LU502444B1 - Application of reagent for detecting sdc1 expression in preparing product for diagnosing drug resistance of patients with cholangiocarcinoma to pemigatinib - Google Patents
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Abstract
The invention provides an application of a reagent for detecting SDC1 expression in preparing a product for diagnosing the drug resistance of patients with cholangiocarcinoma to pemigatinib, belonging to the technical field of biomedicine. The invention claims the application of a reagent for detecting the expression of SDC1 in preparing a product for diagnosing drug resistance of cholangiocarcinoma patients to pemigatinib. The invention also claims to protect a product for diagnosing the drug resistance of cholangiocarcinoma patients to pemigatinib, comprising a reagent for detecting the expression of SDC1. The drug resistance of pemigatinib is an important influencing factor for the poor prognosis of cholangiocarcinoma patients. According to the invention, SDC1 is an important marker for detecting the drug resistance of pemigatinib in cholangiocarcinoma patients, and the detection of the expression of SDC1 can help realize the detection of the drug resistance of pemigatinib in cholangiocarcinoma patients.
Description
DESCRIPTION LU502444 APPLICATION OF REAGENT FOR DETECTING SDC1 EXPRESSION IN
TECHNICAL FIELD The invention relates to the technical field of biomedicine, in particular to the application of a reagent for detecting SDCI1 expression in preparing a product for diagnosing the drug resistance of patients with cholangiocarcinoma to pemigatinib.
BACKGROUND Biliary tract cancer (BTC) is the second most common malignant tumor in hepatobiliary system after hepatocellular carcinoma, mainly including gallbladder cancer and cholangiocarcinoma (CCA). Cholangiocarcinomas are classified into intrahepatic cholangiocarcinoma (ICCA), perihilar cholangiocarcinoma (pCCA) and distal cholangiocarcinoma (dCCA) according to their location. The early clinical features of cholangiocarcinoma are often unclear and the early diagnosis is difficult. About 65% of patients have lost the opportunity of radical surgical resection at the time of confirmed diagnosis. Overall, the S-year survival rate of patients with cholangiocarcinoma is less than 10%. Even after surgical treatment, bile duct cancer is prone to invasion of blood vessels, lymphatic tissues and nerves, distant metastasis and insensitivity to adjuvant treatment such as radiotherapy and chemotherapy, resulting in a high recurrence rate. Even after radical resection, the 5-year survival rate of patients is less than 10%. The 1-year survival rate of patients who can't operate is only 10%-20%.
In recent decades, the treatment options and overall prognosis of cholangiocarcinoma have almost stagnated. Radical resection is one of the most effective methods to treat early bile duct cancer. However, the early clinical symptoms of cholangiocarcinoma are not obvious, and it is usually diagnosed when the biliary tract is blocked, followed by symptoms such as jaundice and abdominal pain, which makes about 65% of patients lose the chance of radical surgery. High postoperative chemical resistance and recurrence rate also affect the prognosis of patients with cholangiocarcinoma. Even if radical resection is performed, the five-year survival rate of patienit$)502444 is less than 10%, while the one-year survival rate of patients who cannot be operated is only 10%-20%. The comprehensive treatment of cholangiocarcinoma, which is dominated by surgery and supplemented by chemotherapy or targeted drugs, has become an international consensus. As far as chemotherapy is concerned, there is currently no standardized and recognized chemotherapy scheme that can prolong the survival time of advanced cholangiocarcinoma. NCCN guidelines recommend gemcitabine combined with cisplatin as the main systemic chemotherapy, but the five-year survival rate of patients with advanced cholangiocarcinoma is less than 5%. Various factors lead to poor prognosis of cholangiocarcinoma.
At present, the research on cholangiocarcinoma is mostly focused on gene level, lacking the verification of protein expression level and large sample multi-center clinical investigation. At present, the detection methods of cholangiocarcinoma are as follows:
1. MRCP, magnetic resonance examination, has a high accuracy, and it is also beneficial to observe the whole biliary stricture or the tumor site, so as to make a clear identification;
2. CT, as one of the common methods in tumor examination, has a high differential rate for bile duct tumors;
3. Duodenal endoscopy is common in clinic, and similar to gastroscopy, which can be performed by retrograde upper digestive tract to biliary tract for endoscopic examination, or even biopsy, to find cancer cells through pathological sections;
4. Combined detection of tumor markers CAS0, CA125, CA242, CA19-9 and CEA to diagnose cholangiocarcinoma.
At present, although the method is effective in detecting cholangiocarcinoma, many patients have pemigatinib resistance during treatment. The drug resistance of pemigatinib is an important factor affecting the poor prognosis of patients with cholangiocarcinoma. At present, there is no effective detection method to predict the drug resistance of patients with cholangiocarcinoma.
SUMMARY The purpose of the present invention is to provide an application of a reagent for detecting SDC1 expression in preparing a product for diagnosing drug resistance of cholangiocarcinoma patients to pemigatinib, so as to solve the problems existing in the prior art. Detection of SDC1 expression can effectively predict the drug resistance of cholangiocarcinoma patients tdJ502444 pemigatinib.
To achieve the above purpose, the present invention provides the following solutions: The invention provides an application of reagent for detecting SDC1 expression in preparing product for diagnosing drug resistance of patients with cholangiocarcinoma to pemigatinib.
Further, the product is a detection reagent or a kit.
Further, when the sample to be detected is a tissue sample, the method for detecting the expression of SDC1 specifically comprises: (1) extracting total RNA from a tissue sample, and performing reverse transcription to obtain cDNA; (2) using the cDNA as a template, using the primer pairs shown in SEQ ID NO.1-2 for fluorescent real-time quantitative PCR detection.
Further, the reaction system of the fluorescent real-time quantitative PCR detection is: 10 uL of SYBR qPCR Mix, 2 pL of cDNA, 0.4 uL of Forward Primer (10 uM), 0.4 uL of Reverse Primer (10 uM), 7.2 uL of RNase Free HO.
Further, the reaction program of the fluorescent real-time quantitative PCR detection is: 94°C for 2-3 min; 94°C for 5-10 s, and 60°C for 30 s, 40 cycles; Dissociation stage.
Further, when the sample to be detected is bile or serum sample, the expression of SDCI is detected by enzyme-linked immunosorbent assay.
The invention also provides a product for diagnosing the drug resistance of patients with cholangiocarcinoma to pemigatinib, characterized by comprising a reagent for detecting the expression of SDC1.
Further, the product is a reagent or a kit.
Further, the reagent for detecting the expression of SDC1 comprises a primer pair as shown in SEQ ID NO.1-2.
Effect of invention The drug resistance of pemigatinib is an important factor affecting the poor prognosis of patients with cholangiocarcinoma. At present, there is no effective detection of pemigatinib resistance. According to the invention, SDC1 is an important marker for detecting the drug resistance of pemigatinib in cholangiocarcinoma patients, and the detection of the expression bË502444 SDC1 can help realize the detection of the drug resistance of pemigatinib in cholangiocarcinoma patients.
BRIEF DESCRIPTION OF THE FIGURES In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without any creative effort.
Fig. 1 shows the results of enzyme-linked immunosorbent assay of bile samples from 10 patients with cholangiocarcinoma; * * indicates significant difference; Fig. 2 shows the results of ELISA of serum samples from 12 patients with cholangiocarcinoma; * * * * means that the difference is extremely significant; Fig. 3 shows the results of fluorescence real-time quantitative PCR detection of tissue samples from 17 patients with cholangiocarcinoma; * * * * means that the difference is extremely significant.
DESCRIPTION OF THE INVENTION Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be taken as a limitation of the present invention, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present invention.
It should be understood that the terms mentioned in the present invention are only used to describe specific embodiments, and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Any stated value or intermediate value within the stated range, and any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
Unless otherwise stated, all technical and scientific terms used herein have the same meanings commonly understood by those of ordinary skill in the field to which this invention relates. Although the present invention only describes preferred methods and materials, at/502444 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.
Without departing from the scope or spirit of the present invention, it is obvious to those skilled in the art that many modifications and changes can be made to the specific embodiments of the present specification. Other embodiments obtained from the description of the present invention will be obvious to the skilled person. The description and embodiment of that invention are only exemplary. As used in this paper, the terms "comprising", "including", "having" and "containing" are all open terms, meaning including but not limited to.
Embodiment 1 I. Sample preparation
1. Tissue samples: (1) Preparation of wax blocks: fix the collected cancer tissues and adjacent tissues of cholangiocarcinoma patients in 4% paraformaldehyde, and embed them into wax blocks with paraffin embedding machine.
(2) RNA sample preparation: 1> Take fresh tissues (or frozen at -80°C) (including CCA tissues and adjacent tissues) and record the patient's name, hospitalization number and other information; 2> Cut out about 50 mg of tissue, wash it with PBS, put it into a 1.5 mL EP tube, add 1 mL TRIzol, and homogenize it with a tissue grinder; 3> Let stand at room temperature for 5 minutes to fully crack the tissue; at this time, the sample can be stored at -80°C for a long time; centrifuge at 12000 rpm, 4°C for 5 minutes, suck the supernatant, transfer it to a new EP tube, and mark the patient information on the side wall of the tube; 4> Add 1/5 volume of chloroform (that is, add at least 200 uL of chloroform to every 1 ml of trizol); close the EP tube cap tightly, shake it by hand for 30 seconds to mix it thoroughly, and let it stand at room temperature for 5 minutes;
5> Centrifuge at 12000 rpm and 4°C for 15 minutes; after centrifugation, it can be seen thkW502444 the liquid is divided into three layers: the upper layer is colorless and nearly transparent RNA, the middle layer is white and thin DNA, and the lower green layer is protein and organic phase; transfer 400 mL of the upper water phase into a new EP tube, add equal volume of isopropanol, shake it up and down for 6-8 times, and stand at room temperature for 10 minutes.
6> Centrifuge at 12000 rpm, 4°C for 15 minutes, carefully discard the supernatant, and a small amount of flocs can be seen settling on the wall or bottom of EP tube; 7> Add I mL absolute alcohol to wash the precipitate, and gently bounce the RNA precipitate by hand or blow it with a pipette; 8> Centrifuge at 12000 rpm at 4°C for 10 minutes, carefully suck up the ethanol with a pipette, and be careful not to absorb the precipitate; invert the EP tube on absorbent paper and let it stand at room temperature to dry.
Blood sample: take 4 ml of fresh blood; 3000 g, 4°C, 15 min, centrifuge to get the supernatant.
Bile sample: take 40 mL of fresh bile; 20000 g, 4°C, 30 min, centrifuge to collect supernatant.
IT Sample testing The expression of SDC1 (polyligand proteoglycan 1) in the sample was detected by the following method:
1. Tissue samples Real-time quantitative PCR detection Firstly, reverse transcribe the obtained RNA sample; then, perform real-time fluorescence quantitative PCR detection.
Primer pair: upstream primer (SEQ ID NO.1): CGTGGGGCTCATCTTTGCT; the downstream primer (SEQ ID NO.2): TGGCTTGTTTCGGCTCCTC.
uL PCR reaction system: 10 uL of SYBR qPCR Mix, 2 pL. of cDNA, 0.4 uL of Forward Primer (10 uM), 0.4 uL of Reverse Primer (10 uM), 7.2 uL of RNase Free HO.
PCR reaction procedure: 94°C for 2-3 min; 94°C for 5-10 s, and 60°C for 30 s, 40 cycles; Dissociation stage.
2. Blood samples and bile samples
The supernatant of fresh samples was taken for enzyme-linked immunosorbent assdy502444 (ELISA) of human soluble polygalacturn 1(sSDC1); 1) Dilution of standard: dilute the original standard in a small test tube according to the following method: 4000 pg/mL: add 150 pL of standard dilution solution (i.e. sterile water without enzyme) to the original standard of No.5 standard (150uL); 2000 pg/mL: add 150 pL of standard dilution solution to 150 pL of No. 4 standard and No. standard; 1000 pg/mL: add 150 pL of standard dilution solution to 150 pL of No. 3 standard and No. 4 standard; 500 pg/mL: add 150 pL of standard dilution solution to 150 pL of No. 2 standard and No. 3 standard; 250 pg/mL: add 150 pL of standard dilution solution to 150 uL of No. 1 standard and No. 2 standard; 2) Sample adding: blank hole (blank control hole does not add sample and enzyme-labeled reagent, and other steps are the same as the sample hole to be tested), standard hole and sample hole to be tested are respectively set. Accurately add 50 uL of the standard sample to the enzyme-labeled plate, and add 40 uL of sample diluent to the hole of the sample to be tested, and then add 10 pL of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-labeled plate hole, try not to touch the hole wall, gently shake and mix.
3) Incubation: seal the plate with a sealing film and incubate at 37°C for 30 min.
4) Liquid preparation: dilute 30 times of concentrated washing liquid with distilled water (20 times of 48 T) for later use.
5) Washing: carefully peel off the sealing film, discard the liquid, spin it dry, fill each hole with washing liquid, let it stand for 30 seconds and discard it. Repeat this process for 5 times and pat it dry.
6) Adding enzyme: 50 uL of horseradish peroxidase (HRP), an enzyme-labeled reagent, is added to each well, except the blank well.
7) Incubation: the operation is the same as 3).
8) Washing: the operation is the same as 5). LU502444 9) Color development: add 50 pL of tetramethylbenzidine (TMB) into each hole, then add 50 uL of tetramethylbenzidine (TMB), gently shake and mix, and develop color at 37°C in the dark for 10 min.
10) Termination: add 50 pL of termination solution to each well to terminate the reaction (at this time, blue turns yellow). 40% sodium hydroxide is used as the termination solution.
11) Measurement: Zero the blank hole, and measure the absorbance (OD value) of each hole in sequence at 450 nm wavelength. The measurement should be carried out within 15min after adding the stop solution.
II. Results SDC1 was detected in bile, serum and tissue samples of 39 patients with cholangiocarcinoma. The results are shown in Figure 1, Figure 2 and Figure 3 respectively.
According to the invention, high expression of SDC1 in bile serum and tissues is found, and the high expression of SDCI1 activates FGFR2, thereby affecting the drug resistance of pemigatinib. According to the invention, 39 patients are detected by combined application of tissues, bile and serum, and 10 patients with high expression of SDC1 have poor prognosis because of pemigatinib resistance.
The above-mentioned embodiments only describe the preferred mode of the invention, but do not limit the scope of the invention. On the premise of not departing from the design spirit of the invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the scope of protection determined by the claims of the invention.
Sequence Listing LU502444 <110> SHANDNG UNIVERSITY <120> APPLICATION OF REAGENT FOR DETECTING SDC1 EXPRESSION IN
CHOLANGIOCARCINOMA TO PEMIGATINIB <130> PT2252 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 catggggctc atetttgct 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 tggcttgttt cggctecte 19
Claims (9)
1. An application of reagent for detecting SDC1 expression in preparing product for diagnosing drug resistance of patients with cholangiocarcinoma to pemigatinib.
2. The application according to claim 1, characterized in that the product is a detection reagent or a kit.
3. The application according to claim 1, characterized in that when the sample to be detected is a tissue sample, the method for detecting the expression of SDC1 comprises: (1) extracting total RNA from a tissue sample, and performing reverse transcription to obtain cDNA; (2) using the cDNA as a template, using the primer pairs shown in SEQ ID NO.1-2 for fluorescent real-time quantitative PCR detection.
4. The application according to claim 3, characterized in that the reaction system of the fluorescent real-time quantitative PCR detection is: 10 uL of SYBR qPCR Mix, 2 pL of cDNA,
0.4 pL of Forward Primer (10 uM), 0.4 uL of Reverse Primer (10 uM), 7.2 uL of RNase Free HO.
5. The application according to claim 3, characterized in that the reaction program of the fluorescent real-time quantitative PCR detection is: 94°C for 2-3 min; 94°C for 5-10 s, and 60°C for 30 s, 40 cycles; dissociation stage.
6. The application according to claim 1, characterized in that when the sample to be detected is a bile or serum sample, the expression of SDC1 is detected by enzyme-linked immunosorbent assay.
7. A product for diagnosing the drug resistance of patients with cholangiocarcinoma to pemigatinib, characterized by comprising a reagent for detecting the expression of SDC1.
8. The product according to claim 7, characterized in that the product is a reagent or a kit.
9. The product according to claim 7, characterized in that the reagent for detecting the expression of SDC1 comprises a primer pair as shown in SEQ ID NO.1-2.
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| Application Number | Priority Date | Filing Date | Title |
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| LU502444A LU502444B1 (en) | 2022-06-30 | 2022-06-30 | Application of reagent for detecting sdc1 expression in preparing product for diagnosing drug resistance of patients with cholangiocarcinoma to pemigatinib |
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| LU502444A LU502444B1 (en) | 2022-06-30 | 2022-06-30 | Application of reagent for detecting sdc1 expression in preparing product for diagnosing drug resistance of patients with cholangiocarcinoma to pemigatinib |
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