CN112941012B - Preparation method and application of purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells - Google Patents
Preparation method and application of purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells Download PDFInfo
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Abstract
The invention discloses a preparation method and application of purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells. The preparation method of the invention overcomes the prior technical problems, can efficiently prepare the uterine cavity epithelial cells, the glandular epithelial cells and the stromal cells which are completely separated and have high purity, the required reagent only comprises digestive enzyme, and the prepared cells are all living cells, thus being further applied to the research of downstream various molecular biology and cell biology.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a preparation method and application of purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells.
Background
Endometrial epithelial cells are derived from the endometrium, which is the mucosa of the lining of the uterus. The endometrium is closely related to the implantation of the embryo and plays an important role in the research of reproductive physiology. The epithelium on the surface of the endometrium sinks deeply into the lamina propria to form a plurality of tubular uterine glands, and the terminal part of the uterine glands is frequently branched near the muscular layer. The surface epithelium is similar to the glandular epithelium in structure and consists of secretory cells and a small number of ciliated cells, but the glandular epithelium distributed in the uterine functional layer is sensitive to ovarian hormone response and has periodic changes. The lamina propria is thicker, the vessels are more abundant and there are a large number of less differentiated spindle or stellate cells, called stromal cells (stroma cells). The endometrium, which constitutes the innermost layer of the uterine wall of a female mammal, is located at the uterine cavity surface and plays an important role in the reproductive physiological activities of the mammal. The endometrium cavity epithelium, glandular epithelium and stroma cell prepared and cultured in vitro by separation have important significance for researching the physiological function, the drug action and the pathophysiology change under the action of various pathogenic factors.
The existing technology can separate and purify the epithelial cells and the stromal cells of the uterus, and culture the living cells in vitro. However, this method does not allow separate preparation of different epithelial cell types, i.e. luminal and glandular epithelium.
Different types of epithelial cells in the uterus can be separated by using a laser fiber cutting technology, but because the technology needs to carry out frozen sectioning on the uterus tissue currently, the separated cells die, and the technology cannot be further applied to the research of cell biology. Therefore, it is highly desired to develop a method for more efficiently and separately preparing living uterine cavity epithelial cells, glandular epithelial cells and stromal cells.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a preparation method of purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells, which can simultaneously, efficiently and respectively prepare high-purity uterine cavity epithelial cells, glandular epithelial cells and stromal cells, and the prepared high-purity uterine cavity epithelial cells, glandular epithelial cells and stromal cells are all living cells and can be further used for research in cell biology.
In a first aspect of the present invention, there is provided a cell digest comprising HBSS (Hank's Balanced Salt Solution), trypsin, and Dispase II.
In some embodiments of the present invention, the cell digest contains 840 to 880. Mu.L HBSS, 20 to 60. Mu.L 5% Trypsin and 80 to 120. Mu.L Dispase II per 1000. Mu.L.
In some preferred embodiments of the invention, the cell digest contains 860 μ L HBSS, 40 μ L5% Trypsin and 100 μ L Dispase II per 1000 μ L.
The solvent of the digestive juice according to the first aspect of the present invention is HBSS, which functions to put cells in a suitable osmotic pressure state, prevent cell rupture, etc.; trypsin is a Trypsin enzyme that breaks the junction between luminal epithelial cells and stromal cells; dispase II was designed to prevent clumping of the detached cells.
In a second aspect of the invention, a cell digest is provided, which contains Fetal Bovine Serum (FBS), collagen hydrolase IV (Collagenase IV) and neutral protease Dispase II.
In some embodiments of the present invention, the cell digest contains 680 to 720. Mu.L of 10% FBS-HBSS, 180 to 220. Mu.L of Collagenase IV and 80 to 120. Mu.L of Dispase II per 1000. Mu.L.
In some preferred embodiments of the present invention, the above cell digest contains 700. Mu.L of 10% FBS-HBSS, 200. Mu.L of Collagenase IV and 100. Mu.L of Dispase II per 1000. Mu.L.
The solvent of the digestive juice according to the second aspect of the present invention is HBSS containing 10% FBS, which functions to protect glandular cells from being over-digested on the basis of bringing the cells into a suitable osmotic state; collagenase IV is used for digesting extracellular matrix between the stromal cells and the glandular epithelial cells and separating the stromal cells and the glandular epithelial cells; dispase II, however, is intended to prevent clumping of the separated cells.
In a third aspect of the present invention, there is provided a method for preparing purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells, comprising the steps of:
(1) Taking a uterine sample, putting the uterine sample into the cell digestive juice according to claim 1 for digestion, and collecting uterine cavity epithelial cells;
(2) Placing the uterine sample with collected uterine cavity epithelium into the cell digestive juice according to claim 2 for digestion, and filtering to obtain filter residue as glandular epithelial cells;
(3) Collecting the filtrate, centrifuging, removing the supernatant, and culturing the precipitated cells to obtain the stromal cells.
In some embodiments of the invention, the uterine epithelial cells are collected in step (1) by washing, wherein the washing comprises HBSS.
In some preferred embodiments of the present invention, the HBSS is subjected to a pre-cooling treatment.
In some embodiments of the present invention, the step (2) further comprises cutting the uterine sample collected from the epithelium of the uterine cavity to a size of 0.4-0.6 mm 3 。
In some preferred embodiments of the present invention, the size after cutting is 0.5mm 3 。
In some embodiments of the invention, the filtration size in step (2) is greater than 200 mesh.
In some preferred embodiments of the present invention, the above-mentioned filtration size is 900 to 1000 mesh.
In some more preferred embodiments of the present invention, the above filtration size is 1000 mesh.
In some embodiments of the present invention, the culturing of the pelleted cells in step (3) is performed using DMEM/F12 medium containing 10% FBS, incubated at 35-38 ℃ for 30-40min, and the supernatant and non-adherent cells are removed.
According to a third aspect of the present invention, in some embodiments of the present invention, the uterine cavity epithelial cells, glandular epithelial cells and stromal cells prepared by the above preparation method have a purity of 90% or more.
According to an embodiment of the present invention, at least the following advantages are provided:
the preparation method overcomes the prior technical problems, can obtain the uterine cavity epithelial cells, the glandular epithelial cells and the stromal cells with high purity with high preparation efficiency, requires only digestive enzyme as a reagent, and can be further applied to the research of downstream various molecular biology and cell biology, and the prepared cells are all living cells.
In a fourth aspect of the present invention, there is provided a cell line comprising at least one of the uterine cavity epithelial cells, glandular epithelial cells or stromal cells prepared by the method of the third aspect of the present invention.
Since the implantation mode and placenta type of mouse and human embryo have great similarity, it has low breeding cost, fast breeding speed and easy gene manipulation, so it is widely used in animal model of human female reproduction and related diseases. However, in mammalian endometrium, there are two different epithelial types of luminal epithelium and glandular epithelium besides stromal cells, which have great difference in morphological structure and physiological function, and the research of differentiation process and function difference has great fundamental theoretical significance for the research of human reproductive health. The uterine cavity epithelial cells, glandular epithelial cells or stromal cells prepared by the preparation method of the third aspect of the invention can be used for constructing endometrial cell-related cell lines for further research and development.
In a fifth aspect of the present invention, there is provided a cell digest according to the first aspect of the present invention, and an application of the cell digest according to the second aspect of the present invention in construction of a germ cell model.
In a sixth aspect of the invention, there is provided the use of the cell line of the fourth aspect of the invention in disease diagnosis or therapeutic drug screening.
Drawings
FIG. 1 is a microscope image (4.7) of uterine cavity epithelial cells prepared in an example of the present invention;
FIG. 2 is a microscope image (. Times.4.7) of the uterine glandular epithelial cells prepared in the example of the present invention;
FIG. 3 is a microscope image (4.7) of stromal cells prepared in an example of the present invention;
FIG. 4 shows the expression of Caib1 (A), foxa2 (B) and Hoxa10 (C) genes in uterine epithelial cells, glandular epithelial cells and stromal cells prepared in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration only.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Preparation of digestive juice of epithelial cells in uterine cavity
The uterine cavity epithelial cell digestive juice is mainly used for separating the cavity epithelial cells in a uterine sample, and comprises the following specific components (per 1000 mu L):
860 μ L HBSS, 40 μ L5% Trypsin and 100 μ L Dispase II.
Preparation of uterus stromal cell digestive juice
The uterine stromal cell digestive juice is mainly used for separating stromal cells in a uterine sample without uterine cavity epithelial cells, and the specific components (per 1000 mu L) of the uterine stromal cell digestive juice are as follows:
700 μ L10% FBS, 200 μ L Collagenase IV and 100 μ L Dispase II.
Preparation of purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells
To verify the effectiveness of the preparation method in this example, the uterus of a mouse is used as the uterus sample, and the uterus sample is not limited to any form of sample, including various uterus tissue slices or other related materials of uterus origin.
The mice in this example were mice of the C57 strain, including pregnant mice (day 4 of pregnancy).
5% Trypsin, collagenase IV in this example were purchased from Gibco; dispase II was purchased from Roche.
The method comprises the following specific steps:
(1) Pretreatment of a uterus sample;
(2) The uterus sample is put into the uterine cavity epithelial cell digestive juice (the dosage is 1 mL/mouse) prepared in the embodiment for water bath digestion (the temperature is 37-40 ℃), and the uterine cavity epithelial cell digestive juice is shaken once at intervals of 30 minutes to ensure uniform digestion;
(3) The digested uterine sample is washed out of the intact uterine cavity epithelial cells from one side by using pre-cooled HBSS using the shear force of water flow and collected;
(4) Longitudinally cutting open the uterine sample on both sides after washing, and cutting into appropriate size (0.5 mm in size) under sterile environment 3 ) The small tissue of (2);
(5) Placing the small piece of tissue in the uterine stromal cell digestive juice (the dosage is 1 mL/mouse) prepared in the above embodiment to digest for 30min;
(6) Filtering with 200 mesh nylon screen to remove residual tissue after digestion, adding appropriate amount of precooled HBSS to wash the screen, and collecting filtrate;
(7) Filtering the filtrate with 1000 mesh screen, collecting the filtrate (the filtrate is stromal cells), and collecting the substance on the screen as glandular epithelial cells;
(8) Repeatedly filtering the glandular epithelial cells by using a 1000-mesh screen to remove residual stromal cells to obtain high-purity glandular epithelium;
(9) Centrifuging the stromal cells collected in the step (7) at 1200rpm for 5min, removing the supernatant, adding DMEM/F12 medium containing 10% FBS, culturing at 37 ℃ for 30-40min, and removing the supernatant and nonadherent cells to obtain high-purity stromal cells.
Wherein, if the uterus of a living mouse is adopted, the pretreatment of the uterus sample in the step (1) comprises the following steps:
(1) Precooling a culture dish filled with sterile HBSS on ice, and taking out a uterus and accessories after a female mouse is killed by a cervical dislocation method;
(2) After the ovaries, oviducts, cervix uteri, and mesentery and fat in the uterus and the attachments obtained in the step (1) are removed, placing the uterus of the mouse in a precooled culture dish filled with sterile HBSS for later use; wherein, if the uterus of the mouse is a pregnant uterus, M2 embryo flushing fluid is also needed to flush embryos in uterine cavities on both sides;
(3) Repeatedly washing uterus with new HBSS twice to obtain uterus sample.
Detection of preparation Effect
The types of cells (uterine cavity epithelial cells, glandular epithelial cells, and stromal cells) prepared in the above examples were observed under a microscope, and the results are shown in FIGS. 1 to 3.
From FIG. 1, it can be seen that the first digestive juice can be separated into clean intact luminal epithelium with no glandular epithelium. In FIG. 2, it can be seen that the second digestive juice separated the luminal epithelium and stromal cells in which the glandular epithelium is in the form of a pillar and has no sheet. From FIG. 3, it can be seen that the stromal cells are in a single cell state and are substantially free of epithelial cells.
Cell purity assay
The purity analysis of the various cells (uterine cavity epithelial cells, glandular epithelial cells and stromal cells) prepared in the above examples is carried out by the following specific steps:
1. the separated luminal epithelium, glandular epithelium and stromal cells are respectively collected in a centrifugal tube without RNase.
2. After adding Trizol to extract RNA and performing reverse transcription to form cDNA, the gene expression level of markers of the luminal epithelium, the glandular epithelium and the stromal cells (the internal references of markers of the luminal epithelium and the glandular epithelium are Rpl7 and the internal reference of a stromal cell marker is Rpl 19) is detected by using real-time quantitative fluorescent RCR.
The results are shown in fig. 4 and table 1.
TABLE 1 purity of various types of cells isolated in the examples of the invention
| Luminal epithelium LE | Glandular epithelium GE | Stromal cell S | |
| Purity of | About 91.4% | About 97.5% | About 80.4% |
FIG. 4 shows the gene expression of different specific genes of Luminal epithelial glandular Epithelium and stromal cells detected by real-time quantitative fluorescence PCR, and the purity of each cell calculated by the gene expression level (Table 1), showing that Luminal epithelial LE (Luminal epithelial) specifically expresses Luminal epithelial specific gene Caib1; the Glandular epithelial GE (Glandular Epithelium) specifically expresses Foxa2 Glandular epithelial specific genes and the stromal cell S (Stroma) specifically expresses Hoxa10 stromal cell specific genes, and the purity of the luminal epithelial Glandular Epithelium can reach more than 90 percent and the purity of stromal cells can reach more than 80 percent.
As can be seen from FIG. 4, the preparation method in the above embodiment can simultaneously obtain high-purity uterine cavity epithelial cells, glandular epithelial cells and stromal cells from a uterine sample, and compared with the conventional method (such as enzymatic method or direct digestion) which can only obtain a mixture of cavity epithelium and glandular epithelium and stromal cells, the preparation method in the above embodiment has better separation effect and more accuracy, and can be used for downstream research of various molecular biology and cell biology, wherein each type of living cells with extremely high purity are obtained.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Claims (6)
1. A preparation method of purified mouse uterine cavity epithelial cells, glandular epithelial cells and stromal cells is characterized by comprising the following steps:
(1) Taking a uterine sample, putting the uterine sample into cell digestive juice 1 for digestion, and collecting uterine cavity epithelial cells, wherein 860 mu L HBSS, 40 mu L5% trypsin and 100 mu L Dispase II exist in every 1000 mu L of the cell digestive juice 1;
(2) Placing the uterus sample with the collected uterine cavity epithelium into a cell digestive juice 2 for digestion and filtration, wherein the filter residue is glandular epithelial cells, and each 1000 mu L of the cell digestive juice 2 contains 700 mu L of 10% fetal calf serum, 200 mu L of collagen hydrolase Collagenase IV and 100 mu L of neutral protease Dispase II;
(3) Collecting the filtrate, centrifuging, removing the supernatant, and culturing the precipitated cells to obtain the stromal cells.
2. The method of claim 1, wherein the step (1) comprises collecting the epithelial cells of the uterine cavity by rinsing, and the rinsing comprises HBSS.
3. The method according to claim 1, wherein the step (2) further comprises cutting the uterine sample collected from the epithelium of the uterine cavity to a size of 0.4 to 0.6mm 3 。
4. The production method according to claim 1, wherein the filtration size in the step (2) is 1000 mesh.
5. The method according to claim 1, wherein the culture of the precipitated cells in step (3) is carried out by incubating the cells in DMEM/F12 medium containing 10% FBS at 35 to 38 ℃ for 30 to 40min and removing the supernatant and the nonadherent cells.
6. The use of the cell digest 1 and the cell digest 2 of claim 1 for constructing a mouse endometrial cell model.
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| US8252591B2 (en) * | 2004-05-07 | 2012-08-28 | Whitehead Institute For Biomedical Research | Hormone responsive tissue culture system and uses thereof |
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| JP2020130055A (en) * | 2019-02-20 | 2020-08-31 | 学校法人東京女子医科大学 | Endometrium-like tissue and method for producing the same |
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