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Prostatic cancer stem cell marker, application of antibody OV6 in preparation of prostatic cancer stem cell marking material and marking method

A technology for stem cell markers and prostate cancer, which is applied in the field of preparation of prostate cancer stem cell marker materials, can solve problems affecting the exploration of prostate cancer stem cells in depth and clinical application, and achieve enhanced labeling effect, important clinical value, and high yield. tumor rate effect

Inactive Publication Date: 2018-11-02
SHANGHAI CHANGHAI HOSPITAL
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

At present, the markers of prostate cancer stem cells mainly include CD133, CD44, integrin α2β1, etc. However, on the one hand, whether the prostate cancer cells characterized by them belong to prostate cancer stem cells is still controversial. Some studies have reported that hidden in CD44 + α2β1 hi CD133 - Cells are the tumor-initiating cells. On the other hand, there are large differences in the expression abundance of these markers in most prostate cancer cell lines and tissues, which have greatly affected the in-depth research of prostate cancer stem cells and the exploration of clinical applications.

Method used

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  • Prostatic cancer stem cell marker, application of antibody OV6 in preparation of prostatic cancer stem cell marking material and marking method
  • Prostatic cancer stem cell marker, application of antibody OV6 in preparation of prostatic cancer stem cell marking material and marking method
  • Prostatic cancer stem cell marker, application of antibody OV6 in preparation of prostatic cancer stem cell marking material and marking method

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Experimental program
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Effect test

Embodiment 1

[0038] Source of cell lines: Prostate cancer cell lines C4-2 and C4-2B were provided by Dr. Leland Chung certified (Cedars-Sinai Medical Center, Los Angeles, CA, USA). C4-2 and C4-2B cells were cultured in RPMI 1640 medium (11835093, Gibco, USA) containing 10% fetal bovine serum (FBS, 10099-141, Gibco, USA), supplemented with 1% penicillin / streptomycin ( 15140122, Gibco, USA).

[0039] Enzalutamide (Enzalutamide, ENZ, S1250, Selleck, USA)

[0040] ① Divide 5×10 6 C4-2 and C4-2B cells were plated in a 10 cm dish (430167, Coring, USA), and enzalutamide (5 nM) was added after 24 hours, and stimulated for 48 hours.

[0041] ② Magnetic-activated cell sorting (MACS) was performed using MiniMACS TM Cell Sorter (Miltenyi Biotec, Bergisch Gladbach, Germany), specifically labeled with mouse-derived OV6 antibody (1:50, MAB2020, R&DSystems, Minneapolis, MN, USA) C4-2 and C4-2B cells treated with ENZ for 30 minutes;

[0042] ③ Incubate C4-2 and C4-2B cells labeled with OV6 primary anti...

Embodiment 2

[0046] OV6 positive prostate cancer cell identification method

[0047] 1. Tissue sample collection and immunohistochemistry

[0048] Between 2012 and 2013, cohort 1: 78 patients with pathologically diagnosed prostate cancer at Changhai Hospital (Shanghai, China) and cohort 2: 67 patients with pathologically diagnosed prostate cancer at Changzheng Hospital (Shanghai, China) were collected , including clinical data, tissue samples and follow-up data.

[0049] The clinical data of prostate cancer patients included age at diagnosis, preoperative PSA (maximum value within 6 months before operation), postoperative pathological status including Gleason score (GS), pathological T stage, pathological N stage, and extracapsular invasion ( CP) Surgical margins (SM), peripheral nerve invasion (PNI) and seminal vesicle invasion (SVI). Time to biochemical recurrence (BCR) (standard: PSA ≥ 0.2 μg / μl), MRI, CT or ECT to determine disease recurrence, clinical observation endpoints include b...

Embodiment 3

[0060] Detection of Stem Cell Characteristics of OV6 Positive Prostate Cancer Cell Lines

[0061] 1. Detection of expression levels of stemness-related genes by real-time polymerase chain reaction

[0062] Total RNA was extracted from C4-2B, C4-2, sorted monocytes or THP-1 using RNAiso Plus (9109, Takara, Japan) according to the manufacturer's protocol. Reverse transcription was performed using PrimeScript One Step RT kit (RR037B, Takara, Japan) in the presence of random primers and reverse transcriptase. Amplification of the generated cDNA was carried out in SYBR GreenRealtime PCR Master Mix (QPK201, Toyobo, Japan) and ABI PRISM 7300HT Sequence Detection System.

[0063] Each measurement was performed in triplicate and the results were normalized by the expression of the β-actin gene. The fold change from the mean by 2 -△△Ct OK, the result is as follows Figure 8 shown, data are expressed relative to the OV6 - Fold change in C4-2B cells, indicating high expression of ste...

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Abstract

The invention relates to the field of medical technique research and discloses a prostatic cancer stem cell marker, application of an antibody OV6 in the preparation of a prostatic cancer stem cell marking material and a marking method. The marking method comprises the following steps: marking a prostate cancer cell line or a prostate tissue cell by virtue of a mouse-source OV6 antibody; incubating the prostate cancer cell line or the prostate tissue cell together with rat anti-mouse IgG and magnetic beads; putting the prostate cancer cell line or the prostate tissue cell on an MACS MS column,and carrying out filtration separation, so as to obtain an OV6 positive prostatic cancer cell; and finally detecting the marking efficiency of the OV6 positive prostatic cancer cell by virtue of a flow cytometry. Furthermore, enzalutamide is utilized for realizing drug stimulation so as to enhance the marking effect before the marking of the mouse-source OV6 antibody. According to the marking method, the prostatic cancer stem cell can be remarkably marked, and the OV6 positive cell has high expression dryness-related gene and high granulation rate and tumor formation rate. The marking methodhas relatively high specificity and sensitivity and important clinical values.

Description

technical field [0001] The invention relates to the field of medical technology research, in particular to the application and marking method of prostate cancer stem cell markers and antibody OV6 in preparing prostate cancer stem cell marker materials. Background technique [0002] Prostate cancer (Prostate Cancer, PCa) is one of the tumors with the highest incidence rate after lung cancer, especially among men in developed countries, prostate cancer has become the tumor with the highest incidence rate. According to the global data released in 2015, it is estimated that 1.112 million new prostate cancer patients are diagnosed each year, and 308,000 people die from prostate cancer. Compared with Western countries, the incidence and mortality of prostate cancer in Asia are lower. In China, the incidence and mortality of prostate cancer are far lower than the world average. However, research data show that the incidence of prostate cancer in China is increasing year by year. T...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/095G01N33/569
CPCC07K16/18C12N5/0695G01N33/56966
Inventor 孙颖浩王超鲁欣高旭黄海孔德沛李惠珍瞿旻
Owner SHANGHAI CHANGHAI HOSPITAL
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