Biomarker applied to early diagnosis and preoperative evaluation of pheochromocytoma/paraganglioma as well as application thereof

A biomarker, pheochromocytoma technology, applied in the field of exosome-derived double-stranded DNA biomarkers, which can solve problems such as unknown genetic information

Inactive Publication Date: 2018-09-28
FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether exosomes derived from pheochromocytoma / paraganglioma patients carry genetic information that reflects the same tumor cells from which they originated remains unknown

Method used

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  • Biomarker applied to early diagnosis and preoperative evaluation of pheochromocytoma/paraganglioma as well as application thereof
  • Biomarker applied to early diagnosis and preoperative evaluation of pheochromocytoma/paraganglioma as well as application thereof
  • Biomarker applied to early diagnosis and preoperative evaluation of pheochromocytoma/paraganglioma as well as application thereof

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Experimental program
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Embodiment 1

[0033]1Collect cases and tissue samples

[0034] 15 sporadic pheochromocytoma / paraganglioma cases and tumor samples were collected for genetic testing. Three of them excluded germline and somatic mutations in 12 known susceptibility genes, and the other 12 had somatic mutations in RET, VHL, HIF2A, and SDHB, respectively. No adjuvant radiotherapy or chemotherapy was given before tumor resection. 10 mL of peripheral blood was collected from each patient. Blood samples were centrifuged at 4°C and 3000g for 10 minutes to separate and obtain serum for exosome and DNA extraction and analysis.

[0035] 2 cell culture

[0036] During the culture of PC12 cells, in order to avoid the contamination of exosomes in the serum, the serum was subjected to ultra-high speed centrifugation at a speed of 100,000 g for 11 hours, and the supernatant was taken. The basal medium RPMI 1640 was supplemented with 10% of horse serum and 5% of fetal bovine serum for removing exosomes, and was used for...

Embodiment 2

[0043] 1. Cell and serum exosome isolation and characterization

[0044] Exosomes were extracted from the collected culture medium of PC12 cells, the serum of nude mice with mutant xenograft tumors, and the serum of patients with pheochromocytoma / paraganglioma.

[0045] 1) For PC12 cells in the logarithmic growth phase, replace the medium with a medium containing exosome-depleted serum (10% horse serum, 5% fetal bovine serum), and centrifuge at 3000rpm at 4°C for 10 minutes to remove cell debris and dead cells. Cells, supernatant was taken and then differential centrifuged at 100,000g for 2.5 hours. The resulting pellet was washed once with PBS and centrifuged again at 100,000g for 70 minutes. The final pellet was resuspended in PBS solution.

[0046] 2) To isolate exosomes in peripheral blood, the sample was first centrifuged at 4°C and 3000rpm for 15 minutes to collect serum. After the collected serum was centrifuged again at 3000 rpm for 10 minutes, the supernatant was t...

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Abstract

The invention discloses a double-chain DNA biomarker applied to early diagnosis and preoperative evaluation of pheochromocytoma / paraganglioma as well as application thereof. The biomarker is a genomedouble-chain DNA fragment specifically derived from pheochromocytoma / paraganglioma serum exosome. The double-chain DNA can represent the variation condition of RET, VHL and HIF2A with high body cell mutation frequency in a pheochromocytoma / paraganglioma susceptible gene as well as SDHB related to metastatic phenotype. The exosome DNA circulated in peripheral blood of patients comprises 97 percentchromosome locus information with the same source tumor cell. The invention provides the existence basis of the double-chain DNA in the pheochromocytoma / paraganglioma serum exosome; the double-chain DNA can be used for authenticating the mutation existing in the tumor cell; and a noninvasive molecular marker is provided for clinical diagnosis and preoperative evaluation of the pheochromocytoma / paraganglioma.

Description

technical field [0001] The invention belongs to the field of tumor diagnosis and treatment, and in particular relates to an exosome-derived double-stranded DNA biomarker for early diagnosis and preoperative evaluation of pheochromocytoma / paraganglioma. Background technique [0002] Pheochromocytoma / paraganglioma is a rare endocrine tumor derived from chromaffin cells of the adrenal medulla. Approximately 50-80% of pheochromocytomas / paragangliomas are associated with germline mutations in at least 12 genes (NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, HIF2A, FH) or somatic mutations. In addition, with the continuous accumulation of mutation detection data, 21 newly described genes have been shown to be strongly associated with pheochromocytoma / paraganglioma development. Compared with other tumors, pheochromocytoma / paraganglioma has a significant genetic predisposition. The genotypes of predisposing gene mutations corresponded one-to-one with their clinical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 刘晶王亮马静云
Owner FIRST AFFILIATED HOSPITAL OF DALIAN MEDICAL UNIV
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