CN106755356A - For detecting arthritic mark, genetic chip and preparation method - Google Patents
For detecting arthritic mark, genetic chip and preparation method Download PDFInfo
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- CN106755356A CN106755356A CN201611103637.9A CN201611103637A CN106755356A CN 106755356 A CN106755356 A CN 106755356A CN 201611103637 A CN201611103637 A CN 201611103637A CN 106755356 A CN106755356 A CN 106755356A
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Abstract
The invention provides one kind for detecting arthritic mark, genetic chip and preparation method；The invention provides mark include 1L1 β, C16 orf35 or 1L 8 etc., it is found through experiments that, several genes are expressed in normal person and having differences property of arthritic, specifically, its trend that low up-regulated expression in various degree is presented, therefore, during detection is arthritic, can be by determining the different genes up-regulated expression situation and then may determine that whether the patient is potential arthritic, judge for potential patient provides early warning, to facilitate patient and early treatment, mitigate ailing.
Description
Technical field
The present invention relates to medical domain, in particular to one kind for detect arthritic mark, genetic chip with
And preparation method.
Background technology
Osteoarthritis (OA) is a kind of degenerated joint disease, is related to the institute in joint in a organized way, can in pathological process
It was observed that the structural change of articular cartilage, synovial membrane, subchondral bone and surrounding soft tissue's (muscle, ligament etc.), shows as joint soft
Bone is not normal, and conduction mechanical load is not normal, causes the biomethanics of cartilage and biological chemical environment to change, and cartilage gradually occurs
Degraded qualitative change, final there is thinning cartilage, fibrosis, erosion, crack, the lower ulcer of naked eyes and holostrome articular surface and disappear etc..With
Focused mostly in the destruction of articular cartilage toward the etiological study to OA, increasing research shows, subchondral bone changes in OA hairs
The generation of positive role, i.e. Subchondral bone sclerosis and OA is played during disease, develops closely related, the knot that more than OA occurs
Really, and, the change of subchondral bone is possible to the change prior to articular cartilage.
Articular cartilage sending down the fishbone includes cortical endplate and against bone trabecula below, the lacuna between blood vessel and girder.It is soft
The basic function of bone sending down the fishbone is the shape for absorbing stress, buffering concussion and maintaining joint.In OA cases, subchondral bone often occurs
Hardening, capsule, aseptic necrosis etc. change.Can trigger articular cartilage damage and progressive there are some researches show subchondral bone changes
Deteriorate, Subchondral bone sclerosis can cause cartilage cell's functional disturbance and OA to occur.Genetic chip is by substantial amounts of target fragment
In an orderly manner, it is arranged in high-density on the carriers such as glass, silicon, then sample fluorochrome label is prepared into probe, with chip
Molecule hybridization reaction is carried out, hybridization signal intensities are detected, and through computer analysis and data processing, so as to gene order and work(
Extensive, high speed, high-throughout research can be carried out.Genetic chip is used for existing scholar the research of osteoarthritis, it was recently reported that
The gene expression profile of OA cartilages or bone tissue is studied using genetic chip, it was found that a series of with the osteoarthritis close phase of morbidity
Close.
Osteoarthritis is a kind of degenerated joint disease, is related to the institute in joint in a organized way, the observable in pathological process
To the structural change of articular cartilage, synovial membrane, subchondral bone and surrounding soft tissue's (muscle, ligament etc.), show as articular cartilage and move back
Change, conduction mechanical load is not normal, causes the biomethanics in joint and biological chemical environment to change, the drop that cartilage gradually occurs
Qualitative change is solved, it is final that thinning cartilage, fibrosis, erosion, crack, the lower ulcer of naked eyes and the disappearance of holostrome articular surface etc. occur.It is reported that
English, the U.S. have 5%-10% adult to suffer from gonitis；In China, the investigation of epidemiology finds that Symptomatic OA suffers from
Between 5.1%-20.8%, Chinese OA patient numbers are estimated to exceed 100,000,000 to sick rate.This multi-factor disease is to cause the elderly to ache
The first cause of pain and disability, directly results in serious individual and social economical burden.At present, the treatment of OA still lacks healing
Effective means, conventional treatment method is mainly physiotherapy, medicine etc., is uniquely to control to terminal stage of a disease surgical operation therapy
Treatment method.
Therefore, how early prediction effectively to be carried out in morbidity early stage to Osteoarthritis, and then warn potential patient to enter
Row treatment ahead of time for Osteoarthritis rehabilitation and to mitigate slight illness be very crucial, but now in the art not yet at present
See the molecular biology method to Osteoarthritis early warning, in view of this, spy proposes the present invention.
The content of the invention
The first object of the present invention is to provide a kind of arthritic mark of detection, the mark in arthritic and
Having differences property expression between normal person, and then can be as the mark of detection arthritic.
The second object of the present invention is to provide a kind of described mark in preparing for detecting arthritis product
Using to realize the application in arthritis context of detection.
The third object of the present invention is a kind of genetic chip, the genetic chip by load specific probe sequence and then
Mark in body can be quickly detected from.
The fourth object of the present invention is to provide a kind of preparation method of described genetic chip, the preparation method, simply
Effectively, by after parameter optimization, it is possible to achieve the accurate Detection results of chip.
In order to realize above-mentioned purpose of the invention, spy uses following technical scheme：
The invention provides one kind arthritic mark of detection, the mark includes 1L1- β, and its gene order number is
NM-000576.2。
It is found through experiments that, the gene is expressed in normal person and having differences property of arthritic, specifically, in its presentation
The trend that mileometer adjustment reaches, the multiple of the gene upregulation is compared compared with normal person, and it can reach more than 12 times, therefore, in detection joint
In scorching process, can be by determining the gene upregulation expression and then may determine that whether the patient is potential joint
Scorching patient, judges for potential patient provides early warning, to facilitate patient and early treatment, mitigates ailing.
Optionally, the mark also include C16orf35 or 1L-8, its gene order number be respectively NM-012075.1 and
NM-000584.2。
In addition, being found through experiment, said gene there is also different degrees of difference between arthritic and normal person
Expression, it hands over normal person to compare respectively, and the multiple of up-regulated expression is above more than 6 times (less than 12 times), therefore it can also be single
Solely or by being combined with 1L1- β as the arthritic mark of detection.
In view of above-mentioned mark effect in terms of arthritis is detected, during it is as preparing for detecting arthritis product
Using ought to also belong within protection scope of the present invention, specifically, these products include：By RT-PCR, real-time quantitative
PCR, immune detection, in situ hybridization chip or marker gene described in high-flux sequence detection of platform express to diagnose osteoarthritis
Product.
Present invention also offers a kind of genetic chip, the genetic chip is loaded with being capable of specific detection claim 1
Or the probe fragment of the mark described in 2.The genetic chip has been also loaded being capable of specific detection NM-001356, NM_
031560th, the probe of NM_031757, NP_001102853, NM_001014008 or NM_001007612；6 kinds of genes its
In the presence of the up-regulated expression of at least more than 2 times (less than 6 times) between arthritic and normal person, therefore the gene that the present invention is provided
The probe sequence of these genes can also be loaded with detecting in chip, to pass through the standard for aiding in detection mode to improve testing result
True property.
A kind of preparation method of described genetic chip, comprises the following steps：
1) chip carrier, is prepared；
2), the chip carrier will be fixed on for the oligonucleotide fragment of specific detection mark.
In order to realize that chip is quickly and efficiently prepared, the present invention is optimized to above-mentioned preparation method, optionally, in step
It is rapid 1) in, specifically include：
Empty vectors are cleaned up, and are soaked in the 3- TSL 8330s (volume content) containing 0.5-0.8%
And mass concentration for 90-94% acetone in 5-6 minutes, then cleaned with the acetone (mass concentration) of 95-96% 3-5 times after
50-60 DEG C dries 40-50 minutes, obtains amido modified carrier；
The carrier includes slide or nylon membrane.
Nylon membrane is porous, with permeability, advantage can be to reuse.Make without infiltration on glass substrate, its surface
With sample-adding amount is low, and non-specific hybridization product is easily removed in chip fabrication process, can be with parallel analysis sample.
Optionally, in step 2) in, specifically include：
Amido modified carrier is processed 1.5-1.8 hours with different two thiocyanates (mass concentration) of 0.25-0.4% benzene；Again
With containing 1- [3- (trimethoxy silicon) propyl group] -1- (4- isocyanides phenyl) thiocarbamide that volume content is 1.5-1.8% 95% third
Ketone soaks 5-8 minutes, is then 1 with volume ratio:1 methyl alcohol and acetone is dried during vacuum desiccator is stored in after washing 5-8 minutes；
The oligonucleotide probe fragment for being modified 5 ' Amino End Groups using point sample instrument is fixed on dried carrier.
The slide of above method treatment is good with amido modified probe combination effect.
Compared with prior art, beneficial effects of the present invention are：
(1), can be with the arthritic mark of effective detection, for arthritic early warning detection is provided there is provided one kind
It is convenient；In addition, the mark is also for the clinical research of arthritis detection provides basic data.
(2), there is provided a kind of new tool of arthritis detection, the mark is used for quickly detecting by genetic chip,
Again by software analysis, determine that whether the differential expression situation of these marker gene judges the body with arthritis.
(3), carried out preferably for the preparation method of genetic chip, carrier selection is proper, and has carried out spy to carrier
Different treatment so that in the carrier after point sample, probe can be securely fixed on carrier, improve the using effect of chip.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted preparation manufacturer person of instrument, are
The conventional products that can be obtained by commercially available purchase.
The invention provides one kind arthritic mark of detection, the mark includes that (its gene order number is 1L1- β 9
NM-000576.2), and preferably, the mark can also be C16orf35 or 1L-8, and its gene order number is respectively NM-
012075.1 and NM-000584.2.Above-mentioned several genes exist different degrees of between arthritic and normal person
Otherness up-regulated expression, therefore can be by detecting the up-regulated expression multiple of above-mentioned several genes so as to whether anticipation individuality suffers from
Arthritis.Further, in order to provide facility, present invention also provides the genetic chip that can detect said gene, the base
It is loaded with being capable of the probe fragment of the above-mentioned mark of specific detection because of chip.Preferably, the genetic chip has been also loaded energy
Enough specific detection NM-001356, NM_031560, NM_031757, NP_001102853, NM_001014008 or NM_
001007612 probe.
Next, enumerating specific embodiment by the specific preparation method to genetic chip to illustrate that the application's is detailed
Technical scheme.
Embodiment 1
Prepare chip carrier；
Blank slide carrier is cleaned up, and is soaked in containing 0.5% 3- TSL 8330s and quality is dense
Spend 5 minutes in the acetone for 90%, then 3 times are cleaned with 95% acetone after 50 DEG C of dryings 50 minutes, obtain amido modified
Carrier；
The chip carrier will be fixed on for the oligonucleotide fragment of specific detection mark.
Amido modified slide is processed 1.8 hours with different two thiocyanates of 0.25% benzene；Again with containing 1.5% 1- [3- (three
Methoxyl group silicon) propyl group] -1- (4- isocyanides phenyl) thiocarbamide 95% acetone soak 5 minutes, be then 1 with volume ratio:1 first
Alcohol and acetone are dried during vacuum desiccator is stored in after washing 5 minutes；
The oligonucleotide probe fragment (can specific recognition 1L1- β) for being modified 5 ' Amino End Groups using point sample instrument is fixed on dry
On slide after dry.
Embodiment 2
Blank slide carrier is cleaned up, and is soaked in containing 0.5% 3- TSL 8330s and quality is dense
Spend 5 minutes in the acetone for 90%, then 3 times are cleaned with 95% acetone after 60 DEG C of dryings 50 minutes, obtain amido modified
Carrier；
Amido modified slide is processed 1.8 hours with different two thiocyanates of 0.25% benzene；Again with containing 1.8% 1- [3- (three
Methoxyl group silicon) propyl group] -1- (4- isocyanides phenyl) thiocarbamide 95% acetone soak 8 minutes, be then 1 with volume ratio:1 first
Alcohol and acetone are dried during vacuum desiccator is stored in after washing 8 minutes；
Oligonucleotide probe (can the specific recognition C16orf35 and 1L-8) piece for being modified 5 ' Amino End Groups using point sample instrument
Section is fixed on dried slide.
Embodiment 3
Blank nylon membrane carrier is cleaned up, and is soaked in containing 0.8% 3- TSL 8330s and quality
Concentration be 94% acetone in 6 minutes, then 5 times are cleaned with 96% acetone after 60 DEG C of dryings 40 minutes, obtain amido modified
Carrier；
Amido modified nylon membrane carrier is processed 1.5 hours with different two thiocyanates of 0.4% benzene；Again with containing 1.8% 1-
The acetone soak of the 95% of [3- (trimethoxy silicon) propyl group] -1- (4- isocyanides phenyl) thiocarbamide 5 minutes, is then 1 with volume ratio:
1 methyl alcohol and acetone is dried during vacuum desiccator is stored in after washing 5 minutes；
5 ' Amino End Groups are modified using point sample instrument oligonucleotide probe fragment (can specific detection NM-001356,
NM_031560, NM_031757, NP_001102853, NM_001014008 and NM_001007612) it is fixed on dried glass
On piece.
Embodiment 4
Blank nylon membrane carrier is cleaned up, and is soaked in containing 0.7% 3- TSL 8330s and quality
Concentration be 92% acetone in 5 minutes, then 4 times are cleaned with 95% acetone after 55 DEG C of dryings 45 minutes, obtain amido modified
Carrier；
Amido modified nylon membrane carrier is processed 1.7 hours with different two thiocyanates of 0.3% benzene；Again with containing 1.6% 1-
The acetone soak of the 95% of [3- (trimethoxy silicon) propyl group] -1- (4- isocyanides phenyl) thiocarbamide 7 minutes, is then 1 with volume ratio:
1 methyl alcohol and acetone is dried during vacuum desiccator is stored in after washing 7 minutes；
The oligonucleotide probe fragment (can specificity low identification 1L1- β) for being modified 5 ' Amino End Groups using point sample instrument is fixed on
On dried slide.
Embodiment 5
Blank nylon carrier is cleaned up, and is soaked in containing 0.55% 3- TSL 8330s and quality is dense
Spend 5 minutes in the acetone for 93%, then 5 are cleaned with 95% acetone for several times after 60 DEG C of dryings 50 minutes, obtain amido modified
Carrier；
Amido modified white nylon carrier is processed 1.6 hours with different two thiocyanates of 0.35% benzene；Again with containing 1.8% 1-
The acetone soak of the 95% of [3- (trimethoxy silicon) propyl group] -1- (4- isocyanides phenyl) thiocarbamide 5 minutes, is then 1 with volume ratio:
1 methyl alcohol and acetone is dried during vacuum desiccator is stored in after washing 6 minutes；
5 ' Amino End Groups are modified using point sample instrument oligonucleotide probe fragment (can specific detection 1L1- β,
C16orf35,1L-8, NM-001356, NM_031560, NM_031757, NP_001102853, NM_001014008 and NM_
001007612) it is fixed on dried slide.
Test example
Genechip detection：5 RA patients and 3 healthy volunteers take peripheric venous blood 3ml, ethylenediamine tetra-acetic acid
(EDTA) anti-freezing, lymphocyte separation medium separates PBMC.Trizol methods extract total serum IgE, and RNA is complete for the identification of 1% agarose electrophoresis
Property.
The RNA of extraction is carried out by reverse transcription and linear amplification using Illumina RNA Amplification Kit.
Comprise the following steps that：Each sample takes 200ng RNA, and with RNAase-free water into 11 microlitres of systems, addition 9 is micro-
Reverse transcription mother liquor is risen, synthesizes cDNA.After crossing purification column cleaning purifying cDNA, the cDNA that will be purified adds 10 microlitres of transcription mother liquors,
In-vitro transcription synthesizes cRNA.Cross purification column purifying cRNA.Each sample applied sample amount is 1500ng cRNA, mixed with hybrid mixed liquid
The genetic chip (each patient correspondence one embodiment) of embodiment 1-5,55 DEG C of reaction 16-22h, with envelope after cleaning are added after conjunction
Close buffer blind.Dyeed with Streptavidin-Cy3, reacted chip is scanned.Bead Studio softwares are carried out
Data analysis, selects the rise situation that RA groups organize omparison purpose gene with healthy volunteer.Statistics is as shown in table 1 below.
The testing result of 1 test example of table 1
Seen to find out by result above, in the result between arthritic and normal person, above-mentioned listed several bases
Therefore regularly difference is clearly present between and raises situation.Wherein, there is more than 12 times of rise in 1L1- β genes in patients
Situation, and C16orf35 or 1L-8 then raise more than 6 times, the rise multiple of other genes is below 6 times therefore above-mentioned all
Gene can be as the mark of detection arthritic, testing result is based primarily upon its rise between normal person times
Depending on number, 1L1- β raise more than 12 times；Or C16orf35 or 1L-8 raises more than 6 times, less than 12 times；Or NM-
001356th, NM_031560, NM_031757, NP_001102853, NM_001014008 or NM_001007612 raise 2 times with
On, qualitative by less than 6 times is arthritic, and then for early warning effect is played in the early diagnosis of patient, is further aided
Patient and early treatment, mitigate ailing.
Although illustrate and describing the present invention with specific embodiment, but will be appreciated that without departing substantially from of the invention
Many other changes and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of to detect arthritic mark, it is characterised in that the mark includes 1L1- β, its gene order number is NM-
000576.2。
2. mark according to claim 1, it is characterised in that the mark also includes C16 orf35 or 1L-8, its
Gene order number is respectively NM-012075.1 and NM-000584.2.
3. application of the mark according to claim any one of 1-2 in preparing for detecting arthritis product.
4. application according to claim 3, it is characterised in that the product includes：By RT-PCR, real-time quantitative PCR,
Immune detection, in situ hybridization chip or marker gene described in high-flux sequence detection of platform express to diagnose the product of osteoarthritis
Product.
5. a kind of genetic chip, it is characterised in that the genetic chip is loaded with being capable of the institute of specific detection claim 1 or 2
The probe fragment of the mark stated.
6. genetic chip according to claim 5, it is characterised in that the genetic chip be also loaded can specificity examines
Survey the spy of NM-001356, NM_031560, NM_031757, NP_001102853, NM_001014008 or NM_001007612
Pin.
7. application of the genetic chip according to claim 5 or 6 in preparing for detecting arthritic product.
8. the preparation method of a kind of genetic chip according to claim 6 or 7, it is characterised in that comprise the following steps：
1) chip carrier, is prepared；
2), the chip carrier will be fixed on for the oligonucleotide fragment of specific detection mark.
9. the preparation method of genetic chip according to claim 8, it is characterised in that in step 1) in, specifically include：
Empty vectors are cleaned up, and are soaked in the 3- TSL 8330s and mass concentration containing 0.5-0.8% and are
5-6 minutes in the acetone of 90-94%, then cleaned with the acetone of 95-96% 3-5 times and dried after 50-60 DEG C 40-50 minutes, obtained
To amido modified carrier；
The carrier includes slide or nylon membrane.
10. the preparation method of genetic chip according to claim 9, it is characterised in that in step 2) in, specifically include：
Amido modified carrier is processed 1.5-1.8 hours with different two thiocyanates of 0.25-0.4% benzene；Use again containing 1.5-1.8%
Acetone soak 5-8 minutes of the 95% of 1- [3- (trimethoxy silicon) propyl group] -1- (4- isocyanides phenyl) thiocarbamide, then uses volume ratio
It is 1:1 methyl alcohol and acetone is dried during vacuum desiccator is stored in after washing 5-8 minutes；
The oligonucleotide probe fragment for being modified 5 ' Amino End Groups using point sample instrument is fixed on dried carrier.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110066866A (en) * | 2019-01-17 | 2019-07-30 | 浙江大学 | The biomarker and purposes of the 4th hypotype of osteoarthritis |
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| CN104870012A (en) * | 2012-06-27 | 2015-08-26 | 吉安特科技公司 | Anti-CXCL9, anti-CXCL 10, anti-CXCL 11, anti-CXCL 13, anti-CXCR3 and anti-CXCR5 agents for inflammatory disorder |
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