CN-121992061-A - Embryonic stem cell differentiation technology and application thereof in unknown risk replacement toxicity screening of novel biological breeding products

Abstract

The invention provides an embryonic stem cell differentiation technology and application thereof in screening unknown risk substitution toxicity of novel biological breeding products, wherein Cry1Ab protein is used as a test object to evaluate development toxicity of the Cry1Ab protein. EBs are produced by hanging drop culture, and then osteodifferentiation inducing substances (beta-glycerophosphate, ascorbic acid and vitamin D3) are added to promote differentiation. After the induced differentiation of the bone cells is finished, the differentiation condition of the bone cells is observed through alizarin red S staining and absorbance value detection, the growth condition of the cells is observed through detection of the total protein concentration and alkaline phosphatase activity of the cells, and then the influence of the test object on the bone differentiation process is analyzed through the gene expression condition of osteogenic differentiation related markers (Runx 2, SPARC and type I collagen). The invention solves the problem of in vitro evaluation of unknown risks of novel biological breeding products, creates an embryonic stem cell bone differentiation test technology, and uses the technology as a screening method of unknown risk substitution toxicity of the novel biological breeding products, and improves the accuracy of predicting the developmental toxicity of a test object by combining with an embryonic stem cell myocardial differentiation experiment.

Inventors

• WANG JUNBO
• JIAN YUANZHI
• Liu anyi
• Xia Fangzhi
• GUO YUXIN
• WANG FEI

Assignees

• 北京大学

Dates

Publication Date

20260508

Application Date

20241105

Claims (4)

1. 1. An embryonic stem cell differentiation evaluation system is characterized in that an embryonic stem cell bone differentiation experimental technique and an embryonic stem cell myocardial differentiation experimental technique are combined and applied to unknown risk substitution toxicity screening of novel biological breeding products.
2. 2. The experimental technique for bone differentiation of embryonic stem cells according to claim 1, wherein EBs are produced by hanging drop culture, and further differentiation is promoted by adding an osteodifferentiation inducing substance (β -glycerophosphate, ascorbic acid and vitamin D3).
3. 3. The method according to claim 2, wherein the amount of the inducing substance used in the bone differentiation assay of embryonic stem cells is 10mM, the amount of the ascorbic acid is 50. Mu.g/ml, and the amount of the vitamin D3 is 500. Mu.M.
4. 4. The test substance for the selection of unknown risk replacement toxicity of a novel biological breeding product according to claim 1, characterized by the major differential component Cry1Ab protein of the transgenic crop concerned and of the non-transgenic crop concerned.

Description

Embryonic stem cell differentiation technology and application thereof in unknown risk replacement toxicity screening of novel biological breeding products Technical Field The invention relates to the technical field of cell biology and nutritional food safety evaluation, in particular to an embryonic stem cell differentiation technology and an unknown risk substitution toxicity screening method for novel biological breeding products. Background In-vitro tests are often one of the test methods preferred by the scholars in performing the toxicity evaluation of new substances. Compared with the traditional developmental toxicity animal test, the in-vitro test method accords with the principle of '3R' (Replacement), reduction and optimization (REFINEMENT), greatly reduces the use of experimental animals, has the advantages of less cost, short period, high accuracy and the like, and can carry out high-flux toxicity test [1]. The embryonic stem cell test (Embryonic STEM CELL TEST, EST) is a common alternative method for evaluating the developmental toxicity of substances, and the method is verified by European alternative method verification centers (European Centre for the Validation of Alternative Methods, ECVAM) and has a standardized test scheme, and a plurality of researches prove that the accuracy of evaluating the developmental toxicity of the substances, particularly the prediction accuracy of the substances with strong embryo toxicity can reach 100 percent [2,3]. ESTs utilize the pluripotency of ES-D3, form embryoid bodies (Embryonic Bodies, EBs) using hanging drop culture, spontaneously differentiate into beating cardiomyocytes without the addition of an inducing substance, and evaluate and predict the developmental toxicity of the substance by combining the inhibition of cell growth and the inhibition of myocardial differentiation by the test substance. As a commonly used developmental toxicity evaluation alternative method, ESTs have the advantages of using only cell lines, adding no inducing substances, having relatively short experimental period and the like, but have certain limitations. The traditional EST test has single differentiation end point and detection, mainly considers the beating condition of myocardial cells, and can not accurately predict the developmental toxicity of substances with specific differentiation toxicity [4]. There was a study [5] to test 19 internal receptor-mediated drugs of known in vivo biotoxicity with ESTs, resulting in an overall accuracy of only 53%. In addition, EST centromere development processes predominate in early stages, with poor prediction of [6] for some later-stage developmental toxic substances. Therefore, it is necessary to add new differentiation endpoints, such as bone cell differentiation, neural cell differentiation, vascular endothelial cell differentiation, etc., to improve the use of EST in the screening of unknown risk replacement toxicity of novel biological breeding products. The most direct effect of substances with developmental toxicity on the body is the possibility of causing birth defects. Birth defects refer to abnormalities in the body structure, function or metabolism that occur in infants before they develop, primarily due to genetic or environmental factors, and generally include congenital anomalies, chromosomal abnormalities, inherited metabolic diseases, and the like. The birth defect monitoring data [7] of China show that in the ten advanced malformations before the perinatal period, the incidence rate of congenital heart disease is the first, and the incidence rate accounts for 26.7% of all the cases found by monitoring, and skeletal dysplasia also has a large proportion, such as multi-toe (16.73%), horseshoe varus (5.17%), parallel toe (4.88%), limb shortness (4.09%), and the like. Considering the high incidence of embryonic skeletal dysplasia, it is important to consider the endpoint of bone differentiation as part of the developmental toxicity assessment. Based on that the bone cells and the myocardial cells are mesoderm differentiated cells, the invention creates an embryonic stem cell bone differentiation test technology (Embryonic Stem Cell Based Osteoblast Differentiation Method, ESTo), and uses the embryonic stem cell bone differentiation test technology as a screening method for unknown risk substitution toxicity of novel biological breeding products, and the screening method is used in combination with an embryonic stem cell myocardial differentiation test, thereby improving the accuracy of predicting the developmental toxicity of a test object. Reference to the literature [1]MEZZOMO B P,MIRANDA-VILELA A L,BARBOSA L C,et al.Hematotoxicity and genotoxicity evaluations in Swiss mice intraperitoneally exposed to Bacillus thuringiensis(var kurstaki)spore crystals genetically modified to express individually Cry1Aa,Cry1Ab,Cry1Ac,or Cry2Aa[J].Environmental toxicology,2016,31(8)：970-8. [2]WALSH M C,BUZOIANU S G,GARDINER G