This patent application claims the benefit and priority of Chinese Patent Application No. 202211247274.1, filed with the China National Intellectual Property Administration on Oct. 12, 2022, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.
A computer readable XML file entitled “Sequence Listing”, that was created on Oct. 12, 2023, with a file size of about 8,303 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety.
The present disclosure belongs to the field of biotechnology, and specifically relates to a method for separating a neural crest derived cell from peripheral blood.
Neural crest cells are a temporary group of cells that are produced by embryonic ectoderm cells during development and can produce a variety of cell types. This cell group originates from the ectoderm at an edge of the neural tube, and can migrate widely to different parts of the body after undergoing epithelial-to-mesenchymal transition (EMT), so as to form a large number of different tissues and organs (Alkobtawi M, Ray H, Barriga E H, et al. Characterization of Pax3 and Sox10 transgenic Xenopus laevis embryos as tools to study neural crest development [J]. Developmental biology, 2018, 444: S202-S208.). After migrating to a target tissue, the neural crest derived cells maintain their pluripotency in the state of neural crest stem cells, and the neural crest stem cells in the tissue further promote the production of neural crest derived derivatives. Neural crest derived cells have been found in the intestine, heart, cornea, and bone marrow of adult mammals, and the neural crest derived cells have been successfully extracted from tissues and then subjected to primary culture for experimental research on the neural crest derived cells.
Neural crest derived cells not only have stem cell characteristics but also participate in tissue repair processes. Research results have shown that when adult tissues derived from different neural crest sources respond to injury or stress, specialized cells derived from neural crest cells dedifferentiate or activate cells with neural crest stem cell characteristics remaining in the tissue. The neural crest derived cells can also repair tissues through paracrine signaling (Parfejevs V, Antunes A T, Sommer L. Injury and stress responses of adult neural crest-derived cells. Dev Biol. 2018; 444 Suppl 1: S356-S365. doi: 10.1016/j.ydbio.2018.05.011.).
A mT/mG;Wnt1-Cre mouse is obtained by crossing a Wnt1-Cre transgenic mouse with an mT/mG double-fluorescent reporter mouse. The Wnt1-Cre transgenic mouse line is widely used to study neural crest and its derivatives; while the mT/mG double-fluorescent reporter mouse has a cell membrane surface marked with a tdTomato protein before Cre recombination occurs, and then the cell membrane surface emits red fluorescence. When the mT/mG double-fluorescent reporter mouse is crossed with the Wnt1-Cre transgenic mouse, a Cre recombinase can cut out two Loxp sites located next to a tdTomato sequence under the control of a Wnt1 gene. The tdTomato sequence is connected to an enhanced green fluorescent protein (EGFP) sequence, thereby expressing an EGFP protein. As a result, the cell membrane glows green at this time. Accordingly, in the mT/mG;Wnt1-Cre mouse, neural crest derived cells are marked with the EGFP protein and then emit green fluorescence (Muzumdar M D, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007; 45(9): 593-605. doi:10.1002/dvg.20335.).
In view of the problems existing in the prior art that collecting neural crest derived cells is cumbersome and invasive, the present disclosure provides a method for separating a neural crest derived cell from peripheral blood.
The present disclosure provides a method for separating a neural crest derived cell from peripheral blood, including separating the neural crest derived cell from peripheral blood of a subject to be separated.
Preferably, the subject to be separated is an experimental animal.
Preferably, the experimental animal is selected from the group consisting of an experimental mouse, an experimental rat, and an experimental monkey.
More preferably, the method for separating a neural crest derived cell from peripheral blood specifically includes the following steps:
More preferably, the peripheral blood is collected from an orbital vein when the experimental animal is the experimental mouse.
More preferably, an mT/mG;Wnt1-Cre mouse obtained by hybridization screening of a Wnt1-Cre transgenic mouse and an mT/mG double-fluorescent reporter mouse is used when the experimental animal is the experimental mouse.
More preferably, the peripheral blood is collected to separate the neural crest derived cell after silicosis fibrosis is induced in the mT/mG;Wnt1-Cre mouse by silica.
Even more preferably, the method for separating a neural crest derived cell from peripheral blood specifically includes the following steps:
The present disclosure has the following advantages:
In the present disclosure, the neural crest derived cell separated by the method has been labeled with EGFP. Under 488-nm wavelength excitation, a cell membrane surface can emit a green fluorescent signal, making it easy to distinguish.
In the present disclosure, a mononuclear cell is separated from the peripheral blood and then directly cultured, thereby maximizing the use of a neural crest stem cell with a differentiation potential to avoid loss of the neural crest stem cell.
In the present disclosure, the method establishes a methodology for the separation and culture of neural crest derived cells from mouse peripheral blood, laying a foundation for such research.
In the method of the present disclosure, a sample to be separated is derived from the peripheral blood. The method shows less trauma and low cost. Most importantly, compared to extracting the neural crest derived cell from tissues, the neural crest derived cell extracted from the peripheral blood can be used clinically as a type of biomarker.
mT/mG;Wnt1-Cre Mice:
The Wnt1-Cre transgenic mouse (129S4.Cg-E2f1 Tg(Wnt1-Cre)2Sor/J; Stock No: 022137) and the mT/mG double-fluorescent reporter mouse (B6.129(Cg)-Gt(ROSA)26Sor tm4 (ACTB-tdTomato-EGFP)Luo/J; Stock No: 007676) both are purchased from Jackson Laboratory. The Wnt1-Cre transgenic mouse line is widely used to study neural crest and its derivatives as well as the development of midbrain, and is currently the most common tool mouse line for studying neural crest derived cells. Before Cre recombination in the mT/mG double-fluorescent reporter mouse, the fluorescent expression of tdTomato (mT) localized on the cell membrane spreads throughout the cells/tissues. Cells expressing Cre recombinase (and cell lineages derived from these cells) have fluorescent expression of EGFP (mG) that is localized to the cell membrane, emitting green fluorescence instead of red fluorescence. Therefore, when 8-12-week-old Wnt1-Cre transgenic mouse is crossed with mT/mG double-fluorescent reporter mouse to obtain the mT/mG;Wnt1-Cre mouse as offspring, the cell membrane surface of cells or tissues derived from neural crest can exhibit green fluorescence. After crossing the Wnt1-Cre transgenic mouse with the mT/mG double-fluorescent reporter mouse, four genetic models of offspring mice can be obtained, including a wild-type mouse, a Wnt1-Cre transgenic mouse, an mT/mG double-fluorescent reporter mouse, and an mT/mG;Wnt1-Cre mouse, where the mT/mG;Wnt1-Cre mouse shows a birth probability of about 20%. Therefore, it is necessary to extract a DNA from offspring mice to allow genotype identification and screening.
The Wnt1-Cre transgenic mouse (129S4.Cg-E2f1 Tg(Wnt1-Cre)2Sor/J; Stock No: 022137) and the mT/mG double-fluorescent reporter mouse (B6.129(Cg)-Gt(ROSA)26Sor tm4 (ACTB-tdTomato-EGFP)Luo/J; Stock No: 007676) both were purchased from Jackson Laboratory. After mating, the mT/mG;Wnt1-Cre transgenic mouse was selected through genetic identification by a Southern method. EGFP-positive cells in this mouse were considered to be neural crest derived cells.
1. Genotype Identification of the mT/mG;Wnt1-Cre Mice
1.1. Extraction of Mouse DNA
1.2. PCR System
A total volume of Wnt1-cre gene and EGFP genotype reaction systems was 25 μL.
A Wnt1-cre gene PCR reaction system included the following components:
template DNA, 1 μL;
2-Hieff HotStart PCR Genotyping Master Mix(with Dye), 12.5 μL;
Transgene Forward 16773, 1 μL;
Transgene Reverse 16774, 1 μL;
Internal Positive Control8744, 1 μL;
Internal Positive Control8745, 1 μL;
ddH20, making up to 25 μL.
An EGFP gene PCR reaction system included the following components:
template DNA, 1 μL;
2-Hieff HotStart PCR Genotyping Master Mix(with Dye), 12.5 μL;
Common12177, 1 μL;
Mutant Forward 30297, 1 μL;
Wild type Forward 30298, 1 μL;
ddH20, making up to 25 μL.
1.3. PCR Reaction Conditions
The reaction conditions for Wnt1-cre gene and EGFP genotype were as follows:
In the present disclosure, the genotype detection of the mT/mG;Wnt1-Cre transgenic mouse was shown in
The mT/mG;Wnt1-Cre mice at 8 to 12 weeks were randomly divided into a physiological saline group and a SiO2 model group. 1 d before modeling, an appropriate amount of SiO2 dust was heated in a clean petri dish at 160° C. for 2 h, cooled slightly and then ground in an agate mortar for 30 min. An obtained powder was mixed with sterile physiological saline to form a 25 mg/mL suspension for later use. The mice were anesthetized by intraperitoneal injection of 4% chloral hydrate at a dose of 0.01 mL/g. After 3 min to 4 min, when the mouse's back-and-forth reflex and toe-pinching reflex disappeared, the mouse's abdomen was placed downward, with its upper incisors suspended on a thin line on an operating table, and its neck was illuminated with a cold light source. The mouse's tongue was pulled out with small tweezers of left hand, the mouse's upper jaw was held using one end of small straight tweezers on right hand, with other end against the base of the mouse's tongue. The field of view in mouse's mouth was exposed as possible to observe the position of vocal cords by assistance of the cold light source. When seeing the trachea being translucent, a small bright spot could be seen if watching closely, which was the glottis. When the small bright spot became larger (that is, when the glottis was most opened), a 22G indwelling needle was promptly inserted into the trachea, the needle core was withdrawn, a 1 mL syringe was inserted into the cap of the 22G indwelling needle, tightened by rotating, such that tracheal intubation was completed. The mice in the model group were perfused with 0.1 mL of a silica suspension through the trachea via the oral cavity according to the above method, while the mice in the physiological saline group were perfused with an equal volume of 0.1 mL of sterile physiological saline. The mice were made stand upright and gently shaken from side to side to help the SiO2 suspension distribute evenly while avoiding suffocation of the mouse's organs due to the dust. On the 30th day after SiO2 treatment, mouse lung tissues were fixated with 4% (mass percentage) paraformaldehyde, dehydrated with 30% (mass percentage) sucrose solution, embedded by OCT, and then cut into 6 m frozen sections to allow immunofluorescence detection. The frozen sections were permeabilized with 0.5% Triton X-100, blocked with 5% BSA at 37° C. for 30 min, incubated with primary antibodies such as SOX10 (1:50, abcam) and Acta2 (1:500, CST) at 4° C. for 12 h, incubated with fluorescent secondary antibody Alexa Flour647 (1:1000, abcam) at room temperature for 1 h, stained with DAPI (Beyotime) on the nuclei for 2 min, and then washed with PBS to remove excess DAPI. After sealing with anti-fluorescence quenching mounting solution, the slides were observed under a Zeiss LSM880 ultra-high resolution inverted confocal microscope.
An abnormally aggregated population of neural crest derived cells was found in silica-induced silicosis fibrosis in mT/mG;Wnt1-Cre mouse. This cell population not only had the characteristics of neural crest stem cells, but also had the characteristics of mesenchymal cells, as shown in
This discovery provided a new idea for exploring the mechanism of neural crest derived cells in silicosis fibrosis. At the same time, flow cytometry analysis was conducted to find the presence of EGFP-positive cells in the peripheral blood of mice, that is, neural crest derived cells (
In order to better and in-depth study the role of this cell population in silicosis fibrosis, EGFP-positive cells were planned to be extracted from the peripheral blood of mT/mG;Wnt1-Cre mouse for in vitro culture. This method had not been reported yet.
The neural crest derived cells were separated from the mT/mG;Wnt1-Cre transgenic mouse, including the following steps:
The separated neural crest derived cells were observed.
Number | Date | Country | Kind |
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202211247274.1 | Oct 2022 | CN | national |