CN113493771A - Human eye orbital fat precursor cell line, construction method and application - Google Patents

Human eye orbital fat precursor cell line, construction method and application Download PDF

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CN113493771A
CN113493771A CN202010197820.XA CN202010197820A CN113493771A CN 113493771 A CN113493771 A CN 113493771A CN 202010197820 A CN202010197820 A CN 202010197820A CN 113493771 A CN113493771 A CN 113493771A
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张芳
金其煌
张玲
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Shanghai First Peoples Hospital
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Abstract

The invention discloses a human eye orbital adipose precursor cell line, a construction method and application thereof. The construction method of the cell line comprises the following steps: step 1, separating and culturing human orbital adipose precursor cells from human orbital adipose tissues; step 2, transfecting the human orbital adipose precursor cells with the vector connected with the hTERT gene to enable the cells to over-express the hTERT; and 3, screening the human orbital fat precursor cells with negative CD90 expression. The cell line can be used as a cell research model for drug screening.

Description

Human eye orbital fat precursor cell line, construction method and application
Technical Field
The invention relates to the field of biotechnology, in particular to a human eye orbital fat precursor cell line, a construction method and application.
Background
Thyroid-associated ophthalmopathy (TAO) is an organ-specific autoimmune Disease with exophthalmos as an important sign, mainly caused by hyperthyroidism (GD). The onset of TAO is closely related to inflammation and proliferation of orbital adipose tissue. The pathological changes of TAO cause the increase of the pressure behind the eyes and press the optic nerve to cause optic neuropathy; the protruding eyeball exposes the cornea and is easily damaged. These pathologies not only cause visual dysfunction, even blindness, but also give rise to a malformed appearance, affecting the psychosocial health of the patient. The quality of life of patients with TAO is severely affected, which is comparable to that of patients with diabetes or certain cancers. The incidence of TAO is high, and is the most prominent orbital disease. Global GD prevalence is between 0.2-1.3%, with proportions that cause mild and moderate-severe TAO of about 20-30% and 5%, respectively. Patients with moderate/severe TAO need to receive medical or surgical treatment to prevent or correct impaired vision and abnormal appearance. However, since the pathogenesis of TAO is still not fully elucidated, the treatment of TAO cannot be symptomatic. Therefore, the deep research on the pathogenesis of the TAO has important significance on pathological understanding of the TAO and development of novel therapeutic drugs.
One important cause of the onset of thyroidism is the proliferation of adipose tissue. In the literature relating to immortalized adipocytes, chinese patent CN100595274C discloses a new human precursor adipocyte cell line that can differentiate into adipocytes, and its use for the development of drugs and food ingredients and supplements against obesity, diabetes and cardiovascular diseases. This patent studies the function of white adipose tissues in the body and provides a method for studying the effect of a new drug or food ingredient on white adipose tissues. Chinese patent CN109628404A discloses a method for establishing a porcine subcutaneous adipose precursor cell immortalized cell line and application thereof. Human orbital adipocytes, which are developed from ectodermal cells and distinguished from other mesodermal-derived adipocytes, differ from other sources of lipoprecursor cells with respect to external stimuli, and thus cannot be used in the development of drugs and food ingredients and supplements for treating thyroidism using the cell lines used in the above patents. Meanwhile, human orbital adipose precursor cells are derived from neural spinal cells of ectoderm and have the capacity of differentiating into nerve cells and corneal epithelial cells, and adipose precursor cells from other sources do not have the function, so that the human orbital adipose precursor cells can also be used as a cell model for researching retinal nerve and corneal regeneration.
The current fat cell model mainly comprises 3T3-L1 or subcutaneous fat cells, and lacks a fat cell model constructed for thyroid-related eye diseases.
Disclosure of Invention
The invention aims to provide a human eye orbital fat precursor cell line, a construction method and application thereof, and the cell line can be used for screening medicaments for thyroid-related eye diseases as an adipocyte research model.
In order to achieve the above object, the present invention provides a human orbital adipose precursor cell line obtained by immortalizing human orbital adipose precursor cells.
Preferably, the cell line is obtained by immortalizing human orbital adipose precursor cells derived from human orbital adipose tissue, wherein the orbital adipose tissue is derived from healthy people or patients with thyroidism.
The invention also provides a construction method of the human orbital adipose precursor cell line, which comprises the following steps: step 1, separating and culturing human orbital adipose precursor cells from human orbital adipose tissues; step 2, transfecting the human orbital adipose precursor cells with the vector connected with the hTERT gene to enable the cells to over-express the hTERT; and 3, screening the human orbital fat precursor cells with negative CD90 expression.
Preferably, the expression vector of the hTERT gene in the step 2 is pBABE-hTERT-Hygro plasmid.
Preferably, step 3 specifically comprises: and adding a CD90 antibody into the human orbital adipose precursor cells, and screening the human orbital adipose precursor cells through anti-IgG microbeads by magnetic beads to obtain human orbital adipose precursor cells with negative CD90 expression.
The invention also provides application of the human orbital fat precursor cell line, which is used for screening medicines capable of preventing or treating thyroid-related eye diseases.
The invention also provides another application of the human orbital adipose precursor cell line, which is used for screening a medicine capable of preventing or treating orbital adipose tissue inflammation.
The invention also provides another use of the human orbital adipose precursor cell line for screening or identifying a compound capable of controlling the differentiation of the human orbital adipose precursor cells into nerve cells or corneal epithelial cells, wherein the compound comprises a protein.
The invention also provides another application of the human orbital adipose-derived precursor cell line, and the application of the human orbital adipose-derived precursor cell line as a human orbital adipose-derived cell differentiation research model.
The invention also provides another application of the human orbital adipose precursor cell line, and the application of the human orbital adipose precursor cell line as a cell model for researching retinal nerve and cornea regeneration.
Has the advantages that:
(1) the human orbital adipose precursor cell line can select orbital adipose tissues from clinical thyroid-associated eye disease patients, and is suitable for screening thyroid-associated eye disease drugs.
(2) The human orbital fat precursor cell line is an immortalized cell and can be passaged for a long time.
(3) The human orbital adipose precursor cell line can be differentiated into mature adipocytes and can be used as a model for researching the differentiation of the adipocytes.
Drawings
Fig. 1 is a flow chart of a method for constructing a human orbital adipose precursor cell line provided by the invention.
Fig. 2A is the microscopic morphology of the primary and immortalized orbital adipose precursor cells of the examples.
Fig. 2B shows the time to doubling of the number of growing cells for 2 primary and immortalized orbital adipose precursor cells of the example.
Fig. 3 is a fluorescence microscope image of cells before and after negative screening of immortalized human orbital adipose precursor cells with CD90 antibody in example, in which CD90 positive cells were detected with CD90 antibody (green) and nuclei were shown by DAPI staining (blue).
FIG. 4A is a microscopic image of the example, which shows no CD90 antibody negative screen and CD90 negative cells, after inducing differentiation into adipocytes, stained with oil red.
FIG. 4B is a graph showing the results of qRT-PCR detection of the expression of the adipocyte marker gene aP2 before and after differentiation of the cells of the examples, and after differentiation of the cells without CD90 antibody negative screen and CD90 negative cells into adipocytes.
FIG. 5 is a graph comparing the results of subcutaneous fat precursor cells and the human orbital fat precursor cell line of the present invention for drug screening.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
Examples
FIG. 1 shows a flow chart of the method for constructing the human orbital adipose precursor cell line of the invention. The human orbital adipocyte precursor cell line of the invention is constructed by the following method.
1) Isolation of primary adipose cells from human orbit
The orbit adipose tissues are from healthy people and patients with thyroidism, the tissues are rinsed with normal saline for 1-2 times, the blood is removed, the tissues are cut into pieces by using sterilized scissors, and the pieces are digested for 30 minutes in a 37-degree water bath kettle. After digestion, the cells are centrifuged by a cell screen of 100um at 600rpm for 5min and then are spread in a culture dish to be cultured to obtain the human eye orbital adipose precursor cells. In the invention, the orbit adipose precursor cell line of the invention can be constructed by adopting the orbit adipose tissues from healthy people and patients with thyromegaly, and the invention does not find that the orbit adipose precursor cell line of the human from healthy people or patients with thyromegaly has different characteristics. The human orbital adipose precursor cell line in the following steps is a human orbital adipose precursor cell line derived from healthy people.
2) Immortalization of primary cells in the orbit
Transfecting pBABE-hTERT-Hygro plasmid into Ampho cells, collecting the supernatant of culture solution of the Ampho cells after 48 hours of incubation, passing the supernatant through a 0.45 mu M filter membrane, adding 8 mu g/ml polybrene (polybrene), then infecting the human orbital adipose precursor cells in the step 1, adding Hygromycin (Hygromycin) after 48 hours of incubation to start screening, and removing antibiotics after two weeks; immortalized human orbital adipose precursor cells were obtained.
As shown in fig. 2A, two sets of cell lines (cell line #1 and cell line #2) were taken for observation for the morphology of primary and immortalized orbital adipose precursor cells under the microscope. Fig. 2B shows the time to doubling of the number of growing cells of 2 primary and immortalized orbital adipose precursor cells of the example, and the results show a decrease in the time to doubling of the number of growing cells of immortalized human orbital adipose precursor cells.
3) CD90 negative human orbital adipogenic precursor cell screen
And (3) mixing 200 mu L of the cells of the human orbit adipose precursor cells subjected to immortalization treatment in the step (2) with the CD90 antibody, incubating for 20min at room temperature, then centrifuging for 5min at 600rpm, removing supernatant, resuspending and precipitating with PBS, adding anti-mouse IgG microbeads, incubating for 20min at room temperature respectively, centrifuging for 5min at 600rpm, resuspending the cells with 500 mu L of culture solution, and blowing and uniformly mixing to obtain a cell resuspension solution. The MACS separator (MACS seater) is wetted by 500 mu L of culture solution before use, 500 mu L of cell resuspension is passed through a MACS seater magnetic column, and the cell suspension obtained by column chromatography is the CD90 negative human orbital fat precursor cell, namely the human orbital fat precursor cell line provided by the invention.
As shown in fig. 3, which is a fluorescence microscope image of cells before and after negative screening with CD90 antibody, CD90 positive cells were detected with CD90 antibody (green), and nuclei were shown by DAPI staining (blue). FIG. 3A is a graph showing the results of the detection of cells before screening with the CD90 antibody, and FIG. 3D is a graph showing CD90 negative human orbital precursor cells obtained after screening, in which no fluorescence is observed; DAPI staining results are shown in fig. 3B and fig. 3E, and cell nuclei both showed blue color; the results of the fusion of the cell images after staining with the CD90 antibody and DAPI are shown in FIG. 3C and FIG. 3F. The results show that successful screening resulted in human orbital fat precursor cells that were negative for CD 90.
4) Differentiation of fat cells;
after the CD90 negative human orbital precursor cells were fully confluent, the following inducers were added for fat differentiation: includes 5. mu.g/ml insulin, 1. mu.M dexamethasone, 0.5mM Isobenzoate (IBMX) and 1. mu.M rosiglitazone. Fresh medium (10% FBS + 5. mu.g/ml insulin + 1. mu.M rosiglitazone) was changed on days 2, 4, 6, 8, 10, 12. After differentiation was complete, oil red was stained and the expression of adipocyte marker gene aP2 was analyzed using qRT-PCR assay.
As shown in fig. 4A, the results of oil red staining after differentiation of the selected CD90 negative human orbital precursor cells and the cells not selected with the CD90 antibody show that the cells selected with the CD90 antibody have stronger adipocyte differentiation ability than the cells not selected with the CD90 antibody.
As shown in fig. 4B, the adipocyte marker gene aP2 was not expressed in the cells that were not induced to differentiate, and the cells that were not selected with the CD90 antibody were contaminated with CD90 positive cells that could not differentiate into adipocytes, and the detected relative expression level of gene aP2 was lower than that of the cells that were selected with the CD90 antibody.
5) Reaction to drugs
The two drugs act on subcutaneous fat precursor cells and human orbital fat precursor cells (by adopting the human orbital fat precursor cell line of the invention) respectively, and the drug response of the two cells is observed.
Drug A (rimonabant ) was added to the cell culture solution at concentrations of 0, 100nM and 250nM, and drug B (pargyline, Youjining) was added at concentrations of 0, 3mM and 6 mM.
Fig. 5 a is a microscope image of subcutaneous fat precursor cells at different drug concentrations, and fig. 5C is a microscope image of human orbital fat precursor cells at different drug concentrations.
FIG. 5B shows the relative expression level of aP2 gene detected by q-PCR assay after differentiation of subcutaneous adipocyte precursor cells into adipocytes. FIG. 5D shows the relative expression level of aP2 gene detected by q-PCR assay after induced differentiation of human orbital adipose precursor cells into adipocytes. Fig. 5B and fig. 5D show that the two cells respond differently to the drug, and therefore different adipocyte models should be used for different experimental purposes in drug screening.
In conclusion, the invention provides a human orbital adipocyte precursor cell line which can be used as a cell model for drug screening.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Claims (10)

1. A human orbital adipose precursor cell line obtained by immortalizing human orbital adipose precursor cells.
2. The cell line according to claim 1, wherein the cell line is derived from immortalized human orbital adipose precursor cells derived from human orbital adipose tissue derived from healthy human or from patients with thyroidism.
3. The method of constructing a human orbital adipogenic precursor cell line as claimed in any of claims 1 to 2, comprising the steps of:
step 1, separating and culturing human orbital adipose precursor cells from human orbital adipose tissues;
step 2, transfecting the human orbital adipose precursor cells with the vector connected with the hTERT gene to enable the cells to over-express the hTERT;
and 3, screening the human orbital fat precursor cells with negative CD90 expression.
4. The human orbital adipogenic precursor cell line of claim 3, wherein the expression vector for the hTERT gene in step 2 is the pBABE-hTERT-Hygro plasmid.
5. The human eye orbital adipogenic precursor cell line of claim 3, wherein step 3 comprises: and adding a CD90 antibody into the human orbital adipose precursor cells, and screening the human orbital adipose precursor cells through anti-IgG microbeads by magnetic beads to obtain human orbital adipose precursor cells with negative CD90 expression.
6. Use of the human orbital adipogenic precursor cell line of claim 1 or 2 for screening for drugs capable of preventing or treating thyroid-related eye diseases.
7. Use of the cell line of human orbital adipose precursors of claim 1 or 2 for screening drugs capable of preventing or treating orbital adipose tissue inflammation.
8. Use of a human orbital adipogenic precursor cell line as claimed in claim 1 or 2 for screening or identifying compounds capable of controlling the differentiation of human orbital adipogenic precursor cells into nerve cells or corneal epithelial cells, said compounds comprising proteins.
9. Use of the human orbital adipocyte precursor cell line of claim 1 or 2 as a model for human orbital adipocyte differentiation studies.
10. Use of the human orbital adipogenic precursor cell line of claim 1 or 2 as a cell model for studying retinal nerve and corneal regeneration.
CN202010197820.XA 2020-03-19 2020-03-19 Human eye orbital fat precursor cell line, construction method and application Pending CN113493771A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050008621A1 (en) * 2001-10-06 2005-01-13 James Kirkland Preadipocyte cell strains and uses therefore
CN109628404A (en) * 2018-12-18 2019-04-16 浙江大学 The construction method and purposes of Preadipocyte immortalized cell line under pigskin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050008621A1 (en) * 2001-10-06 2005-01-13 James Kirkland Preadipocyte cell strains and uses therefore
CN109628404A (en) * 2018-12-18 2019-04-16 浙江大学 The construction method and purposes of Preadipocyte immortalized cell line under pigskin

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GENIECE M. LEHMANN等: "Novel anti-adipogenic activity produced by human fibroblasts", 《AMERCIAN JOURNAL OF PHYSIOLOGY. CELL PHYSIOLOGY》 *
HE LI等: "Independent Adipogenic and Contractile Properties of Fibroblasts in Graves’ Orbitopathy: An In Vitro Model for the Evaluation of Treatments", 《PLOS ONE》 *
SZU-YU CHEN等: "Isolation and Characterization of Mesenchymal Progenitor Cells from Human Orbital Adipose Tissue", 《INVESTIGATIVE OPHTHALMOLOGY AND VISUAL SCIENCE》 *
甘露等: "TAO患者眼眶CD90+和CD90-成纤维细胞亚群脂成纤维细胞转化的研究", 《四川大学学报（医学版）》 *
申煌煊: "《分子生物学实验方法与技巧》", 30 June 2010, 中山大学出版社 *
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Application publication date: 20211012