Application of septin gene shRNA in preparing septin gene activity inhibitor

A technology of sept2-shrna-1 and sept9-shrna-1, which is applied in the field of application of septin gene shRNA in the preparation of septin gene activity inhibitors, can solve problems such as difficult treatment, poor prognosis, difficult resection and treatment, and achieve growth inhibitory effect

Active Publication Date: 2018-09-21
浙江欧赛思生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Excision surgery and treatment are difficult due to the ability of GBM cells to grow and invade the normal brain
GBM was discovered and named by Percival Bailey and Harvey Cushing as early as 90 years ago, but it is still difficult to treat becaus

Method used

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  • Application of septin gene shRNA in preparing septin gene activity inhibitor
  • Application of septin gene shRNA in preparing septin gene activity inhibitor
  • Application of septin gene shRNA in preparing septin gene activity inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Finding and identifying GBM-related genes through multi-omics analysis

[0026] (1) Experimental method: comprehensive multi-omics analysis

[0027] GBM transcriptomic studies were selected based on the following four criteria: 1) samples were obtained from primary tissues with normal controls; 2) more than three cases and controls were used; 3) experiments were run on the same platform; 4) studies Conducted by an independent panel (see Table 1). Proteomic analysis of three different GBM cell lines was performed simultaneously to represent gene expression at the protein level. For example, the multi-omics analysis workflow flow chart ( figure 1), the data generated by proteomics and transcriptomics are input, and the output is high-quality candidate functional genes, which are arranged according to different statistical criteria. Studies of GBM expression data from the Gene Expression Omnibus (GEO) repository were combined for multiplex analysis. For each...

Embodiment 2

[0032] Example 2: Expression of SEPT9 and SEPT2 in GBM tissues and cell lines

[0033] 1. Experimental method:

[0034] 1. Immunohistochemistry

[0035] (1) Normal brain tissue slices (grade I normal human brain tissue, purchased from Rockville, MD, USA), 3 grades of glioma slices (grade II (astrocytoma), grade III (anaplastic astrocytoma) cell tumor) and grade IV (glioblastoma, GBM, purchased from Rockville, MD, USA) were washed in xylene to remove paraffin and rehydrated by serial dilutions of alcohol.

[0036] (2) Rinse the tissue sections twice with PBS, 5 minutes each time.

[0037] (3) Incubate in 3% hydrogen peroxide for 10 minutes at room temperature to block endogenous peroxidase activity.

[0038] (4) Rinse the tissue sections with PBS for 5 minutes.

[0039] (5) Antigen retrieval. Heat the antigen retrieval buffer (purchased from Sangon Biotechnology) to boiling state with a microwave oven, put the tissue slices into the buffer at 95-100°C for 10 min, twice, co...

Embodiment 3

[0084] Example 3: Inhibition of SEPT9 and SEPT2 expression using shRNA in A172 cells

[0085] 1. Experimental method (fluorescent quantitative PCR):

[0086]

[0087] Total RNA was extracted from GBM cells (ie, A172 cells) and analyzed by real-time quantitative PCR (qRT-PCR) using Trizol reagent (Thermo Fisher Scientific). The amplification reaction was completed by the cfx96 real-time PCR detection system (Bio-Rad Company, Hercules, CA, USA) using the SYBR Green fluorescent quantitative PCR kit according to the operating specifications. For SEPT9 and SEPT2 primers (Table 2), the expression of related genes was calculated using the 2ΔΔCT method. All qRT-PCR experiments were repeated three times, and all data used GAPDH as a control.

[0088] Table 2 SEPT2, SEPT9 qPCR primer sequences

[0089]

[0090] 2. Western blot

[0091] 1) Construction of scramble, SEPT2, SEPT9, SEPT2+SEPT9 plasmids (GFP co-expression): The most commonly used interference plasmid construction m...

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Abstract

The invention discloses application of a septin gene shRNA in preparing a septin gene activity inhibitor. The method disclosed by the invention has the advantages that the growth of gliocytoma can beeffectively inhibited and no obvious damage to human normal cells such as epidermal fibroblast cells is found.

Description

(1) Technical field [0001] The invention relates to two subtypes of the gene septin, Septin2 (SEPT2), shRNA of Septin9 (SEPT9), and the application in inhibiting the activity of glioblastoma. (2) Background technology [0002] Glioblastoma (GBM) is the most common primary malignancy of the central nervous system (CNS), with a five-year survival rate of less than 10%. There are about 210,000 new cases worldwide each year, accounting for 81% of brain tumors. According to the origin of glioma, it can be divided into four types of glioma, including astrocytoma (World Health Organization classification astrocytoma I, grade II (astrocytoma), grade III (anaplastic astrocytoma) cell tumor) and grade IV (glioblastoma, GBM)), oligodendroglioma, ependymoma, and mixed glioma. Resection surgery and treatment are difficult due to the ability of GBM cells to grow and invade the normal brain. GBM was discovered and named by Percival Bailey and Harvey Cushing as early as 90 years ago, but...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61K48/00A61P35/00
CPCA61K31/7088A61P35/00
Inventor 谭舟徐栋超刘阿娟王轩陈艺丹沈云芸邱猛生
Owner 浙江欧赛思生物科技有限公司
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