阅读说明：本技术 一种基于肠道类器官筛选药物的方法 (Method for screening drugs based on intestinal organoids ) 是由 秦环龙 蔚青 李曼 于 2020-04-17 设计创作，主要内容包括：本发明公开了一种基于肠道类器官筛选药物的方法,包括如下步骤：用手术切除的新鲜结直肠癌组织或肠镜活检标本构建肠道类器官；将培养好的肠道类器官消化为单细胞,然后将单细胞按照一定数量铺于96孔酶标板中；将待筛选的药物加入孔中,放入培养箱中培养5天；培养完成后,每孔加入CellTiter-Glo 3D细胞活力检测试剂10min后,置于OrionⅡ仪器读值,评估细胞活力。本发明提供的一种基于肠道类器官筛选药物的方法采用肠道类器官模型作为疗效评估的手段,通过术中或肠镜活检标本构建肠道类器官,体外应用抗肿瘤药物及靶向药物等处理肠道类器官,效果数据接近真实效果,可为个体化治疗提供依据。(The invention discloses a method for screening drugs based on intestinal organoids, which comprises the following steps: constructing intestinal organoids by using fresh colorectal cancer tissues or enteroscope biopsy specimens which are excised in an operation; digesting the cultured intestinal organoids into single cells, and then paving the single cells in a 96-hole enzyme label plate according to a certain quantity; adding the drug to be screened into the hole, and putting the hole into an incubator for 5 days; after the culture is completed, CellTiter-Glo 3D cell viability detection reagent is added into each well for 10min, and then the cell viability is evaluated by reading the cell viability in an Orion II instrument. The method for screening the medicines based on the intestinal organoids adopts the intestinal organoid model as a curative effect evaluation means, constructs the intestinal organoids through intraoperative or enteroscope biopsy specimens, treats the intestinal organoids by applying anti-tumor medicines, targeted medicines and the like in vitro, has effect data close to a real effect, and can provide a basis for individualized treatment.)

1. A method for screening drugs based on intestinal organoids is characterized by comprising the following steps:

step one, constructing intestinal organoids by using surgically excised fresh colorectal cancer tissues or enteroscopy biopsy specimens;

digesting the cultured intestinal organoids into single cells, and then paving the single cells in a 96-hole enzyme label plate according to a certain quantity;

adding the drug to be screened into the hole of the 96-hole enzyme label plate, and putting the 96-hole enzyme label plate into an incubator for culture;

step four, after the culture is finished, adding CellTiter-Glo 3D cell viability detection reagent into each hole for 10min, and then placing the hole in an Orion II instrument for reading to evaluate the cell viability.

2. The method for screening a drug based on an intestinal organoid as claimed in claim 1, wherein the drug is 5-Fu, oxaliplatin, SN-38, raltitrexed or oncolytic virus.

3. The method of claim 1, wherein the cell viability is assessed based on ATP values.

4. The method for screening drugs based on intestinal organoids according to claim 1, wherein the specific method for constructing intestinal organoids in step one comprises the following steps:

(1) thoroughly cleaning the fresh colorectal cancer tissues, shearing the fresh colorectal cancer tissues into fragments, placing the fragments into a centrifuge tube containing PBS buffer solution for centrifugation, and discarding the supernatant;

(2) adding digestive juice, resuspending the precipitate, shaking vigorously, and incubating on ice; filtering and centrifuging the incubated supernatant, and discarding the supernatant;

(3) resuspending the cell pellet obtained in step (2), and counting; placing the cell suspension in another centrifuge tube, centrifuging, and removing the supernatant;

(4) mixing the cell sediment obtained in the step (3) with liquid temperature-sensitive matrigel; and (4) incubating, adding a human colorectal organoid culture medium, and culturing in an incubator for 5-7 days.

5. The method for screening drugs based on intestinal organoids according to claim 1, wherein the washing solution used in the step (1) is 1% penicillin/streptomycin in PBS.

6. The method for screening a drug based on an intestinal organoid according to claim 1, wherein the size of the fragment in step (1) is 2-4mm³。

7. The method for screening drugs based on intestinal organoids according to claim 1, wherein the ratio of matrigel to cells is 50 μ l/20000.

8. The method for screening drugs based on intestinal organoids according to claim 1, wherein the matrigel is blown by a precooled gun head before being mixed with the cell pellet.

Technical Field

The invention relates to the field of biological medicine, in particular to a method for screening medicines based on intestinal organoids.

Background

The method for culturing mouse intestinal organoid in vitro is reported in 2009 by Hans Clevers laboratory of the Netherlands Hubrecht research institute, and the technology for culturing 3D cells in vitro is developed¹. Toshiro Sato et al constructs human intestinal organoids (PDOs) from colorectal cancer patient samples in vitro, and the technique can simulate in vivo 3D growth environment in vitro, compared with the traditional two-dimensional culture system,can more truly reflect the functions of intestinal tracts, signal conduction and form in vivo and is highly consistent with the mutation type of primary tumor focus genes². Therefore, scientists use organoids as a research tool to research the pathological mechanism of intestinal cancer development and in-vitro screening of antitumor drugs. GeorgiosVlachogiannis et al use PDOs model to screen in vitro effective drugs for colorectal cancer (CRC) patients, and the results show that the effective drugs in PDOs have an effective rate of 88% after being taken by patients, and if the effective drugs in PDOs model are ineffective, the effective drugs are also ineffective after being taken by patients, thereby proving that PDOs can be used as an effective antitumor drug screening means to realize individualized treatment³。

The invention aims to adopt the PDOs model as a means for evaluating the curative effect and provide a strategy for individualized treatment.

Reference documents:

1.Sato T,Vries RG,Snippert HJ,etal.Single Lgr5 stem cells buildcrypt-villus structures in vitro without a mesenchymal niche.Nature 2009,459(7244):262-265.

2.Sato T,Stange DE,Ferrante M,et al.Long-term expansionof epithelialorganoids from human colon,adenoma,adenocarcinoma,and Barrett'sepithelium.Gastroenterology 2011,141(5):1762-1772.

3.Vlachogiannis G,Hedayat S,Vatsiou A,et al.Patient-derived organoidsmodel treatment response of metastatic gastrointestinal cancers.Science2018,359(6378):920-926.

disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for screening drugs based on intestinal organoids, which constructs PDOs through intraoperative or enteroscope biopsy specimens, treats the PDOs by applying anti-tumor drugs, targeting drugs and the like in vitro, and evaluates the treatment effectiveness through cell viability detection.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a method for screening a medicament based on an intestinal organoid, which comprises the following steps:

step one, constructing intestinal organoids by using surgically excised fresh colorectal cancer tissues or enteroscopy biopsy specimens;

digesting the cultured intestinal organoids into single cells, and then paving the single cells in a 96-hole enzyme label plate according to a certain quantity;

adding the drug to be screened into the hole of the 96-hole enzyme label plate, and putting the enzyme label plate into an incubator for culture;

step four, after the culture is finished, adding CellTiter-Glo 3D cell viability detection reagent into each hole for 10min, and then placing the hole in an Orion II instrument for reading to evaluate the cell viability.

Further, the drug is 5-Fu, oxaliplatin, SN-38, raltitrexed or oncolytic virus.

Further, cell viability was assessed based on ATP values.

Further, the specific method for constructing the intestinal organoids in the first step comprises the following steps:

(1) thoroughly cleaning fresh colorectal cancer tissues, shearing the colorectal cancer tissues into fragments, centrifuging the fragments, and discarding supernatant;

(2) adding digestive juice, resuspending the precipitate, shaking vigorously, and incubating on ice; filtering and centrifuging the incubated supernatant, and discarding the supernatant;

(3) resuspending the cell pellet obtained in step (2), and counting; placing the cell suspension in another centrifuge tube, centrifuging, and removing the supernatant;

(4) mixing the cell sediment obtained in the step (3) with liquid temperature-sensitive matrigel; and (4) incubating, adding a human colorectal organoid culture medium, and culturing in an incubator for 5-7 days.

Further, the washing solution used in the step (1) is a PBS solution containing 1% penicillin/streptomycin.

Further, the size of the pieces in the step (1) is 2-4mm³。

Further, the ratio of matrigel to cells was 50. mu.l/20000.

Further, matrigel needs to be sucked by a pre-cooled gun head before being mixed with the cell pellet.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the method for screening the medicines based on the intestinal organoids adopts the PDOs model as a curative effect evaluation means, constructs PDOs through intraoperative or enteroscopic biopsy specimens, treats the PDOs by applying antitumor medicines, targeted medicines and the like in vitro, has effect data close to real effect, and can provide a basis for individualized treatment.

Drawings

FIG. 1 is a diagram showing the condition of an organoid in vitro growth process of a patient with intestinal cancer according to an embodiment of the present invention;

FIG. 2 is a histogram of the results of in vitro drug screening of PDOs models in one embodiment of the present invention.

Detailed Description

The invention provides a method for screening drugs based on intestinal organoids, which adopts a PDOs model as a means for evaluating curative effect, constructs PDOs through intraoperative or enteroscopy biopsy specimens, treats PDOs by applying anti-tumor drugs, targeted drugs and the like in vitro, and evaluates the effectiveness of the treatment through cell viability detection.

The method for screening the medicine based on the intestinal organoids comprises the following steps:

step one, constructing intestinal organoids by using surgically excised fresh colorectal cancer tissues or enteroscopy biopsy specimens;

digesting the cultured intestinal organoids into single cells, and then paving the single cells in a 96-hole enzyme label plate according to a certain quantity;

adding the drug to be screened into the hole, and putting the hole into an incubator for culture;

step four, after the culture is finished, adding CellTiter-Glo 3D cell viability detection reagent into each hole for 10min, and then placing the hole in an Orion II instrument for reading to evaluate the cell viability.

The present invention will be described in detail and specifically with reference to the following examples so that the present invention may be better understood, but the following examples do not limit the scope of the present invention.