CN111593018A - Timely and accurate tumor drug treatment model based on chick embryo chorion-urotheca - Google Patents
Timely and accurate tumor drug treatment model based on chick embryo chorion-urotheca Download PDFInfo
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Abstract
The invention discloses a timely and accurate drug treatment model for tumors based on chick embryo chorionic urea sac, which comprises the establishment of a chick embryo PDX model, the detection of tumor transplantation drugs and the timely and accurate drug treatment analysis; the establishment of the chick embryo PDX model and the detection of the tumor transplantation medicament comprise the following steps: incubation of fertilized eggs, incubation of fertilized eggs at constant temperature and incubation of embryos; planting tissue/cell with bioactivity on the CAM surface, and adding target detection medicine on the surface; and observing and carrying out HE, IHC and molecular pathology detection. The timely accurate treatment analysis of the medicine comprises the following steps: giving targeted drugs and conventional chemotherapy drug suggestions after the primary treatment of the surgical specimens of the patients, including fresh tissue treatment or cryopreservation of a tissue bank, conventional pathological examination or molecular pathological gene analysis; inoculating chicken embryo PDX model tissue to obtain a drug simulation treatment suggestion; comprehensive analysis and individual accurate treatment; and (5) monitoring and analyzing the curative effect. The invention uses CAM PDX model to monitor the curative effect and side effect of chemotherapy drug tumor treatment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a timely and accurate tumor drug treatment model based on chicken embryo chorionic urea sac and an establishing method thereof.
Background
In recent years, with the increase of tumor treatment means, the effect has not been effectively improved, but the side effect has been troubling patients and medical staff. Therefore, how to perform timely and effective accurate individualized treatment of tumors is the focus of research of scientists.
PDX model is used for cancer research experiment model, and is more popular in recent years. They can be used for the establishment of individual cancer treatments such as tumor markers and biological pathways for research, drug efficacy and the like. At present, immunodeficient mice are mostly applied, but the defects of the immunodeficient mice are that tumors are not visual and the observation period is long. The primary culture of the tumor cells is not easy to operate and has a long period. Over the last century, numerous studies have found that the chick embryo chorioallantoic system is of great value for tumor research, where chick embryos can effectively support the growth of inoculated xenogeneic tumor cells, maintaining rapid tumor formation for several days after cell transplantation. The dense capillary network of the CAM allows incorporation into the host vasculature, thereby providing a unique experimental model for studying sensitive drug screening. In addition, it is cheap, easy to operate and observe, but has the disadvantage that the observable time is only 8-10 days.
If an effective tumor drug sensitivity, drug resistance and side effect monitoring platform is summarized through the chick embryo CAM model, the chick embryo CAM model can provide basis for individualized treatment of patients.
Disclosure of Invention
In order to solve the existing problems, the invention provides a timely and accurate tumor drug treatment model based on chick embryo chorion-urotheca and an establishment method thereof. The invention is realized by the following technical scheme.
The timely and accurate tumor treatment model based on chicken embryo chorionic-urethral sac comprises establishment of a chicken embryo PDX model, detection of tumor transplantation medicines, and timely and accurate treatment and analysis of medicines;
the establishment of the chick embryo PDX model and the detection of the tumor transplantation medicament comprise the following steps:
s1, incubation of fertilized eggs, incubation of the fertilized eggs at constant temperature and incubation of embryos;
s2, planting tissues/cells with biological activity on the surface of the CAM on chick embryo days 8-10, and dripping/carrying out microinjection on target detection drugs on the surface of the transplanted tumor in blood vessels on days 11, 13 and 15;
s3, observing the blood vessel supply condition around the transplanted tumor by using ultrasonic on the 17 th day; harvesting transplanted tumor tissues on day 18 for HE, IHC and molecular pathology detection;
the timely accurate treatment analysis of the drugs comprises the following steps:
SS1, the patient operation specimen is processed primarily, including the following modes:
planting fresh tissues in vitro within 20 minutes or freezing and storing the tissues in a tissue bank for later use;
conventional pathological examination or molecular pathological gene analysis further gives a suggestion of a targeted drug and a conventional chemotherapeutic drug;
inoculating chicken embryo PDX model tissue to obtain drug simulation treatment suggestion or curative effect and side effect monitoring suggestion;
SS2. comprehensive analysis, individual precise treatment;
SS3, monitoring and analyzing the curative effect;
SS4, when the treatment effect is poor, and the tumor cells recur or transfer occurs, free tumor cells or peripheral blood CTC in the body fluid specimen are inoculated with chick embryo PDX model cells to obtain drug simulation treatment or detection of curative effect and side effect, and further, related data are comprehensively analyzed to guide individualized accurate treatment;
SS5, continuing to monitor and analyze the therapeutic effect;
and SS6, timely selecting and analyzing the medicaments, and summarizing.
Preferably, the fertilized egg is incubated in the incubator at constant temperature in step S1 under the incubation conditions of 37.5 ℃ and 62% humidity; the embryo incubation is to place the embryo, the egg white and the egg yolk into a plastic weighing box completely for incubation.
Preferably, in step S2, the fresh tissue is transplanted onto the CAM surface, followed by 3-10mg of Matrigel (the tumor is processed into tumor particles or lumps with a diameter of 2mm, and the addition amount of Matrigel is increased appropriately for the tumor which is not easy to grow, such as brain tumor).
Preferably, in step S3, the CAM surface is marked and frozen tissue sections are taken, if necessary with a small amount of CAM tissue.
Preferably, the HE analysis in step S3 includes microscopic observation of the proportion of living tissue and the proportion of Ki-67 positive cells.
Preferably, the molecular pathological examination of the tumor in step S3 includes histological ultrasound analysis.
The invention has the beneficial effects that:
the invention relates to a timely drug precise treatment model of tumor based on chick chorion-uritophorea, which applies chick chorion-uritophorea (CAM) to timely drug sensitive monitoring of free tumor cells in body fluids such as solid tumor, pleural effusion, ascites and the like and circulating tumor cells in peripheral blood, thereby achieving the clinical goal of precise treatment.
The invention can timely monitor the curative effect and the side effect of the chemotherapy drug tumor treatment, can relieve the pain, the side effect and the economic burden of patients, and has great significance to the progress of the country, the patients and the medical career.
Drawings
FIG. 1 is a flow chart of the operation of the chick embryo PDX model building and tumor transplantation drug detection according to the present invention;
FIG. 2 is a timely medication precision treatment analysis roadmap according to the present invention;
FIG. 3 is a diagram of chicken embryo development;
FIG. 4 embryo incubation and tumor inoculation;
FIG. 5 is a schematic representation of microinjection of tumor drugs;
FIG. 6 is an ultrasonic observation of transplanted tumors;
FIG. 7 shows microscopic observations of tumors;
FIG. 8 is the results observed after treatment of transplanted tumors with different drugs;
FIG. 9 is the percentage of growing cells;
FIG. 10 shows Ki-67 percent;
FIG. 11 tumor necrosis ratio and vascular unit content percentage.
Detailed Description
The technical solution of the present invention will be described in more detail and fully with reference to the accompanying drawings and specific embodiments.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
1. Chicken embryo PDX model establishment
1.1 fertilized egg preparation incubation
The fertilized eggs are placed at about 13 ℃ for incubation, and the temperature cannot be too high or too low, even at about 13 ℃, and the storage time does not exceed 1 week.
1.2 fertilized egg incubation at constant temperature
The incubation temperature of the breeding eggs is 37.5 ℃, and the humidity is about 62%.
1.3 embryo incubation;
and (4) incubating the fertilized eggs of the chickens, incubating the fertilized eggs at constant temperature, and incubating embryos until the eighth day.
The egg shell is opened, the embryo, the egg white and the egg yolk are completely placed in the plastic weighing box, the egg yolk cannot be damaged, and the heart beating from the millet to the mung bean can be seen in the whole process.
The development of chicken embryos is shown in FIG. 3, day E8-16, and the tumor inoculation is the best time for harvesting.
1.4 active tissues/cells were seeded on the CAM surface
On day 8, viable tissue/cells were seeded onto the CAM surface as shown in fig. 4; the viable tissue was transplanted onto the CAM surface followed by 3-10mg Matrigel.
Dripping/injecting target detection medicine into surface blood vessel in 11, 13 and 15 days; the method of operation is shown in figure 5.
When tumor transplantation tissue is collected, the CAM surface is marked, frozen tissue sections are taken, and a small amount of CAM tissue can be carried if necessary.
1.5. Observing the blood vessel supply condition around the transplanted tumor by using ultrasonic on the 17 th day;
the tumor vascularization and tissue necrosis can be real-time and non-invasively analyzed with ultrasound, as shown in fig. 6.
The results of the transplanted tumor directly observed with a stop lens are shown in FIG. 7, and the results of the tumor treated with different drugs are shown in FIG. 8.
FIG. A, B, C shows treatment with PBS on days three and five; FIG. D, E, F is the treatment with 0.03mM cisplatin on days three and five; FIG. G, H, I is a graph showing treatment with 0.3mM cisplatin on days three and five.
In general, as drug concentrations increased, chick embryo PDX tumors progressively presented more tissue lysis, even the last bouillon-like appearance;
from the ultrasound perspective, as the concentration of the drug increases, the blood supply to the tumor and the surrounding area is significantly reduced;
from HE pathological sections of the tissue, the proportion of tissue necrosis is clearly increased.
1.6 harvesting transplanted tumor tissues on day 18 for HE, IHC and molecular pathological detection;
histological HE section analysis and the test data are shown in table 1.
TABLE 1 detection of Lung adenocarcinoma tumor cells with cisplatin (Cis)
With the increase of the drug concentration, the tumor and necrosis ratio is obviously increased, and the ki-67 tumor cell proliferation index is obviously reduced, and the test results are shown in FIG. 9 and FIG. 10.
Histological ultrasound analysis of the tumor tissue and the results are shown in table 2.
TABLE 2
| Treatment of | PBS | 0.03mM Cis | 0.3mM Cis |
| Percentage of tumor volume | 21.9 | 9.27 | 3.14 |
| Unit content of blood vessel (mm)3) | 5.12 | 1.76 | 0.94 |
With the increase of the drug concentration, the ultrasound can find that the tumor and necrosis ratio is obviously increased, and the unit content of blood vessels is also obviously reduced, as shown in figure 11.
The experiment provides an operation example of cisplatin in lung adenocarcinoma drug screening, and the core of the experiment is that the chick embryo egg shell is successfully cultured outside, and the chick embryo egg shell is successfully inoculated with fresh tumor tissues, and then a drug screening experiment is carried out and scientific observation and statistics are carried out. The operation is not complicated, the time is 8 days, and the new tumor tissues in the operation of the patient are combined with the clinical drug spectrum to be screened in advance, so that the accuracy of tumor chemotherapy or immunotherapy can be further improved, and the accurate treatment is further contributed.
With the acceleration of population aging and the change of natural environment and social environment, malignant tumors in China are in a high incidence. However, the treatment is not ideal, and the patient can endure great side effects and can not obtain effective relief of the tumor. Moreover, because of the difference of genes, the polymorphism of the tumor is accepted by the medical workers. That is, there are not two tumors that are identical, and the same tumor will have various differences at different times. Each patient should be individually treated precisely at the right time.
It is to be understood that the described embodiments are merely individual embodiments of the invention, rather than all embodiments. All other implementations made by those skilled in the art without any inventive step based on the embodiments of the present invention belong to the protection scope of the present invention.
Claims (6)
1. A timely and accurate tumor treatment model based on chick embryo chorion-urotheca is characterized in that: establishing a chicken embryo PDX model, detecting a tumor transplantation medicament, and carrying out timely medicament accurate treatment analysis;
the establishment of the chick embryo PDX model and the detection of the tumor transplantation medicament comprise the following steps:
s1, incubation of fertilized eggs, incubation of the fertilized eggs at constant temperature and incubation of embryos;
s2, planting fresh tissues/cells on the surface of the CAM on the chick embryo day 8-10, and dripping/carrying out microinjection on target detection drugs on the surface of the transplanted tumor in a surface vessel on days 11, 13 and 15;
s3, observing the blood vessel supply condition around the transplanted tumor by using ultrasonic on the 17 th day; harvesting transplanted tumor tissues on day 18 for HE, IHC and molecular pathology detection;
the timely accurate treatment analysis of the drugs comprises the following steps:
SS1, the patient operation specimen is processed primarily, including the following modes:
cryopreserving tissues (biologically active tissues or cells) from a fresh tissue or tissue bank;
conventional pathological examination or molecular pathological gene analysis further gives a suggestion of a targeted drug and a conventional chemotherapeutic drug;
inoculating chicken embryo PDX model tissue to obtain drug simulation treatment suggestion or curative effect and side effect monitoring suggestion;
SS2. comprehensive analysis, individual precise treatment;
SS3, monitoring and analyzing the curative effect;
SS4, when the treatment effect is poor, and the tumor cells recur or transfer occurs, free tumor cells or peripheral blood CTC in the body fluid specimen are inoculated with chick embryo PDX model cells to obtain drug simulation treatment or detection of curative effect and side effect, and further, related data are comprehensively analyzed to guide individualized accurate treatment;
SS5, continuing to monitor and analyze the therapeutic effect;
and SS6, timely selecting and analyzing the medicaments, and summarizing.
2. The chicken embryo chorion-urinary capsule based tumor timely drug precision treatment model according to claim 1, characterized in that: in the step S1, the fertilized eggs are incubated in the incubator at the constant temperature and the incubation condition is 37.5 ℃ and the humidity is 62%; the embryo incubation is to place the embryo, the egg white and the egg yolk into a plastic weighing box completely for incubation.
3. The chicken embryo chorion-urinary capsule based tumor timely drug precision treatment model according to claim 1, characterized in that: in step S2, the fresh tissue is transplanted onto the CAM surface, followed by 3-10mg of Matrigel.
4. The chicken embryo chorion-urinary capsule based tumor timely drug precision treatment model according to claim 1, characterized in that: in step S3, when the tumor transplant tissue is harvested, the CAM surface is marked and frozen tissue sections are performed, and a small amount of CAM tissue may be included if necessary.
5. The chicken embryo chorion-urinary capsule based tumor timely drug precision treatment model according to claim 1, characterized in that: HE analysis in step S3 included microscopic observation of the proportion of biopsies and the proportion of immunohistochemical Ki-67 positive cells.
6. The chicken embryo chorion-urinary capsule based tumor timely drug precision treatment model according to claim 1, characterized in that: the pathological examination in step S3 includes molecular pathological examination of tumor and histological ultrasound analysis.
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| CN109929797A (en) * | 2019-02-22 | 2019-06-25 | 南昌乐悠生物科技有限公司 | Chicken embryo Transplanted tumor model method for building up and its application in drug susceptibility detection |
| FR3087792A1 (en) * | 2018-10-29 | 2020-05-01 | Inovotion | ANIMAL MODEL FOR AMPLIFYING HUMAN OR ANIMAL CIRCULATING TUMOR CELLS |
| FR3087793A1 (en) * | 2018-10-29 | 2020-05-01 | Inovotion | USE OF A GRAFT EGG WITH TUMOR CELLS TO STUDY THE ANTI-CANCER EFFECTIVENESS OF IMMUNOTHERAPIES, IN THE ABSENCE OF IMMUNE EFFECTOR CELLS OTHER THAN THOSE OF EGG GRAFTS |
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| CN109294977A (en) * | 2018-09-25 | 2019-02-01 | 广州星燎生物科技有限公司 | A kind of liver cancer patient source chick chorioallantoic membrane M-PDX model building method |
| FR3087792A1 (en) * | 2018-10-29 | 2020-05-01 | Inovotion | ANIMAL MODEL FOR AMPLIFYING HUMAN OR ANIMAL CIRCULATING TUMOR CELLS |
| FR3087793A1 (en) * | 2018-10-29 | 2020-05-01 | Inovotion | USE OF A GRAFT EGG WITH TUMOR CELLS TO STUDY THE ANTI-CANCER EFFECTIVENESS OF IMMUNOTHERAPIES, IN THE ABSENCE OF IMMUNE EFFECTOR CELLS OTHER THAN THOSE OF EGG GRAFTS |
| CN109929797A (en) * | 2019-02-22 | 2019-06-25 | 南昌乐悠生物科技有限公司 | Chicken embryo Transplanted tumor model method for building up and its application in drug susceptibility detection |
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