CN104087682A - Method for detecting astragaloside-induced RMMVECs gene expression profiling by utilizing gene chip - Google Patents

Abstract

The invention provides a method for detecting astragaloside-induced RMMVECs gene expression profiling by utilizing a gene chip. The method comprises the following nine steps: (1) preparing and purifying rat myocardial membrane microvascular endothelial cells RMMVECs; (2) culturing the second-generation RMMVECs, dissolving astragaloside in maintenance medium with the concentration of 10 mu g/ml, and further culturing RMMVECs for 9 hours under the conditions of 37 DEG C and 5 percent of CO2, and the like. The RMMVECs can be successfully cultured, the gene chip is utilized to fully verify that expression of RMMVECs related cell factors can be regulated in a bidirectional mode through traditional Chinese medicine active ingredients Ast or Ber for improving amplitude of microvascular vasomotion from a gene expression level, balance of cell factor secretion is maintained, and normal physiological functions of MVECs are maintained, so that injury of pathogenic factors on microvascular endothelial cells is avoided, and an effect of treating diseases is achieved.

Description

Utilize the method for genechip detection Cyclosiversioside F induction RMMVECs gene expression profile

Technical field

The present invention relates to gene engineering technology field, especially relate to a kind of method of utilizing genechip detection Cyclosiversioside F induction RMMVECs gene expression profile.

Background technology

Gene chip (gene chip) is called again DNA array, is the one of biochip, refers to the probe array of a large amount of DNA or the formation of oligonucleotide probe dense arrangement.The know-why of gene chip is base pairing and the complementation of DNA, and basis is sequencing by hybridization (SBH).Gene chip adopts the method for the synthetic or micro-sampling of photoconduction original position, oligonucleotide or cDNA are solidified to the surface in upholder in an orderly manner, with cDNA or the cRNA hybridization of the biological sample to be measured of mark, by specific installations such as laser confocal scannings, the intensity of hybridization signal is detected to analysis, thus the quantity of target molecule in judgement sample.From the proposition of the SBH concept early 1980s, commercialization gene chip is up till now in this time more than ten years, constantly constantly extend, the range of application of chip constantly expands by concept perfect, chip for the manufacturing technology of chip, make biochip technology on 21st century life science and the development of medical science produce the impact that cannot estimate.Huge learning value, social value and the economic worth that chip brings causes the very big interest of Liao Duojia major company and multinational government organs, and throws considerable financial resources, and this makes again chip technology be developed rapidly.

Gene chip can be divided into expression profiles of gene chip and the large class of DNA sequencing chip two by function at present; Can be divided into long probe chip and short probe chip by the length of probe.

Gene chip is genome times afterwards comprehensively important technologies, its modernization that appears as traditional Chinese medicine and pharmacy provides a good point of penetration, be characterized in analyzing the expression of thousands of genes at synchronization, form complete cellular gene expression, solved long, consuming time many, problem that output is few of traditional Chinese medicine pharmacological research method cycle.Capillary endothelium can be by conduction and the regulatory function of its secreting function performance information, ensure between blood and tissue juice, physiology, biochemistry and the hemodynamic homeostasis of complexity between blood and lymph liquid and between the formed elements of blood itself and blood plasma, secrete cytokine profiles and autacoid simultaneously, participate in a series of physiology and pathologic process.But in the Related Research Domain of capillary endothelium now, the application of biochip technology is not also very general.The applying gene chip technologies such as Zhang Bin detect respectively the gene expression profile of cultivating rear endotheliocyte in normal concentration and high density D-Glucose substratum.Result shows that high sugar may cause vascular endothelial dysfunction by the expression that affects microvascular endothelial substance metabolism, signal transduction, membrane structure and extracellular matrix etc., for further research height is sugared, the impact of capillary endothelium gene expression profile is given information.The employing biochip technologies such as Y μ E Fei have been studied Prostatropin (basic fibroblast growth factor, bFGF) to mouse brain capillary endothelium (microvasc μ lar endothelial cell, MVEC) change of the newborn related gene expression spectrum of strain bEnd.3 medium vessels, results suggest: bFGF has the genetic expression of the angiogenesis promoting of rise, lower the effect of angiogenesis inhibiting genetic expression, both synergies, promote angiogenesis.

Summary of the invention

The technical problem that the present invention solves is exactly the analysis of the differential gen expression spectrum by the RMMVECs of Cyclosiversioside F induction vitro culture is produced, inquire into the molecular mechanism that improves the easypro contracting activity amplitude of capillary blood vessel, fully verify from gene expression dose, improve the expression of the Effective Component of Chinese Medicine Ast energy two-ways regulation RMMVECs relevant cell factor of the easypro contracting activity amplitude of capillary blood vessel, maintain the balance of cytokine secretion, maintain the normal physiological function of MVECs, thereby block the injury of virulence factor to capillary endothelium, the effect of performance treatment disease.

In order to address the above problem, the invention provides a kind of method of utilizing genechip detection Cyclosiversioside F induction RMMVECs gene expression profile, comprise the following steps:

(1) preparation purification of rat myocardium microvascular endothelial cells RMMVECs;

(2) cultivate two generation RMMVECs, Cyclosiversioside F is dissolved in maintain base, concentration is 10 μ g/ml, at 37 DEG C, 5%CO ₂condition under, by RMMVECs continue cultivate 9h;

(3) by abundant RMMVECs cracking, extract total RNA;

(4) purifying, quantitatively total RNA;

(5) the synthetic First-strand cDNA of reverse transcription;

(6) synthetic Second-strand cDNA;

(7) the synthetic cRNA of in-vitro transcription;

(8) cRNA reverse transcription;

(9) after fluorescent mark, carry out gene chip hybridization, finally carry out data analysis.

Described method, adopts differential attachment method to carry out purifying to prepared RMMVECs in step (1).

Described method, in step (3), fully the method for cracking is: the PBS of 37 DEG C of preheatings of RMMVECs is cleaned 3 times, add 2ml Trizol solution, leave standstill 5min, with liquid-transfering gun piping and druming, make the abundant cracking of RMMVECs, proceed to centrifuge tube, stand-by in-80 DEG C of Refrigerator stores;

The method of extracting total RNA is: the cell sample being dissolved in Trizol liquid in-80 DEG C of Refrigerator stores thawed, and 12000rpm, 4 DEG C of centrifugal 5min, abandon precipitation, and supernatant is proceeded in a new centrifuge tube; By Trizol: the volume that chloroform is 5:1 adds chloroform, thermal agitation mixes to powder milk shape, and room temperature leaves standstill 3min, 12000rpm, 4 DEG C of centrifugal 15min, get the new centrifuge tube of supernatant to, add equal-volume Virahol, mix, room temperature leaves standstill 5min, 12000rpm, 4 DEG C of centrifugal 15min, visible white RNA precipitation; Supernatant discarded, adds 75% ethanol 1ml along tube wall lentamente, precipitation is suspended in ethanol, 7500rpm, 4 DEG C of centrifugal 15min; Supernatant discarded, then add 75% ethanol 1ml, repeat step; Then suck all supernatants, after residual ethanol volatilization to the greatest extent, add 50 μ l DEPC water dissolution RNA precipitations ,-20 DEG C of freezing preservations.

Described method, in step (5), the method steps of the synthetic First-strand cDNA of reverse transcription is:

E. get total RNA500ng, adjust volume to 4 μ L;

F. add the outer ginseng of gene expression profile 1 μ l;

G. prepare reverse transcription Master Mix, wherein First Strand B μ ffer Mix4 μ L, First Strand Enzyme Mix1 μ L, mixes of short duration centrifugal being placed on ice gently; Getting the 5 μ L Master Mix that prepare is transferred to respectively in the 0.2mL centrifuge tube that contains total RNA sample;

H. mix gently solution, instantaneous centrifugal rear 42 DEG C of reactions 2 hours; The heat lid temperature of PCR instrument is also arranged to 42 DEG C, avoids solution to steam on tube wall; React rear ice bath 5 minutes.

Described method, in step (6), the method steps of synthetic Second-strand cDNA is:

D. prepare Second-strand Master Mix, wherein N μ clease-free Water13 μ L, Second-strand B μ ffer Mix5 μ L, Second-strand Enzyme Mix2 μ L, mixes gently, of short duration centrifugal rear ice bath; Getting step reacts in complete sample hose and adds the 20 μ L Second Strand Master Mix that prepare;

E. mix gently, 16 DEG C are reacted 1 hour, 65 DEG C of 10min;

F. reaction is placed in reactant to continue building-up reactions on ice after finishing, or frozen in-20 DEG C rapidly.Described method, in step (7), the method steps of the synthetic cRNA of in-vitro transcription is:

E.cRNA is synthetic: preparation in-vitro transcription Master Mix, wherein N μ clease-free Water4 μ L, T7B μ ffer Mix20 μ L, T7Enzyme Mix6L, mix gently, of short duration centrifugal, up in a reaction, in ready sample hose, add 30 μ L Master Mix;

F. softly mix, 40 DEG C are reacted 16.5 hours;

G., after having reacted, prepare purifying cRNA;

H. purifying, quantitative cRNA.

Described method, in step (8), the method steps of cRNA reverse transcription is:

A. get cRNA purified product 5 μ g, adjust volume to 7.5 μ l, join in 0.2ml nuclease free centrifuge tube, add 4 μ l random primers, mix, 65 DEG C are reacted 5 minutes, ice bath 5 minutes;

B. prepare reverse transcription Master Mix, wherein 4 × CbcScript II B μ ffer5 μ L, 0.1M DTT2 μ L, CbcScript II 1.5 μ L, mix gently, of short duration centrifugal; In the sample hose having reacted, add 8.5 μ l Master Mix;

C. mix gently, 25 DEG C are reacted 10 minutes, and 37 DEG C are reacted 1.5 hours;

D. reaction adds Terminate Sol μ tion5 μ l after finishing, and mixes;

B. (5) 65 DEG C are reacted 10 minutes, room temperature 5 minutes;

E. add Ne μ tralize Sol μ tion1 μ l, mix, prepare purifying;

F. purifying, quantitative reverse transcription product.

Described method, in step (9), fluorescently-labeled method steps is:

E. cDNA reverse transcription after purifying being obtained is concentrated in vacuo to 14 μ L, and 60 DEG C, 5min, adds 4 μ L random primers, mixes; 95 DEG C are reacted 3 minutes, ice bath 5 minutes;

F. reaction adds 1 μ L Cy3 after finishing;

G. prepare Mix, wherein 5 × Klenow B μ ffer5 μ L, Klenow Fragment1.2 μ L adds 6.2 μ L Mix in above-mentioned mixed solution, mix, wink from, 37 DEG C reaction 1.5 hours, 70 DEG C reaction 5 minutes, ice bath 5 minutes, prepare purifying;

H. purifying, quantitative mark product;

Described method, in step (9), the method steps of gene chip hybridization is:

A. the preparation of chip:

A. hydration: selected chip is dried up 10cm left and right on 65 DEG C of water-baths, steams 10-15 second; Dry 10-15 second, then steam 10-15 second;

B. UV-crosslinked: to arrange 250;

C. first pass is washed: 0.5%SDS, and microwave-oven-heating is to 42-45 DEG C, and shaking table 10min, washes away cy3;

D. wash for second time: 42 DEG C of pure water, SDS is removed in rinsing, proceeds to next washing tank for films;

E. wash for the 3rd time: 42 DEG C of pure water, shaking table 1-2min, washes away SDS, dries;

B. the preparation of sample:

A. cy3/cy5 is concentrated into respectively to 19.2 about μ L, in cy3 (cy5), adds hybridization solution 41.6 μ L, then cy5 (cy3) is drawn in cy3 (cy5), mix, concussion, wink from;

B. wherein hybridization solution preparation: methane amide 20 μ L, 20*SSC12 μ L, 10*SDS1.6 μ L, 50*Dehart8 μ L;

DEG C c.95,3min, cuts 1 × 4cm filter paper during this time, puts into chip cartridges, adds 100-180 μ L distilled water, prepares washing lotion 1:600mL, washing lotion 2:1L;

D. take out PCR pipe, quenching, puts on shelf, prepares loading;

E. with rubber pipette bulb blow cores sheet, when application of sample, keep left and put into the straight line of 1.5cm left and right, then with rifle head is auxiliary, cover glass is covered;

F. adjust the neat degree of cover glass with rifle head, put into chip cartridges, put into hybridization instrument, trim, puts a water bottle moisturizing; Arrange: 42 DEG C, 23 hours, 6 turned, start Hyb hybridization, hybridize 23 hours;

C. hybridization finishes to prepare read tablet: after hybridization, develop a film, scanner preheating 10min, selects pocket: power-80, and PMT-800, first sweeps red passage, then sweeps green passage, ensures that saturation point (white point number) is less than 5/1000ths, after having scanned, preserves.

Described method, the method steps of data analysis is in step (9): scan image is converted into signal value with L μ xScan3.0 software; Determine genetic expression (P value < 0.05), critical expression (0.05 < P value < 0.065) and do not express (P value > 0.065) according to the P value of probe signals value; Respectively the signal value of 6 chips is done after normalized, by 6 groups respectively with the comparison of blank group, differentiate a certain gene whether occur express change: in the time of signal ratio >=2, be considered as genetic expression raise; In the time of signal ratio≤0.5, be considered as down regulation of gene expression; In the time that ratio falls between, being considered as gene has the expression of biological significance to change; Finally with softwares such as Microsoft Excel, S μ M and Cl μ ster, each group of data are added up and cluster analysis.

The Ast of 10 μ g/mL and Ber act on respectively gene chip result after RMMVECs9h and show, each group compared with blank group, difference expression gene quantity is as follows: totally 2231 of Ast groups, wherein raise 2166, lower 65; Totally 576 of Ber groups, wherein raise 513, lower 63.Show in Ast and Ber group, on most of paths, have that function is close, contrary paired gene of differential expression in conjunction with pathway analytical results, the expression that both can two-ways regulation RMMVECs relevant cell factor is described.Totally 47 of the genes of Ast and the common generation of Ber group differential expression, participate in 22 signal paths, comprise actin cytoskeleton adjusting, calcium signal path, protein mediated signal path, closely connection, the signal paths such as connection of adhering of Notch, relate to blood vessel capacity regulating, the biosynthetic adjusting of NO synthase, endothelial cell differentiation, Growth of Cells and maintaining, the adjusting of smooth muscle cell, protein metabolism and modification, carbohydrate metabolism, the functions such as DNA replication dna.

The present invention has successfully cultivated RMMVECs, and utilize biochip technology fully to verify from gene expression dose, improve the expression of Effective Component of Chinese Medicine Ast and the Ber energy two-ways regulation RMMVECs relevant cell factor of the easypro contracting activity amplitude of capillary blood vessel, maintain the balance of cytokine secretion, maintain the normal physiological function of MVECs, thereby block the injury of virulence factor to capillary endothelium, the effect of performance treatment disease.

Brief description of the drawings

Fig. 1 is RMMVECS normal morphology A: photo under inverted microscope mirror (10 × 4); B: VIII F-Ag immunocytochemistry qualification positive findings (10 × 20);

Fig. 2 is the biological quality qualification of denaturing formaldehyde electrophoresis, wherein: (1-6: control group RMMVECs, 7-9:Ast treatment group RMMVECs; 10-12:Ber treatment group RMMVECs; Upper is 28S, and lower is 18S);

The Ast group of Fig. 3 RMMVECs is than the signal scatter diagram of control group group;

The Ber group of Fig. 4 RMMVECs is than the signal scatter diagram of control group;

The dendrogram that the Ast group of Fig. 5 RMMVECs repeats than 3 biology of control group;

The dendrogram that the Ber group of Fig. 6 RMMVECs repeats than 3 biology of control group.

Embodiment

Hereinafter in connection with accompanying drawing, embodiments of the invention are elaborated.It should be noted that, in the situation that not conflicting, the arbitrary combination mutually of the feature in embodiment and embodiment in the application.

1.1 key instrument equipment

1.2 main agents

The configuration of 1.3 main solution

(1) Cyclosiversioside F solution (10 μ g/ml): by the cell maintain basigamy system containing 2%FBS ,-4 DEG C save backup.

(2) Berberine solution (10 μ g/ml): by the cell maintain basigamy system containing 2%FBS ,-4 DEG C save backup.

1.4 test cells

S-generation rat heart muscle film capillary endothelium.

Cell preparation method

1.4.1RMMVECs separation and Culture

3 of healthy 7 age in days rats, are used 75% ethanol disinfection, open its thoracic cavity in Bechtop, win heart, by D-Hank ' the s liquid rinsing heart tissue of 4 DEG C of precoolings 3 times.Subsequently, with another set of eye scissors clip left ventricular wall, in 75% ethanol, soak 10s, then peel off endocardium and visceral pericardium, collect cardiac muscle rinsing in the D-Hank ' of 4 DEG C of precoolings s liquid, abandon supernatant, add 0.2% II Collagenase Type 4mL of 37 DEG C of preheatings, after shredding in 37 DEG C of CO ₂digestion 20min in incubator.During this time, blow and beat once every 5min, to prevent adhesion between tissue block.After 20min, add the DMEM perfect medium 4mL of 37 DEG C of preheatings to stop digestive process.Then proceeded to centrifuge tube, after the centrifugal 8min of 1200rpm, abandoned supernatant, added DMEM perfect medium 6mL, after mixing, be seeded in 6 porocyte culture plates, be placed in 37 DEG C of CO ₂in incubator, cultivate after 2h, change DMEM perfect medium, every hole 2mL.After this, changed a DMEM perfect medium, the cultivation of later can going down to posterity for about 4 days every 2 days.

1.4.2RMMVECs the cultivation of going down to posterity

To grow to D-Hank ' the s rinsing 2 times of 37 DEG C of preheatings of individual layer primary cell of converging state, to adding 0.25% trypsin in each hole containing EDTA), be placed in 37 DEG C of CO ₂digestion 3min left and right in incubator, micro-Microscopic observation, fluffs when loose when connecting between most cells, adds equivalent DMEM perfect medium to stop digestion immediately in each hole, and cell piping and druming is come off and mixed, and goes down to posterity according to the ratio of 1:2.

1.4.3RMMVECs qualification

Adopt the qualification of S-P immunocytochemical method to RMMVECs factor Ⅷ related antigen (F-VIII RAg), concrete operations are as follows:

(1) grow up to after individual layer until the cell in Tissue Culture Plate, inhale and abandon nutrient solution, and with the PBS liquid of 37 DEG C rinsing 3 times gently, then washing lotion is abandoned in suction;

(2) in cell cultures hole, add 95% ethanol of 4 DEG C of precoolings to cover cell cultures layer completely, and in-20 DEG C of fixing 20min, inhale and abandon ethanol, with the continuous rinsing of PBS 3 times, each 3min;

(3) every hole drips 300 μ l rabbit Chinese People's Anti-Japanese Military and Political College mouse factor Ⅷ related antigen primary antibodies, hatches 3h for 37 DEG C, and PBS rinses 3 times, each 3min;

(4) to drip 200 μ l fluorescence (FITC) mark goat anti-rabbit iggs two anti-in every hole, hatches 45min for 37 DEG C, and PBS rinses 2 times, each 3min, deionized water rinsing once, observation of cell form under microscope, and Taking Pictures recording.

1.4.4 results and analysis

Cell attachment growth, adherent after primary cell 3h, after 24h, substantially can find out cellular form, most cells is short fusiformis and flat polygon.After 3～4d, cell presents typical paving stone sample, reaches converging state (Figure 1A) after 5d.Factor Ⅷ related antigen is the reliable marker of endotheliocyte, carry out after factor Ⅷ related antigen immunofluorescence dyeing, there is yellow-green colour positive reaction (Figure 1B) around in core, in conjunction with growth characteristics and the morphological specificity of cell, proves that institute's culturing cell is endotheliocyte.

In this test, we adopt differential attachment method to carry out purifying to cultivated RMMVECs, because the adherent process of inoblast comparatively fast and more firm, generally can complete adherent process at 10-30min, and that vascular endothelial cell still can not adhere to or adhere to is at short notice stable not; RMMVECs and inoblast are also different to the tolerance of pancreatin simultaneously, in the time carrying out cell dissociation, inoblast just first takes off wall, and capillary endothelium needs the time of growing could take off wall, then processes and just can obtain the RMMVECs that purity is higher through going down to posterity.This method is relatively simple and repeatability is better, and its characteristics of cell biology of the RMMVECs obtaining is comparatively stable, can provide comparatively desirable test materials for follow-up test.In this test, the VIII F-Ag that we detect institute's culturing cell surface identifies it, and this is because VIII F-Ag is vascular endothelial cell marker relatively reliably, therefore, by institute's culturing cell being carried out to the immunocytochemistry qualification of VIII F-Ag, can obtain comparatively believable qualification result.Great vessels is fully removed in this test in tissue sampling, in the process of former culture, utilize again the method for differential velocity adherent to carry out purifying to institute's cultured cells, finally again the cell after purifying is carried out to the qualification of marker antigen, thereby provide solid believable test materials and testing data for follow-up test.

1.5 chip gene expression profile

Rich Austria brilliant core platform rat full genome oligonucleotide micro-array chip (27K) of application Bo Ao company, the gene expression profile of detection rat.Its preparation and detection complete in Boao Biological Co., Ltd's biological detection laboratory.

2 methods

The packet transaction of 2.1 cells

The RMMVECs of two cultures, through qualification after for this test.In the time that RMMVECs grows up to the individual layer of converging state, carry out packet transaction.Control group: perfect medium is replaced by the maintain base containing 2%FBS; Test group: Cyclosiversioside F, Berberine are dissolved in maintain base, concentration is 10 μ g/ml.At 37 DEG C, 5%CO ₂condition under, respectively organize RMMVECs continue cultivate 9h.Then RMMVECs is cleaned 3 times with the PBS of 37 DEG C of preheatings, every plate adds 2ml Trizol solution, leaves standstill 5min, with liquid-transfering gun piping and druming, makes the abundant cracking of RMMVECs, proceeds to centrifuge tube, stand-by in-80 DEG C of Refrigerator stores.

2.2 chip hybridization experiments

In gene chip chamber, veterinary science (Chinese materia medica) laboratory, rich brilliant core platform difficult to understand completes cross experiment, and every group of 3 biology repeats.

2.2.1 the extraction of cell Total RNA

The cell sample being dissolved in Trizol liquid in-80 DEG C of Refrigerator stores is thawed, 12000rpm, 4 DEG C of centrifugal 5min, abandon precipitation, and supernatant is proceeded in a new centrifuge tube.Add chloroform by the volume of Trizol: chloroform=5:1, thermal agitation mixes to powder milk shape, and room temperature leaves standstill 3min, 12000rpm, 4 DEG C of centrifugal 15min, get the new centrifuge tube of supernatant to, add equal-volume Virahol, mix, room temperature leaves standstill 5min, 12000rpm, 4 DEG C of centrifugal 15min, visible white RNA precipitation.Careful supernatant discarded, adds 75% ethanol 1ml (being sure not to touch or dispelling precipitation) along tube wall lentamente, and putting upside down gently washing centrifuge tube tube wall is that precipitation is suspended in ethanol, 7500rpm, 4 DEG C of centrifugal 15min.Careful supernatant discarded, then add 75% ethanol 1ml, repeat step.Then carefully suck all supernatants, after residual ethanol volatilization to the greatest extent, add 50 μ l DEPC water dissolution RNA precipitations ,-20 DEG C of freezing preservations.Get 1 μ l nucleic acid-protein determinator and detect quality, record result.

2.2.2 cell total rna obtains quality evalution

The total RNA of RMMVECs of extracting adopts denaturing formaldehyde agarose gel electrophoresis method to carry out quality examination.Before freezing, from extract, get the total RNA of 0.3 μ g, add 5 × sample loading buffer of 1/5 volume, adjust volume to 7 μ l left and right, for 65 DEG C, mixture is heated 5min, on ice quenchings by the secondary structure of eliminating RNA.Before loading, add 1.0 μ l ethidium bromides (EtBr, concentration 1.0mg/ml) to RNA sample, and do not add EtBr to glue, to reduce the background after electrophoresis.Denaturing formaldehyde sepharose (1.2%) first after prerunning 15min, adds RNA sample, electrophoresis 30min under the voltage of 5-10V/cm, Taking Pictures recording under ultraviolet lamp in 1 × denaturing formaldehyde agarose gel electrophoresis damping fluid.

2.2.3 total RNA purifying, quantitative

Get total RNA10 μ g moisturizing to 100 μ L;

Dosing: 300 μ L RA1:300 μ L dehydrated alcohols are 1:1, mix;

By above two kinds of liquid blendings, add in purification column;

10000rpm, centrifugal 30 seconds, abandons liquid;

Add 600 μ L RA3,10000rpm, centrifugal 30 seconds, abandons liquid;

Add 350 μ L RA3,10000rpm, centrifugal 30 seconds, abandons liquid;

10000rmp, sky gets rid of 1.5min;

Change pipe into 1.5mL PE pipe, add 40 μ L DEPC water, 10000rpm, centrifugal 1min, collects liquid.

Quantitative: to get 1 μ l nucleic acid-protein determinator and detect quality, record result.

2.2.4 the synthetic First-strand cDNA of reverse transcription

(1) get total RNA500ng, adjust volume to 4 μ L;

(2) add brilliant core gene expression profile outer ginseng 1 μ l (test group adds A, and control group adds B);

(3) (following table is depicted as single reaction system consumption to preparation reverse transcription Master Mix, mixes gently, of short duration centrifugal being placed on ice.Getting 5 μ L Master Mix is transferred to respectively in the 0.2mL centrifuge tube that contains total RNA sample.Reverse transcription Master Mix:

Component title	Volume
		First?Strand?Bμffer?Mix	4μL
First?Strand?Enzyme?Mix	1μL

(4) mix gently solution, instantaneous centrifugal rear 42 DEG C of reactions 2 hours.The heat lid temperature of PCR instrument is also arranged to 42 DEG C, avoids solution to steam on tube wall.React rear ice bath 5 minutes.Carry out immediately subsequent step.

2.2.5 synthetic Second-strand cDNA

(1) preparation Second-strand Master Mix (following table is depicted as single reaction system consumption), mixes of short duration centrifugal rear ice bath gently.In reacting complete each sample hose, 4.1.2.5 adds 20 μ L Second Strand Master Mix.Second?Strand?Master?Mix：

Component title	Volume
		Nμclease-free?Water	13μL
Second-strand?Bμffer?Mix	5μL
		Second-strand?Enzyme?Mix	2μL

(2) mix gently, 16 DEG C are reacted 1 hour, 65 DEG C of 10min;

(3) reaction is placed in reactant to continue building-up reactions on ice after finishing, or frozen in-20 DEG C rapidly.

2.2.6 in-vitro transcription is synthesized cRNA

1, cRNA is synthetic

(1) preparation in-vitro transcription Master Mix (following table is depicted as single reaction system consumption), mixes gently, of short duration centrifugal, up in a reaction, in ready each sample hose, adds 30 μ L Master Mix; In-vitro transcription Master Mix:

Component title	Volume
		Nμclease-free?Water	4μL
T7Bμffer?Mix	20μL
		T7Enzyme?Mix	6L

(2) softly mix, 40 DEG C are reacted 16.5 hours;

(3), after having reacted, prepare purifying cRNA.

2, cRNA purifying, quantitative

(1) get total RNA10 μ g moisturizing to 100 μ L;

(2) dosing: 300 μ L RA1:300 μ L dehydrated alcohols are 1:1, mix;

(3) by above two kinds of liquid blendings, add in purification column;

(4) 10000rpm, centrifugal 30 seconds, abandons liquid;

(5) add 600 μ L RA3,10000rpm, centrifugal 30 seconds, abandons liquid;

(6) add 350 μ L RA3,10000rpm, centrifugal 30 seconds, abandons liquid;

(7) 10000rmp, sky gets rid of 1.5min;

(8) pipe is changed into 1.5mL PE pipe, add 40 μ L DEPC water, 10000rpm, centrifugal 1min, collects liquid.

(9) quantitative: to get 1 μ l nucleic acid-protein determinator and detect quality, record result.

2.2.7cRNA reverse transcription

1, cRNA reverse transcription

(1) get cRNA purified product 5 μ g, adjust volume to 7.5 μ l, join in 0.2ml nuclease free centrifuge tube.Add 4 μ l Random Primer, mix, 65 DEG C are reacted 5 minutes, ice bath 5 minutes;

(2) preparation reverse transcription Master Mix (following table is depicted as single reaction system consumption), mixes gently, of short duration centrifugal.In the each sample hose having reacted in 4.1.2.7, add 8.5 μ l Master Mix; CRNA reverse transcription Master Mix:

Component title	Volume
		4×CbcScriptⅡ?Bμffer	5μL
0.1M?DTT	2μL
		CbcScriptⅡ	1.5μL

(3) mix gently, 25 DEG C are reacted 10 minutes, and 37 DEG C are reacted 1.5 hours;

(4) reaction adds Terminate Sol μ tion5 μ l after finishing, and mixes;

(5) 65 DEG C are reacted 10 minutes, room temperature 5 minutes;

(6) add Ne μ tralize Sol μ tion1 μ l, mix, prepare purifying.

2, purifying, quantitative reverse transcription product

Use extract II (MN company, Cat.No.740609.250) test kit.

(1) 26 μ L moisturizing to 50 μ L, concussion, turns bat, adds 2 times of volume NT (100 μ L), and above 150 μ L are transferred in purification column, and 10000rpm, centrifugal 30 seconds, abandons liquid;

(2) add NT3,600 μ L, 10000rpm, centrifugal 30 seconds, abandons liquid;

(3) sky gets rid of 1-2min, changes 1.5mL PE pipe into;

(4) add the 1/2NE30 μ L (1/2NE is NE15 μ L, DEPC water 15 μ L) of 65 DEG C, leave standstill 1min, 10000rpm, centrifugal 1min, collects liquid;

(5) quantitative: to get 1 μ l nucleic acid-protein determinator and detect quality, record result.

2.2.8 fluorescent mark

1, labeled reactant

(1) cDNA reverse transcription after purifying being obtained is concentrated in vacuo to 14 μ L (60 DEG C, 5min), adds 4 μ L Random Primer, mixes.95 DEG C are reacted 3 minutes, ice bath 5 minutes;

(2) reaction finishes to add 1 μ L Cy5 in backward control group, and test group adds 1 μ L Cy3.

(3) preparation Mix (following table is depicted as single reaction system consumption), in above-mentioned mixing also, add 6.2 μ L Mix, mix, wink from, 37 DEG C reaction 1.5 hours, 70 DEG C reaction 5 minutes, ice bath 5 minutes, prepare purifying.

Note: Cy5-dCTP (Cy3-dCTP) uses GE Healthcare company's product (Cat.No.PA55021/PA53021).

Mix：

Component title	Volume
		5×Klenow?Bμffer	5μL
Klenow?Fragment	1.2μL

2, purifying, quantitative mark product

(1) 26 μ L moisturizing to 50 μ L, concussion, turns bat, adds 2 times of volume NT (100 μ L), and above 150 μ L are transferred in purification column, and 10000rpm, centrifugal 30 seconds, abandons liquid;

(2) add NT3,600 μ L, 10000rpm, centrifugal 30 seconds, abandons liquid;

(3) sky gets rid of 1-2min, changes 1.5mL PE pipe into;

(4) add the 1/2NE30 μ L (1/2NE is NE15 μ L, DEPC water 15 μ L) of 65 DEG C, leave standstill 1min, 10000rpm, centrifugal 1min, collects liquid;

(5) quantitative: to get 1 μ l nucleic acid-protein determinator and detect quality, record result.

3, fluorescent substance incorporation

Get 1 μ l nucleic acid-protein determinator and detect incorporation, record result.

2.2.9 prepare before hybridization

1, the preparation of chip

Chip pre-treatment: sweep sheet, develop a film.

(1) hydration: selected chip is dried up 10cm left and right on 65 DEG C of water-baths, steams 10-15 second; Dry 10-15 second, then steam 10-15 second.

(2) UV-crosslinked: to arrange 250.

(3) first pass is washed: 0.5%SDS, and microwave-oven-heating is to 42-45 DEG C, and shaking table 10min, washes away cy3.

Wash for (4) second times: 42 DEG C of pure water, SDS is removed in rinsing, proceeds to next washing tank for films.

Wash for (5) the 3rd times: 42 DEG C of pure water, shaking table 1-2min, washes away SDS, dries.

2, the preparation of sample

(1) cy3/cy5 is concentrated into respectively to 19.2 μ L left and right.In cy3 (cy5), add hybridization solution 41.6 μ L, then cy5 (cy3) be drawn in cy3 (cy5), mix, concussion, wink from.

Hybridization solution preparation:

Component	Volume μ L
		Methane amide	20
20*SSC	12
		10*SDS	1.6
50*Dehart	8μL

(2)95℃，3min。Cut during this time 1*4cm filter paper, put into chip cartridges, add 100-180 μ L distilled water.Prepare washing lotion 1:600mL, washing lotion 2:1L.

(3) take out PCR pipe, quenching, puts on shelf, prepares loading.

(4) with rubber pipette bulb blow cores sheet, when application of sample, keep left and put into the straight line (label is on the right) of 1.5cm left and right, then with rifle head is auxiliary, cover glass is covered.

(5) adjust the neat degree of cover glass with rifle head, put into chip cartridges, put into hybridization instrument, trim, puts a water bottle moisturizing.

Arrange: 42 DEG C, 23 hours, 6 turned (RPM), start (Hyb hybridization)

(6) hybridization is set as 23 hours, spends the night.

3, hybridization finishes to prepare read tablet

(1) after hybridization, develop a film, preparation washing lotion 1, washing lotion 2

Washing lotion 1:400mL20*SSC adds water to 4 liters+80mL10%SDS (2*SSC, 0.2%SDS)

Washing lotion 2:40mL20*SSC adds water to 4 liters (0.2*SSC)

Use system 7 is washed machine.

Pipette is inserted to bottle for handling liquid toilet or cosmetic substance, washing lotion is set, the same, selected heating, dries.

Washing lotion 1:2 time, 42 DEG C, 120 seconds, washing lotion dynamics 5;

Washing lotion 2: the same.

(2) scanner preheating 10min

Select pocket: power-80, PMT-800, first sweeps red passage, then sweeps green passage.Ensure that saturation point (white point number) is less than 5/1000ths.

(3) after having scanned, preserve name: cy3-...., cy5-.... date parameter.Be saved as TIF and jpg form is respectively deposited.

2.3 data analysis

Scan image is converted into signal value with L μ xScan3.0 software.Determine genetic expression (P value < 0.05), critical expression (0.05 < P value < 0.065) and do not express (P value > 0.065) according to the P value of probe signals value.Respectively the signal value of 6 chips is done after normalized, by 6 groups respectively with the comparison of blank group, differentiate a certain gene whether occur express change: in the time of signal ratio >=2, be considered as genetic expression raise; In the time of signal ratio≤0.5, be considered as down regulation of gene expression; In the time that ratio falls between, being considered as gene has the expression of biological significance to change.Finally with and the software such as Microsoft Excel, S μ M and Cl μ ster each group of data are added up and cluster analysis.

3 results and analysis

3.1 total RNA quality examinations

OD (260/280) the value concentration of 12 groups of total RNA is in table 1.Total RNA denaturing formaldehyde electrophorogram of 12 groups of cells as shown in Figure 2.High-visible 28S, 18S rRNA band in figure, and 28S is greater than 1:1 than the brightness of 18S rRNA band.

The total RNA of table 1 extracts result

Sample name	Concentration μ g/ μ l	260/280	Sample name	Concentration	260/280
						MAC1	0.7235	1.98	MAT1	0.5904	1.96
MAC2	0.8391	2.03	MAT2	0.7857	1.99
						MAC3	0.6896	1.99	MAT3	0.6613	1.96
MBC1	0.5656	1.94	MBT1	0.6439	1.99
						MBC2	0.5482	1.98	MBT2	0.7959	2.04
MBC3	0.6553	2.03	MBT3	0.5970	2.01

Note: MAC1, MAC2, MAC3, MBC1, MBC2, MBC3-control group; MAT1, MAT2, MAT3-Ast test group; MBT1, MBT2, MBT3-Ber test group

3.2 difference expression gene

The Ast treatment group of RMMEVCs is compared with control group, and totally 2231 of difference expression genes, raise 2166, lower 65; The obvious differential gene details of biological significance are in shown in Table 5-3.Pathway result shows, the gene of differential expression participates in 169 signal paths altogether, comprises that calcium signal path, arginine and Proline Metabolism path, Jak-STAT (protein tyrosine kinase-signal conductive protein and transcriptional activator) signal path, MAPK signal path, p53 signal path, white corpuscle are through endothelial migration signal path etc.The gene function of differential expression is as follows: Growth of Cells and maintain, the adjusting of MAPK vigor, transcriptional regulatory, signal transduction, metabolism, cell cycle etc.Show that Ast has the blood vessel of adjusting capacity, regulate Growth of Cells, maintain cell normal physiological function, regulate the effects such as metabolism.

The Ber treatment group of RMMEVCs is compared with control group, and difference expression gene totally 576, raises 513, lowers 63.The obvious differential gene details of biological significance are in shown in Table 5-4.Pathway result shows, the gene of differential expression participates in 98 signal paths altogether, consistent with Ast, the signal path of participation comprises that calcium, arginine and Proline Metabolism, Jak-STAT (protein tyrosine kinase-signal conductive protein and transcriptional activator), MAPK, p53, white corpuscle are through endothelial migration etc. equally.The gene function of differential expression also comprise transcriptional regulatory, Growth of Cells and maintain, metabolism etc.In addition, express the gene that changes and also comprise to myocardial cell's calcium ion and the relevant gene of function such as regulate, external stimulus is replied, as Adcy8, Phox2b etc.Show that Ber has the blood vessel of adjusting capacity equally, regulate Growth of Cells, maintain cell normal physiological function, regulate metabolic effect.

After contrasting respectively blank group, Ast and Ber group have 47 co-variation differential genes, participate in 22 signal paths, comprise the protein mediated signal path of actin cytoskeleton adjusting, calcium signal path, Notch, closely connect, the signal path such as adhesion connections, relate to Growth of Cells and maintain, the function such as endothelial cell differentiation, blood vessel capacity regulating, the biosynthetic adjusting of NO synthase, DNA replication dna, protein, carbohydrate metabolism and anoxic are replied.

In chip experimentation after 12 groups of relevant purifying RNA concentration, cRNA concentration, cDNA concentration, fluorescent substance incorporation in table 2.

Correlated results in table 2 chip experimentation

Sample name	RNA after purifying	CRNA after purifying	cDNA	Fluorescent dye incorporation amount	Sample name	RNA after purifying	CRNA after purifying	cDNA	Fluorescent dye incorporation amount
										MAC1	230.8	0.9369	295.6	3.8	MAT1	189.3	0.6674	183.4	3.1
MAC2	154.2	0.9732	247.8	3.4	MAT2	178.4	0.4756	100.6	4.7
										MAC3	143.6	0.8654	209.8	2.8	MAT3	146.8	0.3272	130.2	4.1
MBC1	187.6	0.3546	118.5	3.1	MBT1	189.4	0.4721	166.3	5.1
										MBC2	125.5	0.3172	127.9	4.5	MBT2	220.3	0.6932	159.4	2.8
MBC3	123.7	0.2947	179.8	3.8	MBT3	135.3	0.5453	171.3	2.6

Note: (RNA and cDNA concentration: ng/ μ l; CRNA concentration: μ g/ μ l; Fluorescent dye incorporation amount: pmol/ μ l)

Each cell Ast group and Ber group are than the scatter diagram of blank group difference expression gene respectively as shown in Figure 3, Figure 4.Wherein x and y coordinate represent respectively the fluorescence signal intensity of two groups of genes, and oblique line top is the gene of up-regulated, and oblique line below is the gene of down-regulated expression.As shown in Figure 5, Figure 6, grey (or light/dark balance) represents up-regulated gene to gene dendrogram, and more shallow grey represents down-regulated gene, and black represents not occur the gene of noticeable change.

Totally 2231 of the genes of this experimental result demonstration Ast treatment group cell generation differential expression, wherein raise 2166, lower 65, participate in 169 signal paths, comprise MAPK signal path, calcium signal path, PPAR signal path, cell adhesion molecule signal path, Jak-STAT signal path, arginine and Proline Metabolism signal path etc., the gene of differential expression mostly to protein, carbohydrate metabolism, transcriptional regulatory, Growth of Cells and maintain, the function such as adjusting, apoptosis, endothelial cell differentiation and the growth regulation factor vigor of cell proliferation is relevant.Illustrate that Ast can promote the growth of MVECs and maintain, promote MVECs metabolism, maintain MVECs normal configuration and physiological function.Each gene function and relevant information refer to table 5-3.

Totally 576 of the genes of Ber treatment group cell generation differential expression, wherein raise 513, lower 63, participate in 98 signal paths, comprise MAPK signal path, calcium signal path, cell adhesion molecule signal path, arginine and Proline Metabolism signal path, Jak-STAT signal path etc., the gene of differential expression mostly to protein, carbohydrate metabolism, transcriptional regulatory, DNA replication dna, Growth of Cells and maintain, cell development etc. is relevant.Illustrate that Ber can promote equally the growth of MVECs and maintain, promote MVECs metabolism, maintain MVECs normal configuration and physiological function.Each gene function and relevant information refer to table 5-4.

In addition, pathway analytical results shows have function close on most of paths, contrary paired gene of differential expression, the expression that Ast and Ber can two-ways regulation MVEC relevant cell factors is described, this is also consistent to wearing the result of study of coptis lactone with laboratory early stage.

Ast and Ber group is common there is totally 47 of the genes of differential expression, participates in 22 signal paths, comprises actin cytoskeleton adjustings, calcium signal path, protein mediated signal path, closely connection, the signal paths such as connection of adhering of Notch.Infer that Ast and Ber may be thereby that cytokine acts on again MVEC conversely, maintains structure and the function-stable of MVEC, reaches the object for the treatment of disease by regulating some signal path to regulate the expression of MVEC relevant cell factor.In addition, the gene Jag1 of differential expression and endothelial cell differentiation, somatomedin vigor are relevant; Edn1 is relevant with functions such as blood vessel capacity regulating, blood pressure regulation, glucose transport, cAMP biosynthesizing; μ nc13c, Myh9, Fyn participate in cell communicating; Tmprss8, Phkg2, Mipep participate in protein metabolism; RGD1305664, Rasl11b participate in Growth of Cells and maintain.In addition, also comprise the gene relevant with functions such as transcriptional regulatory, ionic equilibrium, ion transportation, DNA replication dna and reparation, carbohydrate metabolism, proteolysis, as the metabolism of Chst7 involved in sugar; RGD1561694 participates in DNA replication dna and reparation; Prox1 participates in transcriptional regulatory; Bace2 participates in proteolysis.

In sum, these common genes that differential expression occurs all can promote the growth of MVECs, metabolism, maintain the own vitality of MVECs, normal configuration, ensure the normal physiological function of MVECs, the Effective Component of Chinese Medicine treatment disease that can infer the easypro contracting activity amplitude of raising capillary blood vessel may be the expression by regulating MVECs relevant cell factor, Cytokine is in MVECs, maintain the normal structure of MVECs and physiological function, make the autacoid of capillary blood vessel secretion and cytokine in point of equilibrium, block the injury of virulence factor to MVECs, and then the function of performance capillary endothelium, regulate cell micro-environment, reach the object for the treatment of disease.

The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (10)

1. the method for utilizing genechip detection Cyclosiversioside F induction RMMVECs gene expression profile, is characterized in that: comprise the following steps:

(1) preparation purification of rat myocardium microvascular endothelial cells RMMVECs;

(2) cultivate two generation RMMVECs, Cyclosiversioside F is dissolved in maintain base, concentration is 10 μ g/ml, at 37 DEG C, 5%CO ₂condition under, by RMMVECs continue cultivate 9h;

(3) by abundant RMMVECs cracking, extract total RNA;

(4) purifying, quantitatively total RNA;

(5) the synthetic First-strand cDNA of reverse transcription;

(6) synthetic Second-strand cDNA;

(7) the synthetic cRNA of in-vitro transcription;

(8) cRNA reverse transcription;

(9) after fluorescent mark, carry out gene chip hybridization, finally carry out data analysis.

2. method according to claim 1, is characterized in that: in described step (1), adopt differential attachment method to carry out purifying to prepared RMMVECs.

3. method according to claim 1, it is characterized in that: in described step (3), fully the method for cracking is: the PBS of 37 DEG C of preheatings of RMMVECs is cleaned 3 times, add 2ml Trizol solution, leave standstill 5min, blow and beat with liquid-transfering gun, make the abundant cracking of RMMVECs, proceed to centrifuge tube, stand-by in-80 DEG C of Refrigerator stores;

The method of extracting total RNA is: the cell sample being dissolved in Trizol liquid in-80 DEG C of Refrigerator stores thawed, and 12000rpm, 4 DEG C of centrifugal 5min, abandon precipitation, and supernatant is proceeded in a new centrifuge tube; By Trizol: the volume that chloroform is 5:1 adds chloroform, thermal agitation mixes to powder milk shape, and room temperature leaves standstill 3min, 12000rpm, 4 DEG C of centrifugal 15min, get the new centrifuge tube of supernatant to, add equal-volume Virahol, mix, room temperature leaves standstill 5min, 12000rpm, 4 DEG C of centrifugal 15min, visible white RNA precipitation; Supernatant discarded, adds 75% ethanol 1ml along tube wall lentamente, precipitation is suspended in ethanol, 7500rpm, 4 DEG C of centrifugal 15min; Supernatant discarded, then add 75% ethanol 1ml, repeat step; Then suck all supernatants, after residual ethanol volatilization to the greatest extent, add 50 μ l DEPC water dissolution RNA precipitations ,-20 DEG C of freezing preservations.

4. method according to claim 1, is characterized in that: in described step (5), the method steps of the synthetic First-strand cDNA of reverse transcription is:

A. get total RNA500ng, adjust volume to 4 μ L;

B. add the outer ginseng of gene expression profile 1 μ l;

C. prepare reverse transcription Master Mix, wherein First Strand B μ ffer Mix4 μ L, First Strand Enzyme Mix1 μ L, mixes of short duration centrifugal being placed on ice gently; Getting the 5 μ L Master Mix that prepare is transferred to respectively in the 0.2mL centrifuge tube that contains total RNA sample;

D. mix gently solution, instantaneous centrifugal rear 42 DEG C of reactions 2 hours; The heat lid temperature of PCR instrument is also arranged to 42 DEG C, avoids solution to steam on tube wall; React rear ice bath 5 minutes.

5. method according to claim 1, is characterized in that: in described step (6), the method steps of synthetic Second-strand cDNA is:

A. prepare Second-strand Master Mix, wherein N μ clease-free Water13 μ L, Second-strand B μ ffer Mix5 μ L, Second-strand Enzyme Mix2 μ L, mixes gently, of short duration centrifugal rear ice bath; Getting step reacts in complete sample hose and adds the 20 μ L Second Strand Master Mix that prepare;

B. mix gently, 16 DEG C are reacted 1 hour, 65 DEG C of 10min;

C. reaction is placed in reactant to continue building-up reactions on ice after finishing, or frozen in-20 DEG C rapidly.

6. method according to claim 1, is characterized in that: in described step (7), the method steps of the synthetic cRNA of in-vitro transcription is:

A.cRNA is synthetic: preparation in-vitro transcription Master Mix, wherein N μ clease-free Water4 μ L, T7B μ ffer Mix20 μ L, T7Enzyme Mix6L, mix gently, of short duration centrifugal, up in a reaction, in ready sample hose, add 30 μ L Master Mix;

B. softly mix, 40 DEG C are reacted 16.5 hours;

C., after having reacted, prepare purifying cRNA;

D. purifying, quantitative cRNA.

7. method according to claim 1, is characterized in that: in described step (8), the method steps of cRNA reverse transcription is:

A. get cRNA purified product 5 μ g, adjust volume to 7.5 μ l, join in 0.2ml nuclease free centrifuge tube, add 4 μ l random primers, mix, 65 DEG C are reacted 5 minutes, ice bath 5 minutes;

B. prepare reverse transcription Master Mix, wherein 4 × CbcScript II B μ ffer5 μ L, 0.1M DTT2 μ L, CbcScript II 1.5 μ L, mix gently, of short duration centrifugal; In the sample hose having reacted, add 8.5 μ l Master Mix;

C. mix gently, 25 DEG C are reacted 10 minutes, and 37 DEG C are reacted 1.5 hours;

D. reaction adds Terminate Sol μ tion5 μ l after finishing, and mixes;

A. (5) 65 DEG C are reacted 10 minutes, room temperature 5 minutes;

E. add Ne μ tralize Sol μ tion1 μ l, mix, prepare purifying;

F. purifying, quantitative reverse transcription product.

8. method according to claim 1, is characterized in that: in described step (9), fluorescently-labeled method steps is:

A. cDNA reverse transcription after purifying being obtained is concentrated in vacuo to 14 μ L, and 60 DEG C, 5min, adds 4 μ L random primers, mixes; 95 DEG C are reacted 3 minutes, ice bath 5 minutes;

B. reaction adds 1 μ L Cy3 after finishing;

C. prepare Mix, wherein 5 × Klenow B μ ffer5 μ L, Klenow Fragment1.2 μ L adds 6.2 μ L Mix in above-mentioned mixed solution, mix, wink from, 37 DEG C reaction 1.5 hours, 70 DEG C reaction 5 minutes, ice bath 5 minutes, prepare purifying;

D. purifying, quantitative mark product.

9. method according to claim 1, is characterized in that: in described step (9), the method steps of gene chip hybridization is:

A. the preparation of chip:

A. hydration: selected chip is dried up 10cm left and right on 65 DEG C of water-baths, steams 10-15 second; Dry 10-15 second, then steam 10-15 second;

B. UV-crosslinked: to arrange 250;

C. first pass is washed: 0.5%SDS, and microwave-oven-heating is to 42-45 DEG C, and shaking table 10min, washes away cy3;

D. wash for second time: 42 DEG C of pure water, SDS is removed in rinsing, proceeds to next washing tank for films;

E. wash for the 3rd time: 42 DEG C of pure water, shaking table 1-2min, washes away SDS, dries;

B. the preparation of sample:

A. cy3/cy5 is concentrated into respectively to 19.2 about μ L, in cy3 (cy5), adds hybridization solution 41.6 μ L, then cy5 (cy3) is drawn in cy3 (cy5), mix, concussion, wink from;

B. wherein hybridization solution preparation: methane amide 20 μ L, 20*SSC12 μ L, 10*SDS1.6 μ L, 50*Dehart8 μ L;

DEG C c.95,3min, cuts 1 × 4cm filter paper during this time, puts into chip cartridges, adds 100-180 μ L distilled water, prepares washing lotion 1:600mL, washing lotion 2:1L;

D. take out PCR pipe, quenching, puts on shelf, prepares loading;

E. with rubber pipette bulb blow cores sheet, when application of sample, keep left and put into the straight line of 1.5cm left and right, then with rifle head is auxiliary, cover glass is covered;

F. adjust the neat degree of cover glass with rifle head, put into chip cartridges, put into hybridization instrument, trim, puts a water bottle moisturizing; Arrange: 42 DEG C, 23 hours, 6 turned, start Hyb hybridization, hybridize 23 hours;

C. hybridization finishes to prepare read tablet: after hybridization, develop a film, scanner preheating 10min, selects pocket: power-80, and PMT-800, first sweeps red passage, then sweeps green passage, ensures that saturation point (white point number) is less than 5/1000ths, after having scanned, preserves.

10. method according to claim 1, is characterized in that: the method steps of data analysis is in described step (9): scan image is converted into signal value with L μ xScan3.0 software; Determine genetic expression (P value < 0.05), critical expression (0.05 < P value < 0.065) and do not express (P value > 0.065) according to the P value of probe signals value; Respectively the signal value of 6 chips is done after normalized, by 6 groups respectively with the comparison of blank group, differentiate a certain gene whether occur express change: in the time of signal ratio >=2, be considered as genetic expression raise; In the time of signal ratio≤0.5, be considered as down regulation of gene expression; In the time that ratio falls between, being considered as gene has the expression of biological significance to change; Finally with softwares such as Microsoft Excel, S μ M and Cl μ ster, each group of data are added up and cluster analysis.