CN103834714A

CN103834714A - In vitro research on inhibition effects of hydrogen peroxide on surviving promotion performance of Insulin-like Growth Factor1 (IGF-1)

Info

Publication number: CN103834714A
Application number: CN201210474838.5A
Authority: CN
Inventors: 郝智慧; 王德军; 吕海鸾
Original assignee: QINGDAO CONLINENT ANIMAL PHARMACEUTICAL CO Ltd
Current assignee: QINGDAO CONLINENT ANIMAL PHARMACEUTICAL CO Ltd
Priority date: 2012-11-21
Filing date: 2012-11-21
Publication date: 2014-06-04

Abstract

The invention belongs to the technical field of medicine, and discloses inhibition effects of hydrogen peroxide on surviving promotion performance of IGF-1. According to an in vitro research on inhibition effects of hydrogen peroxide on surviving promotion performance of IGF-1, PC12 cell (rat adrenal pheochromocytoma) is taken as a research object, a serum deprived group is taken as a model, IGF-1 is used for protecting PC12 from damages caused by serum deprivation, and promoting surviving of PC12 cell; H2O2 (hydrogen peroxide) of different concentration is added before treatment using IGF-1, MTT (methyl thiazolyl tetrazolium method) is used for detecting cell viability, and it is shown by the results that serum deprivation is capable of reducing cell viability obviously, IGF-1 is capable of protecting PC12 cell from damages caused by serum deprivation in a dose-dependent manner, and H2O2 is capable of inhibiting the surviving promotion performance of IGF-1 in a dose-dependent manner.

Description

Hydrogen peroxide suppresses the in vitro study of the short survival of IGF-1 function

Technical field

The invention belongs to medical technical field, relate to the mechanism that a kind of hydrogen peroxide suppresses PC12 cell viability, suppressed the short survival function of IGF-1.

Background technology

Aerobic cell can produce a series of reactive oxygen specieses (reactive oxygen species, ROS) in metabolic process, comprises superoxide anion [O ₂ ^-], hydrogen peroxide [H ₂o ₂] and hydroxy radical qiao [OH] etc., their character is extremely active, can seriously damage the biomacromolecules such as microbial film, enzyme, protein and nucleic acid, existing research shows that the generations of numerous disease such as tumour, aging, cardiovascular and cerebrovascular and the existence of ROS have close ties.Along with the development of Free Radical Biology research, people recognize apoptosis and the propagation that ROS can some tumour cell of double regulation control gradually, and have found the internal association between active oxygen radical and cell signalling.Current research discovery, the active oxygen radical of lower concentration can affect a series of signal transduction pathway.Active oxygen radical in body the life and death balance of body cell by its concentration adjustment, except causing apoptosis, downright bad function, the ROS of lower concentration widely physiological significance is the promotion of its activation to transcription factor and on cell proliferation, differentiation.

Insulin-like growth factor-i (Insulin-like Growth Factor-1, IGF-1) be a kind of at the synthetic nutritional factor of liver, its biological function is extensive, there is the neurocyte proliferation of promotion, differentiation in central nervous system, promote the function of neuronal survival, and these biological functions all rely on its high-affinity receptor IGF-1R and bring into play.The transgenic mice compared with normal Mouse Weight that IGF-1R knocks out reduces approximately 45%, after birth, die from the heteroplasia of many organs, wherein nervous system development defect is the major reason that causes transgenic mice death, and this demonstrates the vital role of IGF-1 in central nervous system.Short neuronal survival is that IGF-1 is at one of most important biological function of central nervous system, external much research has shown that IGF-1 can resist the cell injury that many reasons causes, the Neuron Apoptosis causing as serum deprivation, the neurotoxicity that 6-hydroxydopamine causes, the apoptosis of low potassium induction, the nerve excitability toxicity of cerebral ischemia and L-glutamic acid etc.

Summary of the invention

The object of the invention is to inquire into the effect of hydrogen peroxide to the short survival function of IGF-1, provide a kind of hydrogen peroxide to reduce the mechanism of PC12 cell survival, cell death inducing.Take PC12 cell (Clonal Rat Pheochromocytoma oncocyte) as research object, to deprive serum as model, IGF-1(insulin-like growth factor-i) avoid depriving for the protection of PC12 the damage that serum causes, promote PC12 cell survival.And give before IGF-1 processes to add in advance the H of different concns ₂o ₂(hydrogen peroxide), MTT(thiazole blue laws) detect cell viability, result shows the survival ability of depriving serum and can significantly reduce cell, and the damage that serum brings is deprived in the protection PC12 cell antagonism that IGF-1 can dose-dependently, and H ₂o ₂this short survival function of inhibition IGF-1 that can dose-dependently.

Embodiment

The concrete operation steps of the present invention is as follows:

1, the cultivation PC12 cell derived adult rat adrenal tissue medullary substance pheochromocytoma of cell, is widely used in the in vitro study of nervous system disorders, is incubated in the DMEM substratum that contains 5%FBS, 5%HS, 1%PSA, in 37 ℃, 5%CO ₂under condition, cultivate, after cell covers with, use containing the DMEM culture medium inoculated of 1%FBS, 1%PSA and in 96 holes with after 24 hours, cell is processed.

2, before cell processing, first cell is changed to liquid and become pure DMEM 1hour, then normal group changes DMEM+1.5% FBS into, deprive serum model group and adopt pure DMEM, IGF-1 administration group gives (2.5ng/ml, 25.0ng/ml, 50.0ng/ml, 100.0ng/ml) IGF-1, continues to cultivate mtt assay after 24 hours and detects cell viability; Investigate H ₂o ₂effect to the short survival of IGF-1 function is the H that first uses different concns (10 μ M, 30 μ M, 100 μ M, 200 μ M, 400 μ M) ₂o ₂pretreatment cell 1 hour, then add IGF-1 to process.

3, MTT detect cell viability by cell with 0.5 × 10 ⁵/ ml is inoculated in 96 well culture plates, and 100 μ l/ holes, at 37 ℃ of 5% CO ₂under condition, after overnight incubation, give different treatment factor by grouping requirement, establish 8 parallel multiple holes for every group.Cultivate after 24 h, it is the MTT 100 μ l of 0.5 mg/ml that every hole adds final concentration, then cultivates 5 h, nutrient solution inclines, add DMSO 100 μ l/ holes, after the crystallization of first a ceremonial jade-ladle, used in libation is dissolved completely, read every hole absorbancy (A) at wavelength 570nm place with enzyme linked immunological instrument.The mean of getting 8 hole A values calculates cell survival rate=(test holes A average/control wells A average) × 100 % by formula.Repeat 3 times.

Claims

1. hydrogen peroxide suppresses the in vitro study of the short survival of IGF-1 function, it is characterized in that take PC12 cell (Clonal Rat Pheochromocytoma oncocyte) as research object, to deprive serum as model, investigate IGF-1(insulin-like growth factor-i) short survival function to PC12 cell, its restraining effect to the short survival of IGF-1 function of different concns hydrogen peroxide Study on pretreatment.

2. in vitro study according to claim 1, it is characterized in that research object is PC12 cell, the cell density that mtt assay detects cell viability is 5000-8000/ hole, normal group is DMEM (basic medium)+1.5% FBS (foetal calf serum), deprive the DMEM that serologic group is equivalent, IGF-1 administration group is the IGF-1+DMEM of 12.5ng/ml, 25.0ng/ml, 50.0ng/ml, 100.0ng/ml, after administration, is the CO that 37 ℃, relative humidity are 95% in temperature ₂in incubator, continue to cultivate 24 hours.

3. in vitro study according to claim 1, is characterized in that investigating the H that make used time different concns of hydrogen peroxide to the short survival function of IGF-1 ₂o ₂(10 μ M, 30 μ M, 100 μ M, 200 μ M, 400 μ M) pretreatment cell 1 hour, and then add IGF-1, continue to cultivate mtt assay after 24 hours and detect cell viability.

4. according to the study in vitro described in claim 2,3, it is characterized in that for the basic medium compound method of cell cultures being: one bag of high glycoform DMEM powder is dissolved in to the ultrapure water of 500～800 mL, separately adds 5.95 g HEPES and 3.7 g NaHCO ₃, magnetic agitation is dissolved dry powder completely, is settled to 1000 mL, regulates pH value to 7.2～7.4, under aseptic condition, is packing after the filtering with microporous membrane degerming of 0.22 μ m with aperture, placement storage in 4 ℃ of refrigerators.

5. according to the study in vitro described in claim 2,3, it is characterized in that for detection of the mtt assay compound method of cell viability as follows: under aseptic condition, take a certain amount of MTT powder, it is 5 mg/mL that PBS is dissolved to concentration, separating device-80 ℃ storage is for subsequent use, before using, is diluted to 0.5 mg/mL with DMEM.