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Application of anti-sense VEGF gene sequence

A gene sequence and antisense technology, which is applied in the field of preparation of eukaryotic expression vectors for VEGF121 gene fragment recombination, can solve the problems of high toxicity and side effects, low remission rate and relapse rate, etc.

Inactive Publication Date: 2005-06-29
INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] The purpose of the present invention is to find an effective gene therapy method to down-regulate the secretion of endogenous VEGF from leukemia cells, so as to inhibit angiogenesis and reduce the tolerance to chemotherapeutic drugs, and promote the apoptosis of leukemia cells to overcome In the current clinical treatment of leukemia, there are disadvantages such as large toxic side effects, strong drug resistance, low remission rate and high relapse rate.

Method used

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  • Application of anti-sense VEGF gene sequence
  • Application of anti-sense VEGF gene sequence
  • Application of anti-sense VEGF gene sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0008] Example 1: Determination of the effect of K562 positive clones expressing antisense VEGF and analysis of the expression of endogenous VEGF at the mRNA level and protein level

[0009] K562-antisense VEGF, K562-empty vector cells were divided into 1.5×10 6 / mL inoculated in a 6-well plate, cultured in serum-free medium for 3 days, centrifuged to pellet the cells in each well, collected the culture supernatant-20 and stored it for later use. The genomic DNA and total cellular RNA of K562-antisense VEGF and K562-empty vector cells were extracted respectively, and the integration of exogenous genes and the mRNA expression of endogenous VEGF were performed by PCR and RT-PCR. Results The target band was detected in K562-antisense VEGF cells, but no specific band was found in K562-empty vector cells, indicating that the antisense gene fragment carried by the recombinant vector had been successfully integrated into the genome of K562 cells. The results of RT-PCR found that the...

Embodiment 2

[0011] Example 2: Effect of culture supernatant of K562 positive clonal cells on proliferation and migration of umbilical vein endothelial cells in vitro

[0012] Umbilical vein endothelial cells in 5×10 3 / mL inoculated in 96-well plate, counted by MTT method after cultured for 2 days in M199 culture medium containing 8% fetal calf serum and 50% volume fraction of K562-antisense VEGF cells or K562-empty vector cell culture supernatant: per well 100 μL MTT was added, and after incubation for 4 hours, 10 μL DMSO was added to each well to terminate the reaction, and the absorbance value was measured at a wavelength of 570 nm ( image 3 ). Add 500 μL K562-antisense VEGF cell culture supernatant or K562-empty vector cell culture supernatant to the lower layer of the perforated plate, 10 5 Umbilical vein endothelial cells were resuspended in 100 μL M199 culture medium, and after incubation for 4 hours, the number of umbilical vein endothelial cells migrating to the lower layer of...

Embodiment 3

[0013] Example 3: Proliferation and apoptosis of K562 positive cloned cells in vitro

[0014] K562-antisense VEGF cells or K562-empty vector cells at 1×10 4 / mL inoculated in a 96-well plate and cultured in serum-free medium. From the first day of inoculation, 4 wells were taken every 24 hours until cultured to 72 hours, and the growth curve of the cells was measured by MTT method. Or K562-antisense VEGF cells or K562-empty vector cells at 1×10 5 / mL inoculated in a 6-well plate, cultured in serum-free medium, 3 wells were taken every 24 hours, and placental blue staining was used to count viable cells until 72 hours. The results showed that the self-proliferation ability of K562 cells expressing antisense VEGF was lower than that of K562 control cells transfected with empty vector ( Figure 5 A).

[0015] K562-antisense VEGF cells or K562-empty vector cells at 1×10 5 / mL inoculated in 6-well plate, cultivated in serum-free medium for 72 hours, centrifuged to pellet cells,...

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Abstract

An antisense VEGF gene sequence resisting blood tumor, its recombinant eucaryon expression carrier, and a eucaryon carrier mediated VEGF gene therapy of leukemia are disclosed.

Description

Technical field: [0001] The present invention relates to nucleic acid antisense technology, in particular to a 444-base VEGF containing reverse sequence 121 Preparation of gene fragment recombinant eukaryotic expression vector and its application in vivo and in vitro. Background technique: [0002] The importance of angiogenesis for the development and metastasis of solid tumors has been well established, but its role in hematological tumors, such as leukemia, is still in the process of exploration. In recent years, a series of studies have found that in the bone marrow of patients with acute myeloid leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia, there is obvious microvascular proliferation, corresponding to this is vascular endothelial growth factor VEGF, a key angiogenesis Growth factor, which is significantly highly expressed in the circulating blood and cells of leukemia patients. VEGF is not only an endothelial growth factor, but also a hematopoi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P35/00C12N15/113C12N15/63
Inventor 韩忠朝何睿
Owner INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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