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EpCAM/PSMA dual-antibody-based functionalized micro-fluidic chip as well as preparation method and application thereof

A microfluidic chip and functionalized technology, applied in biochemical equipment and methods, stress-stimulated microbial growth methods, cell dissociation methods, etc., can solve the problem of slow progress in CTCs research, different, lack of specific target molecules, etc. problem, to achieve high specificity, high sensitivity, high accuracy

Active Publication Date: 2018-06-19
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some technical problems in using tissue for PD-L1 histochemical detection: PD-L1 is expressed sporadically in tumor tissue, that is to say, the expression of PD-L1 in different locations of tumor tissue is not the same, and Interferon secreted by T cells upregulates PD-L1 expression in tumor cells
However, due to the lack of epithelial-mesenchymal transition (EMT, epithelial mesenchymal transition) and specific target molecules, the research on the capture and molecular characterization of CTCs in prostate cancer patients has been slow.

Method used

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  • EpCAM/PSMA dual-antibody-based functionalized micro-fluidic chip as well as preparation method and application thereof
  • EpCAM/PSMA dual-antibody-based functionalized micro-fluidic chip as well as preparation method and application thereof
  • EpCAM/PSMA dual-antibody-based functionalized micro-fluidic chip as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Immunohistochemical detection of PSMA expression level in prostate cancer tissue

[0090] The expression levels of PSMA in tissues of patients with different types of prostate cancer and benign prostatic hyperplasia were detected by immunohistochemical technique.

[0091] 1) Collect concomitant bone metastasis (Bone metastasis), lymph node metastasis (LN metastasis) or different Gleason scores (different scores refer to low scores and high scores, low scores refer to gleason scores less than 7, high scores refer to gleason scores Pathological sections of tumor tissue from patients with prostate cancer with a score greater than 7), and the tissue from BPH patients as a control; the above sections are collectively referred to as paraffin tissue sections;

[0092] 2) Put the paraffin tissue sections obtained in step 1) in an oven at 65°C for 2 hours, dewax to water, wash three times with PBS (concentration: 10mM, pH7.2-7.4), and then place the sections in EDTA b...

Embodiment 3

[0189] Example 3 Validation of Prostate Cancer CTCs Capture Efficiency in Simulated Blood Environment

[0190] Take an appropriate amount of prostate cancer cell LnCAP and LnCAP-EMT stimulated by TGF-β1 and add it to 1mL peripheral whole blood of healthy subjects, and mix well, so as to simulate the detection of circulating tumor cells in the peripheral blood environment. After adding erythrocyte lysate to remove erythrocytes, the EpCAM / PSMA double antibody functionalized microfluidic chip was used to identify and capture prostate cancer cells in the peripheral blood simulated environment, and the captured cells were identified by immunofluorescence.

[0191] 1) 2 mL of peripheral venous blood was collected from 24 healthy individuals, and stored in EDTA (Ethylene diamine tetraacetic acid, ethylenediaminetetraacetic acid) vacuum blood collection tubes for anticoagulation.

[0192] 2) Take 1 mL of the prepared LnCAP-EMT single cell suspension stimulated by LnCAP and TGF-β1, and...

Embodiment 4

[0202] Example 4 Capture and Identification of CTCs in Peripheral Blood of Prostate Cancer Patients

[0203] Collect 1 mL of peripheral blood samples from patients with clinical prostate cancer, add erythrocyte lysate to remove red blood cells, and further identify and capture prostate cancer cells in the peripheral blood environment by EpCAM / PSMA double antibody functionalized microfluidic chips, and capture the captured prostate cancer cells Cells were identified by immunofluorescence.

[0204] 1) Put the peripheral blood of the prostate cancer patient into a centrifuge tube, centrifuge at 3000rpm for 5min, and discard the supernatant.

[0205] 2) Add 5 mL of erythrocyte lysate, mix the cells well, and lyse at room temperature for 5 minutes, shaking 1-3 times during the period.

[0206] 3) Centrifuge at 300g for 5 minutes, remove the supernatant, and observe whether the erythrocytes in the bottom cell pellet are completely lysed. If not, follow the above steps to lyse again...

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Abstract

The invention discloses an EpCAM / PSMA dual-antibody-based functionalized micro-fluidic chip as well as a preparation method and application thereof. The invention also discloses a method for capturingprostatic cancer CTCs by using the EpCAM / PSMA dual-antibody-based functionalized micro-fluidic chip as well as method for analyzing relevant molecular characteristic spectrum for the captured prostatic cancer CTCs. The relevant molecular phenotype is a prostatic cancer transfer relevant marker SChLAP1 / PSA as well as targeting treatment response marker AR / PD-L1, so that a prostatic cancer CTCs molecular characteristic spectrum joint detection method and a detection kit can be constructed. The EpCAM / PSMA dual-antibody-based functionalized micro-fluidic chip is effective, high in specificity, high in sensitivity and high in accuracy, and also has clinical practical value, and the tetramolecular joint detection is used for the early warning of the transfer and reoccurrence and the curative effect evaluation in the prostatic cancer CTCs as well as improves the feasibility and credibility of the clinical use by virtue of the targeting treatment.

Description

technical field [0001] The present invention studies the separation and capture of prostate cancer CTCs and the identification of related molecular phenotype profiles, relates to the field of molecular biology diagnosis and curative effect evaluation of prostate cancer metastasis and recurrence, and can be used for the selection of clinical treatment target response for prostate cancer patients and as a companion diagnosis for tumors Provide evidence-based basis, specifically related to the functionalized microfluidic chip based on EpCAM / PSMA double antibody and its preparation method and application. Background technique [0002] Worldwide, prostate cancer (PCa, prostate cancer) has become one of the most common malignant tumors in men and the second leading cause of cancer death in men. In the United States, its incidence rate is only lower than that of skin cancer, and statistics show that in 2014 there were approximately 230,000 new cases of prostate cancer and approxima...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/6886C12M1/00C12M1/42
CPCC12N5/0693C12Q1/6886C12N2509/00C12Q2600/158
Inventor 汪付兵陈浩尹长青袁纯辉陈明
Owner WUHAN UNIV
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