CN108285887B - Human normal conjunctival epithelial cells and uses thereof - Google Patents

Human normal conjunctival epithelial cells and uses thereof Download PDF

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CN108285887B
CN108285887B CN201810258760.0A CN201810258760A CN108285887B CN 108285887 B CN108285887 B CN 108285887B CN 201810258760 A CN201810258760 A CN 201810258760A CN 108285887 B CN108285887 B CN 108285887B
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叶琳
李晖
王玲
魏高斌
叶立娜
于莉
王媛
殷国干
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SHENZHEN OPHTHALMOLOGY HOSPITAL
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Abstract

The invention discloses human normal conjunctival epithelial cells and application thereof. The cell is named as human normal conjunctival epithelial cell HNCEC/HL-043, and the preservation number is CCTCC NO: C201676. the primary separation culture method comprises the following steps: the preparation method comprises the steps of digesting a normal conjunctiva tissue sample on the temporal side of an eyeball of a patient subjected to pterygium surgery, adding a dispersing enzyme and DNase I, collecting cells through filtration and centrifugation, then resuspending the cells by using an HL culture medium, and inoculating and culturing. The subculture method comprises the following steps: when the cells are proliferated to 70-90% abundance, digesting with pancreatin-EDTA, and then neutralizing with DMEM; the cells were collected by centrifugation, resuspended in HL medium and cultured by inoculation. The cell of the invention can be used for physiological research of human normal cells, drug sensitivity and toxicity detection systems, and pathogenesis research of conjunctival related diseases including xerophthalmia and allergic conjunctivitis.

Description

Human normal conjunctival epithelial cells and uses thereof
Technical Field
The invention belongs to the field of cell biology, and relates to a human normal conjunctival epithelial cell and application thereof.
Background
Important organs of the human body such as the lung, kidney, liver, pancreas and skin are composed of organ-specifically differentiated epithelial cells. These specifically differentiated epithelial cells are directly associated with specific functions of various organs, such as the gas exchange function of the lung, the filtration function of the kidney, the detoxification and neutralization function of the liver, the production of insulin by pancreatic cells, and the protection of the skin from the external environment. Once the vital organs are diseased or degenerated, the health of human beings is threatened, because the vital organs are difficult to replace, and specific cells of different organs cannot mutually replace cells of other organs. These specifically differentiated cells are difficult to regenerate, let alone to proliferate in vitro. This greatly limits the understanding of normal cellular functions of the body, and many descriptions of cell biology knowledge from scientific literature and even textbooks are inaccurate or even wrong. The true reason is that most of the biological knowledge of normal cells is derived from the results of in vitro cultured cell line studies. Although scientists in various countries are constantly trying to culture and proliferate functional epithelial cells of vital organs of the human body, it is still difficult to culture primary epithelial cells isolated from humans and mammals in vitro. By applying the existing serum-free culture medium, some cells can only be subjected to short-term primary culture (survive for a plurality of days, such as epithelial cells of lung, pancreas and the like), some cells can only be subjected to limited subculture (1-3 generations, such as epithelial cells of trachea, cornea, prostate and the like), some cells can not be subjected to in vitro culture (such as epithelial cells of liver, colon, prostate and the like), and only keratinized epithelial cells from human skin can be subjected to subculture for about 10 generations. Moreover, the yield of epithelial cells obtained from each animal or biopsy sample is still low, including the number of cells, the purity of the cells in direct isolation or short culture, and the proliferation rate of these primary and secondary epithelial cells is also extremely limited.
For in vitro epithelial cell culture, foreign attempts have been made to extend the number of in vitro cell survival generations by genetic manipulation, such as transfer into viruses (SV40T or HPV16E6E7) or cellular oncogenes. The greatest disadvantage of genetic manipulation, however, is that the genetic background and phenotype of these cells is altered so that the normal epithelial cells lose their normal physiological functions, e.g., the p53 and pRB signaling pathways are often suppressed. Moreover, these genetically modified cells are not possible to re-transplant into the body. However, due to the lack of effective in vitro culture techniques for epithelial cells, these cells are still gaining favor in medical and life science research in the world today. In the non-cancer research field, they represent the original source of "functional" tissues or organs. At present, no normal cell has been applied to basic and clinical medicine research at home and abroad.
The main functions of normal human conjunctival epithelial cells are to secrete tears and mucus, keeping the eyes moist. The human conjunctival epithelial cells are associated with physiological diseases of the eye, such as allergic conjunctivitis and dry eye. If the human normal conjunctival epithelial cells which are stably passaged in vitro can be obtained, people can more comprehensively research the normal functions of the conjunctival epithelial cells and pathogenesis of conjunctival related diseases, which have great significance for basic and clinical medical research and application.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a human normal conjunctival epithelial cell. The cell is separated and cultured from normal conjunctival tissue of Chinese, is not introduced with any exogenous gene, is a normal diploid cell, and is identified as a human normal cell line which is not registered at home and abroad through genotyping.
Another object of the present invention is to provide the use of the above-mentioned human normal conjunctival epithelial cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a human normal conjunctival epithelial cell, named as human normal conjunctival epithelial cell HNCEC/HL-043, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: c201676; the cells are derived from normal conjunctival epithelial cells of a normal conjunctival tissue sample beside a pterygium surgically excised from a Chinese pterygium patient, the chromosomes are diploid, and STR (short tandem repeat) genotypes are represented by 22 "STR loci/allele length": AMEL/X/Y, D3S1358/15, D13S317/10/11, D7S820/11, D16S539/9/12, Penta E/16/22, D2S441/11/14, TPOX/8/11, TH01/9, D2S1338/23/25, CSF1PO/10/12, Penta D/9/11, D10S1248/15, D19S433/13/15.2, vWA/14/20, D21S11/32.2/33.2, D18S51/13/22, D6S1043/12/14, D8S1179/10/14, D5S818/10/13, D12S391/19/20, FGA/21/24.
The cell morphology of the human normal conjunctival epithelial cells observed under a microscope is compact in arrangement, clear in cell boundary, strong in stereoscopic impression and polygonal.
The culture conditions of the human normal conjunctival epithelial cells are preferably based on 5% CO at 37 ℃ by using HL culture2Culturing; the HL culture medium is as follows: DMEM was mixed with serum-free medium SFM at a volume ratio of 1:3, while adding 5% (v/v) FBS (fetal bovine serum), 0.4. mu.g/mL cortisol (hydrocortisone), 5. mu.g/mL insulin (insulin), 8.4ng/mL cholera toxin (cholera toxin), 10ng/mL Epidermal Growth Factor (EGF), 24. mu.g/mL adenine (adenin), 100U/mL penicillin (penicill), 100. mu.g/mL streptomycin (streptomycin), 0.25. mu.g/mL amphotericin B (fungizone), 30. mu.M Fasudil (faudil), which was filtered through a 0.22. mu.m pore size filter.
The primary separation culture method of the human normal conjunctival epithelial cells comprises the following steps:
(1) normal conjunctival tissue samples were collected around pterygium surgically excised by patients with pterygium under informed consent of the patient or patient guardian.
(2) The separated tissue sample was washed with 95-100% (v/v) ethanol, then washed with PBS (0.01M, pH7.4), and then placed in a sterile petri dish containing precooled PBS, and the residual fat in the tissue sample was removed with dissecting forceps and scissors under a dissecting microscope.
(3) Digesting the tissue sample with a digestive fluid; preferably, the digestive juice is HL culture medium containing collagenase and dispase.
(4) The digested tissue was centrifuged to remove the supernatant and the cell pellet was resuspended in 0.25% (w/v) pancreatin-EDTA for digestion.
(5) DMEM medium containing 10% (v/v) FBS was added, and the supernatant was centrifuged off.
(6) Adding the dispase and DNase I in a warm water bath, and repeatedly blowing and beating the sample by using a gun head.
(7) And adding a DMEM medium containing 10% (v/v) FBS, filtering the cell suspension by using a filter with the pore size of 40-70 mu m, collecting the filtered cell suspension, and centrifuging to remove the supernatant.
(8) And (4) suspending the cell sediment in an HL culture medium, and inoculating the cell sediment in a culture flask for culture to obtain the human normal conjunctival epithelial cells.
The pre-cooling in step (2) is preferably pre-cooling on ice.
The amount of the digestive juice used in step (3) is preferably 10 times the volume of the tissue sample.
The digestion condition in the step (3) is preferably digestion at 37 ℃ for 1-3 hours.
The concentration of collagenase and dispase in step (3) is preferably 0.2mg/mL each.
The digestion in step (4) is preferably carried out on ice for 1 hour or at 37 ℃ for 10 minutes.
The warm water bath in the step (6) is preferably a warm water bath at 37 ℃.
The centrifugation in steps (4), (5) and (7) is preferably carried out at 1000rpm for 5 minutes.
The culture conditions in step (8) are preferably 37 ℃ and 5% CO2。
The subculturing method of the human normal conjunctival epithelial cells comprises the following steps:
(1) when human normal conjunctival epithelial cells proliferated to 70-90% abundance, the cells were washed with 1 × PBS (0.01M, pH7.4), and the monolayer cells were digested with 0.05% (mass to volume) pancreatin-EDTA.
(2) Adding DMEM to neutralize digestion reaction; centrifuging to remove supernatant, resuspending the cell pellet with HL medium, and inoculating to a culture flask for culture.
The time for digestion in the step (1) is preferably 2-5 minutes.
The centrifugation described in step (2) is preferably carried out at 1000rpm for 5 minutes.
The culture conditions in step (2) are preferably 37 ℃ and 5% CO2。
The human normal conjunctival epithelial cells can be used for physiological research of human normal cells, and research on pathogenesis of conjunctival related diseases including xerophthalmia and allergic conjunctivitis in a drug sensitivity and toxicity detection system.
Compared with the prior art, the invention has the following advantages and effects:
(1) the human normal conjunctival epithelial cells provided by the invention are primarily separated and cultured from human normal conjunctival tissues, no foreign gene is introduced into the cells, and the cells are identified as human normal diploid cells through karyotype analysis.
(2) The human normal conjunctival epithelial cells provided by the invention are primarily separated and cultured from normal conjunctival tissues of Chinese people, and are a human normal cell line which is unregistered and registered at home and abroad through STR genotyping identification.
(3) The human normal conjunctival epithelial cells provided by the invention can be subjected to in-vitro continuous subculture for 50 days, and can still keep the normal growth of a proliferation state.
(4) The human normal conjunctival epithelial cells provided by the invention can be used for physiological research of human normal cells and pathogenesis research of conjunctival and conjunctival related diseases including dry eye and allergic conjunctivitis.
The human normal conjunctival epithelial cell is named as 'human normal conjunctival epithelial cell HNCEC/HL-043', is preserved in the China center for type culture Collection (address: China, Wuhan university, Wuhan) in 8-23.2017, and has the preservation number of CCTCC NO: C201676.
drawings
FIG. 1 is a cell morphology map of human normal conjunctival epithelial cells.
FIG. 2 is a graph of growth of normal human conjunctival epithelial cells.
FIG. 3 is a chromosome karyotype analysis chart of human normal conjunctival epithelial cells.
FIG. 4 is a STR genotyping panel for normal human conjunctival epithelial cells.
FIG. 5 is a graph of a Matrigel experiment on human normal conjunctival epithelial cells.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
Example 1 Primary isolation culture of Primary human Normal conjunctival epithelial cells
(1) Samples of normal conjunctival tissue from the temporal side of the eyeball of patients with pterygium surgery were collected with informed consent from the patient or patient guardian.
(2) Preparation of digestive juice: HL culture medium containing 0.2mg/mL both collagenase and dispase; wherein, the HL culture medium is as follows: DMEM (GIBCO #11965-092) was mixed with serum-free medium SFM (GIBCO #10744-019) in a volume ratio of 1:3 while adding 5% (v/v) fetal bovine serum and 0.4. mu.g/mL cortisol (hydrocortisone), 5. mu.g/mL insulin (insulin), 8.4ng/mL cholera toxin (choleratoxin), 10ng/mL Epidermal Growth Factor (EGF), 24. mu.g/mL adenine (adenine), 100U/mL penicillin (penicilin), 100. mu.g/mL streptomycin (streptomycin), 0.25. mu.g/mL amphotericin B (fungizone), 30. mu.M Fasudil (Fasudil), and the medium was filtered through a 0.22. mu.m pore size filter.
(3) The separated tissue samples were washed 1 time with 95-100% (v/v) ethanol, then washed 2 times with PBS (0.01M, pH7.4), and then the tissues were placed in a sterile petri dish containing ice-precooled PBS and the residual fat in the tissues was removed with dissecting forceps and scissors under a dissecting microscope.
(4) 1-2 cm of tissue sample3Placing the mixture into a 14mL or 50mL centrifuge tube containing 10mL of the digestion solution in the step (2), and digesting the mixture at 37 ℃ for 1 to 3 hours.
(5) The digested tissue was centrifuged at low speed (1000rpm) for 5 minutes to remove the supernatant.
(6) The cell pellet was resuspended in 2-5 mL of 0.25% (w/v) pancreatin-EDTA and placed on ice for 1 hour or 10 minutes at 37 ℃.
(7) Then 10mL of DMEM medium containing 10% (v/v) FBS is added, and centrifugation is carried out for 5 minutes at low speed of 1000 rmp; the supernatant was removed as clean as possible.
(8) 5mg/mL of dispase and 200. mu.L of 1mg/mL DNaseI were added to a 2mL warm water bath (37 ℃) and the sample was repeatedly blown with a sterile P1000 disposable plastic tip for 1 minute.
(9) Adding 10mL of DMEM containing 10% (v/v) FBS, filtering the cell suspension by using a filter with the pore size of 40-70 mu m, collecting the filtered cell suspension, centrifuging at low speed of 1000rmp for 5 minutes, and removing the supernatant.
(10) Resuspending the cell pellet in HL medium, and inoculating the cell pellet in T25 or T75Culturing in a flask at 37 deg.C and 5% CO2。
Primary human normal conjunctival epithelial cells successfully cultured were isolated according to the above method, and the morphology of the cells was observed under a microscope as shown in FIG. 1 (dense arrangement, clear cell boundary, strong stereoscopic impression, polygonal epithelial cells). The cell is named as 'human normal conjunctival epithelial cell HNCEC/HL-043', is preserved in China center for type culture Collection (address: China, Wuhan university) in 8-23.2017, and has the preservation number of CCTCC NO: C201676.
[ example 2 ] subculture of human normal conjunctival epithelial cells
(1) When human normal conjunctival epithelial cells cultured in a flask of T25 or T75 proliferated to 70-90% abundance, the cells were washed twice with 1 × PBS (0.01M, pH7.4), and the monolayer cells were digested with 0.05% (mass to volume) pancreatin-EDTA for 2-5 minutes.
(2) Adding 10mL of complete DMEM to neutralize and digest 1-2 minutes.
(3) Centrifugation is carried out at 1000rmp for 5 minutes, supernatant is removed, and cell pellets are resuspended in 10mL of HL medium for inoculation and culture.
(4) If necessary, 1 × 106The epithelial cells were resuspended in 1-2 mL of cell freezing medium (90% fetal bovine serum and 10% DMSO, v/v) and stored in liquid nitrogen for use.
The human normal conjunctival epithelial cells are subcultured according to the method, the cell growth curve of the culture line is shown in figure 2, and the human normal conjunctival epithelial cells can still keep the normal growth in a proliferation state after continuous subculture for 50 days.
Example 3 karyotyping of human Normal conjunctival epithelial cells
(1) When human normal conjunctival epithelial cells (1X 10)6) In exponential growth phase, colchicine was added to a final concentration of 0.2. mu.g/mL, and the culture was continued for 3.5 hours.
(2) The cells were repeatedly aspirated to cause exfoliation, and centrifuged at 2000rpm for 5 minutes to harvest the cells.
(3) The supernatant was discarded, 8mL of 0.075mol/L KCl solution pre-warmed at 37 ℃ was added, the cell pellet was gently blown and mixed, and the mixture was subjected to hypotonic treatment at 37 ℃ for 25 minutes.
(4) Add 1mL of freshly prepared fixative (methanol: glacial acetic acid ═ 3:1, v/v), carefully blow-up, mix well, centrifuge at 2000rpm for 5 minutes.
(5) The supernatant was discarded, 8mL of fixative was added, and after pipetting, the cells were suspended and fixed at room temperature for 20 minutes.
(6) Centrifuge at 2000rpm for 5 minutes, discard the supernatant and repeat the fixation once.
(7) And (3) discarding the supernatant, adding a plurality of drops of fixing agent to prepare cell suspension, and taking 2-3 drops of the cell suspension on a glass slide soaked by ice water.
(8) And (4) putting the glass slide into an oven at 70 ℃ for dry baking for 2 hours, and naturally cooling.
(9) 2.5% (mass/volume ratio) pancreatin solution (pH6.8-7.2) 5mL for 25-45 seconds.
(10) Rinsing with physiological saline at a pre-temperature of 37 ℃, dyeing for 5-10 minutes by Giemsa, and carrying out G banding analysis.
(11) Observing the cell karyotype under a microscope, photographing, and carrying out karyotype analysis; at least more than 20 cells in metaphase are observed and analyzed. The result of representative karyotyping is shown in FIG. 3, and human normal conjunctival epithelial cells are normal diploid, and 46 chromosomes are not abnormally arranged.
Example 4 genotyping of human Normal conjunctival epithelial cells
(1) Adherently growing human normal conjunctival epithelial cells (1X 10)6) The cells were washed twice with 1 × PBS, the monolayer cells were digested with 0.05% pancreatin-EDTA for 2-5 minutes, and the digestion reaction was neutralized with 10mL of complete DMEM.
(2) After centrifugation at 10000rpm for 1 minute, the supernatant was decanted off, and 200. mu.L of buffer GA (cell/tissue genomic DNA extraction kit DP304, Tiangen Co.) was added and shaken until suspension was completed.
(3) Add 20. mu.L of protease K solution and mix well.
(4) 200. mu.L of buffer GB (cell/tissue genomic DNA extraction kit DP304, Tiangen Co.) was added, mixed well by inversion, left at 70 ℃ for 10min, and centrifuged briefly.
(5) Adding 200. mu.L of absolute ethyl alcohol, fully shaking and uniformly mixing for 15 seconds, and centrifuging briefly.
(6) The resulting solution and flocculent precipitate were both put on an adsorption column (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.), centrifuged at 12000rpm for 30 seconds, and discarded.
(7) To the adsorption column was added 500. mu.L of buffer GD (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.), centrifuged at 12000rpm for 30 seconds, and discarded.
(8) To the adsorption column, 600. mu.L of a rinsing solution PW (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.) was added, centrifuged at 12000rpm for 30 seconds, and discarded.
(9) Transferring the adsorption column into another centrifuge tube, dripping 50-200 μ L of elution buffer TE (cell/tissue genome DNA extraction kit DP304, Tiangen corporation) into the middle part of the adsorption membrane, standing at room temperature for 2-5 min, centrifuging at 12000rpm (13400 × g) for 2 min, and collecting the extracted DNA solution into the centrifuge tube.
(10) By using
Figure BDA0001609675830000071
The 16HS system (DC2101, promega corporation) performs DNA multiplex amplification of 21 loci (15 STR loci and 1 idiosyncratic locus).
(11) Using ABI
Figure BDA0001609675830000072
Detection of amplified fragments was performed with model 3100 genetic analyzer (version 1.1 data collection software).
(12) Use of
Figure BDA0001609675830000081
And PowerTyper 16Macro software, automatic genotyping, STR typing results figure 4, detecting 22 STR loci, expressed as "STR locus/allele length": AMEL/X/Y, D3S1358/15, D13S317/10/11, D7S820/11, D16S539/9/12, Penta E/16/22, D2S441/11/14, TPOX/8/11, TH01/9, D2S1338/23/25, CSF1PO/10/12, Penta D/9/11, D10S1248/15, D19S433/13/15.2, vWA/14/20, D21S11/32.2/33.2, D18S51/13/22, D6S1043/12/14, D8S1179/10/14, D5S818/10/13, D12S391/19/20, FGA/21/24.
The normal conjunctival epithelial cells of the invention are identified by STR genotyping, and are a human normal cell line which is not registered at home and abroad.
Example 5 3D matrigel assay of human Normal conjunctival epithelial cells
(1) The chamber was coated with an appropriate volume of matrigel and then left at 37 degrees for 15-30 minutes to allow the gel to set.
(2) Adherent human normal conjunctival epithelial cells were washed twice with 1 × PBS, the monolayer cells were digested with 0.05% pancreatin-EDTA for 2-5 min, and the digestion reaction was neutralized with 10mL of complete DMEM.
(3) Centrifuging at 1000rmp for 5min, removing supernatant, resuspending cell pellet in HL medium, counting cells, and collecting human normal conjunctival epithelial cells (4 × 10)4) And gently seeded along the wall onto the coagulated gel.
(4) Left at 37 ℃ for 10 minutes, and then the non-adherent cells were aspirated (inclined chamber, gently aspirated with a pipette tip)
(5) And (3) gently adding HL medium containing 5% (v/v) matrigel into the wall for culture, changing the medium every other day, and culturing for 7-14 days.
(6) After the incubation was completed, the medium was aspirated and the cells were washed three times with 1 × PBS.
(7) 4% paraformaldehyde fixed for 30min, cells were washed three times with 1 × PBS.
(8) Triton-X-100 (5%) was added and permeabilized at room temperature for 15min, and the cells were washed three times (4 min/time) with 1 XPBS.
(9) DAPI (final concentration 100ng/ml) was added and incubated for 5min in the dark, and the cells were washed three times with 1 XPBS (4 min/time).
(10) The chamber of the slide was examined, the upper layer was infiltrated on a cover glass, and the cell karyotype was observed under a microscope and photographed. Representative karyotyping results are shown in FIG. 5, and the clones grown from human normal conjunctival epithelial cells in 3D matrigel experiments are regular polarized spheres, which indicates that the human normal conjunctival epithelial cells of the invention are normal cell lines.
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 changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (2)

1. The human normal conjunctival epithelial cell is named as human normal conjunctival epithelial cell HNCEC/HL-043, and the preservation number of the human normal conjunctival epithelial cell in China center for type culture Collection is CCTCC NO: c201676; the cells are derived from normal conjunctival epithelial cells of a normal conjunctival tissue sample beside a pterygium surgically excised from a patient with a Chinese pterygium.
2. Use of human normal conjunctival epithelial cells according to claim 1, in a drug sensitivity and toxicity detection system.
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Publication number Priority date Publication date Assignee Title
CN106591216A (en) * 2016-12-12 2017-04-26 深圳市眼科医院 Human normal corneal epithelium cells and application thereof

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CN106591216A (en) * 2016-12-12 2017-04-26 深圳市眼科医院 Human normal corneal epithelium cells and application thereof

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Title
人结膜上皮细胞的培养鉴定及液氮冻存;郑健樑等;《眼科学报》;20001231;第16卷(第2期);摘要，第135页右栏第1段,第136页右栏倒数第1段-第137页右栏第1段 *

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