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CN-121975927-A - Method for detecting early-stage neurodevelopmental toxicity of pollutants based on neural crest specificity molecular markers and application

CN121975927ACN 121975927 ACN121975927 ACN 121975927ACN-121975927-A

Abstract

The invention discloses a method for detecting early-stage neurodevelopmental toxicity of pollutants based on neural crest specificity molecular markers and application thereof, and belongs to the technical fields of environmental toxicology and molecular diagnosis. The method comprises 1) exposing the early embryo to a sample or contaminant to be tested, 2) detecting the change in the neural crest and peripheral neuron specific molecular markers, and 3) assessing early neurodevelopmental toxicity based on the difference in marker expression. The invention breaks through the problems of early warning lag and low sensitivity of the traditional neural behavior end point (zebra fish 6 dpf), can detect the early neurotoxicity of low-concentration pollutants within 24 hours (zebra fish sox10 and prph) of pollutant exposure, and can be used for early toxicity early warning and risk assessment in environmental monitoring and chemical risk assessment.

Inventors

  • ZHANG TING
  • DU SEN
  • ZHANG LI

Assignees

  • 中国科学院南海海洋研究所

Dates

Publication Date
20260505
Application Date
20260204

Claims (8)

  1. 1. A method for rapidly detecting early developmental toxicity of a contaminant based on neural crest cell-specific molecular markers, comprising the steps of: a. embryo exposure, namely placing a model biological embryo in an early development stage into an environmental sample or pollutant to be detected for exposure, and setting up a control group; b. marker detection, namely collecting embryos of a control group and a treatment group at a preset early time point after exposure, and detecting mRNA expression levels of a neural crest cell specific molecular marker and a differentiated cell molecular marker thereof in the sample; c. Toxicity evaluation comparing the expression level of the marker gene in the treated group with a control group, and evaluating the early developmental toxicity potential of the environmental sample or contaminant based on the degree of change in the molecular marker.
  2. 2. The method according to claim 1, wherein in step a, the test model organism is zebra fish or medaka.
  3. 3. The method of claim 1, wherein in step a, the early stage of development is within 2 hours after fertilization of the embryo.
  4. 4. The method of claim 1, wherein in step b, the neural crest cell-specific molecular marker is at least one of sox10 and foxd.
  5. 5. The method of claim 4, wherein the sox10 and foxd primer sequences are: Zebra fish: Z-sox10-F: 5'-ACAGCCATTCGCATTTCAA-3'; Z-sox10-R: 5'-TCGCCTGATATTCCTGCCT-3'; Z-foxd3-F: 5'-GCAATACAAGCCGAAGAGC-3'; Z-foxd3-R: 5'-GGGGTCCAGGGACCAGTAG-3'; sea water medaka: M-sox10-F: 5'-GCCAGCTGCTGATGTGCTA-3'; M-sox10-R: 5'-ATGATCGCTCGTTTGTCGC-3'; M-foxd3-F: 5'-CTACAACGCGCTCATCAGC-3'; M-foxd3-R: 5'-GGTCGAGTAGTTCCCCTAT-3'。
  6. 6. the method of claim 1, wherein in step b, the neural crest differentiated cell molecular markers are prph.
  7. 7. The method of claim 6, wherein the prph primer sequences are: Zebra fish: Z-prph-F: 5'-AGCAATCAGAATCGAGAAC-3'; Z-prph-R : 5'-TCCTTAATCTGAGCCAACT-3'; sea water medaka: M-prph-F: 5'-TTTCCGTTAGGTGCTATGT-3'; M-prph-R: 5'-ATTACAACTATCCATTTCG-3'。
  8. 8. use of the method according to any one of claims 1-7 for environmental media as well as chemical toxicity monitoring and ecological risk assessment.

Description

Method for detecting early-stage neurodevelopmental toxicity of pollutants based on neural crest specificity molecular markers and application Technical Field The invention belongs to the technical field of environmental toxicology and molecular diagnosis, and particularly relates to a method for rapidly and sensitively detecting the developmental toxicity of an environmental pollutant nerve by utilizing a neural crest specific molecular marker and application of the method in environmental detection and risk assessment. Background With the continuous acceleration of the industrialization and urbanization process, a large amount of chemical pollutants enter the water environment. Wherein, many pollutants can penetrate through the blood brain barrier and the placenta barrier and directly or indirectly act on the nervous system or influence on the development of the offspring by parent exposure, so that the individuals and offspring have the symptoms of nerve development disorder, such as cognitive deficiency, decline of learning and memory ability, abnormal behaviors and the like. The current methods for assessing the neurotoxicity of pollutants rely mainly on animal models (e.g. rodents, zebra fish). Conventional assessment endpoints include motor ability detection, behavioral tests (e.g., open field experiments, morris water maze), and end-stage neuropathological examinations, with early warning window delays. The existing in-vitro model and molecular marker cannot simulate in-vivo complex microenvironment, poison metabolic process and the like, and often adopts a high-concentration exposure mode, so that the sensitivity and the specificity are low, the environmental concentration low-dose exposure risk is difficult to evaluate accurately, and the extrapolation and the practical prediction significance of research results are limited. Therefore, there is a need for a toxicity detection tool that can specifically and highly sensitively target early stages of neurodevelopment. Neural crest is a temporary population of stem cells with multipotent differentiation potential specific to vertebrate embryos, whose fate decisions, migration and differentiation are critical for the normal development of the peripheral nervous system. The neural crest development process is precisely controlled by a highly conserved gene regulation network, is particularly sensitive to changes in the cellular microenvironment, and is considered to be one of the earliest critical events causing developmental toxicity such as congenital neural tube defects, peripheral neuropathy and the like. Therefore, dynamic changes in neural crest cells and their differentiated peripheral nervous system constitute a key bridge connecting early embryo damage with developmental defects of the long-term nervous system and motor system. Based on the above, the functional state change of the neural crest cells can reflect the toxic influence of the pollutants on the neural development earlier and more sensitively, and the change of the specific molecular markers can provide ideal biological targets for early and targeted neurotoxicity monitoring and risk assessment of the pollutants. Disclosure of Invention The invention aims to overcome the defects of early warning lag, insufficient sensitivity and specificity, poor environment correlation and the like of an environmental pollutant neurodevelopmental toxicity detection method in the prior art, and provides a detection method based on a neural crest specificity molecular marker. The method selects a neural crest specific molecular marker foxd and sox10 and a neural crest differentiated cell molecular marker prph, establishes the relationship between the indexes and traditional neural behavior end points (motor ability and thigmotaxis) (zebra fish 6 dpf, sea water medaka 21 dpf) and is used for evaluating early developmental toxicity of pollutants. Of these, foxd and sox10 are key transcription factors for neural crest development, prph is a key structural component of sensory and motor neurons derived from neural crest, and is critical for neuronal survival and neural signaling. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: A method for rapidly detecting early developmental toxicity of a contaminant based on neural crest cell-specific molecular markers, comprising the following steps: a. embryo exposure, namely placing a model biological embryo in an early development stage into an environmental sample or pollutant to be detected for exposure, and setting up a control group; b. marker detection, namely collecting embryos of a control group and a treatment group at a preset early time point after exposure, and detecting mRNA expression levels of a neural crest cell specific molecular marker and a differentiated cell molecular marker thereof in the sample; c. Toxicity evaluation comparing the expression level of the marker gene in the treated group wit