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Preparation method of human stem cell for continuously secreting active chondroitinase ABC, kit and clinical use of human stem cell

A technology of chondroitinase and stem cells, applied in the field of stem cells, can solve the problems of ineffectiveness, loss, damage, etc.

Inactive Publication Date: 2015-10-28
陈镇洲
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, it has been found that through genetic engineering technology, if the whole gene sequence is directly transfected into mammalian cells, it is difficult to be secreted outside the cell, so there is no therapeutic effect, or it is secreted outside the cell, but loses the activity of the enzyme
One of the main limitations of chABC therapeutic application is that chABC is very sensitive to temperature, and its activity is basically completely lost at 37°C for 3 to 5 days. Because chABC is sensitive to temperature and needs to be administered for a long time, it needs to be used for treatment. Local continuous infusion or multiple injections, which will greatly increase the risk of trauma and infection for patients
In addition, the current route of administration of chABC mostly adopts intrathecal injection, which will be powerless for deep injuries

Method used

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  • Preparation method of human stem cell for continuously secreting active chondroitinase ABC, kit and clinical use of human stem cell
  • Preparation method of human stem cell for continuously secreting active chondroitinase ABC, kit and clinical use of human stem cell
  • Preparation method of human stem cell for continuously secreting active chondroitinase ABC, kit and clinical use of human stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Construction of recombinant chABC-3F lentiviral vector and preparation of human stromal stem cells

[0052] The chABC-3F sequence was amplified from the pCAGIP-chABC-3xFlag plasmid (gifted by Professor Michael Klüppel of Northwestern University), and the target fragment of chABC-3F and the pLenti6.3_MCS vector were digested with PacI / AscI, and T4DNA ligase (Japan TAKARA company), and double-stranded chABC-3F ligation reaction at 4 ° C for 12 hours to prepare a clone connection solution, and transform the E. coli competent cell DH5a (US Invitrogen company) to carry out positive clone PCR identification and sequencing identification.

[0053] Take 293T cells in good condition and in the logarithmic growth phase. After counting the cells, 6×10 6 The number of cells was seeded in a culture dish at 37°C, 5% CO 2 cultured overnight in an incubator. Remove the culture medium before transfection the next day, and replace with 5ml Opti-MEM culture medium. Add 9ug Pa...

Embodiment 2

[0055] Example 2 Analysis of optimal multiplicity of infection of human stromal stem cells transfected with recombinant chABC-3F lentivirus

[0056] Detect the expression level of the target gene in the cells after pLenti6.3-chABC-3F transfects human BMSCs in Example 1 by qPCR method, and determine the optimal multiplicity of infection (multiplicity) of pLenti6.3-chABC-3F transfected human bone marrow mesenchymal stem cells of infection, MOI) value.

[0057] The day before the experiment, the hBMSC cells were divided into 1 × 10 6 were seeded in each well of a six-well plate and incubated overnight. At the beginning of the experiment, the lentivirus solution was removed from -80°C in advance and thawed at room temperature. According to the number of hBMSCs in the 6-well plate, lentiviral solution was added to make the MOI values ​​= 100, 500, 1000 respectively. Add 8ug / ml polybrene at 37°C, 5% CO 2 Incubate for 4-6 hours in an incubator. Then discard the old medium contai...

Embodiment 3

[0060] Example 3 Detection of chABC-3F gene and protein expression in human stromal stem cells transfected with recombinant chABC-3F lentivirus

[0061] The RNA extracted in the above-mentioned Example 2 was reverse-transcribed and then subjected to fluorescent quantitative PCR detection and protein analysis. The method for detecting chABC-3F gene expression by fluorescent quantitative PCR is the same as that in Example 2.

[0062] The specific steps of western blotting analysis of secreted chABC-3F are as follows:

[0063] Protein extraction: Collect the human BMSCs cell suspension transfected with pLenti6.3-chABC-3F prepared in Example 1 and centrifuge at 1000 rpm for 10 minutes, discard the culture medium, and wash with PBS for 3 times. Add the corresponding extraction buffer according to the amount of cells, and shake gently on ice for 15 minutes. Collect the lysate into an EP tube and centrifuge at 14,000rpm for 15min. Take the supernatant into a new EP tube. Determin...

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Abstract

The invention provides a preparation method of a human stem cell for continuously secreting active chondroitinase ABC. The preparation method comprises constructing an N-terminal signal peptide-free chondroitinase ABC gene, adding a gene segment for transcription and translation improvement to the end 3' of the gene, constructing the recombinant gene to a virus vector, and transfecting a human stem cell to obtain the human stem cell for continuously secreting active chondroitinase ABC. The human stem cell can effectively secrete chondroitinase ABC for a long time. The chondroitinase ABC has enzyme activity at a temperature of -20 DEG C for a long time. The invention provides a clinical use of the human stem cell continuously secreting active chondroitinase ABC. Through seed cells and a microenvironment going against nerve regeneration, the human stem cell can produce dual effects of treating chronic neurodegenerative diseases.

Description

technical field [0001] The present invention relates to a method for preparing human stem cells that continuously secrete active chondroitinase ABC and obtain human stem cells that continuously secrete active chondroitinase ABC through the method. In particular, the present invention relates to the construction of chondroitin without N-terminal signal peptide Enzyme ABC gene and adding gene fragments that improve transcription and translation at the 3' end of the gene, so that human stem cells can continuously secrete active chondroitinase ABC; the human stem cells that continuously secrete active chondroitinase ABC of the present invention can be used for clinical treatment For chronic neurodegenerative diseases, it is expected to achieve a "two-pronged approach" for chronic neurodegenerative diseases by promoting regeneration of damaged axons, sprouting undamaged neural circuits, strengthening connections, and protecting damaged projection neurons. the therapeutic effect. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A61K35/28A61K35/545A61K35/30A61P9/00
Inventor 陈振洲郭阳
Owner 陈镇洲
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