CN108588029B - Prostate epithelial cell malignant change induction method and kit - Google Patents
Prostate epithelial cell malignant change induction method and kit Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and provides a prostate epithelial cell malignant change induction method and a kit, wherein the preparation method comprises the following steps: A) constructing a BPH-1 cell line with a PITX3 gene knocked out to obtain a PITX3-BPH-1 cell with the BPH-1 gene of PITX3 knocked out; B) constructing a PITX3-BPH-1 cell transfer nude mouse model; C) primary culture of the cells of the metastasis range; D) and (4) inducing malignant transformation. The malignant cell model obtained by transforming the benign cells can be applied to the clinical basic research of the prostatic cancer, has the characteristics of simple and convenient operation, strong repeatability, high accuracy and the like, and does not have the problem of difficult operation of the induction process.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a prostate epithelial cell degeneration induction method and a kit for inducing prostate epithelial cell degeneration.
Background
Prostate cancer is one of the most common malignancies in men, with increasing incidence with age, globally accounting for the 1 st and 2 nd mortality in men. With the change of dietary structure, aging population and the popularization of Prostate Specific Antigen (PSA) detection, the incidence rate still continuously and rapidly increases in recent years, and the proportion of middle and late stages in the initial patients is high, which has become one of the important diseases affecting male health. Therefore, constructing an appropriate cell model for clinical basic research of prostate cancer has been one of the hot topics, and there are many prostate cancer cell models isolated from primary foci, metastases and induction anaplasia of prostate. However, no technique for inducing malignant transformation of benign prostate epithelial cells has been reported so far.
BPH-1 is a common cell line in clinical and basic research, come from transurethral prostate gland electrotomy human prostatic hyperplasia tissue, after SV40 immortalizes the study of the prostate hyperplasia, compared with normal prostate epithelial cell, the cell has the characteristics of rapid growth, high tumor formation rate, in addition, as prostate hyperplasia model cell, its metabolic mechanism, growth mode and biological expression are obviously different from normal prostate epithelial cell, therefore, in the prostate cancer research field, BPH-1 and normal prostate epithelial cell (RWPE-1) are regarded as benign cell and tumor cell to contrast together.
In addition, in a big sample second generation sequencing study before the applicant, PITX3 gene is found to be relatively low expressed in 65-pair prostate cancer patient tissues, and the PITX3 protein encoded by the gene belongs to a RIEG/PITX3 homeobox family member, is positioned on the long arm of chromosome 10, and belongs to a bicoid group on the structural domain. The family has transcription factor activity, is expressed in skeletal muscle and testis, and has been reported to participate in the crystal formation in the eye development and the terminal differentiation of dopaminergic neuron in the substantia nigra compacta of the midbrain, and the loss of the gene is related to Parkinson's disease and congenital cataract.
There is no one currently linking the relatively low expression of PITX3 gene in prostate cancer patient tissues with the BPH-1 cell line, and there is also a lack of a model of malignant cells transformed from benign cells, and no technique for inducing malignant transformation of benign prostate epithelial cells has been reported.
Disclosure of Invention
The invention aims to provide a prostate epithelial cell malignant change induction method and a kit for inducing prostate epithelial cell malignant change, wherein a malignant cell model is obtained by transforming benign cells, and the method is applied to clinical basic research of prostate cancer.
The invention provides a prostate epithelial cell degeneration induction method, namely a construction method of a prostate epithelial cell degeneration nude mouse model.
The technical scheme of the invention is as follows: firstly constructing a BPH-1 cell line of a knockout PITX3 gene, then transferring the obtained cell line to a nude mouse to enable the nude mouse to generate a focus, then taking the focus of the nude mouse out to carry out primary culture of a focus cell, and finally injecting the cultured primary cell into a healthy nude mouse to carry out tumor induction to obtain a malignant tumor model. The method specifically comprises the following steps:
A. constructing a BPH-1 cell line with a PITX3 gene knocked out to obtain a PITX3-BPH-1 cell with the BPH-1 gene of PITX3 knocked out;
B. construction of PITX3-BPH-1 cell transfer nude mouse model
PITX3-BPH-1 cells were digested, dispersed, resuspended in PBS, and assigned a 5X 10 cell/mouse5～10×105Injecting 100-200 mu L of cell suspension into the vein of each nude mouse on the order of individual cells, observing the metastasis condition by luciferin small animal imaging every week until organ metastasis occurs and the cell concentration of a metastatic focus is more than 106p/sec/cm2/sr；
C. Primary culture of metastatic focus cells
Removing impurity tissues attached to the organ transfer stove from the organ transfer stove, repeatedly cleaning, and shearing the organ transfer stove into fragments; adding trypsin digestive juice with the mass fraction of 0.25% into the fragments of the transfer stove, washing the fragments for several times by using the inoculating liquid after digestion, then suspending the fragments by using the inoculating liquid, repeatedly blowing tissue blocks in the mixed rotary liquid until the tissue blocks are turbid, and transferring the supernatant into a culture bottle for later use; adding inoculation liquid into the culture bottle, blowing and beating again, gradually digesting the tissue block after repeating for a plurality of times, collecting supernatant containing single cell suspension into the culture bottle, suspending cells again by the inoculation liquid, and counting the cells;
inoculating a certain number of cells into a porous culture plate, culturing for 18-30 hours until the cells adhere to the wall, changing the whole culture solution, culturing for 3-5 days, then culturing for 3-5 days according to the growth condition of BPH-1 cells, changing a kanamycin culture solution for 3-5 days to inhibit the growth of hybrid cells, obtaining primary transfer focus cells which are simply cultured, and then changing the whole culture solution for 1 time every 3-5 days, wherein half of the culture solution is changed every time;
D. induction of malignant transformation
The primary metastatic focus cells were digested and dispersed, resuspended in PBS, and assigned to 5X 10 cells per nude mouse6～10×106Injecting 100-200 mu L of cell suspension into each mouse subcutaneously in the order of magnitude of cells, and culturing for 2-3 weeks to obtain tumor-induced nude mice.
In the invention, the organ metastasis of the nude mouse in the step B can be a lung metastasis, a liver metastasis and other multiple organ metastases, and the organ with the most existing lesion is a lung, so the operation of the embodiment part of the invention is performed according to the lung metastasis.
Preferably, the method for constructing the PITX3 gene-knocked-out BPH-1 cell line in step a comprises:
packaging the PITX3 gene into Cas 9-guideeRNA lentivirus, transfecting BPH-1 cells together with 1-2 mu g of commercial CRISPR-Cas9 virus and Luciferace virus respectively, and after 2-3 days of infection, adding Puromycin for screening for 4-6 days to obtain the cell PITX3-BPH-1 which is successfully infected and used for knocking out the BPH-1 of the PITX 3.
The CRISPR-Cas9 system is derived from Streptococcus pyogenes, contains two nuclease domains, and can cut two single strands of DNA respectively. Cas9 first binds to crRNA and tracrRNA as a complex, then binds to and invades DNA through a PAM sequence to form an RNA-DNA complex structure, and further cleaves a target DNA double strand to break the DNA double strand. The PAM sequence has a simple structure (5-NGG-3'), can find a large number of targets in almost all genes, and is widely applied.
The Luciferace virus was used for post-transfection imaging, and the presence of the virus allowed the transfected mice to be directly tested for transfection level by the imaging system.
In addition, in the present invention, packaging of PITX3 gene as Cas 9-guideeRNA lentivirus and co-transfection of the three viruses were performed by Heyu Biotechnology (Shanghai) Inc.
Preferably, the sequence of the Cas 9-guideeRNA lentivirus is shown as SEQ ID NO. 1 and is CCCGACATGAGCACGCGCG.
Preferably, in the step C, after adding 0.25 mass percent of trypsin digestion solution into the fragments of the transfer focus, digesting the fragments in an incubator at 37 ℃ for 30-40 min, then transferring the fragments of the tissue into a centrifuge tube, discarding the residual digestion solution, and washing the fragments with the inoculation solution.
Preferably, the inoculation liquid is a DMEM liquid culture medium, the whole culture liquid is a DMEM liquid culture medium containing 10% FBS fetal bovine serum by volume fraction, the final concentration of the kanamycin culture liquid is 2.5ug/ml, and the half liquid is replaced.
Preferably, in step C, after BPH-1 cells are observed to be adherent stably, kanamycin culture solution is used for replacement.
Preferably, the organ metastases are lung metastases.
In a second aspect of the invention, a kit for inducing malignant transformation of prostate epithelial cells is provided.
The kit comprises a BPH-1 cell gene knockout system, an organ transfer focus primary cell culture system and a nude mouse injection system, wherein the BPH-1 cell gene knockout system comprises a lentivirus transfection and detection reagent.
The invention has the following beneficial guarantee and effects:
the applicant is a hospital, can obtain a large number of tumor specimens of prostate cancer patients, and provides a powerful guarantee for the research of the invention.
In terms of technology, the prostate epithelial cell degeneration induction method is essentially gene knockout of a BPH-1 cell line and primary cell culture of an organ transfer focus, both the two technologies are mature day by day, and the method has the characteristics of simplicity and convenience in operation, strong repeatability, high accuracy and the like, and does not have the problem of difficult operation of an induction process.
In addition, because the time of the primary cells in vitro is short, the genetic character is similar to the cells in vivo, the biological character is not greatly changed, the tumorigenic capacity of the primary cells is strong, a tumor model with high malignant degree is easily generated through malignant transformation induction, the clinical reference value and the reliability are high, the benign cells are converted into the malignant cell model, and the prostate cancer model can be used for clinical basic research of prostate cancer.
Drawings
FIG. 1 is a graph showing the results of flow detection of metastatic focus cells.
FIG. 2 is a graph showing the results of the detection of invasion ability of metastatic focus cells.
FIG. 3 is a graph showing the results of the measurement of the proliferation potency of metastatic focus cells.
FIG. 4 is a diagram showing the flow analysis of the proliferation ability of metastatic focus cells.
FIG. 5 is a graph showing the ability of metastatic focus cells to induce tumor formation in nude mice, wherein 1-5 are metastatic focus cells and 6 is BPH.
Detailed Description
The present invention will now be described in detail with reference to examples and drawings, but the practice of the invention is not limited thereto.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Experimental procedures without specific conditions noted in the following examples were carried out either under conventional conditions or under conditions recommended by the manufacturer.
Example 1: prostate epithelial cell malignancy induction
(1) Obtaining a PITX3 knockout BPH-1 cell line
Packaging the PITX3 gene into a Cas 9-guideeRNA lentivirus with a sequence of CCCGACATGAGCACGCGCG (SEQ ID NO:1), transfecting BPH-1 cells together with 2 mu g of each of a commercial CRISPR-Cas9 virus and a Luciferace virus, and after 3 days of infection, adding Puromycin for screening for 4 days to obtain the successfully infected BPH-1 cell PITX3-BPH-1 which knocks out PITX 3.
(2) Construction of PITX3-BPH-1 cell transfer nude mouse model
PITX3-BPH-1 cells were digested, dispersed, resuspended in PBS, and assigned a 5X 10 cell/mouse5～10×105Injecting 100-200 mu L of cell suspension into the vein of each nude mouse on the order of individual cells, observing the metastasis condition by luciferin small animal imaging every week until organ metastasis occurs and the cell concentration of a metastatic focus is more than 106p/sec/cm2/sr。
(3) Taking out lung metastasis focus to perform primary cell culture
Rinsing lung lobe tissue, separating from precooled dissection solution to remove impurity tissues such as pleura, blood vessels, bronchus, lung portal connective tissue and the like, and repeatedly shearing the lung lobe into fragments.
Sucking and removing the dissecting liquid, adding 2mL of trypsin solution with the mass fraction of 0.25%, digesting in an incubator at 37 ℃ for 30min, transferring the lung tissue fragments into a centrifuge tube, discarding the residual digestive juice, washing for 3 times by using the inoculation liquid, fully settling the tissue fragments to the bottom of the test tube after washing each time, discarding the supernatant, finally suspending by using 1mL of the inoculation liquid, repeatedly blowing the tissue fragments in the suspension with moderate force, transferring the supernatant into a culture bottle for standby after turbidity, then adding 1mL of the inoculation liquid, blowing again, repeating the steps for 3 times, gradually digesting the tissue fragments, discarding the final residues, collecting 5mL of supernatant containing single cell suspension, placing the supernatant into the culture bottle, re-suspending the cells by using the inoculation liquid, and counting the cells.
Inoculation of 1X 107～2×107Culturing the individual cells in 6-well culture plate for 24h until the cells are attached to the wall, changing the culture medium (DMEM + 10% FBS, gibco), observing the BPH-1 cells attached to the wall after culturing for 3d, and then changing to kanamycin cultureCulturing to final concentration of 2.5ug/ml, changing half liquid, culturing for 3d to inhibit hybrid cell growth, obtaining primary culture transfer focus cell, changing whole culture liquid every 3d for 1 time, and changing half liquid every time.
(4) Induction of malignant transformation
The metastatic focus cells were digested and dispersed, resuspended in PBS, and assigned to 5X 10 cells per nude mouse6The order of magnitude of (2) was that each mouse was injected subcutaneously with 100. mu.L of cell suspension and cultured for 3 weeks to obtain tumor-inducing nude mice.
In this embodiment, primary cell culture is performed by using lung metastasis, or by using other organ metastases, and the conditions of the primary cell culture are finely adjusted according to the conditions of the primary cell culture of the lung metastasis.
In addition, according to the above steps, the present embodiment also provides a kit for inducing malignant transformation of prostate epithelial cells, which comprises a BPH-1 cell gene knockout system, an organ metastasis primary cell culture system, and a nude mouse injection system, wherein the reagents and small instruments required for the whole process are summarized together.
Example 2: transfection assay for metastatic focus cells
Digesting and dispersing the cells of the metastasis, and adding about 10 percent of the cells into a flow detection tube after counting the cells6A number of metastatic focus cells; washing cells with PBS, adding 100uL PBS to suspend the cells, adding different flow antibodies of 1uL each comprising CD105, CD73, CD90, CD44, CD34, CD45 and CD11b into different flow tubes, mixing uniformly on a vortex oscillator, and incubating in a refrigerator at 4 ℃ for 30 minutes; after incubation, cells were washed with PBS and centrifuged to remove unbound flow antibody; finally, 300uL PBS was added to the tube for flow detection.
The data were analyzed using FlowJo software, and the results are shown in fig. 1, which shows FITC staining as CD34, CD105, PE staining as CD44, CD73, and successful cell transfection.
Example 3: detection of invasion ability of metastatic focus cells
The Transwell chamber was placed on a 24-well plate, the chamber was taken up, medium containing 10% serum was added to the well, the chamber was placed on a plate, and 5X 10 cells were placed4Cell of metastatic focusSpreading in each chamber, adding serum-free culture medium into the chamber, covering with a cover, culturing for 72h, sucking the upper and lower culture mediums after the cells penetrate to the lower layer of the chamber, uncovering the cover, wiping off the upper layer cells of the chamber to take up the chamber, adding polyformaldehyde in the lower layer, fixing in the upper chamber for 15min, staining with crystal violet for 30min, cleaning, taking up the chamber, air drying, observing under a microscope, taking a picture, and using BPH cells as a control.
As shown in fig. 2, the color of the metastatic focus cells was much darker than that of BPH cells, indicating that the metastatic focus cells were more invasive.
Example 4: detection of cell proliferation ability of metastatic focus
Diluting the EdU solution (reagent A) with a cell culture medium according to a ratio of 1000:1 to prepare a proper amount of 50 mu M EdU culture medium, replacing the cell culture medium with the EdU culture medium, adding 1mL of the EdU culture medium into each hole, incubating for 2 hours, collecting cells into a flow tube, centrifuging at 1500rpm for 5min, discarding supernatant, resuspending the cells with PBS, centrifuging at 1500rpm for 5min, discarding supernatant, fixing with 4% paraformaldehyde for 15min, neutralizing with 2mg/mL glycine for 5min, washing with PBS for 1 time, incubating with 0.5% TritonX-100 penetrant at room temperature for 10min, washing with PBS for 1 time, adding 500 mu L of 1 sample into each tube
Staining reaction solution, resuspending cells, keeping out of the sun, incubating at room temperature for 10min, centrifuging at 1500rpm for 5min, discarding staining reaction solution, washing with 0.5% TritonX-100 penetrant at room temperature for 3 times, resuspending with PBS, and performing flow detection. As a result, as shown in FIGS. 3 and 4, the proliferation potency of the metastatic focus cells was significantly stronger than that of the PBH cells.
Example 5: detection of metastatic focus cell tumorigenicity ability
The metastatic focus cells were digested and dispersed, resuspended in PBS, and assigned to 5X 10 cells per nude mouse6Of the order of magnitude of 100. mu.L of cell suspension per mouse injected subcutaneously, and observed weekly until tumorigenesis, per mouseEach radial line of the tumor is measured by a vernier caliper and photographed, the nude mouse is sacrificed when the tumor grows to about 2cm multiplied by 2cm, the tumor section is stripped off, the malignant degree of the tissue is observed by immunohistochemical HE staining, and the nude mouse inoculated with BPH cells is used as a control, and the specific result is shown in figure 5.
As shown in FIG. 5, 1-5 are metastatic cells, and 6 are BPH cells. Compared with BPH cells, the metastatic focus cells have obviously better tumorigenic capacity.
The present invention is not limited to the embodiments described above, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are included in the scope defined by the claims of the present application.
Sequence listing
<110> Shanghai Changhai Hospital
<120> prostate epithelial cell malignant transformation induction method and kit
<130> instructions; claims
<141> 2018-04-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cccgacatga gcacgcgcg 19
Claims (7)
1. A method for inducing malignant change of prostate epithelial cells is characterized by comprising the following steps:
A. constructing a BPH-1 cell line with a PITX3 gene knocked out to obtain a PITX3-BPH-1 cell with the BPH-1 gene of PITX3 knocked out;
B. construction of PITX3-BPH-1 cell transfer nude mouse model
PITX3-BPH-1 cells were digested, dispersed, resuspended in PBS, and assigned a 5X 10 cell/mouse5~10×105Cell number ofInjecting 100-200 mu L of cell suspension into the tail of each nude mouse in a graded manner, observing the transfer condition through luciferin small animal imaging every week until organ transfer occurs and the cell concentration of a transfer focus is more than 106p/sec/cm2/sr；
C. Primary culture of metastatic focus cells
Removing impurity tissues attached to the organ transfer stove from the organ transfer stove, repeatedly cleaning, and shearing the organ transfer stove into fragments; adding trypsin digestive juice with the mass fraction of 0.25% into the fragments of the transfer stove, washing the fragments for several times by using the inoculating liquid after digestion, then suspending the fragments by using the inoculating liquid, repeatedly blowing tissue blocks in the mixed rotary liquid until the tissue blocks are turbid, and transferring the supernatant into a culture bottle for later use; adding inoculating liquid into the culture bottle, blowing and beating again, digesting the tissue block gradually after repeating for several times, collecting supernatant containing single cell suspension in the culture bottle, suspending cells again with the inoculating liquid, counting cells,
inoculating a certain number of cells into a porous culture plate, culturing for 18-30 hours until the cells adhere to the wall, changing the whole culture solution, culturing for 3-5 days, then culturing for 3-5 days according to the growth condition of BPH-1 cells, changing a kanamycin culture solution for 3-5 days to inhibit the growth of hybrid cells, obtaining primary transfer focus cells which are simply cultured, and then changing the whole culture solution for 1 time every 3-5 days, wherein half of the culture solution is changed every time;
D. induction of malignant transformation
The primary metastatic focus cells were digested and dispersed, resuspended in PBS, and assigned to 5X 10 cells per nude mouse6~10×106Injecting 100-200 mu L of cell suspension into each mouse subcutaneously in the order of magnitude of cells, and culturing for 2-3 weeks to obtain tumor-induced nude mice.
2. The method for inducing prostatic epithelial cell degeneration according to claim 1, wherein:
the method for constructing the BPH-1 cell line with the PITX3 gene knocked out in the step A comprises the following steps:
packaging the PITX3 gene into Cas 9-guideeRNA lentivirus, transfecting BPH-1 cells together with 1-2 mu g of commercial CRISPR-Cas9 virus and Luciferace virus respectively, and after 2-3 days of infection, adding Puromycin for screening for 4-6 days to obtain the cell PITX3-BPH-1 which is successfully infected and used for knocking out the BPH-1 of the PITX 3.
3. The method for inducing prostatic epithelial cell degeneration according to claim 2, wherein:
wherein the sequence of the Cas 9-guideeRNA lentivirus is shown as SEQ ID NO. 1.
4. The method for inducing prostatic epithelial cell degeneration according to claim 1, wherein:
and C, adding trypsin digestion solution with the mass fraction of 0.25% into the fragments of the transfer stove, digesting for 30-40 min in an incubator at 37 ℃, transferring the fragments of the tissue into a centrifuge tube, discarding the residual digestion solution, and washing with the inoculation solution.
5. The method for inducing prostatic epithelial cell degeneration according to claim 1, wherein:
the inoculation liquid is a DMEM liquid culture medium, the whole culture liquid is a DMEM liquid culture medium containing 10% FBS fetal bovine serum in volume fraction, the final concentration of the kanamycin culture liquid is 2.5ug/ml, and a half liquid is replaced.
6. The method for inducing prostatic epithelial cell degeneration according to claim 1, wherein:
wherein, in the step C, after the stable adherence of the BPH-1 cells is observed, the kanamycin culture solution is used for replacement.
7. The method for inducing prostatic epithelial cell degeneration according to claim 1, wherein:
wherein the organ metastasis is lung metastasis.
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| CN103031335A (en) * | 2011-10-09 | 2013-04-10 | 李川源 | Method for generating dopamine-like neurons from human fibroblasts through direct induction and application of dopamine-like neurons |
| CN104561016A (en) * | 2014-12-29 | 2015-04-29 | 深圳华大基因科技有限公司 | CC (congenital cataract) PITX3 gene novel mutation |
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| CN103031335A (en) * | 2011-10-09 | 2013-04-10 | 李川源 | Method for generating dopamine-like neurons from human fibroblasts through direct induction and application of dopamine-like neurons |
| CN104561016A (en) * | 2014-12-29 | 2015-04-29 | 深圳华大基因科技有限公司 | CC (congenital cataract) PITX3 gene novel mutation |
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