CN108315302A - A kind of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain and its construction method and application - Google Patents
A kind of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain and its construction method and application Download PDFInfo
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Abstract
A kind of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain of present invention offer and its construction method and application, Cell Name are people's B-lineage Acute Lymphocyte Leukemia cell strain NALM 6/HDR, deposit number CCTCCNO：C2017234；First people's B-lineage Acute Lymphocyte Leukemia cell strain NALM 6 is placed under hypoxia condition after cultivating, it is added at one time 0.25 μM of dexamethasone, then proceed to hypoxemia culture, it is finally placed under normoxic condition and expands culture, to obtain people B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain NALM 6/HDR.The present invention provides new cell model for the research and biological medicine research and development for recurring refractory people's acute lymphoblastic leukemia, has larger practical value in the application for exploring the drug resistant pathogenesis of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid, the refractory mechanism of recurrence and reversing drug resistance medicament research and development.
Description
Technical field
The invention belongs to field of biomedicine technology, more particularly to a kind of people's B-lineage Acute Lymphocyte Leukemia sugar cortical hormone
Plain drug-resistant cell strain and its construction method and application.
Background technology
Acute lymphoblastic leukemia（acute lymphoblastic leukemia, ALL）The childhood of being, is most common
Malignant tumour, children ALL cases 85% are B-lineage Acute Lymphocyte Leukemia（B-ALL）.Although nearly 50 years, the prognosis of children ALL
Rapid progress is achieved, 5 years survival rates are increased to 90% from the 10% of 1960s, and 5 years Event-free survival rates are more than 80%, but still
Have that 10 ~ 20% patient is refractory, recurrence, poor prognosis, and still have after 5 years patient because ALL recurrences or due to the second tumour it is dead.Due to
Its high incidence, in even developed country of developing China, refractory, recurrence ALL is still to cause children because of tumor mortality
Primary factor.The B-ALL incidence of teenager and adult are far below children, but its 5 years survival rates only 30 ~ 40%.Therefore, difficult
It controls, recur the difficult point that ALL is current clinical treatment, it would be highly desirable to solve.
Refractory, recurrence ALL common feature is to chemotherapeutics drug resistance, especially to glucocorticoid, glucocorticoid
（Glucocorticoid, GC）Drug resistance is ALL recurrences, refractory one of the major reasons.The specific inducing malignant lymph of GC energy is thin
Born of the same parents' arrest proliferation simultaneously inspires apoptosis, is always one of the choice drug for treating ALL and other lymph source property malignant tumours, Ji Husuo
It includes GC to have ALL Combination chemotherapies.Common GC is prednisone and dexamethasone in chemotherapy regimen at present.GC sensibility is
Children's ALL clinical risk degree is grouped, one of the important indicator of guiding treatment and judging prognosis, and GC drug resistances are clinical urgently to be resolved hurrily
Treat problem.Therefore, T-ALL GC mdr cell models are established, it will help illustrate the drug resistant molecule mechanisms of GC, find early stage
Accurate early warning GC resistant organism markers, improve leukemia risk degree parting, specify the therapy target of reversing drug resistance, establish individual character
Change the chemotherapy regimen of high-efficiency low-toxicity；Advantageously account at present it is refractory, recurrence ALL treatment difficult point, further increase children and at
People ALL or even other lymph source property tumor cure rates improve patient's long term survival quality, great clinical value.
Drug screening method is the common method for establishing tumor drug resistance cell strain, and particular technique is divided into two kinds, first, gradually increasing
Add drug concentration, successive induction method, second is that large dosage impact intermittent administration method or two methods combine practicality.By above-mentioned
Method has been set up the drug-resistant cell strain of kinds of tumors both at home and abroad at present, but longer the time required to obtaining drug-resistant cell strain,
Cumbersome, complicated more than June, toxigenic capacity is high, and the loss of resistant characterization can be caused in succeeding generations, therefore, experiment
It needs to carry out screening pressurization to cell with drug in the process, to maintain resistant characterization.NALM-6 is B-ALL cell strains, 1976
It is established from the non-B Patients With Acute Lymphoblastic Leukemias peripheral blood separation of a non-T of 19 years old male, is research B-ALL common thin
Born of the same parents' strain, and the research common sensitive control strain of GC drug resistances, but NALM-6 cells are highly sensitive to GC, fill in over the ground within 48 hours
At 0.0024 ± 0.0002 μM, conventional medicament screening method needs induce the half-inhibition concentration of meter Song since very low concentration,
The drug domestication time is long, it is difficult to successfully build GC drug-resistant cell strains on the basis of NALM-6 cell strains.
Invention content
The present invention provides a kind of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain, Cell Names
It is people B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR, deposit number is：CCTCC NO：C2017234, preservation day
Phase：On October 25th, 2017, depositary institution：China typical culture collection center.
The present invention also provides people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain NALM-6/HDR
Construction method：Its process is, first by people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6 of exponential phase by 1~3 ×
104A/ml inoculations, it is 1~3 × 10 to be placed in culture to cell density under low-oxygen environment5A/ml（About 4 days time）It is disposable afterwards
0.25 μM of dexamethasone is added, it is 2~10 × 10 to then proceed to culture to cell density5A/ml（About 4 weeks time）, then set
Expand culture under normoxic condition, to obtain people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain NALM-6/
HDR。
In the above method, the hypoxia condition is that simulation B-lineage Acute Lymphocyte Leukemia cell is survived micro-loop in marrow
Border, the normoxic condition are the growing environments simulated B-lineage Acute Lymphocyte Leukemia cell and be released into peripheral blood from marrow.
In the above method, only pass through 0.25 μM of induced by dexamethasone.
Specific steps are as follows：
（1）By people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6 routine cultures in the RPMI 1640 containing 10% fetal calf serum
In complete medium, it is placed in 37 DEG C, 5%CO2, saturated humidity, 21%O2Normal oxygen incubator culture；
（2）Take step（1）It is middle to cultivate to the NALM-6 cells of exponential phase, by 1~3 × 104A/ml is inoculated with into 6 orifice plates,
Simulation B-lineage Acute Lymphocyte Leukemia cell is placed in survive in marrow microenvironment（37℃、5%CO2, saturated humidity, 1%O2）'s
After hypoxemia incubator culture 4 days, 0.25 μM of dexamethasone is added, continues culture 4 weeks, it is spare；
（3）By step（2）The cell that culture obtains is placed in simulation B-lineage Acute Lymphocyte Leukemia cell and is released into periphery from marrow
The growing environment of blood（37℃、5% CO2, saturated humidity, 21%O2）Lower culture, when cell Proliferation to 1 × 106A/ml, it is conventional to pass
In generation, changes liquid, obtains people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain NALM-6/HDR.
People's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain NALM-6/HDR that the present invention is obtained has
Following biological characteristics：
NALM-6/HDR cellular morphologies are complete, and round, size is almost the same, suspension growth, it is seen that packed cell, featheriness dissipate；
In vitro culture well-grown, by 2 × 105/ ml density is inoculated with, and passage in 2 ~ 3 days is primary, continuous in vitro to cultivate more than 8 months,
More than 90 generations, population doublings were more than 280 generations for passage, stablized proliferation, and in good condition, Resistance index is more than 25000.Through liquid nitrogen or surpass
Character is constant after cryopreservation, recovery, keeps good resistant characterization.
The totally 5 weeks time of cell drug-resistant cell strain from handling to obtaining, cell height drug resistance, drug sensitivity test confirm
NALM-6/HDR cells are more than 25000 to the Resistance index of dexamethasone.For population doublings at 10 generation, cell fills in rice over the ground
The Resistance index of pine reaches 40000 or more, slightly falls after rise later, and to after 100 generations, Resistance index is stablized 25000 ~ 30000,
It is not required to use drug screening again, resistant characterization is stablized.
STR is detected and Immunophenotyping analysis confirms that NALM-6/HDR cells and parental cell NALM-6 are same next
Source, immunophenotyping Pre-B；STR results and DSMZ cell bank Comparative results, as a result as shown in table 1 below；
Table 1：NALM-6/HDR and NALM-6 cell STR testing results and with DSMZ cell bank Comparative result results
Chromosomal G-banding karyotyping confirms NALM-6/HDR cells and parental cell NALM-6 is same Clone Origin,
Caryogram is 46, XY, t (5;12)(q33;p13), t(7;19)(q11.1;p13.3).
The growth curve that NALM-6/HDR cells are drawn using Trypan Blue cell count and mtt assay, calculates cell times
It is 18~20 hours to increase the time, and multiplication rate is stablized, it is seen that split coil method.
The NALM-6/HDR cell cycles are detected using PI decoration methods, it is seen that NALM-6/HDR cell S phase ratios are apparently higher than
NALM-6 prompts NALM-6/HDR proliferation more active compared with NALM-6.
Being tested using methylcellulose Semi-solid cell culture confirms that NALM-6/HDR cell strains have high semisolid Clone formation
Ability.
NALM-6/HDR cell inoculation NOD/SCID mouse have high oncogenicity, and tumor cells and in vitro culture
NALM-6/HDR cell origins are consistent.
Transcript profile sequencing finds that NALM-6/HDR cells have 140 gene expression up-regulations, 201 bases compared to parental cell
Because expression is lowered.
People's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR that the present invention is built has to be applied below：
（1）People B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR is establishing the leaching of humanization glucocorticoid drug resistance acute B
Application in bar chronic myeloid leukemia animal model, cell strain NALM-6/HDR have high oncogenicity, NALM-6/HDR cells are connect
Kind NOD/SCID mouse, subcutaneously can touch lump in 10 days, lump mushrooms out after 15 days, tumorigenesis rate 100%, and tumor formation is thin
Born of the same parents are consistent with the NALM-6/HDR cell origins of in vitro culture.
（2）People B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR is resistance in screening or assessment treatment glucocorticoid
Medicine B-lineage Acute Lymphocyte Leukemia drug and application in glucocorticoid medicine is reversed, by NALM-6/HDR cells
Different chemotherapeutics, the changes such as observation growth and proliferation of cell, death, period are added in culture medium, and can clear drug reverse
Glucocorticoid drug resistance obtains preliminary effective drug candidate, then drug candidate is used for above-mentioned B-lineage Acute Lymphocyte Leukemia
Animal model detects the internal effect of drug, observes ordinary circumstance, time-to-live, tumor size change and the drug of animal
After effect in body situations such as apoptosis, necrosis and the change of other associated signal paths, commented to carry out curative effect to drug candidate
Estimate and mechanism analysis.
（3）People B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR is in research glucocorticoid drug resistance acute B lymph
Application in chronic myeloid leukemia mechanism, cytomorphology, Biological characteristics observes cellular morphology knot using light microscopic, Electronic Speculum
Structure and ultra microstructure compare the difference of NALM-6/HDR and its parental cell NALM-6 forms and Biological characteristics, using albumen
The method of group, full-length genome and metabolism group analyzes NALM-6/HDR and NALM-6 cells, it is intended to find tool
Discrepant Disease-causing gene, albumen or metabolite, to the drug resistant pathogenesis of further investigated glucocorticoid.
（4）People B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR is to establish acute lymphoblastic leukemia immune
Learn and hematopoieticmicroenviron-ment research platform in application, NK cells, CAR-T or other immune antiboidies, ligand etc. are applied to
NALM-6/HDR and NALM-6 cells and its humanized animal's model are inquired into immunization therapy and are given birth to sugared cortex drug-resistant leukemia cell
The influence that long, immunity of organism and leukaemia develop and lapse to；Establish simulation marrow hemopoiesis tabernacle dimensional culture environment, compare including
The influence that different pharmaceutical including immunotherapy is treated and lapsed to glucocorticoid drug resistance acute lymphoblastic leukemia.
The invention has the advantages that：
1, people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR of the invention is that the refractory people's acute lymphoblastic of recurrence is white
The research of blood disease and biological medicine research and development provide new cell model, are exploring people's B-lineage Acute Lymphocyte Leukemia sugar cortical hormone
To hold out broad prospects in the application of the drug resistant pathogenesis of element, the refractory mechanism of recurrence and reversing drug resistance medicament research and development with it is larger
Practical value.
2, existing drug screening method builds drug-resistant cell strain, needs to be stepped up drug concentration, carries out successive induction, or
Large dosage impact intermittent administration or two methods combine, the above method is more difficult to cell that drug is highly sensitive filter out it is resistance to
Medicine strain, and the more time is needed to carry out drug domestication to cell.People's B-lineage Acute Lymphocyte Leukemia cell strain of the present invention
The construction method of NALM-6/HDR is impacted after using hypoxia condition culture with dexamethasone large dosage, simulates clinical leukaemia cell
Drug resistance forming process, experimentation is simple, and modeling conditional stability can obtain drug-resistant cell strain in a short time, without using again
Drug carries out screening pressurization to cell, can significantly reduce modeling cost；Meanwhile this method can the cell highly sensitive to drug make
Mould because under hypoxia condition, malignant cell to Treated with Chemotherapeutic Drugs object compared with normoxic condition when be more resistant to, the method can be extensive
For various malignant cells, the drug resistance to different pharmaceutical is obtained, or screened simultaneously to a variety of drugs, it is resistance to obtain multiple medicine
The cell strain of medicine.
3, Resistance index is commonly used in the judgement of drug resistance of tumor cell degree（resistant index, RI）It indicates, RI is
Mdr cell half-inhibition concentration（50% inhibitory concentration, IC50) with the ratio of parental cell IC50.
Drug resistance is divided into minuent by Snow etc. by the height of Resistance index（<5), moderate（5-15), height（> 15).The present invention is established
Drug-resistant cell strain NALM-6/HDR its RI be 25000~30000, i.e., be the 25000 of parental cell to the IC50 of dexamethasone
~30000 times.
Description of the drawings
Fig. 1 is NALM-6, NALM-6/HDR and its monoclonal NALM-6/HDR-C5 cell observations under inverted phase contrast microscope
Figure（×400）；
Fig. 2 is NALM-6, NALM-6/HDR and its monoclonal NALM-6/HDR-C5 cells Switzerland-Ji under ordinary optical microscope
The wooden Sa colored graph（×1000）；
Fig. 3 is the half-inhibition concentration that dexamethasone acts on NALM-6/HDR and its monoclonal cell 48 hours（IC50）And its
Resistance index（RI）, wherein A：Dexamethasone is to 48 hours IC50 of NALM-6/HDR cytosiies；B：NALM-6/HDR is over the ground
The RI of Sai meter Song；C：Dexamethasone acts on NALM-6/HDR monoclonal cells C1, C3, C4, C5, C6 and C9 48 hours
IC50；D：RIs of NALM-6/HDR monoclonal cells C1, C3, C4, C5, C6 and the C9 to dexamethasone；PDL：Cell population doublings
Number（population doubling level）；
Fig. 4 is NALM-6, NALM-6/HDR and its monoclonal NALM-6/HDR-C5 cell growth curves；
Fig. 5 is NALM-6/HDR cell clonal formation observation charts under inverted phase contrast microscope, A：×100；B：×200；
Fig. 6 is NALM-6/HDR and parent's NALM-6 cell chromosome G Banded karyotype analysis result figures（×1000）；
Fig. 7 is to scheme flow cytometer detection NALM-6, NALM-6/HDR and its monoclonal NALM-6/HDR-C5 cell cycles, wherein A：
NALM-6；B：NALM-6/HDR；C：NALM-6/HDR-C5；
Fig. 8 is transcription group sequencing detection NALM-6/HDR and NALM-6 cellular gene expression differences, and wherein X-axis represents A values
(the transformed Average expression levels of log2), Y-axis represent M values (the transformed fold differences of log2)；
Fig. 9 is that NALM-6/HDR cell NOD/SCID mouse subcutaneous tumors form lab diagram.
People B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR, deposit number are：CCTCC NO：C2017234 is protected
Hide the date：On October 25th, 2017, depositary institution：China typical culture collection center preserves unit address：Hubei Province is military
No. 299 Wuhan Universitys of the Wuchang Districts Han Shi Bayi Road are in the school（The first attached primary school of Wuhan University opposite）, Wuhan University's collection.
Specific implementation mode
The present invention is in conjunction with specific embodiments described further invention, but the implementation of the present invention is not limited to that.
Method therefor is conventional method to following embodiments unless otherwise specified
Embodiment 1
The structure of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain NALM-6/HDR of dexamethasone rapid induction
Construction method：
1. cell culture：Recovery people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6 cells, are suspended in containing 10% fetal calf serum
（Thermo companies）1640 cell culture mediums of RPMI（Gibco companies）In, 37 DEG C, 5%CO2, saturated humidity, 21%O2Normal oxygen training
Support case culture.
2. screening and culturing condition：The NALM-6 cells for taking exponential phase in step 1, by 2 × 104A/ml is inoculated with into 6
Orifice plate, the holes 2ml/ are respectively placed in 37 DEG C, 5%CO2, saturated humidity, 21%O2Normal oxygen incubator（Normal oxygen group）And 37 DEG C, 5%CO2, it is full
With humidity, 1%O2Hypoxemia oxygen incubator（Hypoxia group）After 4 days, 0.25 μM of ground plug rice is added in normal oxygen and hypoxia group in culture
Pine, each 3 hole are control with 3 holes of not dosing, continue grouping culture.
About the addition of dexamethasone, the present embodiment be separately added into preliminary experiment 0.01 μM, 0.05 μM, 0.1 μM,
0.25 μM, 0.5 μM and 1 μM dexamethasone, as a result, it has been found that when less than 0.1 μM of concentration, cell can be effective in low-oxygen environment
It is resistant to dexamethasone, cell more difficult recovery proliferative capacity after large dosage is impacted when being more than 0.25 μM, therefore, the present invention uses
The dexamethasone of 0.25 μM of dosage.
3. building resistant models：After dosing 4 days, normal oxygen group complete cell death, hypoxia group after dexamethasone processing exist
Still there is survivaling cell after dexamethasone processing, continues hypoxemia culture, the normal oxygen culture of 4 weeks postpositions, when cell Proliferation to 1 × 106A/
Ml, routine passage change liquid, half-inhibition concentration of the cell to dexamethasone are detected after 1 week, and calculate its Resistance index and be
47750 ± 166, this cell is named as NALM-6/HDR.Methylcellulose culture, pick out monoclonal cell C1, C3, C4,
C5, C6 and C9.Cell doubling time is calculated, and detects the cell cycle.
4. drug resistance tracks：In normal oxygen often by NALM-6/HDR and its monoclonal cell C1, C3, C4, C5, C6 and C9
Rule culture, passage monthly repeat detection cell to 48 hours half-inhibition concentrations of effects of dexamethasone, and calculate Resistance index,
, the results show that after 100 generation of population doublings, NALM-6/HDR and its monoclonal cell press down the half of dexamethasone for it
Concentration processed is stablized at 50~120 μM, and Resistance index is stablized 20000~50000.
5. cell stablizes growth, does not influence its resistant characterization and biological characteristics through cryopreservation resuscitation repeatedly.
Embodiment 2
The growth of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain NALM-6/HDR, biological characteristics and resistance to
Medicine CHARACTERISTICS IDENTIFICATION
（1）Morphological observation：The NALM-6/HDR cells of logarithmic growth phase, monoclonal cell NALM-6/HDR-C5 and
Its parental cell NALM-6 observes living cells form under inverted microscope, and the results are shown in Figure 1.NALM-6/HDR cells and parent
This cell is the same, the suspension growth in 1640 complete mediums of RPMI, round, than more uniform, and agglomerating life is more easy to compared with parental cell
It is long, in vitro culture well-grown.
NALM-6/HDR cells, monoclonal cell NALM-6/HDR-C5 and its parental cell NALM- of logarithmic growth phase
6, routine smear is fixed, and the wooden Sa dyeing of Switzerland-Ji, in just setting microscopically observation cellular morphology, the results are shown in Figure 2：NALM-
6/HDR, NALM-6/HDR-C5 are similar with its parent NALM-6 cell size, morphosis.
（2）Mtt assay detects drug resistance of the cell to glucocorticoid：The NALM-6/HDR that exponential phase is collected by centrifugation is thin
Born of the same parents, monoclonal cell NALM-6/HDR（C1, C3, C4, C5, C6, C9）With its parental cell NALM-6, cell concentration is adjusted separately
It is 5 × 105A cell/ml, is inoculated in 96 orifice plates, per 100 μ l of hole, every group of 3 multiple holes；In NALM-6/HDR, monoclonal cell
NALM-6/HDR（C1, C3, C4, C5, C6, C9）With the dexamethasone that increasing concen-trations are separately added into NALM-6 cells, wherein
0.00001~0.1 μM of dexamethasone is added in NALM-6 cells, is incremented by by 10 times, and NALM-6/HDR and its monoclonal cell are added
0.1~1000 μM of dexamethasone is incremented by by 10 times.Blank control and not dosing control are set, handled 48 hours, 5mg/ml is added
10 holes μ l/ MTT, mix well, 37 DEG C be incubated 4 hours after the SDS of acidification is added（10%SDS contains 0.01MHCl）, 100 μ l
/ hole, mixing.37 DEG C of overnight incubations, the absorbance value of secondary daily multi-function microplate reader detection 570nm wavelength（OD）.According to blank
The OD mean values of group, control group plus Dexamethasone group, calculate the survival rate of cell.Cell survival rate=（OD dexamethasone mean values-OD
Blank mean value）/（OD compares mean value-OD blank mean values）×100%.Curve is drawn according to time, drug concentration, cell survival rate
Calculation of half inhibitory concentration.NALM-6/HDR cells are that NALM-6/HDR cell halves inhibit to the Resistance index of glucocorticoid
The ratio of concentration and parental cell NALM-6 half-inhibition concentrations.The results are shown in Figure 3：NALM-6/HDR cells are in population doublings
When 10 generation of number, cell is up to 138 μM to the half-inhibition concentration of dexamethasone, and Resistance index is up to 47750, to 100 generations with
Afterwards, cell stablizes at 55~70 μM the half-inhibition concentration of dexamethasone, and Resistance index is stablized 25000~30000；
NALM-6/HDR（C1, C3, C4, C5, C6, C9）Monoclonal cell stablizes in 50~120 μ the half-inhibition concentration of dexamethasone
M, Resistance index are stablized 20000~50000.
（3）Growth curve and doubling time measure：NALM-6/HDR, the monoclonal cell of exponential phase is collected by centrifugation
NALM-6/HDR-C5 and NALM-6 cells, adjustment cell concentration are 1 × 105A cell/ml, is inoculated in 6 orifice plates, set 37 DEG C,
5%CO2、21% O2It is cultivated in incubator 7 days, counts cell with Trypan Blue daily, mtt assay is used in combination to detect cell Proliferation feelings
Cell suspension is added in 96 orifice plates in condition, and per 100 μ l of hole, blank control is arranged in every group of 3 multiple holes, and 5mg/mlMTT is added（Sigma
Company）10 holes μ l/, mix well, 37 DEG C be incubated 4 hours after the SDS of acidification is added（10%SDS contains 0.01MHCl）, 100 μ l/
Hole, mixing.37 DEG C of overnight incubations, secondary daily multi-function microplate reader survey the absorbance value of 570nm wavelength（OD）.With the time for horizontal seat
Mark, cell number is ordinate, draws cell growth curve, as shown in Figure 4：NALM-6/HDR, NALM-6/HDR-C5 and NALM-6
Cell growth is almost the same, and NALM-6 cell plateaus occur a little later, and cell doubling time is 18~20 hours.
（4）Methylcellulose Semi-solid cell culture, as shown in Figure 5：NALM-6/HDR cells are trained in methylcellulose semisolid
It supports and cultivates 7 days i.e. visible apparent clone in base, it was demonstrated that NALM-6/HDR cell strains have high semisolid clonality.
（5）STR is detected, and the results are shown in Table 1, the STR results and DSMZ of NALM-6/HDR and its parental cell NALM-6
The result of cell bank compares, it was demonstrated that NALM-6/HDR cells and its parental cell NALM-6 are same source.
（6）Chromosomal G-banding karyotyping, the results are shown in Figure 6：The core of NALM-6/HDR and its parental cell NALM-6
Type is 46, XY, t (5;12)(q33;p13), t(7;19)(q11.1;P13.3), it was demonstrated that NALM-6/HDR and parental cell
NALM-6 is same Clone Origin.
（7）PI decoration methods compare NALM-6/HDR and its monoclonal cell NALM-6/HDR-C5 and parental cell NALM-6
Cell cycle status, the results are shown in Figure 7：NALM-6/HDR and its monoclonal NALM-6/HDR-C5 cell cycle distribution phases
Seemingly, G0/G1Phase ratio is significantly lower than NALM-6, and S phase ratios are apparently higher than NALM-6, prompts NALM-6/HDR and its Dan Ke
The proliferation of grand cell is more active compared with NALM-6.
（8）Flow cytometer detection cellular immunity parting, it was demonstrated that NALM-6/HDR cells and its parental cell NALM-6 are same next
Source expresses CD10, CD19, CD22, cCD79a and HLA-DR, does not express CD20 and sIgM, immunophenotyping Pre-B, NALM-6
Immunophenotyping result is consistent with the result of DSMZ cell banks.
（9）Transcript profile is sequenced, as shown in figure 8, NALM-6/HDR cells have 140 genes compared to parent's NALM-6 cells
Up-regulated expression, 201 down regulation of gene expression.
Embodiment 3
Establish humanization glucocorticoid drug resistance B-lineage Acute Lymphocyte Leukemia animal model
NOD/SCID mouse tumor formations are tested：NALM-6/HDR cells in exponential phase are pressed 6 × 106/ 100 μ l/ only, connect
Kind is in Balb/c (nu/nu) mouse（6 week old, 19 ~ 21g of weight）Apterium is subcutaneous；The next day observation inoculation position tumour generate feelings
Condition.The results are shown in Figure 9, and inoculation can touch grain of rice size lump after 10 days in nude mice by subcutaneous, and tumour is given birth to rapidly after inoculation 15 days
It is long, take tumor mass that cell suspension is made after 15 days, immunophenotyping and STR detections confirm tumor mass separation cell and NALM-6/HDR cells
For same source.
Claims (10)
1. a kind of people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug-resistant cell strain, Cell Name is that people's acute B lymph is thin
Born of the same parents leukemia cell line NALM-6/HDR, deposit number CCTCCNO：C2017234.
2. the construction method of people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR, feature described in a kind of claim 1
It is：It is 1~3 that people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6, which is first placed in culture to cell density under hypoxia condition,
×105After a/ml, it is added at one time 0.25 μM of dexamethasone, it is 2~10 × 10 to then proceed to hypoxemia culture to cell density5
A/ml is finally placed under normoxic condition and expands culture, to obtain people's B-lineage Acute Lymphocyte Leukemia glucocorticoid drug resistance
Cell strain NALM-6/HDR.
3. the construction method of people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR according to claim 2, special
Sign is：The hypoxia condition refers to that simulation B-lineage Acute Lymphocyte Leukemia cell is survived microenvironment in marrow, the normal oxygen
Condition refers to the growing environment simulated B-lineage Acute Lymphocyte Leukemia cell and be released into peripheral blood from marrow.
4. the construction method of people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR according to claim 2, special
Sign is：People's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6 is the NALM-6 cells of exponential phase, by 1~3 ×
104A/ml inoculations, are placed in hypoxia condition culture.
5. the construction method of people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR according to claim 3, special
Sign is：Existence microenvironment of the simulation B-lineage Acute Lymphocyte Leukemia cell in marrow refers to 37 DEG C, 5%CO2, saturation
Humidity, 1%O2Environment.
6. the construction method of people's B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR according to claim 3, special
Sign is：The simulation B-lineage Acute Lymphocyte Leukemia cell be released into from marrow the growing environment of peripheral blood refer to 37 DEG C, 5%
CO2, saturated humidity, 21%O2Environment.
7. people B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR is establishing the leaching of humanization glucocorticoid drug resistance acute B
Application in bar chronic myeloid leukemia animal model.
8. people B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR is acute in screening or assessment treatment glucocorticoid drug resistance
Application in bone-marrow-derived lymphocyte leukemia medicament.
9. people B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR is in research glucocorticoid drug resistance acute B lymphocyte
Application in leukaemia mechanism, cytomorphology, Biological characteristics.
10. people B-lineage Acute Lymphocyte Leukemia cell strain NALM-6/HDR establish acute lymphoblastic leukemia immunology and
Application in hematopoieticmicroenviron-ment research platform.
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