1. The extraction method of bile exosomes for bile duct cancer is characterized by comprising the following steps: the method comprises the following steps:

the method comprises the following steps: collecting a specimen: step 1.1: taking 10ml-20ml of bile sample of a bile duct cancer patient, wherein the bile sample is taken from ERCP or PTCD; step 1.2: collecting bile, immediately placing in a dry test tube, keeping out of the sun, placing in a refrigerator at 4 deg.C, centrifuging at 4000r/min for 10min within 2h, collecting supernatant, and placing in a centrifuge tube without RNA enzyme; step 1.3: before operation, extracting 5ml-10ml of fasting venous blood, standing a blood specimen at room temperature for 2h, centrifuging at 4000r/min for l0min, and taking upper serum in a centrifuge tube without RNA enzyme; step 1.4: after the bile and serum samples are collected, putting the collected bile and serum samples into a refrigerator for freezing and storing for later use;

step two: extraction of exosomes: step 2.1: one day before centrifugation, taking out the bile specimen from a refrigerator, and placing the bile specimen in a 4-degree refrigerator for melting to obtain 50ml of yellow bile; step 2.2: centrifuging bile at 4 deg.C, 300g, 5min for 1 time, and collecting supernatant; step 2.3: centrifuging the supernatant at 4 deg.C for 1 time at 2000g for 10min, and collecting the supernatant; step 2.4: diluting the obtained 50mL of supernatant to 60mL, centrifuging for 30min at 4 degrees and 12500g, and taking the supernatant after centrifugation is finished; step 2.5: diluting the obtained bile supernatant to 70mL, sieving with 0.22um sieve, centrifuging for 2h at 4 degrees and 120000g to obtain bottom precipitate, repeatedly blowing and dissolving the precipitate with sterile D-PBS, transferring to an EP tube to obtain 200uL of exosome suspension, detecting NTA, and storing in a-80 ℃ refrigerator;

step three: identification of exosomes: observing the form of the extracted bile exosome particles by adopting a transmission electron microscope; measuring the grain size of the exosome by adopting a nanoparticle tracking analysis technology; and detecting the expression condition of the exosome surface protein marker by Western blotting.

2. The method for extracting bile exosomes for cholangiocarcinoma according to claim 1, characterized in that: in the step 1.1, the bile juice samples are extracted before injecting the contrast medium after the bile duct is inserted successfully.

3. The method for extracting bile exosomes for cholangiocarcinoma according to claim 1, characterized in that: the temperature for freezing and standby in the step 1.4 is-80 ℃.

4. The method for extracting bile exosomes for cholangiocarcinoma according to claim 1, characterized in that: the bottom precipitate in step 2.5 is an exosome.

5. The method for extracting bile exosomes for cholangiocarcinoma according to claim 1, characterized in that: the antibody used in the third step comprises CD63, TSG101 and Hsp 70.

Background

Cholangiocarcinoma is the most common malignant tumor of biliary tract system, and at present, no effective biological marker is available for diagnosis and prognosis prediction. Therefore, the discovery of new reliable bile duct cancer markers has important significance for primary screening, early diagnosis, recurrence monitoring and prognosis prediction. Exosome is a lipid bilayer vesicle with the diameter of about 30-150 nm secreted and released by cells, contains specific protein and nucleic acid, and is widely present in various body fluids such as blood, urine, bile and the like. Recent researches show that exosomes and non-coding RNAs (such as microRNAs, lncRNAs, circRNAs and the like) contained in the exosomes in body fluid can reflect the progress and prognosis of tumors and can be a new tumor marker, but the research on the aspect is less at present and the application cannot be better.

Disclosure of Invention

In order to overcome the above problems, the present invention provides a method for extracting bile exosomes of bile duct cancer.

The technical scheme adopted by the invention is as follows:

the extraction method of bile exosomes for bile duct cancer comprises the following steps:

the method comprises the following steps: collecting a specimen: step 1.1: taking 10ml-20ml of bile sample of a bile duct cancer patient, wherein the bile sample is taken from ERCP or PTCD (bile is extracted before a contrast agent is injected after the bile duct is inserted successfully); step 1.2: collecting bile, immediately placing in a dry test tube, keeping out of the sun, placing in a refrigerator at 4 deg.C, centrifuging at 4000r/min for 10min within 2h, collecting supernatant, and placing in a centrifuge tube without RNA enzyme; step 1.3: before operation, extracting 5ml-10ml of fasting venous blood, standing a blood specimen at room temperature for 2h, centrifuging at 4000r/min for l0min, and taking upper serum in a centrifuge tube without RNA enzyme; step 1.4: collecting bile and serum samples, and freezing at-80 deg.C;

step two: extraction of exosomes: step 2.1: one day before centrifugation, the bile specimen is taken out from a refrigerator with the temperature of-80 ℃ and placed in a refrigerator with the temperature of 4 ℃ for melting, and 50ml of yellow bile is obtained; step 2.2: centrifuging bile at 4 deg.C, 300g, 5min for 1 time, and collecting supernatant; step 2.3: centrifuging the supernatant at 4 deg.C for 1 time at 2000g for 10min, and collecting the supernatant; step 2.4: diluting the obtained 50mL of supernatant to 60mL, centrifuging for 30min at 4 degrees and 12500g, and taking the supernatant after centrifugation is finished; step 2.5: diluting the obtained bile supernatant to 70mL, sieving with a 0.22um sieve, centrifuging for 2h at 4 degrees and 120000g to obtain a bottom precipitate which is an exosome, repeatedly blowing and dissolving the precipitate with sterile D-PBS, transferring the dissolved precipitate into an EP tube to obtain 200uL exosome suspension, and storing in a-80-degree refrigerator after NTA detection;

step three: identification of exosomes: observing the form of the extracted bile exosome particles by adopting a transmission electron microscope; measuring the grain size of the exosome by adopting a nanoparticle tracking analysis technology; western blotting is adopted to detect the expression of the exosome surface protein marker, wherein the antibodies used comprise CD63, TSG101 and Hsp 70.

The invention has the following advantages:

through the collection of specimens, the extraction of exosomes and the identification of exosomes, the extraction of bile exosomes of bile duct cancer can be realized, and the method has great potential application value in clinical diagnosis and treatment.

Detailed Description

The present invention will be further described below, but the present invention is not limited to these.

Example 1

The extraction method of bile exosomes for bile duct cancer comprises the following steps:

the method comprises the following steps: collecting a specimen: step 1.1: taking 10ml of bile sample of a bile duct cancer patient, wherein the bile sample is taken from ERCP (bile is extracted before injecting contrast agent after the bile duct is inserted successfully); step 1.2: collecting bile, immediately placing in a dry test tube, keeping out of the sun, placing in a refrigerator at 4 deg.C, centrifuging at 4000r/min for 10min within 2h, collecting supernatant, and placing in a centrifuge tube without RNA enzyme; step 1.3: before operation, 5ml of fasting venous blood is extracted, a blood specimen is stood for 2 hours at room temperature, is centrifuged for l0min at 4000r/min, and upper serum is taken out to be put in a centrifuge tube without RNA enzyme; step 1.4: collecting bile and serum samples, and freezing at-80 deg.C;

step two: extraction of exosomes: step 2.1: one day before centrifugation, the bile specimen is taken out from a refrigerator with the temperature of-80 ℃ and placed in a refrigerator with the temperature of 4 ℃ for melting, and 50ml of yellow bile is obtained; step 2.2: centrifuging bile at 4 deg.C, 300g, 5min for 1 time, and collecting supernatant; step 2.3: centrifuging the supernatant at 4 deg.C for 1 time at 2000g for 10min, and collecting the supernatant; step 2.4: diluting the obtained 50mL of supernatant to 60mL, centrifuging for 30min at 4 degrees and 12500g, and taking the supernatant after centrifugation is finished; step 2.5: diluting the obtained bile supernatant to 70mL, sieving with a 0.22um sieve, centrifuging for 2h at 4 degrees and 120000g to obtain a bottom precipitate which is an exosome, repeatedly blowing and dissolving the precipitate with sterile D-PBS, transferring the dissolved precipitate into an EP tube to obtain 200uL exosome suspension, and storing in a-80-degree refrigerator after NTA detection;

step three: identification of exosomes: observing the form of the extracted bile exosome particles by adopting a transmission electron microscope; measuring the grain size of the exosome by adopting a nanoparticle tracking analysis technology; western blotting is adopted to detect the expression of the exosome surface protein marker, wherein the antibodies used comprise CD63, TSG101 and Hsp 70.

Example 2

The extraction method of bile exosomes for bile duct cancer comprises the following steps:

the method comprises the following steps: collecting a specimen: step 1.1: taking 20ml of bile sample of a bile duct cancer patient, wherein the bile sample is taken from PTCD (all bile is extracted before injecting contrast medium after bile duct is inserted successfully); step 1.2: collecting bile, immediately placing in a dry test tube, keeping out of the sun, placing in a refrigerator at 4 deg.C, centrifuging at 4000r/min for 10min within 2h, collecting supernatant, and placing in a centrifuge tube without RNA enzyme; step 1.3: before operation, 10ml of fasting venous blood is extracted, a blood specimen is stood for 2h at room temperature, is centrifuged for l0min at 4000r/min, and upper serum is taken out in a centrifuge tube without RNA enzyme; step 1.4: collecting bile and serum samples, and freezing at-80 deg.C;

step two: extraction of exosomes: step 2.1: one day before centrifugation, the bile specimen is taken out from a refrigerator with the temperature of-80 ℃ and placed in a refrigerator with the temperature of 4 ℃ for melting, and 50ml of yellow bile is obtained; step 2.2: centrifuging bile at 4 deg.C, 300g, 5min for 1 time, and collecting supernatant; step 2.3: centrifuging the supernatant at 4 deg.C for 1 time at 2000g for 10min, and collecting the supernatant; step 2.4: diluting the obtained 50mL of supernatant to 60mL, centrifuging for 30min at 4 degrees and 12500g, and taking the supernatant after centrifugation is finished; step 2.5: diluting the obtained bile supernatant to 70mL, sieving with a 0.22um sieve, centrifuging for 2h at 4 degrees and 120000g to obtain a bottom precipitate which is an exosome, repeatedly blowing and dissolving the precipitate with sterile D-PBS, transferring the dissolved precipitate into an EP tube to obtain 200uL exosome suspension, and storing in a-80-degree refrigerator after NTA detection;

step three: identification of exosomes: observing the form of the extracted bile exosome particles by adopting a transmission electron microscope; measuring the grain size of the exosome by adopting a nanoparticle tracking analysis technology; western blotting is adopted to detect the expression of the exosome surface protein marker, wherein the antibodies used comprise CD63, TSG101 and Hsp 70.

Example 3

The extraction method of bile exosomes for bile duct cancer comprises the following steps:

the method comprises the following steps: collecting a specimen: step 1.1: taking 15ml of bile sample of a bile duct cancer patient, wherein the bile sample is taken from ERCP (bile is extracted before injecting contrast agent after the bile duct is inserted successfully); step 1.2: collecting bile, immediately placing in a dry test tube, keeping out of the sun, placing in a refrigerator at 4 deg.C, centrifuging at 4000r/min for 10min within 2h, collecting supernatant, and placing in a centrifuge tube without RNA enzyme; step 1.3: before operation, 7ml of fasting venous blood is extracted, a blood specimen is stood for 2 hours at room temperature, is centrifuged for l0min at 4000r/min, and upper serum is taken out to be put in a centrifuge tube without RNA enzyme; step 1.4: collecting bile and serum samples, and freezing at-80 deg.C;

step two: extraction of exosomes: step 2.1: one day before centrifugation, the bile specimen is taken out from a refrigerator with the temperature of-80 ℃ and placed in a refrigerator with the temperature of 4 ℃ for melting, and 50ml of yellow bile is obtained; step 2.2: centrifuging bile at 4 deg.C, 300g, 5min for 1 time, and collecting supernatant; step 2.3: centrifuging the supernatant at 4 deg.C for 1 time at 2000g for 10min, and collecting the supernatant; step 2.4: diluting the obtained 50mL of supernatant to 60mL, centrifuging for 30min at 4 degrees and 12500g, and taking the supernatant after centrifugation is finished; step 2.5: diluting the obtained bile supernatant to 70mL, sieving with a 0.22um sieve, centrifuging for 2h at 4 degrees and 120000g to obtain a bottom precipitate which is an exosome, repeatedly blowing and dissolving the precipitate with sterile D-PBS, transferring the dissolved precipitate into an EP tube to obtain 200uL exosome suspension, and storing in a-80-degree refrigerator after NTA detection;

step three: identification of exosomes: observing the form of the extracted bile exosome particles by adopting a transmission electron microscope; measuring the grain size of the exosome by adopting a nanoparticle tracking analysis technology; western blotting is adopted to detect the expression of the exosome surface protein marker, wherein the antibodies used comprise CD63, TSG101 and Hsp 70.

It is noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.