CN112369369B - Construction method and application of human melanoma cell A375/DR5 stable transgenic nude mouse transplantation tumor model - Google Patents

Abstract

The invention relates to the field of construction of animal models, in particular to a construction method and application of a human melanoma cell A375/DR5 stable transgenic nude mouse transplantation tumor model. According to the invention, by constructing the human melanoma cell A375/DR5 stable transgenic nude mouse transplanted tumor model, the problems of large DR5 expression difference, low reproducibility, difficult target evaluation and the like in the original animal model are improved, an ideal experimental animal platform is provided for target drug evaluation by taking DR5 as a target, the reliability and effectiveness of preclinical research of malignant melanoma target drugs are improved, and the method has a certain practical application value. Also can provide research thinking and experimental basis for the research of experimental animals of other tumors and the evaluation of anti-tumor targeted drugs, and has important scientific significance.

Description

Construction method and application of human melanoma cell A375/DR5 stable-transgenic nude mouse transplantation tumor model

Technical Field

The invention relates to the field of construction of animal models, in particular to a construction method and application of a human melanoma cell A375/DR5 stable transgenic nude mouse transplantation tumor model.

Background

Malignant Melanoma (MM), abbreviated as malignant melanoma, is a tumor with high malignancy degree, is one of the most invasive and lethal tumors in skin tumors, has the characteristics of poor prognosis, easy transfer, easy relapse, easy drug resistance and the like, and has high treatment difficulty and extremely high mortality rate. To date, there is no effective treatment to prolong the survival of MM patients. The anti-tumor active targeting drug is considered to be one of the most promising drugs with high efficiency and low toxicity for curing malignant tumor such as MM at present. However, there are many problems and challenges in the research and development of active targeting drugs against MM, and one of the key problems is the lack of an ideal animal model for malignant melanoma which can be used to evaluate the efficacy of active targeting drugs. Therefore, the construction of stable and reliable MM animal models is an urgent problem to be solved in the process of researching and developing anti-melanoma targeted drugs, and is a research hotspot in recent years.

One of receptors of TRAIL is high expressed on melanoma cell and most tumor cells, and the combination of TRAIL and DR5 agonist antibody and DR5 can kill tumor cells and promote tumor cell death effectively and specifically without cytotoxicity to normal cell. Therefore, DR5 can be considered as a highly potential anti-melanoma target spot, and the construction of a stable and high-expression human melanoma cell line transplanted tumor animal model is undoubtedly an important link in the research process of developing an active targeting drug targeting DR 5. Although various melanoma models are established at home and abroad, few reports are given about establishment of a human melanoma transplantation tumor model, and particularly, the research on a melanoma animal model lacking a specific high-expression related target is rarely reported about the research on a melanoma model for evaluating a specific targeted drug. Therefore, it is necessary to develop an ideal tumor animal model for evaluating the targeting property and the synergistic attenuation characteristics of the anti-melanoma targeting drug.

Disclosure of Invention

The invention aims to provide a construction method of a nude mouse orthotopic transplantation tumor model of a human melanoma A375 cell strain stably transfected with a DR5 target, and provides a tumor animal model with stable target expression, small individual difference, strong controllability and reliability, and good repeatability and practicability for extensive researchers.

The invention provides a method for constructing a nude mouse transplantation tumor model of a human melanoma cell A375/DR5 stable transformant, which comprises the steps of selecting a human melanoma high-metastasis cell line A375 as a tumor forming cell, stably transferring a DR5 gene into the human melanoma cell A375, screening to obtain a DR5 stable high-expression A375/DR5 cell line for molding, and then injecting the A375/DR5 stable transformant nude mouse transplantation tumor model of the human melanoma cell A375/DR5 stable transformant subcutaneously at the back.

Furthermore, the nucleotide sequence of the DR5 gene is shown in SEQ ID NO. 1.

Furthermore, the preparation method of the A375/DR5 cell strain with high and stable DR5 expression comprises the following steps:

A. construction of a plasmid carrying the Gene of interest

pHBLV-CMVIE-Luc-T2A-Puro vector map construction: designing a target fragment according to a DR5 sequence, inserting the target fragment into an MCS region, regulating and expressing by a CMVIE promoter, and co-expressing ZsGreen gene and Puro regulated and controlled by an EF1 promoter in the vector;

B. lentiviral packaging

Preparing a lentivirus shuttle plasmid and an auxiliary packaging original vector plasmid thereof, respectively carrying out high-purity endotoxin-free extraction on three plasmid vectors, co-transfecting 293T cells, replacing the transfected cells with a complete culture medium for 6h, culturing for 48h and 72h, collecting cell supernatant rich in lentivirus particles, filtering virus supernatant by a 0.45um filter, and concentrating the virus by ultracentrifugation of the virus supernatant. Concentrating to obtain high-titer lentivirus concentrated solution. The virus pellet was resuspended in 500ul fresh medium and stored at-80 ℃ or even in liquid nitrogen. And (4) determining the titer.

C. TNFRSF10B lentivirus A375 cell line

A375 cells were plated in 6-well plates at 5X 10 per well ⁵ Cells, different lentiviruses (pHBLV-CMVIE-Luc-T2A-Puro and TNFRSF 10B) were added to each well, MOI =6, and the fluid was changed after 24 hours.

After 48 hours, the culture medium containing 1ug/ml puromycin was replaced for selection, and after the cells had stabilized growth, they were passaged. After 2 generations, the cells were frozen until the number of cells reached the requirement. The cell morphology after establishment of the line is shown in FIG. 4.

D. Verification of stable transgenic plant

The screened cell lines are identified to obtain the A375/DR5 cell line with high and stable DR5 expression.

Further, the construction method comprises the following steps: placing A375/DR5 cell strain with high and stable DR5 expression in RPMI-1640 culture solution containing 10% Fetal Bovine Serum (FBS), at 37 deg.C and 5% CO ₂ Culturing; taking cells in logarithmic growth phase to make the concentration of the cells to be 1 multiplied by 10 ⁷ Placing the cell suspension of each ml in a pre-cooling way at 4 ℃, adding matrigel with the same volume, uniformly mixing, wherein a suction pipe and the like which are contacted with the matrigel in the whole uniformly mixing process can be used after the pre-cooling at 4 ℃, and the operation process is carried out on ice; after mixing, ice-bath for standby; selecting a BALB/c nude mouse, and inoculating 0.2ml of cell suspension to the back subcutaneous part of the nude mouse; after 15-20 days of breeding, selecting nude mice with mellow and compact tumor shape and tumor diameter of 10mm as melanoma animal models.

The Matrigel is a commercially available product, and the Matrigel Type 2 can be selected

(Merck, darmstadt, germany, matrigel was added to facilitate in vitro cell culture and differentiation, to aid in neoplasia.

In a second aspect of the present invention, there is provided a human melanoma cell a375/DR5 stable transformant nude mouse graft tumor model constructed by the method described above.

In a third aspect of the invention, the application of the human melanoma cell A375/DR5 stable transformant nude mouse transplantation tumor model constructed by the method in the evaluation of anti-melanoma active targeting drugs is provided.

The invention has the advantages that:

aiming at the main problems existing in the current tumor animal model and melanoma model, the invention aims to construct a melanoma in-situ transplantation tumor model with stable DR5 target expression, small individual difference, strong reliability and good repeatability, and the melanoma in-situ transplantation tumor model is used for evaluating the anti-melanoma active targeting drug. The method comprises the steps of selecting a human melanoma high-transfer cell strain A375 as a tumorigenic cell, adopting a lentivirus vector, stably transferring a DR5 gene into the human melanoma cell A375, screening to obtain a DR5 stably and highly expressed A375/DR5 cell strain for molding, establishing a nude mouse transplanted tumor model through back subcutaneous injection, and researching the tumorigenic rate, the growth speed, the expression of DR5, the sensitivity of a DR5 monoclonal antibody (DR 5 mAb) and other biological characteristics of the transplanted tumor.

According to the invention, by constructing the human melanoma cell A375/DR5 stable transgenic nude mouse transplanted tumor model, the problems of large DR5 expression difference, low reproducibility, difficult target evaluation and the like in the original animal model are improved, an ideal experimental animal platform is provided for target drug evaluation by taking DR5 as a target, the reliability and effectiveness of the MM target drug preclinical research are improved, and the method has a certain practical application value. Also can provide research thinking and experimental basis for the research of experimental animals of other tumors and the evaluation of anti-tumor targeted drugs, and has important scientific significance.

Drawings

FIG. 1.PHBLV-CMVIE-Luc-T2A-Puro vector map.

FIG. 2.DR5 monoclonal PCR identification results. Lane7: geneRay 1kb DNA MarkerLane1-6: the PCR products were identified by DR5 monoclonal (Marker: 12000bp,8000bp,6000bp,5000bp,4000bp,3000bp,2500bp,2000bp,1500bp,1000bp,750bp, 5000bp, 250bp, in this order from the top to the bottom).

FIG. 3 is a graph of cell morphology by titer determination.

FIG. 4 is a cell morphology map of A375/DR5 stable transformant.

Figure 5.Dr5 gene lysis curve.

Figure 6.Dr5 gene amplification curve.

Figure 7.Hgapdh gene lysis curve.

FIG. 8.HGAPDH gene amplification curve.

FIG. 9 tumor-bearing nude mouse model constructed using A375/DR5 cells.

FIG. 10 shows a tumor-bearing nude mouse model constructed using A375 cells.

FIG. 11A 375/DR5 cell constructed tumor-bearing nude mouse model. (A: nude mice bearing normal A375 cell tumor, BCDE: A375/DR5 cell tumor).

FIG. 12 HE staining pattern of A375/DR5 tumor tissue.

FIG. 13 immunohistochemical staining of tumor tissue DR5 (scale 50 μm).

FIG. 14 tumor-bearing nude mouse model constructed using A375/DR5 cells.

FIG. 15 tumor-bearing nude mouse model constructed using A375 cells.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example 1: construction and screening of human melanoma cell A375/DR5 stable transformant

1.1 vectors and Gene information of interest

1.1.1pHBLV-CMVIE-Luc-T2A-Puro vector map construction

According to the sequence of DR5, a target fragment is designed, an MCS region is inserted, the expression is controlled by a CMVIE promoter, and the vector contains the ZsGreen gene controlled by an EF1 promoter and the Puro for co-expression, as shown in figure 1.

1.1.2DR5 Gene sequence information

The DR5 gene nucleotide sequence is shown in SEQ ID NO. 1.

1.1.3 recovery of the gel after completion of the digestion of the vector

And (3) recovering glue after the plasmid vector is subjected to enzyme digestion, and verifying to show that the plasmid vector is successfully constructed.

1.1.4DR5 fragment PCR recovery

And (3) recovering glue after the DR5 plasmid is digested, and verifying that the DR5 plasmid is successfully constructed.

1.1.5 competent cell preparation and monoclonal PCR product identification

And (3) selecting transformed DR5 plates, shaking the plates at 37 ℃ at 250 rpm for 14 hours, carrying out PCR identification on the plates by using bacterial liquid, and sending the positive clone bacterial liquid to a sequencing company for sequencing. The results of the monoclonal PCR assay (fig. 2) show: the DR5 monoclonal construction was successful.

1.1.6DR5 overexpression vector sequencing results

DR5 overexpression vector sequencing results: the reverse complementary sequence is shown in SEQ ID NO.2, wherein the 115 th site to the 1437 th site are target sequences, and the target sequence is shown in SEQ ID NO. 1.

Construction and verification of 1.2A375/DR5 stable transformant

1.2.1 Lentiviral vectors, packaging cells and strains

The virus packaging system is a three-plasmid system and comprises pSPAX2, pMD2G and pHBLV-CMVIE-Luc-T2A-Puro.

Cell line 293T, packaging cell for lentivirus, anchorage-dependent epithelioid cell, growth medium DMEM (10% FBS-containing). Adherent cells are cultured, grown and proliferated to form a monolayer of cells.

Strain escherichia coli strain DH5- α. For amplification of lentiviral vectors and helper packaging vector plasmids.

1.2.2 Lentiviral packaging

Preparing a lentivirus shuttle plasmid and an auxiliary packaging original vector plasmid thereof, respectively carrying out high-purity endotoxin-free extraction on three plasmid vectors, co-transfecting 293T cells, replacing the transfected cells with a complete culture medium 6h, culturing for 48h and 72h, collecting cell supernatant rich in lentivirus particles, filtering virus supernatant by a 0.45um filter (Millpore company), and concentrating the virus supernatant through ultracentrifugation. Concentrating to obtain high-titer lentivirus concentrated solution. The virus pellet was resuspended in 500ul fresh medium and stored at-80 ℃ or even in liquid nitrogen.

1.2.3 Titer assay (dilution count method)

Cell preparation: the 293T cells with good growth state are digested and counted, and then diluted to 1x10 ⁴ Perml, add to 96 well plates, 100 ul/well, prepare 6 wells for each virus. Put at 37 ℃ and 5% CO ₂ Culturing in an incubator.

Adding viruses: the following day, 3-fold gradient dilutions were made in EP tubes for 6 serial dilutions. The dilution method is as follows: each virus was prepared into 6 1.5ml EP tubes, 90ul of culture medium was added to each tube, 10ul of virus stock solution was added to the first tube, and after mixing, 10ul of virus stock solution was aspirated and added to the second tube. By analogy, 6 dilutions (10-3.3 x 10) were made ^-2 ) And adding the diluted solution into the corresponding 96-well.

The results were observed and titers calculated: on the fifth day, the virus titer was calculated in wells with a fluorescence percentage of 10-30% as observed under a fluorescence microscope. Titer (PFU/ml) = percent fluorescence = MOI (1) × viral dilution fold × 10 ³ . The titer assay results were as follows: control group titer =4*10 ⁴ *10% MOI (1) viral dilution multiple (30) 10% ³ ＝1*10 ⁸ TU/ml (0.033 ul virus well selected) TNFRSF10B titer =4 x10 ⁴ *20% MOI (1) viral dilution factor (30) 10 ³ ＝2*10 ⁸ TU/ml (0.033 ul virus wells selected) and the assay chart is shown in FIG. 3.

1.2.4TNFRSF10B lentivirus A375 cell line

A375 cells were plated in 6-well plates at 5X 10 per well ⁵ Cells, different lentiviruses (pHBLV-CMVIE-Luc-T2A-Puro and TNFRSF 10B) were added to each well, MOI =6, and the fluid was changed after 24 hours.

After 48 hours, the culture medium containing 1ug/ml puromycin was replaced for selection, and after the cells had stabilized growth, they were passaged. After 2 generations, the cells were frozen until the number of cells reached the requirement. The cell morphology after establishment is shown in FIG. 4.

1.2.5 Stable transformant validation

The screened cell lines are identified, the overexpression condition of DR5 is examined, and the result shows that: hGAPDH and DR5 solubility curves were good (fig. 5, fig. 7), demonstrating that the RNA extraction and QPCR processes were not problematic. hGAPDH and DR5 amplification curves were better (fig. 6, fig. 8), demonstrating that DR5 was overexpressed in a375 cells.

Example 2: establishment and evaluation of human melanoma cell A375 nude mouse transplantation tumor model and A375/DR5 stable transgenic nude mouse transplantation tumor model

2.1 model construction

The constructed high-expression DR5 human malignant melanoma cell strain A375/DR5 and normal human melanoma cell A375 are used for inducing and forming a malignant melanoma nude mouse subcutaneous transplantation tumor animal model.

The A375/DR5 cell line was placed in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS), at 37 ℃ and 5% CO ₂ And (4) culturing. Taking cells in logarithmic growth phase to make the concentration of the cells be 1 multiplied by 10 ⁷ The cells/ml suspension was placed at 4 ℃ for pre-cooling, and an equal volume of Matrigel (Matrigel Type 2) was added

(Merck, darmstadt, germany), mixing homogeneously, the whole being mixedThe suction pipe and the like which are contacted with the matrigel in the homogenizing process can be used after precooling at 4 ℃, and the operation process is carried out on ice. After mixing uniformly, ice-bath for standby; selecting a BALB/c nude mouse, and taking a cell suspension to inoculate the cell suspension to the subcutaneous back (0.2 ml/mouse) of the nude mouse; after the nude mice are carefully bred for 15-20 days, the nude mice with mellow and compact tumor shapes and tumor diameters of 10mm are selected as experimental models.

10 tumor-bearing nude mice model nude mice constructed by the A375/DR5 cells, and 5 tumor-bearing nude mice model nude mice constructed by the A375/DR5 cells are used for comparison. After inoculation of malignant melanoma A375/DR5 cells or A375 cells, and 2 weeks of rearing, tumor-bearing nude mice with a tumor volume average diameter of about 10mm were selected as an experimental model, see FIG. 9, FIG. 10.

2.2 in vivo imaging verification

Selecting 4 tumor-bearing nude mice and 1 normal A375 cell-bearing nude mouse, anesthetizing, injecting pGL-3 luciferase reaction solution through tail vein, observing after 30min, as shown in figure 11, wherein DR5 high expression plasmid loaded by nude mice emits fluorescence after reacting with the reaction solution in blood due to the pGL-3 luciferase sequence, and is detected by a living body imaging detection instrument. As shown in FIG. 11, the fluorescence was clearly detected at the tumor site, and the activity of the tumor tissue emitted fluorescence of different intensities according to the size of the tumor, providing a basis for the growth determination of the tumor. And the nude mice with normal A375 cells do not see fluorescence.

2.3 evaluation of tumor indicators

2.3.1HE staining

Taking 4 constructed nude mice models in the 1, killing the nude mice by using a cervical dislocation method, obtaining tumor tissues, fixing, dehydrating, embedding, slicing and carrying out conventional hematoxylin-eosin staining by using neutral formalin, observing the growth condition of the tumor tissues, and showing in figure 12 that the tumor tissues of the 4 nude mice grow vigorously, thereby proving that the modeling is successful, and the method can be used for the subsequent construction and application of A375/DR5 related tumor animal models.

2.3.2 antibody staining

After the nude mice were sacrificed, the tumor tissues were removed along with the skin, and after being split longitudinally in half, the sections were deparaffinized to water conventionally. Immunohistochemical staining is carried out, the expression condition of the DR5 receptor is observed, as shown in figure 13, under different magnification (scale of 50 mu m), a yellow part can be clearly observed, the expression staining of the DR5 is indicated, and the fact that a large amount of DR receptors are expressed after tumor inoculation is proved, so that the method can be used for subsequent construction and application of A375/DR5 related tumor animal models.

Example 3: in vivo targeting property, anti-tumor effect and safety evaluation of DR5mAb in A375/DR5 stable transgenic nude mouse transplantation tumor model

3.1 construction of models of transplantation tumor of different nude mice

The constructed high-expression DR5 human malignant melanoma cell line A375/DR5 and normal human melanophore A375 are used for inducing to form a malignant melanoma nude mouse subcutaneous transplantation tumor animal model.

The A375/DR5 cell line was placed in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS), at 37 ℃ and 5% CO ₂ And (5) culturing. Taking cells in logarithmic growth phase to make the concentration of the cells be 1 multiplied by 10 ⁷ Placing the cell suspension per ml, precooling at 4 ℃, adding matrigel with the same volume, uniformly mixing, wherein a suction pipe and the like which are in contact with the matrigel in the whole uniform mixing process can be used after precooling at 4 ℃, and the operation process is carried out on ice. After mixing, ice-bath for standby; selecting a BALB/c nude mouse, and taking a cell suspension to inoculate the cell suspension to the subcutaneous back (0.2 ml/mouse) of the nude mouse; after the nude mice are carefully bred for 15-20 days, the nude mice with mellow and compact tumor shapes and tumor diameters of 10mm are selected as experimental models.

10 tumor-bearing nude mouse models constructed by the A375/DR5 cells, and 10 tumor-bearing nude mouse models constructed by the A375/DR5 cells are used for comparison. After inoculation of malignant melanoma A375/DR5 cells or A375 cells, and 2 weeks of rearing, tumor-bearing nude mice with a tumor volume average diameter of about 10mm were selected as experimental models, see FIG. 14, FIG. 15.

3.2 tumor inhibition index investigation

3.2.1 tumor inhibition Rate

From the constructed nude mouse transplantation tumor model, 5 mice were selected per group. The specific administration method comprises the following steps: DR5mAb was treated 1 time at the same dose on days 1,3,5 and 7 of treatment. The grouping is as follows: group A: group A375; group B: group A375/DR 5.

On day 16, the mice were sacrificed by dislocation of cervical vertebrae, and serum was taken from nude mice to detect glutamic-pyruvic transaminase, muscular intoxication, and blood leukocyte count. And (4) weighing the stripped tumor body, and calculating the tumor weight inhibition rate. Tumor weight inhibition rate = (average tumor weight of control group-average tumor weight of experimental group)/average tumor weight of control group × 100%.

The results are shown in table 1, and show that the tumor inhibition rate of the group B and the group A375/DR5 is significantly higher than that of the group A and the group A375, and P is less than 0.05, which has statistical significance, and probably because the binding of tumor cells DR5 is increased after the tumor cells DR5 are over-expressed in the group A375/DR5, so that the DR 5-mediated apoptosis process is widely opened. Shows a high inhibitory effect on tumor cells.

TABLE 1 tumor inhibition rate of nude mice transplantation tumor model of different groups

3.2.2 evaluation of treatment safety

The analysis results in Table 2 show that there is no significant difference among the 2 groups in the measurement results of leucocyte number, alanine aminotransferase and creatinine of nude mice with tumor, and the P values are all greater than 0.05, which indicates that the antibody treatment has no obvious liver and kidney toxicity and high safety.

TABLE 2 liver and kidney function of nude mice transplantation tumor model of different groups

Although there are many models established at present, none of the models can completely meet the needs of researchers, and relatively speaking, transgenic models and gene reconstruction are more ideal models among many models at present. The transgenic animal is a biological high and new technology to establish various transgenic animal models of human diseases and study the expression regulation rule of exogenous genes in the whole animal, thereby playing a great role in promoting the etiology, pathogenesis and therapeutics of the human diseases. At present, the genetic animal model is mainly used for researching disease pathogenesis, detecting a new treatment scheme, evaluating drug effect and screening drugs. Therefore, various animal models established by molecular and genetic means will be one of the important contents for tumor research in the present and future. With the development of research and the improvement of various technologies, people will gradually find the pathogenesis of tumors, the relationship between tumors and hosts, and the invasion and metastasis processes of tumors, so as to establish an animal model more similar to human tumor diseases.

The construction of the animal experiment model provides a powerful method for identifying pharmacodynamics and pharmacokinetics of the targeting preparation. The targeted preparation is studied more and more deeply, and the main problem of experimental discussion is how to verify the targeting property of the preparation and the characteristics of the targeted preparation in the aspect of synergy and attenuation. The tumor targeting preparation requires accurate killing of target tumor cells, and uses the concept of missile guidance technology to reduce the damage of surrounding normal tissues to the maximum extent. The characteristics of the targeting preparation provide tests for a verification method of the targeting preparation, a large number of reports are reported for the research of applying various tumor models to pharmacodynamic experiments at present, and in the pharmacokinetic research, the targeting preparation is verified to be a normal animal commonly used, but the report adopting the tumor animal model verification method is rare. If the tumor animal model can provide the tumor biological characteristics similar to those of tumor patients, the pharmacodynamics and pharmacokinetic research of the tumor targeting preparation can provide more reliable guarantee.

In conclusion, the invention successfully constructs a human melanoma cell A375 nude mouse transplanted tumor model and an A375/DR5 stable transgenic nude mouse transplanted tumor model by a back subcutaneous injection method, and then measures the tumorigenicity rate, the growth speed, the tumor size and the DR5 expression of the transplanted tumor, and the result shows that the A375/DR5 cell strain has the characteristics of high tumorigenicity rate and rapid tumor growth, maintains the biological characteristics of over-expression of DR5, and has small individual difference, strong controllability and reliability. And further using anti-DR 5 monoclonal antibody DR5mAb to treat the transplantation tumor model, finding that the treatment of the antibody has no obvious liver and kidney toxicity and higher safety. And the A375/DR5 tumor is sensitive to the antibody reaction, shows high inhibition effect on tumor cells, and can completely meet the requirements of preclinical research of the antibody medicament. In addition, the establishment of a human melanoma cell A375/DR5 stable transgenic nude mouse transplantation tumor model can lay a good foundation for efficiently, practically and reliably evaluating the pharmacological action of an active targeting tumor drug targeting DR5 in the future, and can provide an ideal experimental animal platform for evaluating the pharmacological action of an anti-tumor targeting drug.

While the preferred embodiments of the present invention have been illustrated and described, 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 as defined by the appended claims.

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Claims (2)

1.A human melanoma cell A375/DR5 stable transfer strain nude mouse transplantation tumor model construction method is characterized in that a human melanoma high transfer cell line A375 is selected as a tumor forming cell, DR5 gene is stably transferred into the human melanoma cell A375, an A375/DR5 cell line with DR5 stable high expression for molding is obtained after screening, and then the human melanoma cell A375/DR5 stable transfer strain nude mouse transplantation tumor model is established by back subcutaneous injection; the nucleotide sequence of the DR5 gene is shown as SEQ ID NO. 1; the construction method comprises the following steps: placing the A375/DR5 cell line with high and stable DR5 expression in RPMI-1640 culture solution containing 10% fetal bovine serum, and making the content of CO at 37 deg.C and 5% ₂ Culturing; taking cells in logarithmic growth phase to make the concentration of the cells be 1 multiplied by 10 ⁷ Placing the cell suspension per ml for precooling at 4 ℃, adding matrigel with the same volume, uniformly mixing, wherein a suction tube which is in contact with the matrigel in the whole uniformly mixing process can be used after precooling at 4 ℃, and the operation process is carried out on ice; after mixing uniformly, ice-bath for standby; selecting a BALB/c nude mouse, and taking 0.2ml of cell suspension per mouse to inoculate the cell suspension to the back subcutaneous part of the nude mouse; after breeding for 15-20 days, selecting a nude mouse with a mellow and compact tumor shape and a tumor diameter of 10mm as a melanoma animal model; the preparation method of the A375/DR5 cell strain with the DR5 stably and highly expressed comprises the following steps:

A. construction of a plasmid carrying the gene of interest: pHBLV-CMVIE-Luc-T2A-Puro vector map construction: designing a target fragment according to a DR5 sequence, inserting the target fragment into an MCS region, regulating and expressing by a CMVIE promoter, wherein the vector contains ZsGreen gene regulated and controlled by an EF1 promoter and Puro coexpression;

B. and (3) slow virus packaging: preparing a lentivirus shuttle plasmid and an auxiliary packaging original vector plasmid thereof, respectively performing high-purity endotoxin-free extraction on three plasmid vectors, co-transfecting 293T cells, replacing the cells with a complete culture medium after 6h of transfection, culturing for 48h and 72h, collecting cell supernatant rich in lentivirus particles, filtering virus supernatant by a 0.45um filter, and concentrating viruses by ultracentrifugation of the virus supernatant; concentrating to obtain high-titer lentivirus concentrated solution; resuspending virus precipitate in 500ul fresh culture medium, and storing at-80 deg.C or even in liquid nitrogen; measuring the titer;

C. TNFRSF10B lentivirus a375 cell line was established: a375 cells were plated in 6-well plates at 5X 10 per well ⁵ Cells, different lentiviruses pHBLV-CMVIE-Luc-T2A-Puro and TNFRSF10B are added into each well, MOI =6, and the solution is changed after 24 hours; after 48 hours, replacing culture solution containing 1ug/ml puromycin to carry out screening, and carrying out cell passage after the cells grow stably; after 2 generations, freezing and storing the cells when the number of the cells meets the requirement;

D. and (3) verifying the stable transgenic plant: the screened cell lines are identified to obtain the A375/DR5 cell line with high and stable DR5 expression.

2. The use of the human melanoma cell A375/DR5 stable transformant nude mouse graft tumor model constructed by the method of claim 1 in the evaluation of anti-melanoma active targeting drugs.

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