A mouse experimental method for detecting tissue distribution of exogenous oxytocin

Abstract

This invention discloses a mouse experimental method for detecting the tissue distribution of exogenous oxytocin, belonging to the field of biotechnology. Specifically, oxytocin is injected into or nasally sprayed into oxytocin knockout mice, and body tissues, including but not limited to brain tissue, mammary gland tissue, blood, heart tissue, uterine tissue, bone tissue, and muscle tissue, are collected at different time points. The tissue distribution and content of oxytocin are then detected using liquid chromatography-mass spectrometry (LC-MS / MS). This experimental method allows for a wider range of experimental samples, not just focusing on brain tissue and blood; it also has higher sensitivity and stronger specificity; and by utilizing oxytocin gene knockout mice, it eliminates the influence of endogenous oxytocin, providing certain assistance for subsequent animal experiments and clinical research on oxytocin.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a mouse experimental method for detecting the tissue distribution of exogenous oxytocin. Background Technology

[0002] Oxytocin, primarily synthesized in neurons of the paraventricular nucleus and supraoptic nucleus of the hypothalamus, is composed of nine amino acids and is released into the bloodstream via the posterior pituitary gland after synthesis. Oxytocin acts as both a hormone in the peripheral blood and a neurotransmitter in the brain, causing a wide range of physical effects (e.g., uterine contractions during childbirth and milk production during breastfeeding). Recent research on oxytocin has mainly focused on its effects on social behavior and anxiety-related behaviors.

[0003] Liquid chromatography-mass spectrometry (LC-MS) is an analytical technique that combines the ability of liquid chromatography to separate mixtures with the ability of mass spectrometry to identify the characteristics of compounds. LC-MS combines the advantages of both techniques: first, liquid chromatography is used to preliminarily separate the components in a complex mixture; then, the separated components are directly introduced into a mass spectrometer for precise mass measurement and structural analysis. This approach not only improves the selectivity and sensitivity of the analysis but also effectively reduces background interference, making it suitable for the analysis of trace and even ultra-trace substances.

[0004] As research into the social role of oxytocin deepens, more and more studies are focusing on oxytocin administration for treatment. This research faces challenges related to the distribution and concentration of exogenous oxytocin in tissues. Current studies on the detection of exogenous oxytocin suffer from insufficient sensitivity (it may still be undetectable at very low concentrations) and cross-reactivity (due to the structural similarity between oxytocin and other related peptides (such as vasopressin), some detection methods may exhibit cross-reactivity, affecting the accuracy of the results). Therefore, a method with higher sensitivity and specificity is needed. Liquid chromatography-mass spectrometry (LC-MS) can analyze trace amounts of this substance. Furthermore, we have constructed our own oxytocin gene knockout mice (KO mice) to achieve systemic knockout of the oxytocin gene in these mice, eliminating interference from endogenous oxytocin in the detection of exogenous oxytocin and facilitating the study of its distribution in mouse tissues. Summary of the Invention

[0005] To address the aforementioned problems, this invention discloses a mouse experimental method for detecting the tissue distribution of exogenous oxytocin.

[0006] The first step in this invention is to confirm that the oxytocin gene knockout mouse (KO mouse) has indeed had the oxytocin gene knocked out systemically, which is mainly confirmed through genotype identification.

[0007] Furthermore, exogenous oxytocin was injected or sprayed nasally into KO mice. The injection was mainly carried out through two routes: subcutaneous injection and intraperitoneal injection.

[0008] Furthermore, animal tissues were collected at different time points after injection or nasal spray of exogenous oxytocin. The collected tissue samples included, but were not limited to, brain tissue, mammary gland tissue, blood, heart tissue, uterine tissue, bone tissue, and muscle tissue.

[0009] Further, the tissue samples undergo preliminary processing. This includes the following steps: grinding the tissue on ice and then treating it with different reagents to remove large molecular protein impurities.

[0010] Further, the tissue sample is purified. This includes the following steps: passing the pre-treated sample through a solid-phase extraction (SPE) column for further purification, followed by vacuum drying.

[0011] Further, the purified tissue sample is reconstituted to prepare a test solution. This includes the following steps: reconstitute the purified tissue sample with an acetonitrile:water solution containing 0.1% formic acid (v:v = 5:95), and then filter the insoluble substances using a positive pressure manifold to prepare the test solution.

[0012] Furthermore, the test solution is analyzed by high-performance liquid chromatography (HPLC). This includes the following steps: the test solution is passed through a reverse-phase silica gel column, using a 0.1% formic acid aqueous solution as mobile phase A and a 0.1% formic acid acetonitrile solution as mobile phase B to determine the oxytocin content in the test solution.

[0013] Further, mass spectrometry analysis was performed. This included the following steps: using a mass spectrometer in heated electrospray ionization positive ion mode, oxytocin was detected by timed multiple reaction monitoring. Attached Figure Description

[0014] Figure 1 Gene knockout diagram

[0015] Figure 2 Genotyping results of oxytocin gene knockout mice (KO mice)

[0016] Figure 3 Standard curve of oxytocin analyzed by HPLC

[0017] Figure 4 Systemic suitability chromatogram of oxytocin analyzed by HPLC

[0018] Figure 5 Systemic adaptability map of oxytocin analyzed by mass spectrometry Detailed Implementation

[0019] The invention will now be described in detail with reference to examples. Unless otherwise specified, all experimental methods are conventional and all experimental materials are purchased from commercial sources.

[0020] 1. Genotyping of oxytocin gene knockout mice (KO mice) includes the following steps: DNA products extracted from KO mice are subjected to PCR, followed by agarose gel electrophoresis.

[0021] The results are as follows Figure 2 As shown, homozygous KO mice show a band at 302bp, while heterozygous mice show bands at both 302bp and 417bp.

[0022] 2. Inject or nasally spray exogenous oxytocin into KO mice.

[0023] 3. Collect animal tissue samples at different time points. Animal tissues were collected at different time points after injection or nasal spray of exogenous oxytocin. The collected tissue samples included, but were not limited to, brain tissue, mammary gland tissue, blood, heart tissue, uterine tissue, bone tissue, and muscle tissue.

[0024] 4. Pretreatment of the tissue samples obtained in step 3. This includes the following steps: treating the tissue samples with acetic acid, acetonitrile, and ammonium acetate respectively, then further purifying them by passing them through a solid-phase extraction (SPE) column, followed by vacuum drying. Finally, the purified tissue samples are reconstituted with an acetonitrile:water solution containing 0.1% formic acid (v:v = 5:95), and the insoluble substances are filtered using a positive pressure manifold to prepare the test solution.

[0025] 5. High-performance liquid chromatography (HPLC) analysis, including the following steps:

[0026] 5.1. After preparing oxytocin standard solutions with different concentration gradients, the oxytocin standard curve was obtained by high performance liquid chromatography.

[0027] 5.2. Pass the test solution obtained in step 4 through a reverse silica gel column, using a 0.1% formic acid aqueous solution as mobile phase A and a 0.1% formic acid acetonitrile solution as mobile phase B to determine the content of oxytocin in the test solution.

[0028] The results are as follows Figure 3 , 4 As shown, the retention time of oxytocin is 2.93 min, and the standard curve is y = 1856.4x - 1855.4, R0. 2 =0.8776.

[0029] 6. Mass spectrometry analysis. This includes the following steps: oxytocin is detected using a timed multiple reaction monitoring method in heated electrospray positive ion mode.

[0030] The results are shown in the figure. The mass-to-charge ratio of the results in the figure is consistent with the mass-to-charge ratio of oxytocin, proving that oxytocin was successfully detected.

Claims

1. A mouse experimental method for detecting the tissue distribution of exogenous oxytocin, characterized in that, The experimental steps include the following: (1) We commissioned Jiangsu Jicui Yaokang Biotechnology Co., Ltd. to construct oxytocin gene knockout mice (KO mice). (2) Inject or nasally spray exogenous oxytocin into KO mice. (3) Animal tissues were collected at different time points after injection or nasal spray of exogenous oxytocin. (4) The exogenous oxytocin in the tissue sample described in (3) was quantitatively analyzed using liquid chromatography-mass spectrometry.

2. The mouse experimental method for detecting the tissue distribution of exogenous oxytocin according to claim 1, characterized in that, The oxytocin gene knockout mice (KO mice) described in experimental step (1) have three exons of the oxytocin gene deleted.

3. The mouse experimental method for detecting the tissue distribution of exogenous oxytocin according to claim 1, characterized in that, The injection methods described in experimental step (2) are extensive, with the main methods including subcutaneous injection, intraperitoneal injection, and nasal spray.

4. The mouse experimental method for detecting the tissue distribution of exogenous oxytocin according to claim 1, characterized in that, The body tissues collected in experimental step (3) include, but are not limited to, brain tissue, breast tissue, blood, heart tissue, uterine tissue, bone tissue, and muscle tissue.

5. The mouse experimental method for detecting the tissue distribution of exogenous oxytocin according to claim 1, characterized in that, The tissue sample described in experimental step (4) should be kept at a low temperature as much as possible, and the sample solution should be prepared after pretreatment using a solid phase extraction (SPE) column.

6. The mouse experimental method for detecting the tissue distribution of exogenous oxytocin according to claim 1, characterized in that, The liquid chromatography-mass spectrometry (LC-MS) method described in experimental step (4) uses a reverse-flow silica gel column with a 0.1% formic acid aqueous solution as mobile phase A and a 0.1% formic acid acetonitrile solution as mobile phase B. High-performance liquid chromatography (HPLC) is used to detect the oxytocin content in the test solution. Mass spectrometry mainly uses electrospray ionization to transfer ions from the non-volatile solution phase to the gas phase, making it suitable for compounds that are difficult to volatilize and thermally unstable.

7. Tissue distribution data of exogenous oxytocin in mice obtained by the method according to any one of claims 1-6.

8. An application of tissue distribution data of exogenous oxytocin in mice obtained by the method according to any one of claims 1-6, characterized in that, The tissue distribution data of exogenous oxytocin in mice can be used for relevant animal experiments or clinical trials.

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