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Method for separating rat hypothalamic arcuate nucleus neurons

A technique for the arcuate nucleus and hypothalamus, applied in the field of isolating the arcuate nucleus neurons of the rat hypothalamus, can solve the problems of lack, affecting the spatial structure of the marker protein connection end, low dependence on instruments and platforms, etc., and achieve simple operation Effect

Pending Publication Date: 2022-04-05
上海昆盟生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are some false positives in this method, and the fusion protein will affect the spatial structure of the marker protein junction, which may destroy the normal function of the marker, and the preparation cycle of transgenic animals is long, expensive, and requires continuous feeding and maintenance. This will increase the cost of manpower and financial resources, which cannot be accepted by general scientific research units
[0005] All in all, there is currently a lack of a method that can effectively and accurately separate different types of neuron nuclei in the hypothalamus, and this method is simple to operate, inexpensive, and less dependent on instruments and platforms, and can be popularized in a large number of scientific research institutions and accept

Method used

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  • Method for separating rat hypothalamic arcuate nucleus neurons
  • Method for separating rat hypothalamic arcuate nucleus neurons
  • Method for separating rat hypothalamic arcuate nucleus neurons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The invention provides a method for isolating neurons of the arcuate nucleus of the rat hypothalamus, specifically as follows.

[0043] Step 1. Identify several markers with the most significant differential expression of arcuate nucleus neuron nuclei in the hypothalamus; the specific method is as follows:

[0044] S11. Take the SD rat whole brain slice, place it on the capture area of ​​the glass slide, fix and permeabilize the whole brain slice, and the probe on the capture area captures the RNA released by the cells;

[0045] S12. Dimensional reduction and clustering of the cell populations on the slide into 14 cell populations, among which the position of the No. 9 population is consistent with the arcuate nucleus located on the brain map; find the top 9 cells with the most significant differential expression in the No. 9 cell population markers, respectively Fezf1, Kiss1, Ghrh, Sox14, S100g, Ces1d, Gck, Crhr2 and Six6; figure 1 It is a violin plot of the expressio...

Embodiment 2

[0060] Step 1 and step 2 in Embodiment 2 are completely the same as Embodiment 1, and will not be described again in this embodiment.

[0061] Step 3. Use flow cytometry to sort the positive cells of the S100g marker in the hypothalamus, which is the arcuate nucleus neuron nucleus. The protein expressed by the S100g marker is located on the cell membrane of the neuron. The protein expressed by the S100g marker is as follows: The nucleotide sequence shown in SEQUENCE LISTING ID NO.6. The specific method is as follows:

[0062] S31. Take out 30 parts of the hypothalamus of SD rats 45 days after birth, and prepare a single cell suspension; place the single cell suspension in ice phosphate buffered saline containing 10% fetal bovine serum and 1% sodium azide Resuspend in solution to approximately 5x 10 6 cells / mL;

[0063] S32. Add 30 μL of 100 g antibody to each resuspended cell solution, add an equal volume of ice ethanol, fix overnight at -20 ° C, take it out the next day, c...

Embodiment 3

[0067] Step 1 and step 2 in Embodiment 3 are completely the same as Embodiment 1, and will not be described again in this embodiment.

[0068] Step 3, using flow cytometry to sort the positive cells of the Gck marker in the hypothalamus, which is the arcuate nucleus neuron nucleus, the protein expressed by the Gck marker is located inside the neuron cell, and the protein expressed by the Gck marker is as follows: The nucleotide sequence shown in SEQUENCE LISTING ID NO.5. The specific method is as follows:

[0069] S31. Take out 30 parts of the hypothalamus of SD rats 45 days after birth, and prepare a single cell suspension; place the single cell suspension in ice phosphate buffered saline containing 10% fetal bovine serum and 1% sodium azide Resuspend in solution to approximately 5x 10 6 cells / mL;

[0070] S32. Add 30 μL of Gck antibody to each resuspended cell solution, add an equal volume of ice ethanol, fix overnight at -20°C, take it out the next day, centrifuge at 400...

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Abstract

The invention discloses a method for separating rat hypothalamic arcuate neurons. The method comprises the following steps: finding out a plurality of markers with most significant differential expression of arcuate neuron nuclei in hypothalamus; according to the expression difference of the markers in other cell populations, screening out unique markers with significant high expression in the arcuate nucleus region, wherein the markers are respectively Gck, S100g, Six6 and Sox14; positive cells containing four markers Sox14, S100g, Gck and Six6 in hypothalamus are sorted by adopting flow cytometry, and the concentration of arcuate nucleus neuron nuclei in the obtained positive cell population is relatively high; based on a space transcriptome technology, separation is realized through flow cytometry sorting, so that the separation of the arcuate nucleus neuron nuclei is realized; according to the method for separating the hypothalamic arcuate nucleus neuron nuclei, transgenic rats do not need to be cultured, the operation cost is low, the dependence degree on instruments and platforms is low, and the method is suitable for being popularized and popularized in research and development institutions.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for isolating neurons of the arcuate nucleus of the hypothalamus of rats. Background technique [0002] Neurons are the basic functional units that make up the structure of the nervous system. Neurons in the central nervous system have two typical organizational structures, one is a layered structure, such as the cerebral cortex; the other is a nucleus structure, such as the hypothalamus. The hypothalamus is a relatively conserved and highly heterogeneous region in the evolution of the animal brain. Its internal neuron nuclei are diverse in types and are critical to endocrine, sleep and circadian rhythms, reproduction and sexual behavior, and diet and energy balance. Regulation. [0003] The hypothalamus is located on the ventral surface of the brain, below the subthalamic hook, forms the lower wall of the third ventricle, and extends downward to connect with the p...

Claims

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Application Information

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IPC IPC(8): C12N5/0793G01N15/14
Inventor 沈根宏沈益行胡延平周莎莎臧少莲余金成董卓
Owner 上海昆盟生物科技有限公司
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