阅读说明：本技术 胰腺癌稳转细胞系及其构建方法和应用 (Pancreatic cancer stable cell line and construction method and application thereof ) 是由 郝继辉 高松 周博栋 袁帅 陈志强 朱文博 于 2021-07-27 设计创作，主要内容包括：本发明公开了胰腺癌稳转细胞系及其构建方法和应用,包括：(1)构建过表达OVA基因的质粒载体；(2)将过表达OVA基因的质粒载体扩增后转染到工具细胞中进行病毒包装得到病毒上清液；(3)将病毒上清液转染到目的细胞中进行筛选得到稳定的胰腺癌细胞系。本发明所建立的胰腺癌细胞系具有生长速度稳定、可稳定传代、小鼠成瘤性好等特点,能用于建立胰腺癌细胞模型和动物模型,进而为胰腺癌的研究提供良好的肿瘤模型,应用于胰腺癌的发生发展及转移机制、免疫治疗及其相关联合治疗、耐药原理及新药筛选等方面。(The invention discloses a pancreatic cancer stable cell line and a construction method and application thereof, wherein the pancreatic cancer stable cell line comprises the following steps: (1) constructing a plasmid vector for over-expressing OVA genes; (2) after amplifying the plasmid vector of the over-expression OVA gene, transfecting the amplified plasmid vector into tool cells to carry out virus packaging to obtain virus supernatant; (3) and (4) transfecting the virus supernatant into target cells for screening to obtain a stable pancreatic cancer cell line. The pancreatic cancer cell line established by the invention has the characteristics of stable growth speed, stable passage, good mouse tumorigenicity and the like, can be used for establishing a pancreatic cancer cell model and an animal model, further provides a good tumor model for the research of pancreatic cancer, and is applied to the aspects of the occurrence and development and transfer mechanism of pancreatic cancer, immunotherapy and related combined therapy thereof, drug resistance principle, new drug screening and the like.)

1. A method for constructing a pancreatic cancer cell line, comprising: (1) constructing a plasmid vector for over-expressing OVA genes; (2) after amplifying the plasmid vector of the over-expression OVA gene, transfecting the amplified plasmid vector into tool cells to carry out virus packaging to obtain virus supernatant; (3) and (4) transfecting the virus supernatant into target cells for screening to obtain the stable pancreatic cancer cell line.

2. The method of claim 1, wherein the plasmid vector for over-expression of OVA gene is pHS-AVC-0700, and the sequence of elements is pLV-EF1a-hluc-P2A-mNeongreen-CMV-OVAL-3 Xflag-P2A-puro.

3. The method according to claim 1, wherein the plasmid obtained by amplification is transfected into the tool cell by lipofectamine3000 transfection in step (2).

4. The method of claim 3, wherein the tool cell is 293T.

5. The method of claim 1, wherein the target cell in the step (3) is Pan 02.

6. The method of claim 1, wherein the screening in step (3) comprises: puromycin is added into the culture solution of the transfected target cells for screening to obtain a stable pancreatic cancer cell line.

7. A pancreatic cancer cell line obtained by the construction method according to any one of claims 1 to 6.

8. The pancreatic cancer cell line according to claim 7, wherein the microorganism collection number is CGMCC No. 22352.

9. The pancreatic cancer cell line of claim 7, as a pancreatic cancer cell model for the development and metastasis mechanism of pancreatic cancer, immunotherapy and its related combination therapy, drug resistance principle and new drug screening.

10. The use of the pancreatic cancer cell line of claim 7 in the construction of an animal model of pancreatic cancer, comprising inoculating a mouse with said pancreatic cancer cell line to construct an animal model of pancreatic cancer.

Technical Field

The invention relates to the construction of a tumor cell line, in particular to a pancreatic cancer stable cell line and a construction method thereof, and further relates to application of the pancreatic cancer stable cell line as a tool cell in the aspects of pancreatic cancer occurrence, development and metastasis mechanism, immunotherapy and related combined therapy thereof, drug resistance principle, new drug screening and the like, belonging to the field of pancreatic cancer stable cell lines and application thereof.

Background

Pancreatic cancer is a malignant tumor that occurs in exocrine pancreatic glands. Pancreatic malignancies can be derived from exocrine, endocrine or non-epithelial tissues of the pancreas, 95% of which are pancreatic cancers with the worst prognosis, high morbidity and mortality, and less than 8% survival rate for 5 years. Mainly adopts the methods of operation, radiotherapy and chemotherapy, etc. to treat the disease, but the treatment effect is not satisfactory.

Therefore, in response to this situation, there is a need for an innovation in alternative therapies such as immunotherapy and related combination therapies. The key link in the research of immunotherapy and related combined therapy is the establishment of a suitable tumor cell line. In view of the current situation of lack of research cell lines for immunotherapy of murine pancreatic cancer and related combination therapy thereof, the need of constructing a pancreatic cancer stable cell line as a tool cell for tumor etiology and prevention, detection kit, new drug development and the like is urgently needed.

Disclosure of Invention

One of the objectives of the present invention is to provide a pancreatic cancer stable cell line and a method for constructing the same;

the invention also aims to apply the pancreatic cancer stable cell line as a tool cell to the aspects of pancreatic cancer generation, development and metastasis mechanism, immunotherapy and related combined therapy thereof, drug resistance principle and related combined therapy thereof and the like.

The invention also provides a construction method of the pancreatic cancer cell line, which comprises the following steps:

(1) constructing a plasmid vector for over-expressing OVA genes; (2) after amplifying the plasmid vector of the over-expression OVA gene, transfecting the amplified plasmid vector into tool cells to carry out virus packaging to obtain virus supernatant; (3) and (4) transfecting the virus supernatant into target cells for screening to obtain the stable pancreatic cancer cell line.

As a preferred embodiment of the invention, the plasmid vector for over-expressing OVA gene is pHS-AVC-0700, and the element sequence is pLV-EF1a-hluc-P2A-mNeongreen-CMV-OVAL-3 Xflag-P2A-puro.

As a preferred embodiment of the present invention, in step (2), the plasmid obtained by amplification is transfected into the tool cell by lipofectamine3000 transfection; wherein, the tool cell is preferably 293T.

As a preferred embodiment of the present invention, the objective cell described in the step (3) is Pan 02;

as a preferred embodiment of the present invention, the screening in step (3) comprises: puromycin is added into the culture solution of the transfected target cells for screening to obtain a stable mouse pancreatic cancer Pan02-OVA cell line.

The invention uses a laser confocal microscope and a flow cytometer to observe the virus packaging level of the obtained cells, and uses a small animal in vivo imaging system IVIS Spectrum and the like to prove that the cell line obtained by screening is a stable pancreatic cancer Pan02-hluc-mNeongreen-OVA cell line.

The invention submits the stable pancreatic cancer Pan02-hluc-mNeongreen-OVA cell line obtained by screening to a patent approved organization for preservation, and the microorganism preservation numbers are as follows: CGMCC No.22352, classification name is: mouse pancreatic cancer stable cell line Pan02, with a preservation date of: 6/3/2021, storage unit: china general microbiological culture Collection center, preservation Address: xilu No. 1, Beijing, Chaoyang, Beijing, and institute of microorganisms, China academy of sciences.

The constructed stable pancreatic cancer Pan02-hluc-mNeongreen-OVA cell line is inoculated to the subcutaneous and pancreatic in-situ formed tumors of a mouse; therefore, the pancreatic cancer stable cell line constructed by the invention can be used as a tool cell or a pancreatic cancer cell model to be applied to the aspects of researching the occurrence, development and transfer mechanism of pancreatic cancer, immunotherapy and related combined therapy thereof, a drug resistance principle, new drug screening and the like.

Preferably, the mouse is a C57/BL6 mouse.

The pancreatic cancer cell model includes a cell model of pancreatic cancer development, progression, or metastasis.

The pancreatic cancer cell line established by the invention can be stably passaged, has good tumorigenicity, and can be suitable for establishing cell models and animal models. The pancreatic cancer cell line constructed by the invention can be used for researches on occurrence, development and transfer mechanisms of pancreatic cancer, immunotherapy and related combined therapy thereof, drug resistance principle, new drug screening and the like. The immune preparation and the combined treatment medicine thereof are applied to a pancreatic cancer animal model, and the weight and the tumor growth condition of the animal are observed and measured; substances to be detected which, after administration, lead to an improvement or cure in the symptoms of pancreatic cancer in animal models of pancreatic cancer are candidate substances for the treatment of pancreatic cancer.

The pancreatic cancer cell line constructed by the invention has the characteristics of stable growth speed, high mouse tumor formation rate and the like, provides a good tumor model for the research of pancreatic cancer, can be used as tool cells for tumor etiology and prevention, detection kits, new drug research and development and the like, and can assist in cancer prevention and treatment.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

The term "polynucleotide" or"nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al, J. Biol. Chem. 260:2605-,Mol Cell. Probes 8:91-98 (1994)）。

the term "host cell": refers to a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may be integrated into the host genome.

The term "transfection": it means that microorganisms such as viruses proliferate or replicate in host cells.

The term "passage": when the cells proliferate to reach a certain density, a part of the cells need to be separated and the nutrient solution needs to be updated, otherwise the continuous survival of the cells is influenced.

The term "adherent": when the cell suspension is inoculated into a culture vessel, adhesion occurs first, binding to the surface of the growth substrate to form an adherent.

The term "coding gene": a nucleic acid sequence transcribed into RNA.

Drawings

FIG. 1 shows the results of examining Pan02 cells into which the desired plasmid had been successfully introduced.

FIG. 2 is a schematic diagram showing the pancreatic cancer stable cell line obtained by confocal laser microscopy.

FIG. 3 shows that the pancreatic cancer stable cell line obtained by screening is detected by flow cytometry.

FIG. 4 is a graph of the results of day 3 in vivo imaging of cells obtained from pancreatic cancer stable cell lines obtained by in situ inoculation and screening of mouse pancreas.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

EXAMPLE 1 transformation

A plasmid vector pHS-AVC-0700 for over-expressing OVAL-Gallus Gallus and other genes is constructed, and the element sequence is pLV-EF1a-hluc-P2A-mNeongreen-CMV-OVAL-3 Xflag-P2A-puro.

Example 2 plasmid amplification

1. Experimental Material

Tryptone

Yeast extract

Sodium chloride

Agar-agar

Beaker

Glass rod

Volumetric flask

Conical bottle

Sealing film

Kraft paper

Rubber band

Sterilization pot

Super clean bench

PH meter

HCl

NaOH

Ampicillin

10cm culture dish

Water bath pot

Competent Escherichia coli stbl3

Destination plasmid pHS-AVC-0700

Packaging plasmids psPAX2, pMD2G

Liquid transfer device

Float for angling

EP pipe support

Shaking table

Coated glass rod

Centrifugal tube

Long forceps

Water-absorbing paper

TIANGEN small-and medium-amount extraction kit for endotoxin-free plasmid

2. Experimental procedure

2.1 preparation of LB Medium

S1, weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride by balance, dissolving in a clean beaker by adding a proper amount of ultrapure water with the conductivity of 18.25M omega cm, transferring the mixed solution in the beaker into a 1000ml volumetric flask, rinsing the beaker and a glass rod with the ultrapure water for 2-3 times, transferring the rinsing solution into the volumetric flask, and fixing the volume of the liquid in the volumetric flask to 1000ml by using the ultrapure water;

s2, subpackaging 700ml of LB culture medium prepared in S1 into a clean 1000ml reagent bottle (used for extracting plasmids subsequently); the remaining 300ml was transferred to a clean 500ml Erlenmeyer flask (for plating) and 4.5g agar was added; sealing with sealing film, covering bottle mouth with kraft paper, and fastening the reagent bottle and the conical flask with rubber band, placing in a high pressure steam sterilizing pot, sterilizing at 121 deg.C for 20min, transferring to a clean bench, measuring pH of the culture medium with a pH meter, and adjusting pH of the culture medium to 7.40 with HCl and NaOH;

s3, operation in a super clean bench: cooling agar LB culture medium to 40-50 deg.C, adding 300 μ l ampicillin solution (100 mg/ml), mixing, taking 10 clean culture dishes with diameter of 10cm, pouring 15ml agar LB culture medium into each culture dish, cooling and solidifying, sealing the culture dish with sealing film, and placing in 4 deg.C refrigerator;

2.2 plasmid amplification

S1, preparing a box of ice, and opening the water bath kettle at the same time, wherein the water bath kettle is set to be 42 ℃;

s2, taking out 4 tubes of competent escherichia coli stbl3 from a refrigerator at the temperature of-80 ℃, inserting and melting the escherichia coli stbl3 in ice, and marking the target plasmid pHS-AVC-0700 to be amplified and the names of packaging plasmids psPAX2 and pMD2G on the tubes;

s3, operation in a super clean bench: 2.5 microliter (100-;

s4, inserting competent escherichia coli stbl3 with S3 added into plasmids into ice for 20-30 min;

s5, fastening the sample obtained in the S4 by using a buoy, and then placing the sample in a water bath kettle at 42 ℃ for heat shock for 60-90S;

s6, immediately afterwards, transferring and inserting the sample obtained in S5 into ice for 3-5 min;

s7, operation in a super clean bench: adding 700 mul of LB culture medium into each sample tube obtained in S6, tightly covering a cover, and tightly wrapping the sample tube with a sealing film;

s8, placing the sample tube obtained in S7 on a buoy, then reversely buckling the sample tube on an EP tube frame, firmly binding the sample tube by using a rubber band, transversely placing the sample tube on a shaking table, and amplifying plasmids at 37 ℃ and 200rpm for 40-60 min;

s9, operation in a super clean bench: marking 4 agar LB culture dish plates (information such as plasmid names and the like), tearing off a sealing film, blowing and beating a sample obtained by uniformly mixing S8 by using a 1000-microliter pipette tip, then sucking 500-microliter of the sample, discarding, sucking 400-microliter of LB culture medium to a sample tube, blowing and uniformly mixing, sucking 600-microliter of the sample by using a pipette, adding the sample to the agar LB culture dish plate with the corresponding mark, and uniformly spreading the sample in the culture dish on the agar LB culture medium by using a coated glass rod;

s10, after paving corresponding samples on the 4 culture dishes according to the S9 step, half opening the cover of the culture dishes, naturally blowing air in a super clean bench until the samples are dry and not dry (about 5-10 min), closing the cover of the culture dishes, transferring the culture dishes to a 37 ℃ incubator, inversely placing the culture dishes for culture for 12-16h, taking out the culture dishes, and thus, colonies with the size of a uniform white needle point grow on each dish of agar LB culture medium;

s11, preparing 8 clean 50ml centrifuge tubes, marking the tube bodies and covers of the centrifuge tubes (2 tubes for each plasmid) according to the names of the plasmids, adding 15ml of LB culture medium and 15 mul of ampicillin solution (100 mg/ml) into each tube, and covering the covers to fully mix the mixture;

s12, selecting escherichia coli: taking a long tweezer, fully burning the head of the long tweezer by the outer flame of an alcohol lamp for sterilization, cooling for about 10 seconds, clamping the tail of a 10 microliter suction head by the long tweezer, holding a culture dish obtained by S10 with one hand to align light, picking 1 bacterial colony by the head of the suction head through light, then placing the suction head containing the bacterial colony into a corresponding 50ml centrifugal tube obtained by S11, screwing the cover and then turning the cover back for a half-circle, treating the 8 centrifugal tubes by the same steps, placing the centrifugal tubes on a shaking table in a slightly inclined way, and shaking at 37 ℃ and 200rpm for 12-16 hours;

s13, taking out the centrifugal tube containing the sample obtained in S12, screwing the cover of the centrifugal tube, centrifuging at 3000rpm for 10min, discarding the supernatant, and then reversely buckling the centrifugal tube on clean absorbent paper at room temperature for 20-30 seconds to fully draw out the supernatant in the centrifugal tube as far as possible;

s14, extracting plasmids: extracting target plasmid pHS-AVC-0700, packaging plasmid psPAX2 and pMD2G from the thallus precipitate obtained from S13 by using a TIANGEN small-extraction medium-amount plasmid kit, and measuring the concentration: the plasmid of interest pHS-AVC-0700=927.4 ng/. mu.L, the packaging plasmid psPAX2=1907.7 ng/. mu. L, pMD2G =841.5 ng/. mu.L.

Example 3 viral packaging

1. Experimental Material

Super clean bench

293T cells (70% -90% confluency) passaged in 6-well plate are revived in advance

Thermo Fisher liposome lipofectamine3000 kit

Destination plasmid pHS-AVC-0700

Packaging plasmids psPAX2, pMD2G

Pipette and tip

1.5ml clean centrifugal tube

Cell culture 6-well plate

Cell culture box

DMEM basal medium

DMEM complete medium

2. Experimental procedure (inner operation of cell super clean bench)

S1, transfection tool cell 293T:

each part of the system is as follows:

prepared in a 1.5ml centrifuge tube:

tube 1: 125 μ L of DMEM basal medium +7.5 μ L lipofectamine3000

Tube 2: 125 μ L of DMEM basal medium +2.5 μ g of DNA +5 μ L of P3000

Wherein the DNA of tube 2 contains the target plasmid pHS-AVC-0700: packaging plasmid psPAX 2: packaging plasmid pMD2G mass ratio = 6: 4.5: 1.5;

tubes 1, 2 were incubated for 5min at room temperature, respectively, and then tubes 1, 2 were mixed together and incubated for 10-15min at room temperature.

While incubating, 293T cells were replated with 1.75ml of DMEM basal medium.

After the incubation of the mixed solution (DNA-liposome complex) in the tubes 1 and 2, the DNA-liposome complex is sucked by a pipette, carefully and dropwise added to 293T cells, placed in an incubator at 37 ℃ and 5% CO₂Culturing in a humidifying incubator.

S2, after 12h of culture, 2ml of DMEM complete medium is replaced.

EXAMPLE 4 transfection of cells of interest

1. Experimental Material

Super clean bench

Pipette and tip

Cell culture 6-well plate

15ml centrifuge tube

Cell culture box

DMEM complete medium

10ml syringe

Filter membrane with pore size of 0.45 μm

Pan02 cells revived in advance (confluence degree of 70% -80%)

Infection enhancer polybrene (1000X)

2. Experimental procedure (inner operation of cell super clean bench)

S1, collecting culture solution supernatant (containing virus) after 48 hours, sucking the supernatant by using a 10ml syringe, filtering the supernatant by using a 0.45 mu m filter membrane to remove impurities such as cell debris and the like, and collecting the supernatant into a 15ml centrifuge tube;

s2, adding 1/10 volumes of DMEM fresh culture solution into the supernatant in each 15ml centrifugal tube, adding 2.2 mu l of infection enhancer polybrene, and then blowing and mixing uniformly by using a pipette;

s3, taking out target cells Pan02 with 70% -80% of adherence confluency to be transfected, and observing the cell state. The original liquid in the culture dish was removed by suction, and the mixed solution obtained in S2 was added to each culture dish, and the mixture was labeled on the culture dish and placed at 37 ℃ in 5% CO₂Culturing in a humidifying culture box for 12h, and then replacing 2ml of fresh DMEM culture solution to be placed in the culture box for culturing;

after liquid changing for 36h at S4 and S3, the growth states of the target cells introduced with the plasmids and the normal cells of the same species without the plasmids were observed, and the ratio of 1: 2 passage and then placing in an incubator at 37 ℃ and 5% CO₂And (5) culturing.

Example 5 screening of pancreatic cancer Stable cell line Pan02-OVA cell line

1. Experimental Material

Puromycin solution (5 mg/ml)

Pipette and tip

10cm culture dish

15ml centrifuge tube

Cell culture box

DMEM complete medium

2. Experimental method and experimental results

S1, previous pre-experiment: the puromycin solution kills target cells Pan02 without the introduction of a target plasmid, and the minimum total lethal concentration is 1.75 mu g/ml;

s2, example 4 step S3 after 36h of liquid change, the growth state of the plasmid-introduced target cells and the plasmid-uninduced normal allogeneic cells (control cells) were observed, and each well was subcultured with DMEM medium containing 2.00. mu.g/ml puromycin at 37 ℃ and 5% CO₂Culturing in a humidifying incubator. The DMEM culture solution containing 2.00 mu g/ml puromycin solution is replaced every 24h, and after the control cells are all dead, the target plasmid-introduced cell Pan02 is subcultured and screened for 5 times by using the DMEM culture solution containing 2.00 mu g/ml puromycin solutionThereafter, it was found that all Pan02 cells into which the plasmid of interest had been successfully introduced in the culture dish had Puro resistance.

As can be seen from the results of FIG. 1, Pan02 cells have successfully introduced the desired plasmid.

Example 6 detection and comparison of pancreatic cancer Stable cell line Pan02-OVA cell line of homologous interest without transfected Virus

The cells obtained in example 5 were observed for their virus packaging level by confocal laser microscopy and flow cytometry.

The cells obtained from example 5 were observed with confocal laser microscopy and the results are shown in FIG. 2 where almost all cells in the field of view were visible as green fluorescence, indicating that lentiviruses successfully transfected the mNeonGreen gene into Pan02 cells.

The cells obtained in example 5 were detected by flow cytometry:

and (3) staining by using a Flag flow antibody, detecting by using a flow cytometer, comparing positive cells expressing Flag genes (which can reflect the expression level of OVA genes under the same promoter as the OVA genes) in Pan02 and Pan02-hluc-mNeon Green-OVA cells of the same target cells of the untransfected viruses with the OVA genes to 87.93%, and indicating that the Pan02-hluc-mNeon Green-OVA cells can stably express the OVA (figure 3).

Example 7 tumor formation assay for pancreatic cancer stable cell lines

The Pan02-hluc-mNeon Green-OVA cells obtained in example 5 were subjected to 6X 10⁴After resuspension of individual cells with PBS, the cells were mixed with matrigel 1: 1 mixed and inoculated to C57/BL6 mice pancreas in situ to form tumors, and FIG. 4 is a graph of the in vivo imaging results of cells obtained by inoculating S5 mice pancreas in situ on day 3.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing description, it will be apparent to one skilled in the art that various changes, modifications, equivalents, and improvements may be made without departing from the spirit and scope of the invention.