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Insulin-expressing human islet cell lines capable of reversibly proliferating and use thereof

一种胰岛细胞、可逆性的技术，应用在胰腺细胞、人工细胞构建体、遗传修饰的细胞等方向，能够解决导入基因困难、无法实现无限增殖等问题

Inactive Publication Date: 2006-12-13
KURARAY CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as of today, many researchers are actively working on the immortalization of human islet cells, but there is no report on an insulin-producing human islet cell line
It is considered that this is because 1) it is difficult to introduce the gene because insulin-producing β cells exist inside the islets, and 2) in order to establish an immortal cell line, the use of virus-derived oncogenes (simian virus 40 tumor antigen, etc.) can make the cells Extended lifespan, but cannot achieve full infinity

Method used

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  • Insulin-expressing human islet cell lines capable of reversibly proliferating and use thereof
  • Insulin-expressing human islet cell lines capable of reversibly proliferating and use thereof
  • Insulin-expressing human islet cell lines capable of reversibly proliferating and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046]Example 1: Reversible immortalized human islet cell line NAKT-13 (deposited in the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology, the address is Central No. 6, East 1-1-1, Tsukuba City, Ibaraki, Japan (postal code 305-8566), the deposit date is September 4, 2003, and the deposit number is the establishment of FERM BP-08461)

[0047] Islet cells isolated from healthy individuals provided by the University of Alberta, Canada, by hand pickup under a stereomicroscope (STEMI, Carl Zeiss, Germany) (University of Alberta, Canada, Human Islet Transplantation Protocol, courtesy of Jonathan RT. Lakey 10 islet cells with spherical shape and clear shape were selected from the public and available to the public), and they were inoculated into T25 culture flasks. Use by adding 10% fetal calf serum (FCS, SIGMA company) in WILLIAM'S medium E (SIGMA company, St.Louis, U.S.), 10 -7 mol / l insulin (SIGMA company), 10 -6 mol / l dex...

Embodiment 2

[0051] Example 2: Removal of hTERT gene and SV40T gene from reversibly immortalized human islet cell line

[0052] The reversible immortalized human islet cell line obtained in Example 1 was cultured at 100,000 cells / ml in low-glucose DMEM (Giboco, Oakland, New Jersey) with 10% FCS, 10 mM nicotinamide, penicillin G / streptomycin medium. At 24 hours from the start of the culture, 1 MOI (MOI: multiplicity of infection) of the adenoviral vector AxCANCre expressing Cre recombinase was added to the medium, and infection was continued for 48 hours thereafter. The cells were washed twice with PBS to replace the medium. Such as Figure 5 As shown, the obtained recovered NAKT-13 cells have the characteristic that the cells naturally aggregate with each other to form a morphology similar to the normal islet structure.

Embodiment 3

[0053] Example 3: Insulin expression and morphology

[0054] The cells (each 50,000 cells) obtained in Example 1 and 2 were inoculated on a position covered with coverslips in a 6-well plate, using low glucose DMEM (Giboco Company, Oakland, New Jersey) with 10% FCS , 10 mM nicotinamide, and penicillin G / streptomycin as the culture medium, cultured for 24 hours until the cell density reached 60%. Next, the medium was replaced with a medium composed of high glucose DMEM (Giboco, Oakland, New Jersey) added with 10% FCS, 10 mM nicotinamide, penicillin G / streptomycin, and cultured for 6 hours.

[0055] After the culture, the expression of insulin in the cells was studied by immunostaining using a rabbit anti-human insulin antibody (manufactured by Dako Cytomation Co., Ltd., Kyoto, Japan). The staining results are shown in image 3 and 5 middle. and, Figure 4 and 6 respectively image 3 and 5 schematic diagram. image 3 and 5 Respectively represent the NAKT-13 cells obtai...

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Abstract

Reversibly immortalized human islet cell lines containing an hTERT gene and an SV40T gene each located between a pair of LoxP sequences, characterized by being capable of producing insulin and the expression of insulin being enhanced after removing the hTERT gene and the SV40T gene, in particular, NAKT-13 (having been deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Address: Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, 305-8566 JAPAN, Deposition date: 04 September, 2003, Deposition No.: FERM BP-08461) or passage cell lines thereof; human islet cells obtained by removing the hTERT gene and the SV40T gene from the reversibly immortalized human islet cell lines or passage cell lines thereof as described above; and use of these cells. By using the reversibly immortalized human islet cell lines as described above, insulin-producing cells can be easily and surely obtained in a number enough to meet demand.

Description

technical field [0001] The invention relates to a human islet cell line expressing insulin capable of reversible proliferation. The present invention also relates to a therapeutic agent for diabetes and a method for producing insulin. Background technique [0002] According to the cause of decreased insulin activity, diabetes can be divided into two categories: type 1 diabetes (juvenile diabetes) and type 2 diabetes. [0003] Type 1 diabetes occurs due to autoimmune abnormalities that specifically damage insulin-producing pancreatic β cells (see Atkinson MA, Maclaren NK., N. Engl. J. Med. 331:1428-1436, 1994). Transplantation, one of the regenerative alternative therapies of pancreatic beta cells, is considered to be fundamentally curative. Such methods include pancreas transplantation and islet transplantation. The goal of both transplants is to regulate blood sugar very tightly to avoid hypoglycemia and thus avoid long-term complications. The aim is not just to improve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N5/10A61P3/10C12P21/02A61K35/12A61K35/39C12N5/071
CPCC12N2510/04A61K35/12C12N5/0676A61P3/10
Inventor 田中纪章小林直哉成岛道树田中斋仁
Owner KURARAY CO LTD
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