Substance for inhibiting XPB expression and endothelial cell line with XPB low expression

An endothelial cell, low expression technology, applied in the fields of medical molecular biology and genetic engineering, to achieve the effect of improving and inhibiting endothelial mesenchymalization

Pending Publication Date: 2022-07-29
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] The current prevalence and mortality of cardiovascular diseases are high worldwide, and the existing diagnosis and treatment methods need to be further updated to improve the prognosis of various cardiovascular diseases

Method used

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  • Substance for inhibiting XPB expression and endothelial cell line with XPB low expression
  • Substance for inhibiting XPB expression and endothelial cell line with XPB low expression
  • Substance for inhibiting XPB expression and endothelial cell line with XPB low expression

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preparation example Construction

[0076] The present invention also provides a method for preparing a recombinant lentiviral expression vector, comprising the following steps:

[0077] Step 1: annealing the single-stranded DNA molecule shown in SEQ ID NO: 11 and the single-stranded DNA molecule shown in SEQ ID NO: 12 to form a DNA double-stranded fragment with sticky ends;

[0078] Step 2: The lentiviral vector pHBLV-U6-MCS-PGK-NEO was digested by restriction endonuclease 1 and restriction enzyme 2, and the DNA double-stranded fragment obtained in step 1 was connected by T4 ligase vector to obtain a recombinant lentiviral vector.

[0079] The present invention also provides a method for constructing a microvascular endothelial cell line with low expression of XPB, comprising the following steps:

[0080] Step 1: Combining the recombinant lentiviral vector with the viral packaging auxiliary plasmids pSPAX2 and pMD2G to form a three-plasmid lentiviral system for viral packaging, and then transfecting 293T cells...

Embodiment 1

[0113] Example 1. Design and synthesis of siRNA targeting XPB gene mRNA (referred to as XPB siRNA)

[0114] The inventor of the present invention designs the siRNA targeting XPB gene by searching the sequence of human XPB in the NCBI GenBank database (www.ncbi.nlm.nih.gov / gene), and the design mainly follows the following principles: (1) In The cDNA of XPB is designed around 50-100 nt downstream of the initiation codon (AUG), and avoids non-coding regions such as introns, 5'UTR, 3'UTR; (2) avoids more than 4 identical bases and SNP single nucleotide polymorphism sites; (3) try to control the content of G and C bases between 30% and 60%; (4) carry out homology alignment after the design is completed.

[0115] Following the above principles, three pairs of XPB siRNA were designed and named as XPB siRNA1, XPB siRNA2 and XPBsiRNA3. The sense and antisense strands of 3 pairs of XPB siRNA were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. siRNA NC was donated by Sangon...

Embodiment 2

[0120] Example 2. Construction of lentiviral vector targeting XPB gene and identification of its interference effect on XPB gene

[0121] 1. Construction of XPB shRNA and shRNA NC recombinant lentiviral vectors

[0122] (1) Determine the interference target and design and synthesize primers

[0123] Short hairpin RNAs (shRNAs) were designed based on XPB siRNA1, XPB siRNA2 and XPB siRNA3, respectively.

[0124] ① Design a short hairpin RNA (XPB shRNA1) sequence based on the XPB siRNA1 sequence and synthesize primers. The upstream primer is: 5'-GATCCGCCTTCTCTCCAGTTTACAAATATGCTCGAGCATATTTGTAAACTGGAGAGAAGGCTTTTTTG-3' (SEQ ID NO: 9), and the downstream primer is: 5'-AATTCAAAAAAGCCTTCTCCAGTTTACAAATATGCTCGAGCATATTTGTAAACTGGAGAGAAGGCG-3' (SEQ ID NO: 10);

[0125] ② Design and synthesize short hairpin RNA (XPB shRNA2) sequence based on XPB siRNA2 sequence and synthesize primers. The upstream primer is 5'-GATCCGCCAAGACTTCTTGGTGGCTATTGCACTCGAGTGCAATAGCCACCAAGAAGTCTTGGTTTTTTG-3' (SEQ ID...

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Abstract

The invention discloses a substance for inhibiting XPB expression and an endothelial cell line for inhibiting XPB low expression of the substance, the endothelial cell line comprises any one of small interfering RNA and shRNA, the small interfering RNA is any one of siRNA1, siRNA2 and siRNA3, and the XPB siRNA2 and the XPB shRNA2 can inhibit expression of XPB in human microvascular endothelial cells; the XPB shRNA2 can be used for inhibiting endothelial mesenchymal transdifferentiation of the HMEC-1 under an anoxic condition and inhibiting the migration capability of cells; the RNAi technology inhibits the expression of XPB genes in microvascular endothelial cells, constructs a microvascular endothelial cell model with low XPB expression, studies the important role of XPB in the generation and development of endothelial interstitization, is beneficial to reveal the potential mechanism of endothelial interstitization, and provides useful clues for treating cardiovascular diseases caused by endothelial interstitization. The method has an important application value.

Description

technical field [0001] The invention relates to the technical fields of medical molecular biology and genetic engineering, in particular to an endothelial cell line for inhibiting the expression of XPB and the low expression of XPB. Background technique [0002] The universal transcription factor TFIIH (transcription factor IIH) plays an important role in transcription initiation and DNA damage repair, and is involved in cell cycle regulation and chromosome segregation. TFIIH is a multi-protein complex composed of 10 subunits, which is structurally divided into XPB / ERCC3, XPD / ERCC2, TTDA / p8 / GTF2H5, p34 / GTF2H3, p44 / GTF2H2, p52 / GTF2H4 and p62 / GTF2H1. The core subcomplex and the cyclin-dependent kinase (CDK) activating kinase (CDK-activating kinase, CAK) subcomplex composed of CDK7, Cyclin H and MAT1. Human xeroderma pigmentosum group B (Xeroderma pigmentosum group B, XPB) gene, also known as excision repair cross complementing 3 (excision repair cross complementing 3, ERCC3) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N5/10C12N15/867C12N15/66A61K31/713A61P9/00A61P9/10
CPCC12N15/113C12N5/069C12N15/86C12N15/66A61K31/713A61P9/10A61P9/00C12N2310/141C12N2740/15043C12N2740/15052C12N2510/00
Inventor 李华方哲彦葛均波
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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