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A kind of human acute lymphoblastic leukemia glucocorticoid-resistant cell line and its construction method and application

A technology of B lymphocytes and glucocorticoids, applied in the field of biomedicine, can solve problems such as difficulty, long time required, cumbersome operation, etc., and achieve the effects of reducing modeling costs, stable modeling conditions, and simple experimental procedures

Active Publication Date: 2019-01-29
THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Through the above method, drug-resistant cell lines of various tumors have been established at home and abroad, but it takes a long time to obtain drug-resistant cell lines, more than 6 months, the operation is cumbersome and complicated, and the cost of culture is high, and it will cause damage during the passage process. Loss of drug-resistant properties, therefore, cells need to be screened and stressed with drugs during the experiment to maintain drug-resistant properties
NALM-6 is a B-ALL cell line, which was isolated from the peripheral blood of a 19-year-old male patient with non-T non-B acute lymphoblastic leukemia in 1976. It is a commonly used cell line for the study of B-ALL and a sensitive cell line for the study of GC drug resistance. The control strain, but NALM-6 cells are highly sensitive to GC, and its 48-hour half-inhibitory concentration of dexamethasone is 0.0024±0.0002μM. Traditional drug screening methods need to be induced from a very low concentration, and the drug acclimation time is long, which is difficult Successful construction of GC drug-resistant cell line based on NALM-6 cell line

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  • A kind of human acute lymphoblastic leukemia glucocorticoid-resistant cell line and its construction method and application
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  • A kind of human acute lymphoblastic leukemia glucocorticoid-resistant cell line and its construction method and application

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Embodiment 1

[0047] Construction method of dexamethasone rapidly induced human acute lymphoblastic leukemia glucocorticoid-resistant cell line NALM-6 / HDR:

[0048] 1. Cell culture: resuscitate human acute lymphoblastic leukemia cell line NALM-6 cells, suspend in RPMI 1640 cell culture medium (Gibco Company) containing 10% fetal bovine serum (Thermo Company), 37°C, 5% CO 2 , saturated humidity, 21%O 2 Normoxic incubator culture.

[0049] 2. Screening culture conditions: Take the NALM-6 cells in the logarithmic growth phase in step 1, and press 2×10 4 Inoculated into 6-well plate, 2ml / well, placed at 37℃, 5%CO 2 , saturated humidity, 21%O 2 Normoxic incubator (normal oxygen group) and 37°C, 5% CO 2 , saturated humidity, 1%O 2 Hypoxic incubator (hypoxic group) culture, after 4 days, add 0.25μM dexamethasone to both normoxia and hypoxia group, each 3 wells, with no drug added as control, continue group culture.

[0050] Regarding the amount of dexamethasone added, in this example, 0.01 μ...

Embodiment 2

[0055] Identification of growth, biological characteristics and drug resistance of human acute lymphoblastic leukemia glucocorticoid-resistant cell line NALM-6 / HDR

[0056] (1) Observation of cell morphology: NALM-6 / HDR cells in the logarithmic growth phase, monoclonal cells NALM-6 / HDR-C5 and its parent cell NALM-6 were taken to observe the morphology of live cells under an inverted microscope. The results are as follows: figure 1 shown. NALM-6 / HDR cells, like the parental cells, grow in suspension in RPMI 1640 complete medium, are round and uniform, and are easier to grow in clusters than the parental cells, and grow well in vitro.

[0057] Take NALM-6 / HDR cells in the logarithmic growth phase, monoclonal cells NALM-6 / HDR-C5 and its parental cell NALM-6, fix them with routine smears, stain with Swiss-Gimsa, and observe the cell morphology under an upright microscope , the result is as figure 2 Shown: NALM-6 / HDR, NALM-6 / HDR-C5 and their parent NALM-6 cells are similar in si...

Embodiment 3

[0067] Establishment of an animal model of humanized glucocorticoid-resistant acute B-lymphoblastic leukemia

[0068] Tumor formation experiment in NOD / SCID mice: NALM-6 / HDR cells in the logarithmic growth phase were divided into 6×10 6 / 100µl / mouse, inoculated subcutaneously in the bare area of ​​Balb / c (nu / nu) mice (6 weeks old, body weight 19-21g); observe the tumor formation at the inoculation site every other day. The result is as Figure 9 As shown, 10 days after inoculation, the size of rice grains could be touched under the skin of nude mice. After 15 days of inoculation, the tumor grew rapidly. After 15 days, the tumor mass was taken to make cell suspension. HDR cells are from the same source.

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Abstract

The invention provides a human acute B lymphocytic leukemia glucocorticoid drug-resistant cell strain as well as an establishment method and application thereof. The strain has a cell name of human acute B lymphocytic leukemia cell strain NALM-6 / HDR, and the preservation number is CCTCCNO: C2017234. The establishment method comprises the following steps: firstly, culturing the human acute B lymphocytic leukemia cell strain NALM-6 / HDR under a hypoxia condition, adding 0.25mu M of dexamethasone at one time, performing hypoxia culture continuously, and performing amplified culture under a normoxia condition, thereby obtaining the human acute B lymphocytic leukemia glucocorticoid drug-resistant cell strain NALM-6 / HDR. By adopting the strain, a novel cell model for study and biological medicineresearch and development for refractory-relapsed human acute lymphocytic leukemia, and the strain has relatively great practical values in application to study on pathogenesis, refractory-relapsed mechanisms and drug resistance reversing medicine development for human acute B lymphocytic leukemia glucocorticoid drug resistance.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a human acute B lymphocytic leukemia glucocorticoid-resistant cell line and a construction method and application thereof. Background technique [0002] Acute lymphoblastic leukemia (ALL) is the most common malignant tumor in childhood, and 85% of ALL cases in children are acute lymphoblastic leukemia (B-ALL). Although, in the past 50 years, the prognosis of children with ALL has made great progress, the 5-year survival rate has increased from 10% in the 1960s to 90%, and the 5-year event-free survival rate exceeds 80%, but there are still 10-20% of patients Refractory, relapsed, poor prognosis, and 5 years later, patients still died of ALL recurrence or second tumor. Due to its high incidence, refractory and relapsed ALL is still the leading cause of childhood cancer deaths in developing China and even in developed countries. The incidence of B-ALL in adolescen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09C12Q1/02A01K67/027C12R1/91
CPCA01K67/027A01K2207/12A01K2227/105A01K2267/0331C12N5/0694G01N33/5011G01N33/5044
Inventor 顾玲张妍乐左川黄凌依
Owner THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN