprising a mitochondrial fluorescent probe.
  • the detection composition contains the mitochondrial fluorescent probe and a culture medium for culturing germ cells or embryos.
  • the concentration of the mitochondrial fluorescent probe in the detection composition is 0.01 ⁇ M-2 ⁇ M, for example, 1 ⁇ M, 0.5 ⁇ M, 0.25 ⁇ M, 0.1 ⁇ M, 0.05 ⁇ M, 0.025 ⁇ M, 0.01 ⁇ M.
  • the mitochondrial fluorescent probe is selected from MG-Rho-2, MitoTracker® Deep Red FM, MitoTracker Green FM, MitoTracker Orange CMTMRos1, MitoTracker Orange CMTMRos 2, TPP-KIz, TPP-KIzTZ, MCY-BF2, SiR-Mito 8, SiR-Mito 11.
  • MG-Rho-2 is as shown in Formula I: ;
  • MitoTracker® Deep Red FM is shown in Formula II: ;
  • MitoTracker Green FM is shown in Formula III: III;
  • MitoTracker Orange CMTMRos1 is shown in Formula IV: IV;
  • MitoTracker Orange CMTMRos 2 is shown in Formula V: V;
  • TPP-KIz is shown in Formula VI: VI;
  • TPP-KIzTZ is shown in Formula VII: VII;
  • MCY-BF2 is represented by Formula VIII: VIII;
  • SiR-Mito 8 is shown in Formula IX: IX;
  • SiR-Mito 11 is shown in Formula X: X.
  • mitochondrial fluorescent probes and the above-mentioned detection compositions are also provided for use in the preparation of reagents for detecting the distribution status of mitochondria during the development of germ cells and embryos; and for the preparation of reagents for evaluating the development process and quality of germ cells and embryos.
  • the mitochondrial fluorescent probe may also be a composition comprising the mitochondrial fluorescent probe, wherein the composition comprising the mitochondrial fluorescent probe comprises the mitochondrial fluorescent probe and a culture medium for culturing germ cells and embryos.
  • the germ cells and embryos are selected from humans or mammals, and the mammals are common mammals or mammals on the list of national key protected wild animals; common mammals are preferably mice, rats, humans, rabbits, cattle, sheep, pigs, horses, monkeys, dogs, and pandas. Mammals on the list of national key protected wild animals are, for example, Vietnamese antelopes, wild donkeys, yaks, Przewalski's gazelles, snow leopards, giant pandas, and Siberian tigers.
  • the reproductive cell is selected from oocytes and sperm.
  • the oocyte is selected from immature oocytes or mature oocytes.
  • the embryo is selected from the group consisting of a fertilized egg, a 2-cell embryo, a 4-cell embryo, an 8-cell embryo, a morula, and a blastocyst.
  • the embryos can be used in other experiments or undergo embryo transfer to further evaluate reproductive outcomes.
  • the detection of mitochondrial status is selected from the detection and monitoring of the mitochondrial distribution status of mature oocytes, the mitochondrial distribution status of various stages of preimplantation embryo development, and the mitochondrial distribution status of preimplantation embryos in the blastocyst stage.
  • the method for detecting the state of mitochondria is to observe the distribution state of mitochondria using a laser confocal imaging system.
  • the method for detecting mitochondrial status further includes the step of further analyzing the mitochondrial distribution status after obtaining the mitochondrial distribution status.
  • the analysis is to analyze the fluorescence intensity and aggregation state of the mitochondrial probe.
  • the present invention provides a use of a mitochondrial fluorescent probe in preparing a reagent for screening embryos in assisted reproductive therapy.
  • the mitochondrial fluorescent probe is selected from MG-Rho-2, MitoTracker® Deep Red FM, MitoTracker Green FM, MitoTracker Orange CMTMRos1, MitoTracker Orange CMTMRos 2, TPP-KIz, TPP-KIzTZ, MCY-BF2, SiR-Mito 8, and SiR-Mito 11.
  • the present invention provides a method for screening germ cells, preimplantation embryos and embryonic blastocysts using a mitochondrial fluorescent probe, which comprises the following steps:
  • step S2 placing the germ cells, preimplantation embryos and embryonic blastocysts in the detection composition obtained in step S1) for incubation for 0.1-12 hours or culturing for 12-72 hours;
  • the preimplantation embryos are fertilized eggs, 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae and blastocysts;
  • the germ cells are selected from oocytes and sperms;
  • step S3) observing the distribution of mitochondria in the culture obtained in step S2); the observation is one-time or is performed two or more times along with the culture or incubation in step S2); the method for observing the distribution of mitochondria in step S3) is to use a laser confocal imaging system for imaging observation.
  • step S4) analyzing the distribution of mitochondria obtained in step S3), evaluating or screening the quality and vitality of the corresponding germ cells, preimplantation embryos, and embryonic blastocysts; the evaluation or screening method is to evaluate the distribution of mitochondria obtained in step S3), and the germ cells, preimplantation embryos, and embryonic blastocysts with uniform mitochondrial distribution have higher quality and vitality than those with uneven mitochondrial distribution.
  • the embryonic blastocyst can also be transplanted into the subject.
  • step S4) the means of analysis and evaluation is to use Image J software to detect the fluorescence intensity in the specified fluorescence area of the image, obtain the fluorescence intensity parameter results, and import the parameter results into Graphpad to generate a result schematic diagram.
  • the present invention provides a method for screening embryonic blastocysts for transplantation, the screening method comprising the following steps:
  • step S02 placing the embryonic blastocyst in the detection composition obtained in step S01) for incubation or culture;
  • step S04 analyzing the distribution status of mitochondria obtained in step S02) to evaluate the quality and vitality of the corresponding embryonic blastocyst;
  • Step 1 Prepare concentrated stock and working solutions of fluorescent probes
  • Step 2 Collect samples and incubate with fluorescent probes
  • the fluorescent probe of the required working solution concentration is prepared with culture medium and placed in a 37°C carbon dioxide incubator for equilibrium for 3-5 hours.
  • the collected germ cells, fertilized eggs or embryos are then placed in the culture medium containing the fluorescent probe of the working solution concentration for culture.
  • Step 3 Observe and acquire images
  • the sample can be directly placed under a laser confocal microscope for rapid observation and imaging.
  • the mitochondria of normal oocytes are evenly distributed, without obvious irregular aggregation, and the fluorescence intensity in the region is moderate and stable, while the mitochondria of low-quality oocytes have serious irregular aggregation, and the fluorescence intensity in the region is enhanced.
  • the mitochondria of normal embryos are evenly distributed, with clear cytoplasm and nuclear outlines, no obvious aggregation, and moderate and stable fluorescence intensity in the region.
  • the mitochondria of low-quality embryos have serious irregular aggregation, with blurred cytoplasm and nuclear outlines, and enhanced fluorescence intensity in the region.
  • Image J software was used to detect the fluorescence intensity in the specified fluorescence area of the image, obtain the fluorescence intensity parameter results, and the parameter results were imported into Graphpad to generate a result schematic diagram.
  • Step 5 Conduct various follow-up experiments
  • Oocytes and embryos screened using mitochondrial probes can be used for other experiments or embryo transfer and further evaluation of reproductive outcomes.
  • the following examples mainly use MG-Rho-2 and MitoTracker® Deep Red FM mitochondrial targeted probes to evaluate the distribution of mitochondria in germ cells and early embryos, but are not limited to these two probes.
  • the following examples mainly detect, monitor and analyze the mitochondrial distribution status of mouse and yak mature oocytes and in vitro fertilized embryos, but are not limited to this species and this type of germ cells and early embryos.
  • mice aged 8-12 weeks were superovulated and injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). 48-52 hours later, 7.5 IU human chorionic gonadotropin (hCG) was injected again. The vaginal plug was checked the next morning, and the female mice with plugs were cervically dislocated. Mature oocytes were collected from the magnum of the fallopian tube, digested with 0.1% hyaluronidase, and mitochondrial probe staining was performed after the cumulus cells around the oocytes fell off.
  • PMSG pregnant mare serum gonadotropin
  • hCG human chorionic gonadotropin
  • the fluorescent probe working solution is made into droplets, it is covered with mineral oil and placed in a carbon dioxide incubator for overnight equilibrium (this step must be performed after hCG injection). Then, the prepared mature oocytes are placed in the balanced mitochondrial fluorescent probe droplets and incubated at 37°C for 0.5-1 hour. The mitochondrial distribution status of mature oocytes is directly observed using laser confocal microscopy and images are obtained.
  • Image J software was used to detect the fluorescence intensity in the specified fluorescence area of the image, and the fluorescence intensity parameter results were obtained.
  • the parameter results were imported into Graphpad to generate a result diagram. Normal oocyte mitochondria were evenly distributed, without obvious aggregation, and the fluorescence intensity in the region was moderate and stable. Low-quality oocyte mitochondria had severe aggregation, and the fluorescence intensity in the region was enhanced.
  • the 1 mM probe dye concentrated stock solution was diluted with KSOM embryo culture medium to a 0.1 ⁇ M working solution for later use.
  • mice Female mice aged 8-12 weeks were superovulated and injected with 7.5 IU pregnant mare serum gonadotropin (PMSG) 48-52 hours later. Then, 7.5 IU human chorionic gonadotropin (hCG) was injected again and the mice were caged together. The vaginal plug was checked the next morning. 2-cell embryos were collected from the dilated part of the oviduct at 1.5 days from the female mice with plug.
  • PMSG pregnant mare serum gonadotropin
  • hCG human chorionic gonadotropin
  • the collected 2-cell embryos were placed in KSOM culture medium containing Mito Tracker mitochondrial fluorescent probe or MG-Rho-2 mitochondrial fluorescent probe and incubated in a 37°C carbon dioxide incubator. Embryos at any development stage (2-cell, 4-cell, 8-cell, morula and blastocyst) could then be placed directly under a laser confocal imaging system to observe the mitochondrial distribution of the embryos and acquire images.
  • Mitochondria in normal embryos are evenly distributed without obvious aggregation, and the fluorescence intensity in the region is moderate and stable. Mitochondria in low-quality embryos have serious aggregation, and the fluorescence intensity in the region is enhanced.
  • mitochondrial fluorescent probe reagent and method of the present invention can be used to evaluate the embryo status, and the embryo division and growth are not restricted. Therefore, each stage of embryo development can be continuously observed in real time.
  • mice aged 8-12 weeks were superovulated and injected with 7.5 IU pregnant mare serum gonadotropin (PMSG) 48-52 hours later. Then, 7.5 IU human chorionic gonadotropin (hCG) was injected again and the mice were caged together. The vaginal plug was checked the next morning. When the plugged female mice were 1.5 days old, 2-cell embryos were collected from the dilated part of the oviduct. The 2-cell embryos were then placed in KOSM embryo culture medium and cultured in a 37°C carbon dioxide incubator. Blastocysts were collected 72 hours later.
  • PMSG pregnant mare serum gonadotropin
  • hCG human chorionic gonadotropin
  • the collected blastocysts were placed in KSOM culture medium containing probe dye and incubated in a 37°C carbon dioxide incubator for 0.5-1 hour.
  • the blastocyst mitochondrial distribution status can be directly observed using laser confocal microscopy to obtain images.
  • the mitochondrial distribution status of the blastocyst was detected using the MG-Rho-2 probe, and embryos with normal and abnormal mitochondrial distribution were transplanted respectively.
  • the reproductive results showed that compared with the blastocysts with normal mitochondria, the live birth rate of the blastocysts with uneven mitochondrial distribution and severe aggregation was significantly reduced ( Figure 7 ).
  • Example 3 shows that the method for evaluating normal and abnormal embryos used in Example 3 is highly accurate, and the mitochondrial distribution can be used to evaluate the live birth rate of blastocysts.
  • the present invention overcomes the bias in the prior art, and the reproductive performance of blastocysts screened by mitochondrial probes is not affected and can be used for further transplantation.
  • the present invention provides a new option for assisted reproductive screening by overcoming the above technical bias.

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Abstract

本发明公开一种基于线粒体靶向有机荧光探针用于评估活生殖细胞和早期胚胎线粒体状态的应用方法。一种线粒体荧光探针在制备用于检测生殖细胞、胚胎发育过程中线粒体分布状态；或在制备用于评估生殖细胞、胚胎发育过程和质量的试剂中的应用；还公开了一种以线粒体荧光探针对生殖细胞、着床前胚胎以及用于移植胚胎囊胚的筛选方法。

Description

一种基于有机荧光探针评估活生殖细胞及其早期胚胎线粒体状态的应用方法 技术领域
本发明属于生物技术领域，涉及一种新型荧光探针评估生殖细胞和早期胚胎线粒体状态的应用方法。
 背景技术
光动力疗法(Photodynamic therapy，PDT)和声动力疗法(Sonodynamic therapy ,SDT)，是较有前景的癌症治疗方法，它们分别是通过光敏剂或声敏剂在光照或超声激发下，通过与环境中的水分子或氧分子发生化学反应，产生单线态氧和自由基等活性氧物种(ROS)，从而杀伤肿瘤细胞。与传统的癌症治疗方法(如手术、化疗、放疗等)相比，PDT有对靶组织选择性高、毒副作用小、对内脏器官无损伤等优点，但穿透力较差；与PDT相比，SDT还具有穿透性强、无创等优点，但对靶组织选择性较低。声动力治疗利用超声的组织强穿透能力，使聚集在深层肿瘤中的声敏剂产生 ROS 或空化效应来治疗肿瘤，成功吸引了人们的广泛关注。目前，肿瘤声动力治疗已用于临床，结合免疫疗法和内分泌疗法成功治愈了三例晚期乳腺癌患者。
虽然声动力治疗在恶性肿瘤研究领域中有很大优势，并已取得了令人瞩目的成就，但是仍存在二大瓶颈问题限制其在临床治疗中广泛应用：
第一，传统声敏剂生物利用率低。声敏剂作为声动力治疗的三大要素之一，其超声波吸收特性、声动力活性以及靶向性在很大程度上决定了声动力疗效及声动力疗法在临床的可用性和适用范围。当前普遍使用的声敏剂分子有：卟啉、卟啉衍生物、化疗药物DOX、酸性黄、亚甲辅助生殖技术是解决人类不孕不育，家畜扩繁和野生动物濒危物种人工繁育的重要手段之一，而筛选高质量活生殖细胞和早期胚胎对辅助生殖技术的成功率至关重要。
线粒体是胞质中重要和独特的细胞器，在生殖细胞和早期胚胎发育过程中，线粒体经历一系列的形态和数量的变化，其在细胞中的结构、分布和数目与线粒体的功能密切相关。在线粒体内，通过氧化磷酸化产生ATP（三磷酸腺苷）为生殖细胞减数分裂、细胞质成熟、植入前胚胎卵裂、谱系分化等过程提供能量。动物模型已证实，生殖细胞或胚胎线粒体活性状态和功能与胚胎质量和发育潜能有很大关系，线粒体异常会导致囊胚发育率降低、着床潜力受损、胎盘发育改变和胎儿生长速率异常。因此，通过检测线粒体的结构、分布和数目的异常可作为判断生殖细胞和早期胚胎的质量好坏以及发育潜能的一个重要指标。
目前，尽管许多探针可以检测生殖细胞和早期胚胎细胞线粒体功能及状态，但其因为半衰期短，生物相容性差，水溶性差，细胞通透性不佳等限制了其对活生殖细胞和早期胚胎线粒体状态检测的效果。如传统线粒体探针罗丹明123（Rhodamine 123）虽然可立即标记在功能线粒体上，然而，一旦线粒体膜电位丧失，这些探针就很容易离开细胞。此外，在活细胞内含影响膜电位药物的实验中，这些探针将受到限制，效果欠佳。又如商品化MitoTracker （Red）荧光探针分析衰老小鼠MII期卵母细胞线粒体的分布时，M2培养基中需要加入500 nM的MitoTracker 染料，并在37 ℃，5% CO 2的培养箱中孵育30 min，孵育结束后再次需要M2培养基清洗3遍后，最后将卵母细胞固定在玻片上，在激光共聚焦显微镜下成像观察。以上探针的使用过程繁琐，并且线粒体探针检测完成后的样品其质量有所下降，不利于持续追踪观察同一样品后续线粒体的变化情况以及线粒体状态与后续发育潜能之间的关系。
因此，探索一种使用简单、无损且可长期检测监测活生殖细胞和早期胚胎发育的线粒体靶向探针，以及开发也适用于其他可检测监测活生殖细胞和早期胚胎线粒体分布状态的应用方法，对高效率筛选高质量活生殖细胞和早期胚胎至关重要。
 技术问题
以上探针的使用过程繁琐，并且线粒体探针检测完成后的样品其质量有所下降，不利于持续追踪观察同一样品后续线粒体的变化情况以及线粒体状态与后续发育潜能之间的关系。
 技术解决方案
本发明目的是提供可使用一种线粒体靶向荧光探针在检测和监测人类和动物活生殖细胞和早期胚胎线粒体分布状态中的应用，为人类辅助生殖治疗和动物繁育相关技术和科学研究中获取高质量生殖细胞和早期胚胎提供有效手段。
本发明一个方面提供了一种线粒体荧光探针在制备用于检测生殖细胞、胚胎发育过程中线粒体分布状态；或在制备用于评估生殖细胞、胚胎发育过程和质量的试剂中的应用。
进一步地，所述的线粒体荧光探针为靶向线粒体，且显示线粒体分布状态的荧光探针，具体地，所述线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、 MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11。
更进一步地，所述的线粒体荧光探针为包含线粒体荧光探针的组合物，所述包含线粒体荧光探针的组合物中包含线粒体荧光探针以及培养生殖细胞、胚胎的培养基。
进一步地，生殖细胞、胚胎选自来自人或哺乳动物，所述哺乳动物为普通哺乳动物或国家重点保护野生动物名录中哺乳动物；普通哺乳动物优选为小鼠、大鼠、人、兔子、牛、羊、猪、马、猴、狗、熊猫。国家重点保护野生动物名录中哺乳动物例如为藏羚羊、野驴、牦牛、普氏原羚、雪豹、大熊猫、东北虎。
进一步地，所述生殖细胞选自卵母细胞、精子；更进一步地，所述卵母细胞选自未成熟卵母细胞或成熟卵母细胞。
进一步地，所述胚胎选自受精卵、2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚和囊胚。
进一步地，所述胚胎发育过程选自体内自然受精胚胎、孤雌激活胚胎、体外受精胚胎，单精子注射胚胎，以及体细胞核移植胚胎中任意一种。
进一步地，所述胚胎能够用于其他实验或进行胚胎移植，以进一步评估生殖结果。
进一步地，检测线粒体状态选自对成熟卵母细胞线粒体分布状态、着床前胚胎发育各阶段线粒体分布状态，以及对着床前胚胎囊胚阶段线粒体分布状态进行检测和监测。
进一步地，检测线粒体状态的方法为采用激光共聚焦成像系统进行观察线粒体分布状态。
更进一步地，检测线粒体状态的方法还包括在获得线粒体分布状态后进一步对线粒体分布状态进行分析的步骤。
更进一步地，所述的分析为分析线粒体探针的荧光强度以及聚集状态。
本发明另一个方面提供了一种线粒体荧光探针在制备用于辅助生殖治疗中筛选的胚胎的试剂中的用途。
进一步地，所述的线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、 MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11。
本发明再一个方面提供了一种包含线粒体荧光探针的检测组合物，所述的检测组合物中含有线粒体荧光探针以及用于培养生殖细胞或胚胎的培养基。
进一步地，所述检测组合物中线粒体荧光探针的浓度为0.01μM -2μM，例如为1μM、0.5μM、0.25μM、0.1μM、0.05μM、0.025μM、0.01μM。
进一步地，所述线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、 MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11。
本发明再一个方面提供了一种以线粒体荧光探针对生殖细胞、着床前胚胎以及胚胎囊胚进行筛选的方法，其包括如下步骤：
S1) 配制上述包含线粒体荧光探针的检测组合物；
S2) 将生殖细胞、着床前胚胎以及胚胎囊胚置于步骤S1)获得的检测组合物中孵育或培养；
S3) 观察步骤S2)得到的培养物中线粒体的分布状态；
S4) 分析步骤S3）获得的线粒体的分布状态，评估或筛选对应的生殖细胞、着床前胚胎以及胚胎囊胚的质量和活力。
进一步地，在步骤S2)中，着床前胚胎为受精卵、2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚和囊胚。
进一步地，在步骤S2）中生殖细胞选自卵母细胞、精子。
进一步地，在步骤S2）中孵育的时间为0.1-12小时。
进一步地，在步骤S2) 中培养时间为12-72小时，更进一步地，培养着床前胚胎至任意阶段的胚胎，更进一步地，任意阶段的胚胎选自2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚或囊胚。
进一步地，在步骤S3）中，观察为一次性的或者是随着步骤S2）的培养或孵育进行2次以上的观察。
进一步地，在步骤S3)中观察线粒体的分布状态的方法为采用激光共聚焦成像系统进行成像观察。
进一步地，在步骤S4)中，胚胎囊胚在经过步骤S4）评估后，还能够移植到受试者体内。
进一步地，在步骤S4)中，评估或筛选的方法为评价步骤S3）获得的线粒体的分布状态，线粒体分布均匀的生殖细胞、着床前胚胎以及胚胎囊胚相较于线粒体分布不均匀的生殖细胞、着床前胚胎以及胚胎囊胚的质量和活力更高。
更进一步地，着床前胚胎以及胚胎囊胚的评估和筛选中，评估线粒体是否呈均匀分布，以及细胞质与核轮廓是否清晰；线粒体分布越均匀且细胞质与核轮廓越清晰则着床前胚胎以及胚胎囊胚的质量和活力越高。
更进一步地，在步骤S4）中，分析和评估的手段为使用Image J软件对图片指定荧光区域内进行荧光强度检测，获得荧光强度参数结果，并将参数结果导入Graphpad中以生成结果示意图。
本发明再一个方面提供了一种用于移植的胚胎囊胚的筛选方法，所述筛选方法包括以下步骤：
S01) 配制上述包含线粒体荧光探针的检测组合物；
S02) 将胚胎囊胚置于步骤S01)获得的检测组合物中孵育或培养；
S03) 观察步骤S02)得到的胚胎囊胚中线粒体的分布状态；
S04) 分析步骤S02)获得的线粒体的分布状态，评估对应的胚胎囊胚的质量和活力；
S05) 选择线粒体的分布状态均匀的胚胎囊胚，并用于移植。
进一步地，在步骤S02)中，着床前胚胎为受精卵、2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚和囊胚。
进一步地，在步骤S02）中生殖细胞选自卵母细胞、精子。
进一步地，在步骤S02）中孵育的时间为0.1-12小时。
进一步地，在步骤S02)中培养时间为12-72小时，更进一步地，培养着床前胚胎至任意阶段的胚胎，更进一步地，任意阶段的胚胎选自2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚或囊胚。
进一步地，在步骤S03）中，观察为一次性的或者是随着步骤S02）的培养或孵育进行2次以上的观察。
进一步地，在步骤S03)中观察线粒体的分布状态的方法为采用激光共聚焦成像系统进行成像观察。
进一步地，在步骤S04)中，胚胎囊胚在经过步骤S04）评估后，还能够移植到受试者体内。
进一步地，在步骤S04)中，评估或筛选的方法为评价步骤S03）获得的线粒体的分布状态，线粒体分布均匀的生殖细胞、着床前胚胎以及胚胎囊胚相较于线粒体分布不均匀的生殖细胞、着床前胚胎以及胚胎囊胚的质量和活力更高。
更进一步地，着床前胚胎以及胚胎囊胚的评估和筛选中，评估线粒体是否呈均匀分布，以及细胞质与核轮廓是否清晰；线粒体分布越均匀且细胞质与核轮廓越清晰则着床前胚胎以及胚胎囊胚的质量和活力越高。
更进一步地，在步骤S04）中，分析和评估的手段为使用Image J软件对图片指定荧光区域内进行荧光强度检测，获得荧光强度参数结果，并将参数结果导入Graphpad中以生成结果示意图。
 有益效果
本发明人意外发现本发明所使用的探针能够实现在激光共聚焦下进行快速观察和成像，荧光强度强。更重要的是，本发明使用的线粒体荧光探针检测试剂克服了现有技术的偏见，能够在判断卵母细胞、胚胎、囊胚的质量后不影响其进一步生长分化，对于细胞的副作用极低，使得胚胎和生殖细胞能够进一步在后续试验中存活，并且该探针能够在进一步的活体实验中保持体外类似的效果。这一点克服了现有技术中检测试剂仅能评价生殖细胞或胚胎质量，但无法将其应用于筛选过程的问题，对辅助生殖技术具有开拓性影响。
 附图说明
图1为Mito Tracker线粒体荧光探针检测牦牛成熟卵母细胞线粒体分布状态代表性图像。
图2为MG-Rho-2线粒体荧光探针检测小鼠成熟卵母细胞线粒体分布状态代表性图像。
图3为MG-Rho-2探针评估成熟卵母细胞正常和异常线粒体分布状态代表性图像。
图4为MG-Rho-2探针对着床前胚胎发育过程中线粒体分布状态持续监测检测代表性图像。
图5为Mito Tracker探针对着床前胚胎发育过程中线粒体分布状态持续监测代表性图像。
图6为MG-Rho-2探针评估囊胚正常和异常线粒体分布状态代表性图像。
图7为利用MG-Rho-2探针筛选正常和异常线粒体囊胚后移植结果代表性图像。
实施例
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
本发明提供了一些具体的技术方案，首先提供了一种包含线粒体荧光探针的检测组合物，所述的检测组合物中含有线粒体荧光探针以及用于培养生殖细胞或胚胎的培养基；所述检测组合物中线粒体荧光探针的浓度为0.01μM -2μM，例如为1μM、0.5μM、0.25μM、0.1μM、0.05μM、0.025μM、0.01μM；所述线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、 MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11；
其中，MG-Rho-2如式I所示： ；
MitoTracker® Deep Red FM如式II所示： ；
MitoTracker Green FM如式III所示： III；
MitoTracker Orange CMTMRos1如式IV所示： IV；
MitoTracker Orange CMTMRos 2如式V所示： V；
TPP-KIz如式VI所示： VI；
TPP-KIzTZ如式VII所示： VII；
MCY-BF2如式VIII所示： VIII；
SiR-Mito 8如式IX所示： IX；
SiR-Mito 11如式X所示： X。
在一些具体方案中还提供了线粒体荧光探针以及上述检测组合物在制备用于检测生殖细胞、胚胎发育过程中线粒体分布状态；在制备用于评估生殖细胞、胚胎发育过程和质量的试剂中的应用。
所述的线粒体荧光探针也可以是为包含线粒体荧光探针的组合物，所述包含线粒体荧光探针的组合物中包含线粒体荧光探针以及培养生殖细胞、胚胎的培养基。
在一些具体方案中所述生殖细胞、胚胎选自来自人或哺乳动物，所述哺乳动物为普通哺乳动物或国家重点保护野生动物名录中哺乳动物；普通哺乳动物优选为小鼠、大鼠、人、兔子、牛、羊、猪、马、猴、狗、熊猫。国家重点保护野生动物名录中哺乳动物例如为藏羚羊、野驴、牦牛、普氏原羚、雪豹、大熊猫、东北虎。
在一些具体方案中所述生殖细胞选自卵母细胞、精子。所述卵母细胞选自未成熟卵母细胞或成熟卵母细胞。
在一些具体方案中所述胚胎选自受精卵、2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚和囊胚。
在一些具体方案中所述胚胎发育过程选自体内自然受精胚胎、孤雌激活胚胎、体外受精胚胎，单精子注射胚胎，以及体细胞核移植胚胎中任意一种。
在一些具体方案中所述胚胎能够用于其他实验或进行胚胎移植，以进一步评估生殖结果。
在一些具体方案中检测线粒体状态选自对成熟卵母细胞线粒体分布状态、着床前胚胎发育各阶段线粒体分布状态，以及对着床前胚胎囊胚阶段线粒体分布状态进行检测和监测。
在一些具体方案中，检测线粒体状态的方法为采用激光共聚焦成像系统进行观察线粒体分布状态。
在一些具体方案中，检测线粒体状态的方法还包括在获得线粒体分布状态后进一步对线粒体分布状态进行分析的步骤。
在一些具体方案中，所述的分析为分析线粒体探针的荧光强度以及聚集状态。
本发明在一些具体方案中提供了一种线粒体荧光探针在制备用于辅助生殖治疗中筛选的胚胎的试剂中的用途。
在一些具体方案中，所述的线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、 MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11。
本发明在一些具体方案中提供了一种以线粒体荧光探针对生殖细胞、着床前胚胎以及胚胎囊胚进行筛选的方法，其包括如下步骤：
S1) 配制上述包含线粒体荧光探针的检测组合物；
S2) 将生殖细胞、着床前胚胎以及胚胎囊胚置于步骤S1)获得的检测组合物中孵育0.1-12小时或培养12-72小时；着床前胚胎为受精卵、2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚和囊胚；生殖细胞选自卵母细胞、精子；
S3) 观察步骤S2)得到的培养物中线粒体的分布状态；观察为一次性的或者是随着步骤S2）的培养或孵育进行2次以上的观察；在步骤S3)中观察线粒体的分布状态的方法为采用激光共聚焦成像系统进行成像观察。
S4)分析步骤S3）获得的线粒体的分布状态，评估或筛选对应的生殖细胞、着床前胚胎以及胚胎囊胚的质量和活力；评估或筛选的方法为评价步骤S3）获得的线粒体的分布状态，线粒体分布均匀的生殖细胞、着床前胚胎以及胚胎囊胚相较于线粒体分布不均匀的生殖细胞、着床前胚胎以及胚胎囊胚的质量和活力更高。胚胎囊胚在经过步骤S4）评估后，还能够移植到受试者体内。
在一些具体方案中，着床前胚胎以及胚胎囊胚的评估和筛选中，评估线粒体是否呈均匀分布，以及细胞质与核轮廓是否清晰；线粒体分布越均匀且细胞质与核轮廓越清晰则着床前胚胎以及胚胎囊胚的质量和活力越高。
在一些具体方案中，在步骤S4）中，分析和评估的手段为使用Image J软件对图片指定荧光区域内进行荧光强度检测，获得荧光强度参数结果，并将参数结果导入Graphpad中以生成结果示意图。
本发明再一个方面提供了一种用于移植的胚胎囊胚的筛选方法，所述筛选方法包括以下步骤：
S01) 配制上述包含线粒体荧光探针的检测组合物；
S02) 将胚胎囊胚置于步骤S01)获得的检测组合物中孵育或培养；
S03) 观察步骤S02)得到的胚胎囊胚中线粒体的分布状态；
S04) 分析步骤S02)获得的线粒体的分布状态，评估对应的胚胎囊胚的质量和活力；
S05) 选择线粒体的分布状态均匀的胚胎囊胚，并用于移植。
在一个具体的技术方案中，通过应用MG-Rho-2线粒体荧光探针染料为例，具体方法步骤如下：
第一步：制备荧光探针浓储液和工作液
将8mg荧光探针分析纯溶解在1mL超纯水中，制备为1mM浓储液并置于-20℃长期保存，使用时将浓储液用培养基稀释为所需的工作液浓度。
第二步：收集样品与荧光探针共孵育
用培养基配制所需工作液浓度的荧光探针，并置于37℃二氧化碳培养箱平衡3-5小时，再将收集的生殖细胞，受精卵或胚胎放入包含工作液浓度的荧光探针的培养基中进行培养。
第三步：观察及获取图像
线粒体荧光探针染料孵育后的样品可直接置于激光共聚焦显微镜下进行快速观察和成像。例如正常卵母细胞线粒体呈均匀分布状态，无明显无规则聚集现象，区域内荧光强度适中、稳定，而低质量卵母细胞线粒体存在严重的无规则聚集现象，区域内荧光强度增强。此外，正常胚胎线粒体呈均匀分布状态，细胞质与核轮廓清晰，无明显聚集现象，区域内荧光强度适中、稳定，低质量胚胎线粒体存在严重的无规则聚集现象，细胞质与核轮廓模糊，区域内荧光强度增强。
第四步：分析
使用Image J软件对图片指定荧光区域内进行荧光强度检测，获得荧光强度参数结果，并将参数结果导入Graphpad以生成结果示意图。
第五步：开展各类后续实验
利用线粒体探针筛选后的卵母细胞和胚胎可用于其他实验或进行胚胎移植，并进一步评估生殖结果。
以下实例主要利用MG-Rho-2和MitoTracker® Deep Red FM线粒体靶向探针评估生殖细胞和早期胚胎线粒体分布状态为例，但并不仅限于这两种探针。
以下实例主要对小鼠和牦牛成熟卵母细胞和体外受精胚胎线粒体分布状态进行检测，监测和分析，但不仅限于此物种和此类型生殖细胞和早期胚胎。
以下实例中的实验均设置了三次重复。
实施例1 对不同物种成熟卵母细胞线粒体分布状态的评估
1.1 收集成熟卵母细胞
对8-12周龄雌性小鼠进行超数排卵，注射7.5IU孕马血清促性腺激素(PMSG) 48-52小时后，再次注射7.5IU人绒毛膜促性腺激素(hCG)，次日早晨检查阴栓，对见栓雌鼠进行颈椎脱臼，从输卵管膨大部收集成熟卵母细胞，利用0.1%透明质酸酶对成熟卵母细胞进行消化，待卵母细胞周围卵丘细胞脱落干净后进行线粒体探针染色。
1.2. 制备含有探针染料的培养液
分别将1mM Mito Tracker线粒体荧光探针或MG-Rho-2线粒体荧光探针浓储液用M2培养基稀释为0.1μM的工作液，备用。
1.3. 对成熟卵母细胞线粒体进行探针染色
将荧光探针工作液制成液滴后用矿物油覆盖，并置于二氧化碳培养箱过夜平衡（此步骤须在hCG注射后进行），再将准备好的成熟卵母细胞放入平衡好的线粒体荧光探针液滴中，37℃孵育0.5-1小时，直接利用激光共聚焦观察成熟卵母细胞线粒体分布状态，并获得图像。
1.4. 对成熟卵母细胞线粒体成像进行分析（ 图1，2，3）
使用Image J软件对图片指定荧光区域内进行荧光强度检测，获得荧光强度参数结果，并将参数结果导入Graphpad以生成结果示意图。正常卵母细胞线粒体呈均匀分布状态，无明显聚集现象，区域内荧光强度适中、稳定。低质量卵母细胞线粒体存在严重的聚集现象，区域内荧光强度增强。
实施例2. 不同探针对着床前胚胎各阶段线粒体分布状态进行持续监测
2.1. 制备含有探针染料的培养液
将1mM探针染料浓储液用KSOM胚胎培养液稀释为0.1μM的工作液，备用。
2.2 收集2-cell胚胎
对8-12周龄雌性小鼠进行超数排卵，注射7.5IU孕马血清促性腺激素(PMSG) 48-52小时后，再次注射7.5IU人绒毛膜促性腺激素(hCG)后进行合笼，次日早晨检查阴栓，见栓雌鼠1.5天时从输卵管膨大部收集2-cell胚胎。
2.3. 对胚胎发育各阶段线粒体状态进行持续监测
将收集2-cell胚胎放入含有Mito Tracker线粒体荧光探针或MG-Rho-2线粒体荧光探针的KSOM培养液，并置于37℃二氧化碳培养箱进行孵育培养，随后可将发育到任意阶段的胚胎（2-cell、4-cell、8-cell、桑葚胚和囊胚）直接放置在激光共聚焦成像系统下观察胚胎线粒体分布状态，并获取图像。
2.4. 对各阶段胚胎线粒体分布状况进行实时观测 （图4，5）
正常胚胎线粒体呈均匀分布状态，无明显聚集现象，区域内荧光强度适中、稳定，低质量胚胎线粒体存在严重的聚集现象，区域内荧光强度增强。
说明采用本发明的线粒体荧光探针试剂和方法能够评估胚胎状态，且胚胎分裂生长不受限制，因此，可以实时地持续观察胚胎发育的各阶段。
实时例3. 利用MG-Rho-2探针检测胚胎囊胚阶段的线粒体分布状态
3.1. 制备含有探针染料的培养液
将1mM Mito Tracker线粒体荧光探针或MG-Rho-2线粒体荧光探针浓储液用KSOM胚胎培养液稀释为0.1μM的工作液，备用。
3.2 收集囊胚阶段胚胎
对8-12周龄雌性小鼠进行超数排卵，注射7.5IU孕马血清促性腺激素(PMSG) 48-52小时后，再次注射7.5IU人绒毛膜促性腺激素(hCG)后进行合笼，次日早晨检查阴栓，见栓雌鼠1.5天时从输卵管膨大部收集2-cell胚胎，再将2-cell胚胎放入KOSM胚胎培养液，并置于37℃二氧化碳培养箱进行培养，72小时后收集囊胚。
3.3. 对囊胚进行线粒体探针染色
将收集的囊胚放入含有探针染料的KSOM培养液中，在37℃二氧化碳培养箱孵育0.5-1小时后，可直接利用激光共聚焦观察囊胚线粒体分布状态，获取图像。
3.4. 对囊胚线粒体分布成像进行分析（ 图6）
使用Image J软件对图片指定荧光区域内进行荧光强度检测，获得荧光强度参数结果，并将参数结果导入Graphpad以生成结果示意图。正常囊胚线粒体呈均匀分布状态，内细胞团和滋养外胚层轮廓清晰，无明显聚集现象，区域内荧光强度适中、稳定，低质量囊胚线粒体存在严重的聚集现象，内细胞团和滋养外胚层轮廓模糊，区域内荧光强度增强。
实施例4. MG-Rho-2探针筛选可移植囊胚
按照实施例3方法，利用MG-Rho-2探针对囊胚线粒体分布状态进行检测，将线粒体分布状态正常和异常的胚胎分别进行移植，生殖结果显示，与线粒体正常囊胚相比，线粒体分布不均匀和严重聚集的囊胚活产率显著降低（ 图7）。
本实施例说明对于实施例3采用的评价胚胎正常与异常的评价方法准确性高，通过线粒体分布情况可以用于评估囊胚活产率。同时本发明克服了现有技术中的偏见，经过线粒体探针筛选的囊胚的繁殖性能未受影响，可以用于进一步移植。本发明通过克服上述技术偏见为辅助生殖筛选提供了新选择。

Claims (10)

  1. 一种线粒体荧光探针在制备用于检测生殖细胞、胚胎发育过程中线粒体分布状态；或在制备用于评估生殖细胞、胚胎发育过程和质量的试剂中的应用；
    优选地，所述的线粒体荧光探针为靶向线粒体，且显示线粒体分布状态的荧光探针；
    更优选地，所述线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、 MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11。
  2. 根据权利要求1所述的应用，其特征在于，所述的线粒体荧光探针为包含线粒体荧光探针的组合物，所述包含线粒体荧光探针的组合物中包含线粒体荧光探针以及培养生殖细胞或胚胎的培养基。
  3. 根据权利要求1所述的应用，其特征在于，所述生殖细胞、胚胎选自来自人或哺乳动物，所述哺乳动物为普通哺乳动物或国家重点保护野生动物名录中哺乳动物；优选地，所述生殖细胞选自卵母细胞、精子；更优选地，所述卵母细胞选自未成熟卵母细胞或成熟卵母细胞；优选地，所述胚胎选自受精卵、2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚和囊胚；优选地，所述胚胎发育过程选自体内自然受精胚胎、孤雌激活胚胎、体外受精胚胎，单精子注射胚胎，以及体细胞核移植胚胎中任意一种。
  4. 根据权利要求1所述的应用，其特征在于，所述胚胎或生殖细胞能够用于其他实验或进行胚胎移植，以进一步评估生殖结果。
  5. 根据权利要求1所述的应用，其特征在于，检测线粒体分布状态选自对成熟卵母细胞线粒体分布状态、着床前胚胎发育各阶段线粒体分布状态，以及对着床前胚胎囊胚阶段线粒体分布状态进行检测和监测。
  6. 一种线粒体荧光探针在制备用于辅助生殖治疗中筛选的胚胎的试剂中的用途；所述的线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、 MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11。
  7. 一种包含线粒体荧光探针的检测组合物，其特征在于，所述的检测组合物中含有线粒体荧光探针以及用于培养生殖细胞或胚胎的培养基；
    优选地，所述检测组合物中线粒体荧光探针的浓度为0.01μM -2μM；
    优选地，所述线粒体荧光探针选自MG-Rho-2、MitoTracker® Deep Red FM、MitoTracker Green FM、MitoTracker Orange CMTMRos1、MitoTracker Orange CMTMRos 2、TPP-KIz、TPP-KIzTZ、MCY-BF2、SiR-Mito 8、SiR-Mito 11。
  8. 一种以线粒体荧光探针对生殖细胞、着床前胚胎以及胚胎囊胚进行评估或筛选的方法，其特征在于，其包括如下步骤：
    S1)配制权利要求7所述的包含线粒体荧光探针的检测组合物；
    S2) 将生殖细胞、着床前胚胎以及胚胎囊胚置于步骤S1)获得的检测组合物中孵育或培养；
    S3) 观察步骤S2)得到的培养物中线粒体的分布状态；
    S4)分析步骤S3）获得的线粒体的分布状态，评估或筛选对应的生殖细胞、着床前胚胎以及胚胎囊胚的质量和活力。
  9. 一种用于移植的胚胎囊胚的筛选方法，其特征在于，所述筛选方法包括以下步骤：
    S01) 配制权利要求7所述的包含线粒体荧光探针的检测组合物；
    S02) 将胚胎囊胚置于步骤S01)获得的检测组合物中孵育或培养；
    S03) 观察步骤S02)得到的胚胎囊胚中线粒体的分布状态；
    S04) 分析步骤S02)获得的线粒体的分布状态，评估对应的胚胎囊胚的质量和活力；
    S05) 选择线粒体的分布状态均匀的胚胎囊胚，并用于移植。
  10. 根据权利要求8的方法或者根据权利要求9所述的筛选方法，其特征在于，在步骤S2)或S02)中，着床前胚胎为受精卵、2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚和囊胚；
    优选地，在步骤S2）或S02)中生殖细胞选自卵母细胞、精子；
    优选地，在步骤S2）或S02)中孵育的时间为0.1-12小时；
    优选地，在步骤S2) 或S02)中培养时间为12-72小时，更优选地，培养着床前胚胎至任意阶段的胚胎，更优选地，任意阶段的胚胎选自2细胞胚胎、4细胞胚胎、8细胞胚胎、桑葚胚或囊胚；
    优选地，在步骤S3）或S03)中，观察为一次性的或者是随着步骤S2）或S02)的培养或孵育进行2次以上的观察；
    优选地，在步骤S3) 或S03)中观察线粒体的分布状态的方法为采用激光共聚焦成像系统进行成像观察；
    优选地，在步骤S4) 或S04中，胚胎囊胚在经过步骤S4）或S04)评估后，还能够移植到受试者体内；
    优选地，在步骤S4) 或S04)中，评估或筛选的方法为评价步骤S3）或S03)获得的线粒体的分布状态，线粒体分布均匀的生殖细胞、着床前胚胎以及胚胎囊胚相较于线粒体分布不均匀的生殖细胞、着床前胚胎以及胚胎囊胚的质量和活力更高；
    更优选地，着床前胚胎以及胚胎囊胚的评估和筛选中，评估线粒体是否呈均匀分布，以及细胞质与核轮廓是否清晰；线粒体分布越均匀且细胞质与核轮廓越清晰则着床前胚胎以及胚胎囊胚的质量和活力越高；
    更优选地，在步骤S4）或S04)中，分析和评估的手段为使用Image J软件对图片指定荧光区域内进行荧光强度检测，获得荧光强度参数结果，并将参数结果导入Graphpad中以生成结果示意图。
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