Method for preparing anti-prostate cancer medicine by co-culturing human embryonic stem cells and prostate cancer cellsTechnical Field
The invention belongs to the technical field of biological medicines, and in particular relates to a method for preparing an anti-prostate cancer medicine by co-culturing human embryonic stem cells and prostate cancer cells.
Background
Prostate cancer (PCa) is a malignancy that is well developed in older men, with its incidence increasing with age. Prostate cancer has a high incidence worldwide. There are more than 90 tens of thousands of new cases worldwide each year, of which 26 tens of thousands die from the exacerbation of prostate cancer. The incidence rate of prostate cancer in European and American countries is obviously higher than that in subnon-regions. Prostate cancer is the leading cause of tumor incidence in men in the united states. In China, the incidence rate of the prostate cancer is increased by nearly 70 percent compared with the prior art. According to the report of the current situation and the epidemic trend analysis of the prostate cancer in China in the 04 th stage of the journal of clinical oncology, the incidence of the prostate cancer in China shows a remarkable continuous increasing trend, and the prostate cancer is becoming a malignant tumor of the urinary system which seriously affects the male health in China. As the aging phenomenon of our society becomes more serious, the incidence rate of prostate cancer in our country may enter a peak period. The clinical manifestations of prostate cancer are mainly difficult urination, with symptoms such as frequent urination, urgent urination, hematuria and the like, and with pain or burning sensation during urination, continuous pain in the lower back, upper thigh or pelvis. The early stage of the prostate cancer tumor has no clinical manifestation, and the symptoms of metastasis are often the earliest reason for treatment. The prostate cancer is difficult to detect due to its long latency, and it has generally progressed to advanced stages.
The growth and metastasis of prostate cancer is closely dependent on androgens (e.g., dihydrotestosterone). About 90% of androgens are secreted from the testes. Androgen is secreted into the cell and binds to the androgen receptor. Androgen receptor is an important nuclear transcription factor that regulates the expression of numerous genes and proliferation of prostate cancer cells. When androgens bind to the androgen receptor, the androgen receptor enters the nucleus to initiate gene expression, thereby promoting prostate cancer growth and metastasis. Thus, de-androgenic therapy (including removal of testes and drug inhibition of androgen levels) has significant efficacy for most prostate cancers. However, the control of tumors by androgen ablation therapy is generally maintained for only 1-4 years, and almost all prostate cancer patients eventually turn into androgen-independent prostate cancer and further develop castration-resistant prostate cancer. At this point, the antiandrogen therapy is lost. Castration-resistant prostate cancer is a significant cause of death in prostate cancer patients. There are various mechanisms of occurrence of castration-resistant prostate cancer, including androgen receptor mutation, disappearance of androgen receptor expression, and expansion of androgen-insensitive cell populations in a low androgen environment. Although the castration therapy is not effective, the androgen receptor also plays an important role in most castration-resistant prostate cancers, and thus is an important target for the current treatment of common and castration-resistant prostate cancers.
There is no single drug available in the world medical community to cure prostate cancer. The main treatment means of the prostate cancer comprise operation, radiotherapy, chemotherapy and biological treatment, so that patients have pain in nine lives, the family economic pressure is huge, the liabilities are tired, and people are least acceptable, and the patients finally have the suffering of death after suffering from great pain. The drugs recently approved by the U.S. Food and Drug Administration (FDA) for the treatment of castration-resistant prostate cancer include Abiraterone, which inhibits androgen synthesis, and Enzalutamide, which is an antagonist of the androgen receptor, both of which are directed against the androgen receptor signaling pathway and have significant efficacy in the treatment of castration-resistant prostate cancer, but after endocrine treatment and targeted androgen receptor drug treatment, patients still develop drug resistance, which is very limited in therapeutic effect and costly. In addition, side effects caused by radiotherapy and chemotherapy are also bottlenecks in clinical treatment.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for preparing an anti-prostate cancer drug by co-culturing human embryonic stem cells and prostate cancer cells. The inventor unexpectedly discovers that when human embryonic stem cells and prostate cancer cells are subjected to direct contact co-culture, the microenvironment of the human embryonic stem cells is stimulated by the prostate cancer cells, the human embryonic stem cells can generate a cancer suppression biological effect to generate a 'prostate cancer suppression' molecule, and then the supernatant is extracted and applied to the treatment of tumors. The invention uses special embryo stem cell culture medium in the co-culture process to keep the characteristics of human embryo stem cells as far as possible, utilizes the embryo stem cells in proper state and proper ratio of embryo stem cells to prostate cancer cells to co-culture, and cannot make the human embryo stem cells produce strong biological effect for inhibiting tumor growth when the ratio of two cells in the co-culture system is improper and the time point of adding the prostate cancer cells is improper.
Except for special descriptions, the parts are parts by weight, and the percentages are mass percentages.
The invention provides a method for preparing an anti-prostate cancer drug by co-culturing human embryonic stem cells and prostate cancer cells.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for preparing an anti-prostate cancer drug by co-culturing human embryonic stem cells and prostate cancer cells, which is characterized by comprising the following steps:
(1) Pre-paving Matrigel matrix glue to prepare a special culture plate for human embryonic stem cells;
(2) Performing digestion and passage on the human embryonic stem cells;
(3) Preparing a culture plate special for human embryonic stem cells by using the Matrigel pre-laid in the step (1) for human embryonic stem cell inoculation;
(4) Taking prostate cancer cells in logarithmic growth phase, sucking and removing the culture medium, then adding DPBS buffer solution to clean the cells, digesting the cells for 2-6 min at 32-42 ℃ by using EDTA, sucking the supernatant, re-suspending the prostate cancer cells into single cell suspension by using a complete culture medium of human embryonic stem cells, and counting the number of the prostate cancer cells according to the number of the human embryonic stem cells in a culture system of 1:1-1:5 proportion, adding into human embryo stem cell culture system, adding two kinds of cells at 32-42deg.C CO2 Co-culturing in a cell incubator;
(5) And (5) co-culturing for 24-96 hours, and collecting a co-culture solution.
The human embryonic stem cells of the invention are established stem cell lines.
During the course of the study, the inventors found that using FBS-containing medium for co-culturing human embryonic stem cells with cancer cells induced differentiation of human embryonic stem cells, thereby inducing increased cell growth. After a large number of experiments, the inventor develops biological effects of inhibiting tumor growth around the embryo microenvironment after being stimulated by tumor cells, optimizes a co-culture method of human embryonic stem cells and cancer cells, and extracts a co-culture supernatant to find the effect of resisting the prostate cancer.
The culture medium comprises PSCeasy cube human pluripotent stem cell culture medium and mTESRTM1 One or two of culture medium, essential 8 ™ culture medium and DMEM/F12 culture medium.
The human embryonic stem cell and cancer cell co-culture solution is preferably supernatant of the human embryonic stem cell and cancer cell co-culture.
The method for preparing the culture plate special for the human embryonic stem cells by pre-paving Matrigel is to mix Matrigelgel dilution and then cell culture pore plate pre-spreading, constant temperature CO at 37 DEG C2 The cells were placed in the incubator for 4-12 hours.
Matrigel dilution to Matrigel was diluted with DPBS, wherein Matrigel: dpbs=1: 30-1:50.
the method for carrying out digestion and passage on the human embryonic stem cells comprises the steps of selecting the human embryonic stem cells in the subculture, sucking and discarding a culture medium, then adding a DPBS buffer solution to clean the cells, adding a cell dissociation reagent stem cell digestive solution, placing the cells into an incubator for incubation, observing that most cloning edges start to separate from the bottom of a dish under a microscope, observing that more obvious gaps appear among most cells in the clone, and observing that cell colonies become opaque and whitish by naked eyes, so that the cell digestion time is ideal, sucking and discarding the stem cell digestive solution, adding the stem cell culture medium, and scraping the cells into dozens of cell clusters.
The method comprises the steps of selecting human embryonic stem cells in subculture, namely selecting culture holes which have coverage rate of 80% -90%, clone edges are smooth and no differentiated cells, and the subculture times are 2-10 generations.
The method for inoculating the human embryonic stem cells comprises the steps of taking the pore plate in the step (1), sucking and discarding the basic working solution for preparing Matrigel, adding a DPBS buffer solution for cleaning the prepared machine-made glue, adding a stem cell culture medium, uniformly inoculating the cell mass suspension in the step (2) into the prepared Matrigel pore plate, and changing the liquid for 24 hours.
The co-culture method comprises the following steps: when human embryo stem cell state is mature, taking tumor cells in logarithmic growth phase, absorbing and discarding culture medium, adding DPBS buffer solution to clean the cells twice, digesting with EDTA at 37deg.C, sucking supernatant, re-suspending cancer cells into single cell suspension with stem cell culture medium, adding the number of cancer cells into human embryo stem cell culture system, and keeping the two cells constant at 37deg.C and CO2 Co-cultivation was performed in a cell incubator.
The human embryonic stem cell state matures to the point that when the cell confluence of the embryonic stem cells reaches 50% -60%, the cells Long Bianyuan are smooth and have no conditions of differentiated cells, dispersion among cells and no fusion.
Collecting co-culture solution, co-culturing for 24-96 hr, collecting co-culture supernatant with centrifuge tube, centrifuging, filtering with microporous membrane, and collecting supernatant.
Advantageous effects
Human embryonic stem cells can be cultured in vitro, have developmental totipotency, and have high self-renewal, unlimited proliferation and multidirectional differentiation capabilities. The inventor unexpectedly discovers that when human embryonic stem cells and prostate cancer cells are subjected to direct contact co-culture, the microenvironment of the human embryonic stem cells is stimulated by other tumor cells, the human embryonic stem cells can generate a cancer suppression biological effect to generate tumor suppression molecules, so that supernatant can be extracted and applied to the treatment of the prostate cancer.
The co-culture supernatant of the invention shows remarkable treatment effect in inhibiting proliferation, migration and invasion of prostate cancer cells; more importantly, the co-culture supernatant also has obvious advantages in inhibiting the growth of tumors in vivo, and has the potential of preparing an anti-prostate cancer drug.
Drawings
FIG. 1 is a graph showing the results of inhibition of proliferation of prostate cancer cells in vitro from co-culture supernatants;
FIG. 2 is a graph showing the effect of co-culture supernatant on inhibiting the clonality of prostate cancer cells, specifically, the results of cell plate cloning experiments;
FIG. 3 is a statistical chart of the clonality inhibitory effect of co-culture supernatants on prostate cancer cells, specifically cell plate cloning experiments;
FIG. 4 shows the inhibition of prostate cancer cell migration invasiveness by co-culture supernatant, specifically a submicroscopic image of the results of cell transwell experiments;
FIG. 5 shows the inhibition of prostate cancer cell migration invasiveness by co-culture supernatant, specifically cell transwell experimental statistics;
FIG. 6 is a graph showing the inhibition of the co-culture supernatant on the tumor in mice of a model of human prostate cancer nude mice transplanted tumor, specifically the comparison of the tumor sizes in mice;
FIG. 7 shows the inhibition effect of co-culture supernatant on tumor in mice of a model of human prostate cancer nude mice transplanted tumor, specifically a graph of the statistical result of tumor weight in mice.
Detailed Description
The present invention is described in detail below by way of specific examples, which are given herein for the purpose of further illustration only and are not to be construed as limiting the scope of the present invention, as many insubstantial modifications and variations of the present invention will become apparent to those skilled in the art in light of the foregoing disclosure. The raw materials and the reagents used in the invention are all commercial products. Wherein the prostate cancer is a PC-3 cell strain of the prostate cancer in the examples, and the PC-3 cell strain is preserved by a biochemical and molecular pharmacological laboratory of Chongqing medical university; in the examples, the human embryonic stem cells are established stem cell lines, and in particular, the human embryonic stem cell H9 cell lines are purchased from stem cell banks of the national academy of sciences.
Example 1
Preparation of supernatant of co-culture of human embryonic stem cells and prostate cancer cells
Pre-paving Matrigel matrix gel to prepare a special culture plate for human embryonic stem cells: pre-plating diluted Matrigel (DPBS: matrigel=50:1) in a six-well plate at a volume of 1 mL/well, placing the plate back into a constant temperature CO2 cell incubator at 37 ℃ for 4 hours;
digestion and passage: taking out PSCeasy complete culture medium of human pluripotent stem cells, balancing to room temperature, taking out the culture dish coated with Matrigel, sucking the coating liquid, adding appropriate amount of PSCeasy complete culture medium of human pluripotent stem cells, and placing at 37deg.C constant temperature CO2 In a cell incubator. Selecting good-state human embryonic stem cells (namely, the cell coverage rate reaches about 80%, the stem cell colony is large, the cloning edge is regular and hESC without differentiated cells), adding 2mL of DPBS to clean the cells twice after sucking and discarding the culture medium, adding 1mL of PSCeasy < human pluripotent stem cell digestive juice after sucking and discarding, then placing the cells into a constant-temperature CO2 cell incubator at 37 ℃ for incubation, observing that most of the cloning edge begins to separate from the bottom of a dish under a microscope, and observing that more obvious gaps appear among most of the cells in the clone, and the cell colony becomes opaque and whitish by naked eyes, thus indicating that the cell digestion time is ideal. After sucking out the digestive juice, adding 1mL PSCeasy human pluripotent stem cell complete culture medium into each hole, and scraping the cells to obtain fine cellsCells, which make cells into small cell clusters.
Inoculating: the well plate pre-plated with Matrigel was removed and then the cell pellet suspension was inoculated evenly into the prepared Matrigel well plate.
Co-cultivation: taking prostate cancer cells in logarithmic growth phase, sucking and removing culture medium, adding DPBS buffer solution to clean the cells twice, digesting for 4 min at 37 ℃ by EDTA, sucking supernatant, re-suspending the cancer cells into single cell suspension by PSCeasy human pluripotent stem cells complete culture medium, adding the single cell suspension into human embryonic stem cell culture system according to the number of prostate cancer cells and the ratio of 1/3 of human embryonic stem cells in the culture system, and keeping the two cells constant in constant temperature CO at 37 DEG C2 Co-cultivation was performed in a cell incubator.
Co-culture supernatant harvest: after co-culturing for 72h, collecting the co-culture supernatant with a 15ml centrifuge tube, centrifuging, filtering with a 0.22 m microporous filter membrane, subpackaging the filtered supernatant, storing in 5ml sterile EP, and storing at-80 ℃.
Example 2
The supernatant of the co-culture of human embryonic stem cells and prostate cancer cells can inhibit the growth of the prostate cancer cells
1. Cell proliferation assay
Taking prostate cancer cells in logarithmic growth phase, and counting 5×10 per cell3 Each was seeded in 96-well plates. 100. Mu.L of the co-culture supernatant was added at 0%, 20%, 40%, 60%, 80% by volume, respectively. The 96-well plates were placed in an incubator at 37 ℃. After 24 hours, 10 μl CCK8 working fluid was added to each well for about 2 hours, and the absorbance value of each well was measured with a microplate reader at 450 nm. Cell viability = (experimental OD-blank OD)/(control OD-blank OD) ×100%. The results show (FIG. 1) that the co-culture supernatant inhibited prostate cancer cell proliferation and that the inhibition was concentration-dependent.
2. Cell colony formation assay
The growth pairs of prostate cancer cells were taken for digestion and resuspended, the cell concentration was adjusted to 100 cells/mL, and added to 6-well plates, 3 mL/well, by group. After 24h of cell attachment, the medium was replaced with the corresponding co-culture supernatant and hESC supernatant, and cultured for 15d, and the cell colonies were visualized. The medium was aspirated, washed twice with PBS, fixed with 4% paraformaldehyde for 20min, stained with 0.1% crystal violet for 10min, and photographed. The results demonstrate (FIGS. 2-3) that the co-culture supernatant inhibited the clonogenic capacity of tumor cells in vitro compared to the placebo and hESC supernatants.
3. Migration experiment
And (3) migration: cells from log phase prostate cancer cells were harvested by pancreatin digestion centrifugation after starvation culture of 24h in serum-free medium. Prostate cancer cells were resuspended with co-culture supernatant and human embryonic stem cell supernatant and inoculated into the upper chamber of a Transwell. Then medium containing 20% fetal bovine serum was added to the lower chamber. After 24 hours incubation, adherent cells were stained with a dye solution containing 0.05% crystal violet, and five areas (200×) of stained cells were randomly selected under an optical microscope for counting and photographing. The results demonstrate (FIGS. 4-5) that the co-culture supernatant inhibited the clonogenic capacity of tumor cells in vitro compared to the placebo and hESC supernatants.
4. In vivo tumor inhibiting Activity of Co-culture supernatant
Taking cancer cells in logarithmic growth phase, digesting prostate cancer cells, and re-suspending with PBS to obtain 1×10 extract6 Cancer cells were subcutaneously injected into Balb/c nude mice. When the tumor volume reaches about 100 mm3 About, the inventors randomly selected a PBS-treated group, a co-culture supernatant-treated group, and a human embryonic stem cell supernatant-treated group (n=5/group). Mice were euthanized after 11 treatment cycles with 2 different sites around the tumor every 48 hours. The results showed that the co-culture supernatant treated group had significantly inhibited tumor growth compared to the PBS and hESC supernatant treated group (fig. 6-7), indicating that the co-culture supernatant had tumor-inhibiting activity in mice.