Pathological angiogenesis inhibitor and application thereof
An angiogenesis and pathological technology, applied in the field of medicine, can solve problems such as the absence of diseases related to abnormal blood vessels, and achieve the best therapeutic effect and prolong the survival time.
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Embodiment 1
[0086] Example 1 shRNA-mediated knockdown of IDI1
[0087] Two primer sequences ShIDI1-1 and ShIDI1-2 targeting different sites of human IDI1 gene (NCBI gene accession number 3422) were synthesized and cloned into the retroviral shRNApLKO.1 vector (Addgene). in,
[0088] ShIDI1-1 sequence SEQ ID NO: 1:
[0089] 5'-CCGGCGTGTTGTAGTCATCCATTAACTCGAGTTAATGGATGACTACAACACGTTTTTG-3';
[0090] ShIDI1-2 sequence SEQ ID NO: 2:
[0091] 5'-CCGGCCAGATCCCAATGAGATTAAACTCGAGTTTAATCTCATTGGGATCTGGTTTTTG-3'.
[0092] In HEK293 cells ( CRL-1573 TM ) using the PEI method to synthesize retroviruses. Human umbilical vein endothelial cells (HUVEC) were transfected with the virus produced ( CRL-1730 TM ). After 48 hours or 72 hours after transfection, the cell protein was harvested and the knockdown efficiency of the IDI1 gene was detected by Western blot. The results were as follows figure 2 As shown in A. Depend on figure 2 A It can be determined that the IDI1 gene is knocked down ef...
Embodiment 2
[0095] Example 2 Effect of IDI1 overexpression on primary human endothelial cells
[0096]In order to study whether the overexpression of IDI1 will affect the function of endothelial cells, the inventors cloned and constructed the retroviral vector of IDI1 overexpression and expressed it in HEK293 cells ( CRL-1573 TM ) to synthesize the retrovirus. HUVEC cells were transfected with the virus produced. After 48 hours or 72 hours after transfection, the expression level of IDI1 was significantly increased, and the results were as follows: image 3 As shown in A. image 3 A shows the expression levels of IDI1 in the control (control) and IDI1 overexpression group cells (OE, overexpression) detected by Western blot. image 3 B shows the migration ability of control and IDI1 overexpressed endothelial cells using the wound healing method, which shows the healing situation of control and IDI1 overexpressed endothelial cells (OE) at 0 hours, 6 hours and 10 hours after wound forma...
Embodiment 3
[0097] Example 3 idi1 zebrafish mutant model
[0098] In order to study the function of idi1 gene in vivo, a zebrafish idi1 gene mutant line was identified and phenotyped. The allele (allele) La014590Tg of this mutation was obtained from the Zebrafish Information and Research Center (ZIRC). A pair of primers SEQ ID NO:3 and 4 are used to determine the allele of mutation, wherein the structure of wild type and mutant idi1 allele is as follows Figure 4 As shown in A.
[0099] Forward primer sequence SEQ ID NO:3:
[0100] 5'-AAAGACCCCCACCTGTAGGTTTG-3';
[0101] Reverse primer sequence SEQ ID NO:4:
[0102] 5'-ACCGTTACCAGAGAGCTAGT-3'.
[0103] Selfing of Idi1 heterozygotes was used to generate mutant zebrafish embryos. The zebrafish (2dpf, days post-hybridization) control and mutant embryos developed to 2 days were collected, and the expression level of IDI1 protein was detected by Western Blot using an anti-idi1 antibody (GeneTex, catalog number GTX106100), and it was foun...
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