CN108685936A

CN108685936A - Application of a kind of adenosine derivative in human muscle creatine kinase cell

Info

Publication number: CN108685936A
Application number: CN201810769802.7A
Authority: CN
Inventors: 程娇; 王黎; 崔昌浩; 刘胜先; 潘慧; 林凡琳
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2018-07-13
Filing date: 2018-07-13
Publication date: 2018-10-23

Abstract

The present invention provides application of a kind of adenosine derivative in human muscle creatine kinase cell, belong to Biochemistry and Molecular Biology technical field.The data of the present invention are established in adenosine derivative oTR, KR, BAR, iPR to the in-vitro multiplication inhibitory activity that the 9 big tumors of people are that 60 kinds of tumor cell lines have highly significant, wherein on basis strongest to the drug susceptibility of leukemia cell line.

Description

Application of a kind of adenosine derivative in human muscle creatine kinase cell

Technical field

The present invention relates to adenosine derivative ortho-Topolin Riboside (oTR), Kinetin riboside (KR), N⁶- Benzyladenosine (BAR), N6-Isopentenyladenosine (iPR) are to human acute myeloid leukemia cell strain Rush break up antitumor action, belong to Biochemistry and Molecular Biology technical field.

Background technology

At present more and more the study found that the basic element of cell division not only adjusts the growth and development of plant simultaneously to zooblast Proliferation and growth also play an important roll.Research find basic element of cell division ortho-Topolin Riboside (oTR), Kinetin riboside(KR),N⁶- Benzyladenosine (BAR), N6-Isopentenyladenosine (iPR) are in body There is very strong toxicity and apoptosis-promoting effect to cancer cell outside.Structurally, understanding that these basic elements of cell division belong to adenosine Derivative.Cause after handling cancer cell with these basic elements of cell division the cell cycle it is different degrees of be obstructed and (or) apoptosis, This depends primarily on susceptibility of the different cell line to different adenosine derivatives.Although the analog of natural CK and they The effect of zooblast is repeatedly reported, but there is presently no the rush to these adenosine derivatives of a system point Change effect is studied.

Acute myeloid leukemia (AML) is a kind of Clonal hematologic malignancy developing into bone marrow cell, It is characterized in that uncontrolled proliferation and blocking in normal hematopoetic cells differentiation, all-trans retinoic acid (ATRA) can be by luring Differentiation is led to cure unique AML hypotypes --- acute promyelocytic leukemia (APL).However, the differentiation therapy that ATRA is mediated It is not suitable for other kinds of AML and ATRA antilepsises and still has the problems such as curative effect is unstable, some patientss easily recur.Cause This, the AML treatments lower therapeutic agent of novel toxicity that there is an urgent need to differentiation can be overcome to stagnate.

Invention content

For the above-mentioned prior art, the present invention provides a kind of adenosine derivative ortho-Topolin by the present invention Riboside(oTR),Kinetin riboside(KR),N⁶-Benzyladenosine(BAR),N6- Isopentenyladenosine (iPR) promotees the application of differentiation in human leukemia cell line U937.

Technical scheme of the present invention:

Application of a kind of adenosine derivative in human muscle creatine kinase cell, steps are as follows:

(1) cell culture:People's acute leukemia cells strain U937 is suspended in containing the sterile of inactivated fetal bovine serum In DMED culture solutions, it is placed in a concentration of 5%CO₂, relative humidity 90%, cultivate in the incubator that temperature is 37 DEG C, cell is long 70%~80% to culture bottle floor space passes on 1 time;

(2) drug is prepared:OTR, KR, BAR, iPR are dissolved with 0.1%DMSO, and continuing will be molten with sterile DMEM media OTR, KR, BAR, the iPR solved is diluted to a concentration of 0.1 μM -30 μM, filtration sterilization, room temperature preservation;

(3) cell is handled:Step (1) is in the 5 × 10 of exponential phase⁴~1 × 10⁵A cell inoculation contains in every hole Have in 24 orifice plates of 1mlDMEM culture solutions, 8 groups of experiments of setting, the 1st group:Blank control group;2nd group:10 μM of oTR groups;3rd group: 10μM KR;4th group:10μM BAR;5th group:10 μM of iPR groups;Corresponding drug is added into each group, is placed in a concentration of 5% CO₂, relative humidity 90%, be incubated 36 hours in the incubator that temperature is 37 DEG C, collect cell;

(4) division guideline is checked:The cell of collection step (3) processing is simultaneously washed three times with the PBS containing 0.2%FBS;It will be thin Born of the same parents and blocking antibody are incubated at room temperature 15 minutes, then 4 DEG C in the dark with anti-human CD11b-PE antibody incubations 30 minutes After wash cell, and pass through flow cytometry;

(5) detection cellular morphology variation:The cell of collection step (3) processing is simultaneously washed three times with the PBS containing 0.2%FBS; Glass slide is fixed with methanol, is used in combination Lai Te-Giemsa staining solution to dye 20 minutes, is rinsed with distilled water, air-dries and uses Micro- sem observation.

Beneficial effects of the present invention:The data of the present invention are established big to the 9 of people in adenosine derivative oTR, KR, BAR, iPR Tumor is the in-vitro multiplication inhibitory activity that 60 kinds of tumor cell lines have highly significant, wherein to the medicaments insensitive of leukemia cell line Property it is strongest basis on.

Description of the drawings

Fig. 1 is the influence that CD11b is expressed in normal U937 cells.

Fig. 2 is the influence that oTR expresses CD11b in U937 cells.

Fig. 3 is the influence that KR expresses CD11b in U937 cells.

Fig. 4 is the influence that BAR expresses CD11b in U937 cells.

Fig. 5 is the influence that iPR expresses CD11b in U937 cells.

Specific implementation mode

Below in conjunction with attached drawing and technical solution, the specific implementation mode that further illustrates the present invention.

Cell, kit and the reagent that the present invention uses:Human leukemia U937 cell strains, fetal calf serum, dual anti-(mould Element/streptomysin), DMEM culture mediums, CD11b antibody, Wright-Giemsa dye liquors, oTR, KR, BAR, iPR.

Embodiment 1

The influence that oTR, KR, BAR, iPR express CD11b in U937 cells:By human leukemia cell's U937 trainings of recovery It supports in the sterile DMEM culture solutions containing 10%FBS inactivated fetal bovine serums, the streptomysin (dual anti-) of 1% penicillin/1%, is placed in In suspension cell bottle, in 37 DEG C, 5%CO₂And cultivated in the incubator under saturated humidity, cell is grown to 70%-80% or so biography Generation 1 time.Continue culture and obtain the active human leukemia cell of growth conditions, collects cell.By 5 in exponential phase × 10⁴A cell inoculation contains in every hole in 24 orifice plates of 1mlDMEM culture solutions, according to experiment demand, is separately added into pair in each group OTR, KR, BAR, the iPR for answering concentration are put into and are collected after being incubated 36 hours in incubator and wash three with the PBS containing 0.2%FBS It is secondary.Cell and blocking antibody are incubated at room temperature 15 minutes, then 4 DEG C in the dark with anti-human CD11b-PE antibody incubations Cell is washed after 30 minutes, and passes through flow cytometry.The results are shown in Figure 1.Fig. 1 is as it can be seen that oTR, KR, BAR, iPR make The expression of CD11b significantly increases in U937 cells.This shows oTR, KR, BAR, iPR induction of U937 cells to ripe granulocyte Differentiation.

Embodiment 2

OTR, KR, BAR, iPR induce the variation of U937 cellular morphologies:The U937 cells that growth conditions are enlivened are with 10 μM After oTR, KR, BAR, iPR handle 36h, collects cell and washed three times with the PBS containing 0.2%FBS.Glass slide methanol is consolidated It is fixed, it is used in combination Lai Te-Giemsa staining solution to dye 20 minutes, is rinsed with distilled water, air-dry and use micro- sem observation.As a result such as Shown in Fig. 2, Fig. 2 is round as it can be seen that cell of the untreated U937 cells as high malignancy, the big and nucleus of circle and sparse Cytoplasm.The cytoplasm ratios of nucleus are reduced with oTR, KR, BAR, iPR processing and change the shape of a hoof of nucleus State.OTR, KR, BAR, iPR are further demonstrated by using the morphological analysis of Wright Giemsa dyeing to break up induction Effect.

Claims

1. application of a kind of adenosine derivative in human muscle creatine kinase cell, which is characterized in that steps are as follows:

(1) cell culture:People's acute leukemia cells strain U937 is suspended in the sterile DMED trainings containing inactivated fetal bovine serum In nutrient solution, it is placed in a concentration of 5%CO₂, relative humidity 90%, cultivate in the incubator that temperature is 37 DEG C, cell is grown to culture 70%~80% passage of bottom of bottle area 1 time;

(2) drug is prepared:OTR, KR, BAR, iPR are dissolved with 0.1%DMSO, continue to be dissolved with sterile DMEM media OTR, KR, BAR, iPR be diluted to a concentration of 0.1 μM -30 μM, filtration sterilization, room temperature preservation;

(3) cell is handled:Step (1) is in the 5 × 10 of exponential phase⁴~1 × 10⁵A cell inoculation contains in every hole In 24 orifice plates of 1mlDMEM culture solutions, 8 groups of experiments of setting, the 1st group:Blank control group;2nd group:10 μM of oTR groups;3rd group:10 μM KR;4th group:10μM BAR;5th group:10 μM of iPR groups;Corresponding drug is added into each group, is placed in a concentration of 5% CO₂, relative humidity 90%, be incubated 36 hours in the incubator that temperature is 37 DEG C, collect cell;

(4) division guideline is checked:The cell of collection step (3) processing is simultaneously washed three times with the PBS containing 0.2%FBS;By cell with Blocking antibody is incubated at room temperature 15 minutes, is then washed after 30 minutes with anti-human CD11b-PE antibody incubations in the dark at 4 DEG C Cell is washed, and passes through flow cytometry;

(5) detection cellular morphology variation:The cell of collection step (3) processing is simultaneously washed three times with the PBS containing 0.2%FBS;It will carry Slide is fixed with methanol, is used in combination Lai Te-Giemsa staining solution to dye 20 minutes, is rinsed with distilled water, is air-dried and using micro- Sem observation.