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Anti blood tumorous reverse VEGF gene sequence and expression carrier of recombination eukaryon

A gene sequence and anti-blood technology, which is applied in the field of preparation of recombinant eukaryotic expression vectors of VEGF121 gene fragments, can solve the problems of large toxic and side effects, low remission rate and recurrence rate, etc.

Inactive Publication Date: 2003-07-23
INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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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 in leukemia cells, so as to inhibit angiogenesis and reduce the tolerance to chemotherapeutic drugs, promote the apoptosis of leukemia cells, and overcome the current leukemia cells. In clinical treatment, there are disadvantages such as large toxic and side effects, strong drug resistance, low remission rate and high recurrence rate.

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  • Anti blood tumorous reverse VEGF gene sequence and expression carrier of recombination eukaryon
  • Anti blood tumorous reverse VEGF gene sequence and expression carrier of recombination eukaryon
  • Anti blood tumorous reverse VEGF gene sequence and expression carrier of recombination eukaryon

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Embodiment 1

[0007] Some examples are listed below to further illustrate the present invention, but not to limit the present invention. 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

[0008] 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 rec...

Embodiment 2

[0010] 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 the perforated plate was counted by flow cytometry. The results showed that the culture supernatant of K562-antisense VEGF cells promoted the in vitro prol...

Embodiment 3

[0010] 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 the perforated plate was counted by flow cytometry. The results showed that the culture supernatant of K562-antisense VEGF cells promoted the in vitro prol...

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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 Applications(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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