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Application of g6pd gene and its expression product in the treatment of colorectal cancer

A colorectal cancer and gene technology, applied in the field of gene therapy, can solve problems such as recurrence and metastasis, the effect of chemotherapy needs to be improved, and the mechanism of tumor drug resistance is complex, so as to prevent recurrence and metastasis, inhibit recurrence and metastasis, and prolong survival. Effect

Active Publication Date: 2020-08-14
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the effect of chemotherapy still needs to be improved, and a large part of the reason is also attributed to the recurrence and metastasis caused by drug resistance.
However, the mechanism of tumor drug resistance is very complex, and it is still one of the most important problems faced by current clinical treatment. A comprehensive and in-depth study of it will help the treatment of malignant tumors including colon cancer.

Method used

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  • Application of g6pd gene and its expression product in the treatment of colorectal cancer
  • Application of g6pd gene and its expression product in the treatment of colorectal cancer
  • Application of g6pd gene and its expression product in the treatment of colorectal cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The preparation of embodiment 1 gene carrier

[0055] 1. Taking polyaspartic acid as the main body, using acid-sensitive phenylimine bonds to connect polyethylene glycol on the main chain, and introducing low-molecular-weight polyethyleneimine to amine the side chains, forming a new type of pH-sensitive Type-responsive polycationic gene carrier PEG-PAsp-PEI (PPP).

[0056] Specifically, the synthesis method is as follows:

[0057] (1) Using the Fuchs-Farthing method to synthesize N-carboxy-L-aspartic acid-benzyl ester-cyclic anhydride (BLA-NCA), using benzylamine as an initiator, ring-opening polymerization to obtain polybenzyl aspartate ;

[0058] (2) polyethylene glycol (PEG) reacts with p-carboxybenzaldehyde under the catalysis of catalyst, and synthesizes and obtains PEG-CHO;

[0059] (3) Then react polybenzyl aspartate with PEG-CHO to form an acid-sensitive structure of phenylimine, use low molecular weight polyethyleneimine for aminolysis, and synthesize the ge...

Embodiment 2

[0066] Preparation of embodiment 2 interference material (PPP / DNA nano-gene complex)

[0067] 1. Design G6PD-specific shRNA sequence

[0068] A specific interference sequence targeting the G6PD gene is designed and synthesized by a biological company.

[0069] The specific interference sequence is as follows:

[0070] shRNA-1 (shown as SEQ ID NO.1): GCCTTCCATCAGTCGGATA;

[0071] shRNA-2 (shown as SEQ ID NO. 2): CCTCATGGTGCTGAGATTT.

[0072] 2. Preparation of PPP / DNA nanogene complexes

[0073] (1) Cloning the above shRNA sequence (shRNA-1 or shRNA-2) into a vector to form plasmid DNA.

[0074] (2) Dissolve the PPP polymer and plasmid DNA in ultrapure water to obtain a mother solution with a concentration of 5mg / mL, and then dilute it with PBS buffer (pH 7.4, 10mM) to obtain a 1mg / mL PPP polymer solution and DNA solution; then according to the PPP / DNA N / P ratio of 30, mix the PPP polymer solution and DNA solution and incubate at room temperature for 30 minutes, and form PP...

Embodiment 3

[0075] In vitro cell transfection of embodiment 3 PPP / DNA nano gene complex

[0076] In this example, the PEG-PAsp-PEI (PPP) vector was used to deliver the interference sequence against the G6PD gene, and the colorectal cancer HCT116 and DLD-1 cells were transfected, and the effect on the expression of G6PD was analyzed by Western blotting.

[0077] 1. The specific method is as follows:

[0078] (1) HCT116 and DLD-1 colorectal cancer cells were divided into 4×10 cells per well 5 Each cell was seeded in a 6-well plate and cultured overnight in RPMI-1640 medium containing 10% FBS (v / v) to allow it to grow adherently.

[0079] (2) The original medium was removed, and while fresh medium was added, PPP / plasmid DNA nanocomplex containing shRNA and PPP / control plasmid DNA nanocomplex were added respectively, and incubated for 48 hours.

[0080] (3) Collect cells, add 500 μL RIPA protein lysate to lyse on ice for 15 min, then collect by centrifugation (13000 rpm, 15 min) and quantif...

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Abstract

The invention discloses application of a G6PD gene and an expression product of the G6PD gene to colorectal cancer treatment. The invention provides a novel genetic vector targeting the G6PD gene and a covering method for an interference gene fragment by the genetic vector. The obtained product can realize the specific silencing suppression on the expression of the G6PD gene, and is combined with chemotherapeutic medicine of oxaliplatin for cooperated inhibition of colorectal cancer cell proliferation and cell apoptosis induction; the colorectal cancer chemotherapy medicine resistance is overcome; the goal of treating the colorectal cancer is achieved. The invention provides a gene treatment method aiming at the colorectal cancer. Compared with a conventional treatment method of the colorectal cancer, the method provided by the invention has the advantages that the gene technology is used for preventing the recurrence and metastasis of the colorectal cancer; the specificity and the sensitivity are realized. The novel scheme of combination of G6PD targeted gene treatment and conventional chemotherapeutic medicine of oxaliplatin provided by the invention has the advantages that the chemotherapy medicine resistance can be overcome; the disease development is delayed; good clinic application prospects are realized.

Description

technical field [0001] The invention belongs to the technical field of gene therapy. More specifically, it relates to the application of G6PD gene and its expression product in the treatment of colorectal cancer. Background technique [0002] Colorectal cancer is a common malignant tumor. The early symptoms are not obvious. As the cancer grows larger, symptoms such as changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain appear. symptom. Its morbidity and mortality are second only to gastric cancer, esophageal cancer and primary liver cancer among digestive system malignant tumors. The incidence of colorectal cancer in my country is increasing year by year, seriously affecting human health and life. [0003] According to the American Cancer Society estimates, more than 90% of tumor patients died of different degrees of drug resistance, and the problem of tumor drug resistance has become a key factor for the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K45/00A61K31/7105A61K48/00A61P35/00
CPCA61K31/7105A61K45/00C12N15/1137C12N2310/141
Inventor 徐瑞华鞠怀强陈雅
Owner SUN YAT SEN UNIV CANCER CENT
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