JPS60161922A - B lymphocyte cell strain derived from patient of bloom syndrome - Google Patents

Abstract

PURPOSE:The titled cell strain having normal karyotype and sister chromatid exchange of high frequency, established by sensitizing EB virus in a lymphocyte cell separated from a patient of Bloom syndrome (BS). CONSTITUTION:A mononuclear cell separated from blood collected from peripheral blood of BS patient by heparin blood-collecting is sterily washed. EB virus is absorbed in the mononuclear cell, a lymphocyte cell is sterily washed away, it is blended with medium RMPI1640 containing 30wt% bovine fetal serum, and cultivated at 37 deg.C by a carbonic acid gas culture container for about 60 days, to establish B lymphocyte cell strains (BS-SY1, BS-SY2, BS-SY3, or BS-SY4). But, BS-SY4 has somewhat extraordinary chromosome construction, and the other three are preferable cell strains. They are observed to produce immunoglobulin on the surface of cytoplasm and cell membrane. 100% cell shows high frequency SCE by taking BrdU in two cell generations.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a BUun/e cell line established from phospho/9-cell cells of patients with plume syndrome.

[Technology background]

Bloom syndrome (hereinafter referred to as BS), if there is a high frequency of chromosome breakage in cultured somatic cells, 4 and sister chromatid exchange (51 atar ahrom)
atidexchange (hereinafter referred to as SCE), and is one of the chromosome breakage syndromes characterized by high carcinogenicity that spreads across multiple organs.

BS was known as a genetic disease characteristic of Jews living in the United States, but in recent years cases of BS have also been found in Japanese people. This genetic disease has attracted attention from cancer researchers around the world due to its extremely high cancer incidence within its patient population. Fancon i anemia (hereinafter referred to as FA) and ataxia telangiectasia (hereinafter referred to as AT), which are classified as chromosome breakage syndromes along with BS, are also known to be highly carcinogenic diseases, but these are more common than BS. cancer is concentrated in specific areas. For example, in AT, phosphorus/
There are many cancers in the Q reticuloendothelial system, and even in FA there are many types of cancer that are very rare in the general population. On the other hand, BS is characterized by the fact that cancers of the type seen in normal people occur more than 100 times more frequently in young people. Therefore, it is possible to think that carcinogenesis in BS is basically the same as or similar to that in normal people. It will also make a major contribution.

From this point of view, the present inventors developed B'Junno9 derived from BS patients.
A cell line (BSl-2-8Y) was established and proposed previously (Japanese Patent Application Laid-open No. 152477/1983). This B51-2-8Y is important in that it has an SCE marker, but its chromosomal structure is not normal and it has many marker chromosomes. Therefore, for example, when attempting to mass-produce immunoglobulin preparations containing normal immunoglobulin factors, it is not preferable to use a B. linnoea cell strain having such an abnormal chromosomal structure.

The purpose of the present invention is to establish a Blin A cell line derived from a BS patient with a normal chromosomal structure.

[Structure of the invention]

The present invention is a method of developing SC cells with a normal nuclear history and a high frequency of SC, which is established by sensitizing lymphocytes isolated from BS patients with EB virus.
This is a BS patient-derived B'Jnnoq cell line with E ability.

The B lymphocyte cell line of the present invention is established as follows.

First, blood was collected from the peripheral blood of a BS patient (
30m) in a test tube under sterile conditions on top of Ficolnowq solution (7-) and centrifuged at 1600 rpm for 30 minutes. Mononuclear cells (mostly T and B'J (N/Q ball) Gather a group. The isolated mononuclear cells are aseptically washed twice with RPMI 1640 medium by centrifugation.

When sensitizing with the EE virus, rather than using the above-mentioned Ficoll/Qtx solution and using a mixture of nine B and T cells/Q cells, it is preferable to remove as much T cells as possible to enrich the B cells. It is desirable to use a cell population containing the present invention to achieve the objectives of the present invention.

In order to obtain a cell population rich in B cells, a mononuclear cell population (50x1
(suspended in RPMI 1640 medium at a concentration of 0'/d or less) is centrifuged on a Percall density gradient. The nine-call density gradient is created as follows. /(-pool (density of Jli fluid 1.13 f/at) 9 volumes with 10 times concentration of P
Add 1 volume of BS and mix this with 100% Norq-col solution (
stock solution). Add this stock solution to normal concentration of PB.
90.80.70.60.50 and 40 diluted with S
% 79-Cole solution is prepared. A 9-col density gradient was prepared as described above in a 40-centrifuge tube starting from the bottom.
00.90%80.70.60.50 and 40chi,
It is created by carefully layering 41R1 of Q-Cole liquid (so that the liquid level is not disturbed).

40 of the /e-call density gradients thus created.
% Percoll solution directly above 2-0B, T lymph mixed solution (I
Gently place the OXIO' cells or higher at 3000 rpm.
Centrifuge at 20° C. for 0 min. Lymph 9 sphere separation layer separation layer between Percoll concentration 409G and 50T, 5
If the phosphorus/q sphere separation layer between 0% and 60% is the phosphorus/q sphere separation layer, and the phosphorus/q sphere separation layer between 60% and 70% is the ■ layer, then 1
In the layer, 72 or more are B IJn/Q spheres, and T phosphorus/q spheres can be separated from layers II and II with a purity of 77% and 75%, respectively. Therefore, in the present invention, cells containing a large number of B lymphocytes separated in one layer (2X10'
It is preferable to use E
Mix with B virus solution (3 Go). Approximately 2 hours B phosphorus/Q
After absorbing the EB virus into the spherical cells, the lymphocytes were washed away aseptically and treated with RPMI containing 30% tallow serum.
Mix with 1640 medium (adjust the total volume to 10 mL) and add 37
B lymphoid cell lines (BS-8Y1, BS5-8Y, B
5-8Y3. B5-8Y4) was established.

The OB Lin IQ cell line of the present invention obtained as described above has the following characteristics.

(2) Cell morphology and characteristics: ① B phosphorus/resistant cells with immunoglobulin production observed in the cytoplasm and cell membrane surface.

■ Have a normal nuclear shop. However, some abnormalities are included in the present invention.

(2) By injecting bromodeoxyuridine (13rd U) into two cell generations, 100-cells exhibit a high frequency of SCE.

Illustratively, the obtained cell line of the present invention B5-5y,
, B5-8Y*, B5-8Ys, and B5-8Y4 are shown in Table 1. Among these, B5-8Ya has a slight abnormality in chromosome structure, and the other three have normal nuclei, so B5-8Yz, B5-8Ys, and B5-8Yj are more preferable cells of the present invention. It is a stock.

Table 1 B5-8YI IgM 72.3±2.42 46. X
XB5-8Ys IgM 74.9±2.30 46.
XYBS-8Ys IgG 69.4±2.91 46
．． XY (Publisher) Cultured cell density: 2 x 10' cells/10m medium (medium consists of RPMI 1640 culture solution 8.51E/ and fetal bovine serum 1.5-) (O subculture: Subculture is possible without limit) .

Usually, when 2×10” cells are cultured in 710 mj medium at 37° C. in a carbon dioxide gas incubator, subculturing is performed every 2 to 3 days.

(To) Storage: Sufficiently suspend 5 x 10' cells in the culture medium prepared above ((2) plus 10% dimethyl sulfoxide, then -
After freezing for one day in an 80°C freezer, it can be permanently stored in -190°C liquid nitrogen.

Among the cell lines obtained in the present invention, B5-8Yt, BS-S
Yz, B S -S Y4 is completely 1gM positive and has the potential to differentiate into IgD, IgA, and IgG, and is significant for the production of a cyimunoguyi purine preparation.

Since the cell line of the present invention is highly sensitive to carcinogens, it can be used for assaying carcinogens.

Agent 11 Moto Imamura

Claims (1)

[Claims] (B Lin/q cell line derived from a plume syndrome patient with normal plasticity and high frequency sister chromatid exchange ability, established by sensitizing Lin/9 cells isolated from a plume syndrome patient with EB virus.