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Gene medication in use for treating tumor from epithelium, and preparation method

A gene drug and source technology, applied in antitumor drugs, gene therapy, drug combination and other directions, can solve the problems of recurrence and metastasis that plague surgeons, cause tumor tissue treatment resistance, and cannot completely cure tumors, etc., and achieve tumor suppression. Extensive, precise tumor suppressor effect, small adverse effects

Inactive Publication Date: 2007-02-21
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Traditional tumor treatment methods include chemotherapy, radiotherapy and surgery, but from the perspective of clinical treatment effects, none of them can completely cure the tumor
Moreover, due to the non-specific killing effect of radiotherapy and chemotherapy, the toxicity to the body is relatively high, and long-term use will cause treatment tolerance of tumor tissues.
The curative effect of surgery on patients with early tumors is obvious, but many tumors are found late and cannot be implemented, and the problem of postoperative recurrence and metastasis has always troubled surgeons
Our previous studies have shown that 14-3-3σ overexpression can inhibit the proliferation and tumorigenesis of Her2 overexpressed breast cancer cell lines MCF-7 and Her18, but whether 14-3-3σ can inhibit the epithelial-derived nasopharyngeal carcinoma cells and Akt overexpression cells, especially breast cancer MDA-MB-361 cells have not been reported at home and abroad

Method used

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  • Gene medication in use for treating tumor from epithelium, and preparation method
  • Gene medication in use for treating tumor from epithelium, and preparation method
  • Gene medication in use for treating tumor from epithelium, and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Recombination of 14-3-3σ gene and adenovirus

[0036] refer to figure 1 As shown, the cDNA sequence of 14-3-3σ was obtained by RT-PCR method. After the sequencing was correct, the full-length gene was amplified by PCR method in vitro, and the amplified product was cut with specific endonuclease and adenovirus shuttle Plasmid, construct a shuttle plasmid carrying the 14-3-3σ gene, subclone the amplified 14-3-3σ gene and signal peptide HA gene into the prokaryotic plasmid PUC19, and then use Cla1 and BamH1 to subclone into pQB1-AdCMV5 (containing gland Virus E1A), co-transfected with QB1-viralDNA, positive virus clones were screened by PCR, and Ad-HA-14-3-3σ gene recombinants were obtained, amplified, transfected into HEK293 cells, and crudely isolated 14-3- The 3σ gene recombinant adenovirus is purified by ultracentrifugation, sterilized, subpackaged, added with additives, and stored at low temperature.

Embodiment 2

[0037] Example 2 14-3-3σ applied to the treatment of poorly differentiated nasopharyngeal carcinoma

[0038] 1. Tetramethylazolazolium blue (MTT) assay to detect cell viability

[0039] Diluted to 3×10 with calf serum-free RPIM1640 medium 7 cells / L cell suspension, then transfect cells with Ad-14-3-3σ and Ad-β-gal or PBS (equal amount) at MOI of 5, 10, 15, and inoculate 96-well culture plate (100 μl / well), with 5 replicate wells in each group. Set at 37°C, 5% CO 2Cultivate under concentration conditions for 2 h, add 100 μl of RPIM1640 culture solution containing 20% ​​calf serum to each well, continue to culture for 22 h, 46 h, 70 h and 94 h, add 3% MTT 20 μl / well, incubate at 37°C for 4 h, discard the supernatant, and Add 100 μl of DMSO to the well, mix for 15 minutes, detect the A value at 590 nm, and calculate the tumor inhibition percentage, that is, the tumor inhibition percentage (%)=(A value of the treatment group-A value of the negative control group) / (A value of t...

Embodiment 3

[0062] Example 3 14-3-3σ applied to the treatment of Akt overexpressed tumors

[0063] 1. Expression and relationship of 14-3-3σ and p-Akt or p27 proteins in breast cancer tissues or cells

[0064] 1.1 Immunohistochemical method to detect the expression of 14-3-3σ and p-Akt protein in breast cancer tissues of patients

[0065] The paraffin specimens of 33 patients with primary breast cancer were collected and made into slices. After deparaffinization to water with xylene, the sections were placed in citrate buffer, adjusted to pH 6.0, and heated to facilitate antigen retrieval. The monoclonal antibody 14-3-3σ (c-18) was diluted 1:50, dropped onto the glass slide, and incubated at room temperature for 1 h, and the phosphorylated Akt (ser473) was diluted 1:100, and added to the glass slide for incubation. Then stain with the general immunohistochemical staining scheme, use the ABC enzyme substrate kit to label the protein, counterstain the slide with hematoxylin, dehydrate, tr...

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Abstract

A genetic medicine for treating the epithelium originated tumor, such as breast cancer, stomach cancer, is a recombinant of gene14-3-3 alpha and adenovirus. Its preparing process includes such steps as obtaining the cDNA sequence of 14-3-3 alpha by RT-PCR method, testing the sequence, externally amplifying its whole length by RCR method, severing the amplified product and the shuttle plasmid of adenovirus by specific endonuclease, transinfecting HEK293 cell strain, and encapsulating the recombinant virus.

Description

technical field [0001] The invention relates to a gene medicine, in particular to a gene medicine for treating epithelial-derived tumors such as breast cancer, gastric cancer, oral squamous cell carcinoma, and nasopharyngeal carcinoma. The invention also relates to a preparation method of the gene medicine. Background technique [0002] Traditional tumor treatment methods include chemotherapy, radiotherapy and surgery, but from the perspective of clinical treatment effects, none of them can completely cure the tumor. Moreover, due to the non-specific killing effect of radiotherapy and chemotherapy, the toxicity to the body is relatively high, and long-term use will cause treatment tolerance of tumor tissues. Surgery has obvious curative effect on patients with early tumors, but many tumors are found late and cannot be implemented, and the problem of postoperative recurrence and metastasis has always troubled surgeons. If the difference in gene expression between tumor cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/00
Inventor 杨惠玲夏云飞李梦宏黄文林苏勇夏洪平
Owner SUN YAT SEN UNIV
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