CN114807043A

CN114807043A - Human-derived vestibular schlemma immortalized cell line and construction method thereof

Info

Publication number: CN114807043A
Application number: CN202210296351.6A
Authority: CN
Inventors: 吴皓; 汪照炎; 薛璐; 何维维; 舒文莹; 王耀萱
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-07-29

Abstract

The invention has provided a human source vestibular schlemma immortalized cell line and its construction method, this vestibular schlemma immortalized cell line JEI-002, obtain primary cell of schlemma through the sporadic vestibular schlemma cell of in vitro culture, and utilize the SV40 gene in the cell of slow virus transfection technique over-expression, in the course of transfection technique, the purpose sequence of the carrier is shown as SEQ ID No.1, make it obtain the immortalized characteristic, namely overcome the limited characteristic of benign tumor proliferation ability, offer the cell carrier basis for in vivo in vitro experiment; JEI-002 is a human-derived cell line from sporadic vestibular neuroma patients, provides different vestibular sphingomyma cells, and provides various material bases and research platforms for the future mechanistic research of vestibular sphingomyma.

Description

Human-derived vestibular schlemma immortalized cell line and construction method thereof

Technical Field

The invention belongs to the technical field of cells, and particularly relates to a human-derived vestibular schlemma immortalized cell line and a construction method thereof.

Background

JEI-002 is a stable human cell line established by culturing tumor cells of patients with sporadic Vestibular Schwannoma (sVS).

The vestibular schwannoma is a benign tumor originated from vestibular schwannoma cells, grows in the area of the pontine and cerebellum horn, and the tumor body is gradually enlarged to press peripheral cranial nerves and brain tissues, so that corresponding clinical symptoms such as hearing loss, tinnitus, dizziness, facial paralysis and the like are generated, and even the life of a patient is threatened. The etiology of the disease is divided into two types, sporadic vestibular schwannoma type2 and neurofibromatosis type2 (NF 2), and the latter is a familial hereditary rare disease. According to long-term clinical follow-up observation of large-scale cases abroad, the clinical biological behaviors of different acoustic neuromas show larger difference, about half of tumors can grow continuously, and the other half of tumors grow statically or even shrink. The difference of the clinical biological behaviors of the vestibular sphingomyma can determine that different treatment schemes are adopted. At present, a lot of unknowns still exist in the mechanism exploration of the vestibular sphingomyma.

This mechanism has been particularly difficult to explore, in part because there are no corresponding cell lines. Cell line (cell line) refers to the cell population propagated after successful first passage of a primary cell culture. The cell line provides an experimental object with more convenient operation, faster growth speed and more stable property in an in vitro experiment stage of an exploration mechanism; meanwhile, a good experimental basis is provided for in vivo experiments of animal tumors and the like. The existing cell lines related to auditory neuroma are only two types, one is auditory neuroma cells from experimental animals, such as RT4-D6P2T, which are rat-derived nerve Schwann cells; second, HEI193, a human schwannoma cell, is a cultured cell line from NF2 patients by the Gene Hung team in 1999. JEI-002 is a human cell line derived from sporadic vestibular schwannoma patients. Provides a material basis and a research platform for the mechanism research of the vestibular schlemma in the future.

The disadvantages are as follows: the same tumor of different individuals has difference, namely JEI-002 only represents the characteristics of vestibular nerve sheath tumor of the patient and is common to all cell lines.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a human-derived vestibular schwannoma immortalized cell line and a construction method thereof.

The main principle of culturing the human neurothecioma immortalized cell line JEI-002 is as follows:

primary schwannoma cells are obtained by culturing sporadic vestibular schwannoma cells in vitro, and SV40 genes in the cells are over-expressed by using a lentivirus transfection technology, so that the immortalized characteristic is obtained, namely the characteristic of limited proliferation capacity of benign tumors is overcome, and a cell vector basis is provided for in vivo and in vitro experiments.

In order to achieve the above purpose, the solution of the invention is as follows:

in a first aspect, the invention provides a human-derived vestibular schwannoma immortalized cell line, which is JEI-002, is derived from a vestibular schwannoma tissue of a clinical patient, and helps to provide a basis for vestibular schwannoma in-vitro research.

In a second aspect, the invention provides a method for constructing the human-derived vestibular sphingomyelinoma immortalized cell line, which comprises the following steps:

(1) separating and culturing vestibular nerve sheath membrane tumor cells;

(2) performing immunofluorescence identification;

(3) overexpression of SV40 gene in cells by using a lentivirus transfection technology;

(4) screening the transfected cells with puromycin;

(5) the screened cells are expanded, and JEI-002 are subjected to cell identification.

Preferably, in step (2), the immunofluorescence assay comprises cell slide, fixation, rupture of membrane, incubation and embedding.

Preferably, in step (3), the target sequence of the vector is shown in SEQ ID NO.1 during the transfection technique.

Preferably, in step (4), puromycin is selected for transfected cells at concentrations of 1. mu.g/mL, 2. mu.g/mL, 3. mu.g/mL, 4. mu.g/mL, 5. mu.g/mL, 6. mu.g/mL and 7. mu.g/mL.

Preferably, in the step (5), the cell identification is carried out by staining JEI-002 with a cell-specific marker protein using immunocytochemistry.

Due to the adoption of the scheme, the invention has the beneficial effects that:

JEI-002 is a human-derived cell line from sporadic vestibular neuroma patients, provides different vestibular sphingomyma cells, and provides various material bases and research platforms for the future mechanistic research of vestibular sphingomyma.

Drawings

FIG. 1 is a diagram showing the cell culture in example 1 of the present invention.

FIG. 2 is a graph showing the result of the identification in example 2 of the present invention.

FIG. 3 is a map of a vector in example 3 of the present invention.

FIG. 4 is a diagram showing transfection of cells in example 3 of the present invention.

FIG. 5 is a diagram of cells immortalized in example 5 of the present invention.

FIG. 6 is a Sanger sequencing chart in example 6 of the present invention.

Detailed Description

The invention provides a human-derived vestibular schlemma immortalized cell line and a construction method thereof.

Culturing human immortalized cell line JEI-002 of vestibular schlemma

Example 1:

first, separation culture of human origin vestibular nerve sheath membrane tumor cell

1. Experimental reagent:

(1) complete medium: DMEM high sugar +1 XN 2 additive + Insulin 5. mu.g/mL + 10% FBS +1 XPicillin/streptomycin solution (P/S);

(2) basic culture medium: DMEM high sugar;

(3) buffer solution: sterile and Ca-free ²⁺ And Mg ²⁺ 1 × PBS +1 × P/S, pH 7.4;

(4) digestion solution: 0.125% trypsin + 0.1% collagenase type ii;

(5) coating liquid: dissolving sterile Laminin (Laminin) by 10 mu g/mL of 1 multiplied by PBS;

(6) 75% medical alcohol;

(7) fetal Bovine Serum (FBS).

2. Isolated culture

(1) Placing the vestibular nerve sheath membrane tumor tissue taken out in the operation into a sterile PBS buffer solution for low-temperature transportation;

(2) taking out tissue from the clean bench, soaking in 75% alcohol for about 2min, and placing in PBS containing P/S;

(3) cutting the tissue blocks into squares with side length of about 0.1cm, repeatedly cleaning, discarding supernatant, placing in a culture dish containing complete culture medium, and incubating at 37 deg.C for 30-60 min;

(4) clamping into a culture flask with tweezers, and inverting in 5% CO ₂ Incubating for 2h in an incubator;

(5) adding 2mL of tumor cell complete culture medium respectively, infiltrating tissue mass but not floating tissue mass, placing in 5% CO ₂ A cell incubator;

(6) changing the liquid every 3 days, removing the tissue block when the cells growing around the tissue block are fused into pieces, digesting the cells with trypsin, and re-bottling. The cell culture pictures are shown in FIG. 1, indicating the morphological characteristics of the cells.

Example 2:

second, immunofluorescence assay

(1) Cell climbing sheet

3 glass plates were placed in a 24-well plate, 1mL of medium was added per well, 0.02 million cells/well was added, and the plate was placed in an incubator for 2h or overnight.

(2) Fixing

After cell mounting, the medium was aspirated, washed 1 time with PBS, and fixed with 4% PFA at 4 ℃ for 30 min. Wash 3X 5 min/time with PBS. The PBS was not aspirated for the last time and left overnight at 4 ℃.

(3) Rupture membrane closure

Preparing a glass sheet sealing liquid: 0.5% Trition X-100 was mixed with PBS 1:1, followed by 10% fetal bovine serum.

The slide was dehydrated, placed on a petri dish support, 50 μ L of membrane-rupture blocking solution was dropped onto the waterproof membrane, and the side of the slide with the cells was covered for 2 h.

(4) Primary antibody incubation

Preparing a primary antibody: dilution of S100 antibody with PBS 1:100(200)

After rupture of the membrane and sealing, 50. mu.L of primary antibody was applied to a waterproof membrane (wet box), and the slide (side with cells) was covered and placed at 4 ℃ for up to one week.

(5) Incubation with secondary antibody

After incubating the secondary antibody (secondary antibody: PBS 1:500) at room temperature in the dark for 2h, the cells were washed with PBS 3X 5 min/time, stained with DAPI (DAPI: PBS 1:1000) for 5min, and washed with PBS 3X 5 min/time.

(6) Embedding

On the slide, 1 drop of Fluorocount-G was added, and the side with the cells was covered.

Identification of cells as P1 passage cells

(7) The results are shown in FIG. 2, and the isolated cells were identified to have characteristics of schwannomas.

Example 3:

III, transfection

(1) SV40 overexpresses essential lentivirus information

SV40 overexpressing lentiviruses

1) Carrier original information: EF1 alpha-SV 40-IRES-puromycin;

2) the carrier carries a fluorescent label: none;

3) vector resistance gene markers: puromycin (puromycin);

4) the vector map is shown in FIG. 3, and the characteristics of the immortalized virus are defined;

5) the sequence information of the vector is as follows:

the underlined region is the target sequence region

GTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGATCTATTTCCGGTGAATTCATGGATAA AGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGCCTGGGGGAATATTCCT CTGATGAGAAAGGCATATTTAAAAAAATGCAAGGAGTTTCATCCTGATAAAGGAGGAGATGAAGAAAAAATGAAGA AAATGAATACTCTGTACAAGAAAATGGAAGATGGAGTAAAATATGCTCATCAACCTGACTTTGGAGGCTTCTGGGA TGCAACTGAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTT TGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGA GAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAAC TCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTCT GTAACCTTTATAAGTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGT CTGCTATTAATAACTATGCTCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTT GATGTATAGTGCCTTGACTAGAGATCCATTTTCTGTTATTGAGGAAAGTTTGCCAGGTGGGTTAAAGGAGCATGAT TTTAATCCAGAAGAAGCAGAGGAAACTAAACAAGTGTCCTGGAAGCTTGTAACAGAGTATGCAATGGAAACAAAAT GTGATGATGTGTTGTTATTGCTTGGGATGTACTTGGAATTTCAGTACAGTTTTGAAATGTGTTTAAAATGTATTAA AAAAGAACAGCCCAGCCACTATAAGTACCATGAAAAGCATTATGCAAATGCTGCTATATTTGCTGACAGCAAAAAC CAAAAAACCATATGCCAACAGGCTGTTGATACTGTTTTAGCTAAAAAGCGGGTTGATAGCCTACAATTAACTAGAG AACAAATGTTAACAAACAGATTTAATGATCTTTTGGATAGGATGGATATAATGTTTGGTTCTACAGGCTCTGCTGA CATAGAAGAATGGATGGCTGGAGTTGCTTGGCTACACTGTTTGTTGCCCAAAATGGATTCAGTGGTGTATGACTTT TTAAAATGCATGGTGTACAACATTCCTAAAAAAAGATACTGGCTGTTTAAAGGACCAATTGATAGTGGTAAAACTA CATTAGCAGCTGCTTTGCTTGAATTATGTGGGGGGAAAGCTTTAAATGTTAATTTGCCCTTGGACAGGCTGAACTT TGAGCTAGGAGTAGCTATTGACCAGTTTTTAGTAGTTTTTGAGGATGTAAAGGGCACTGGAGGGGAGTCCAGAGAT TTGCCTTCAGGTCAGGGAATTAATAACCTGGACAATTTAAGGGATTATTTGGATGGCAGTGTTAAGGTAAACTTAG AAAAGAAACACCTAAATAAAAGAACTCAAATATTTCCCCCTGGAATAGTCACCATGAATGAGTACAGTGTGCCTAA AACACTGCAGGCCAGATTTGTAAAACAAATAGATTTTAGGCCCAAAGATTATTTAAAGCATTGCCTGGAACGCAGT GAGTTTTTGTTAGAAAAGAGAATAATTCAAAGTGGCATTGCTTTGCTTCTTATGTTAATTTGGTACAGACCTGTGG CTGAGTTTGCTCAAAGTATTCAGAGCAGAATTGTGGAGTGGAAAGAGAGATTGGACAAAGAGTTTAGTTTGTCAGT GTATCAAAAAATGAAGTTTAATGTGGCTATGGGAATTGGAGTTTTAGATTGGCTAAGAAACAGTGATGATGATGAT GAAGACAGCCAGGAAAATGCTGATAAAAATGAAGATGGTGGGGAGAAGAACATGGAAGACTCAGGGCATGAAACAG GCATTGATTCACAGTCCCAAGGCTCATTTCAGGCCCCTCAGTCCTCACAGTCTGTTCATGATCATAATCAGCCATA CCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACAGAGCAAAAGCTC ATTTCTGAAGAGGACTTGTAATCTAGACACAGTGCAGCACTCTCAACGTTCAAGGACACTACGCGTCTGGAACAATCAACC。

(2) Transfection

1) Cells were seeded in 6-well plates, approximately 1X 10 cells per well ⁵ A plurality of;

2) on the next day, after the cells adhere to the wall, the liquid is changed;

3) 1mL of complete medium is added, and then 20 mu L of SV40 overexpression lentivirus is added;

4) mixing and culturing;

5) observing the cell state after 12h, and replacing with a fresh culture medium;

6) and when the cells grow to the bottom of the plate, the cells are transferred to a T25 culture flask.

The image of cell transfection is shown in FIG. 4, which shows immortalized cells before specific selection.

Example 4:

fourth, screening

(1) Determination of the kill Curve

1) Spreading untransfected cells into a 24-well plate by 0.05 million per well, and incubating overnight;

2) the next day, old medium was removed from the 24-well plates;

3) fresh medium containing puromycin at different concentrations (1. mu.g/mL, 2. mu.g/mL, 3. mu.g/mL, 4. mu.g/mL, 5. mu.g/mL, 6. mu.g/mL, 7. mu.g/mL) was added to the cell-plated 24-well plates;

4) changing fresh screening culture medium every 2 days;

5) observing the survival rate of the cells every day;

6) the minimum puromycin concentration used was the lowest screening concentration that killed all cells within 1-4 days from puromycin screening.

As a result: puromycin was used at a concentration of 2. mu.g/mL for a duration of 2 days.

(2) Puromycin screening transfected cells

1) The first day, the transfected cells were plated in 24-well plates at 0.05 million per well and incubated overnight;

2) the next day, old medium was removed from the 24-well plates;

3) adding a screening culture medium containing puromycin (2 mu g/mL), and incubating;

4) changing fresh screening culture medium every 2 days;

5) observing the survival rate of the cells every day;

6) the cells which survive at the same time point (2d) are the cells which are successfully transfected;

7) and amplifying the screened cells.

Example 5:

fifth, cell expansion

And expanding the screened cells, wherein the screened cells are P1 generation and are expanded to at least 12 generation.

The picture of immortalized cells is shown in FIG. 5.

Example 6:

sixthly, cell identification

And (3) identifying STR:

tissues and established cell lines were subjected to STR testing separately and cell line alignments were performed using DSMZ tools (containing 2455 cell line STR data from ATCC, DSMZ, JCRB and RIKEN databases) identified: this cell line DNA typing did not find a matching cell line in the cell line search.

Sanger sequencing:

as shown in FIG. 6, both in the tissue sample and in the JEI-002 cell line: regarding the mutational characteristics of NF2 in cells, the NF2 gene has heterozygous mutation c.240G > A at exon 2.

And (3) carrying out immunocytochemistry identification:

in order to determine that the immortalized cell strain is derived from primary schwann cell, the characteristic of the schwann cell is utilized, JEI-002 is stained with cell specific marker protein by using an immunocytochemistry method, and the aim of identification is achieved: wherein S100 is taken as (+). The protein expression characteristics of the human sphingomyma neuroma cells are met.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.

Sequence listing

<110> Shanghai university of traffic medical college affiliated ninth people hospital

<120> human source vestibular schwannoma immortalized cell line and construction method thereof

<141> 2022-03-24

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2157

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa 60

aggagtgcct gggggaatat tcctctgatg agaaaggcat atttaaaaaa atgcaaggag 120

tttcatcctg ataaaggagg agatgaagaa aaaatgaaga aaatgaatac tctgtacaag 180

aaaatggaag atggagtaaa atatgctcat caacctgact ttggaggctt ctgggatgca 240

actgagattc caacctatgg aactgatgaa tgggagcagt ggtggaatgc ctttaatgag 300

gaaaacctgt tttgctcaga agaaatgcca tctagtgatg atgaggctac tgctgactct 360

caacattcta ctcctccaaa aaagaagaga aaggtagaag accccaagga ctttccttca 420

gaattgctaa gttttttgag tcatgctgtg tttagtaata gaactcttgc ttgctttgct 480

atttacacca caaaggaaaa agctgcactg ctatacaaga aaattatgga aaaatattct 540

gtaaccttta taagtaggca taacagttat aatcataaca tactgttttt tcttactcca 600

cacaggcata gagtgtctgc tattaataac tatgctcaaa aattgtgtac ctttagcttt 660

ttaatttgta aaggggttaa taaggaatat ttgatgtata gtgccttgac tagagatcca 720

ttttctgtta ttgaggaaag tttgccaggt gggttaaagg agcatgattt taatccagaa 780

gaagcagagg aaactaaaca agtgtcctgg aagcttgtaa cagagtatgc aatggaaaca 840

aaatgtgatg atgtgttgtt attgcttggg atgtacttgg aatttcagta cagttttgaa 900

atgtgtttaa aatgtattaa aaaagaacag cccagccact ataagtacca tgaaaagcat 960

tatgcaaatg ctgctatatt tgctgacagc aaaaaccaaa aaaccatatg ccaacaggct 1020

gttgatactg ttttagctaa aaagcgggtt gatagcctac aattaactag agaacaaatg 1080

ttaacaaaca gatttaatga tcttttggat aggatggata taatgtttgg ttctacaggc 1140

tctgctgaca tagaagaatg gatggctgga gttgcttggc tacactgttt gttgcccaaa 1200

atggattcag tggtgtatga ctttttaaaa tgcatggtgt acaacattcc taaaaaaaga 1260

tactggctgt ttaaaggacc aattgatagt ggtaaaacta cattagcagc tgctttgctt 1320

gaattatgtg gggggaaagc tttaaatgtt aatttgccct tggacaggct gaactttgag 1380

ctaggagtag ctattgacca gtttttagta gtttttgagg atgtaaaggg cactggaggg 1440

gagtccagag atttgccttc aggtcaggga attaataacc tggacaattt aagggattat 1500

ttggatggca gtgttaaggt aaacttagaa aagaaacacc taaataaaag aactcaaata 1560

tttccccctg gaatagtcac catgaatgag tacagtgtgc ctaaaacact gcaggccaga 1620

tttgtaaaac aaatagattt taggcccaaa gattatttaa agcattgcct ggaacgcagt 1680

gagtttttgt tagaaaagag aataattcaa agtggcattg ctttgcttct tatgttaatt 1740

tggtacagac ctgtggctga gtttgctcaa agtattcaga gcagaattgt ggagtggaaa 1800

gagagattgg acaaagagtt tagtttgtca gtgtatcaaa aaatgaagtt taatgtggct 1860

atgggaattg gagttttaga ttggctaaga aacagtgatg atgatgatga agacagccag 1920

gaaaatgctg ataaaaatga agatggtggg gagaagaaca tggaagactc agggcatgaa 1980

acaggcattg attcacagtc ccaaggctca tttcaggccc ctcagtcctc acagtctgtt 2040

catgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc 2100

acacctcccc ctgaacctga aacagagcaa aagctcattt ctgaagagga cttgtaa 2157

Claims

1. A human source vestibular schwannoma immortalized cell line is characterized in that: the cell line is JEI-002.

2. The method for constructing the human-derived vestibular schlemma immortalized cell line according to claim 1, which is characterized by comprising the following steps: which comprises the following steps:

(1) separating and culturing vestibular nerve sheath membrane tumor cells;

(2) performing immunofluorescence identification;

(4) screening the transfected cells with puromycin;

3. The construction method according to claim 2, wherein: in the step (2), the immunofluorescence identification process comprises cell slide climbing, fixing, membrane rupture sealing, incubation and embedding.

4. The construction method according to claim 2, wherein: in the step (3), in the transfection technology process, the target sequence of the vector is shown as SEQ ID NO. 1.

5. The construction method according to claim 2, wherein: in step (4), puromycin is selected at a concentration of 1. mu.g/mL, 2. mu.g/mL, 3. mu.g/mL, 4. mu.g/mL, 5. mu.g/mL, 6. mu.g/mL and 7. mu.g/mL when transfected cells are selected.

6. The construction method according to claim 2, wherein: in the step (5), in the cell identification process, JEI-002 is subjected to staining cell specific marker protein by using an immunocytochemistry method for identification.