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Application of human CSPG5 gene and related product

A technology of genes and uses, applied in medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc., can solve the problem of lack of CSPG5 gene

Pending Publication Date: 2020-04-28
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no relevant report about the application of CSPG5 gene in the treatment of liver cancer

Method used

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  • Application of human CSPG5 gene and related product
  • Application of human CSPG5 gene and related product
  • Application of human CSPG5 gene and related product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1 Preparation of RNAi lentivirus for human CSPG5 gene

[0107] 1. Screening for effective siRNA targets against the human CSPG5 gene

[0108] Retrieve CSPG5 (NM_006574) gene information from Genbank; design effective siRNA targets for CSPG5 gene. Table 1-1 lists the screened effective siRNA target sequences against CSPG5 gene.

[0109] Table 1-1 is targeted at the siRNA target sequence of human CSPG5 gene

[0110] SEQ ID NO TargetSeq(5'-3') 1 TCCCAAGTTACTGTCACAA

[0111] 2. Preparation of lentiviral vector

[0112] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.

[0113] Table 1-2 Double-stranded DNA Oligo with sticky ends containi...

Embodiment 2

[0131] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0132] Human liver cancer SMMC-7721 cells in the logarithmic growth phase were digested with trypsin to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection value (MOI, SMMC-7721:20, the following examples are based on this multiplicity of infection), add an appropriate amount of the lentivirus prepared in Example 1, and replace the medium after 24 hours of cultivation, and wait until the infection time reaches 5 Days later, cells were collected. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then ...

Embodiment 3

[0140] Example 3 Celigo detects the proliferation ability of tumor cells infected with CSPG5-shRNA lentivirus

[0141] Human liver cancer SMMC-7721 cells in the logarithmic growth phase were digested with trypsin to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection, an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 3 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 2500 / well. 5 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2 Incubator cultivation. From the second day after plating, the plate was detected and read once a day wi...

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Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of a human CSPG5 gene as a target in preparation of liver cancer treatment drugs. Through wide and deep research, the invention finds that proliferation of liver cancer cells can be effectively inhibited, apoptosis is promoted and the growth process of liver cancer can be effectively controlled afterexpression of the human CSPG5 gene is down-regulated by adopting an RNAi method. The siRNA or a nucleic acid construct and lentivirus containing siRNA sequence provided by the invention can specifically inhibit the proliferation rate of the liver cancer cells, promote apoptosis of the liver cancer cells, inhibit cloning of the liver cancer cells, inhibit tumor formation of the liver cancer cellsand inhibit growth of the liver cancer, so that the liver cancer is treated, and a new direction is opened up for treatment of the liver cancer.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human CSPG5 gene and related products. Background technique [0002] CSPG5 is a protein-coding gene (Uniprot) that encodes a proteoglycan that may play a role in nerve growth and differentiation factors, and that proteoglycan stimulates the complexity of dendritic trees in primary cultured hippocampal neuronal cells. sex. Studies have found that CSPG5, PHF6, and PAF1 are components of the intrinsic transcriptional pathway in cells, which coordinates the migration of neurons in the brain, and is of great significance to the pathogenesis of cognitive developmental disorders. During retinal degeneration in Rpe65- / - mice, NGC was induced to express in the neural retina, but was not further induced to express in the retinal pigment epithelium (RPE), which originally expressed the highest level of NGC. Studies have found that CSPG5 is highly expressed in BRCA1...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867A61K45/00A61K31/713A61P35/00
CPCC12N15/113C12N15/86A61K45/00A61K31/713A61P35/00C12N2310/14C12N2740/15043C12N2800/107C12N2310/531Y02A50/30
Inventor 陶开山汪建林张洪涛彭伟李艺杰林志斌杨龙
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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