CN105256039A - Osteoporosis diagnosis marker - Google Patents
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Abstract
The invention discloses a molecular marker which utilizes a PIGK gene to carry out osteoporosis early diagnosis. With the adoption of a method using a gene chip and QPCR (Quantitative Polymerase Chain Reaction), when blood of normal people and blood of osteoporosis patients have remarkable difference, whether tested people have osteoporosis or not can be judged through detecting an expression condition of the PIGK gene in the blood. According to the correlation of the gene chip and the QPCR, the invention develops a kit for diagnosing the osteoporosis; the kit is used for diagnosing the osteoporosis through detecting the expression of the PIGK gene. The diagnosis kit provided by the invention can be used for early diagnosis of diseases and has a wide clinical application prospect.
Description
Technical field
The invention belongs to molecular diagnosis field, relate to a kind of molecular marker for diagnosis of osteoporosis, be specifically related to the application of molecular marker-PIGK gene in the product preparing Diagnosis of osteoporosis in blood.
Background technology
Osteoporosis (osteoporosis, OP) is one group of osteopathy that many reasons causes, and osseous tissue has normal calcification, and calcium salt and matrix are normal rates, and the metabolic osteopathy being reduced to feature with unit volume inner bone tissues amount becomes.In most osteoporosis, caused by the minimizing of osseous tissue increases mainly due to bone absorption.With skeleton pain, be easy to fracture for feature.
Osteoporosis has been classified as the disease of the second largest harm humans health being only second to cardiovascular disorder by the World Health Organization.Promote " 2013 Chinese osteoporotic fracture control blue Book " display of the up-to-date issue of foundation according to China's Healthy, osteoporotic fracture is a kind of fragility fractures, is namely being subject to suffering low-yield wound and generable fracture in minor trauma or daily routines.The common site of osteoporotic fracture is vertebra, hipbone and distal forearm.China more than 50 years old crowd about 6,944 ten thousand people suffers from osteoporosis, and about 2.1 hundred million people's bone amount are on the low side.The regional epidemiology survey display such as Beijing, the morbidity of more than 50 years old women's spinal fracture is 15%, and being equivalent to just has one spinal fracture occurred in every 7 women of more than 50 years old.
The detection of osteoporosis comprises detection and the auxiliary detection of laboratory checking index.
Laboratory checking index:
Patients with osteoporosis part serum studentization index can change (comprising bone forming and bone resorption) state by reactive bone, under the high transformation condition (such as I type osteoporosis) of bone, these indexs can raise, and also can be used for the early response of monitor therapy.But its clinical meaning in osteoporosis still awaits further research.These Biochemistry measurement indexs comprise: the alkaline phosphatase (Bone-specificalkalinephosphatase that bone is special, reactive bone is formed), Tartrate resistant acid phosphatase (tartratedresistantacidphosphatse, reactive bone absorbs), Bone Gla protein (Osteocalcin, reactive bone is formed), I type tropocollagen peptide (TypeIprocollagenpeptidase, reactive bone is formed), Pyridinoline (Urinarypyridinoline) and Deoxypyridinoline (Urinarydeoxypyridinoline, reactive bone absorbs), the N-C-end of NTx is cross-linked peptide (cross-linkedN-andC-telopeptideoftypeIcollagen, reactive bone absorbs).As previously noted, use the tolerance range of biochemical indicator detection osteoporosis inadequate.
Auxiliary examination: comprise bone imaging examination and Bone mineral density.The object of auxiliary examination is all generally the patients with terminal of osteoporosis, can not be used for the examination of early stage patients with osteoporosis.
Based on the limitation of means detecting osteoporosis in prior art, find that a kind of effectively can to get final product the method that Diagnosis of osteoporosis occurs in early days be problem demanding prompt solution.
Summary of the invention
In order to make up the deficiencies in the prior art, one is the object of the present invention is to provide to can be used for the molecular marker of osteoporosis (Osterarthritis, OA) early diagnosis.Compare the diagnostic method of traditional osteoporosis, what use gene marker to carry out Diagnosis of osteoporosis has promptness, specificity and susceptibility, thus make patient just can know disease risks in early days in disease, for risk height, take corresponding prevention and therapy measure.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides the application of product in the instrument preparing Diagnosis of osteoporosis detecting PIGK genetic expression.
Further, the product of the detection PIGK genetic expression mentioned above comprises: by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, chip or high-flux sequence detection of platform PIGK gene expression dose with the product of Diagnosis of osteoporosis.
Further, the product of described RT-PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified PIGK gene; The product of described real-time quantitative PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified PIGK gene; The product of described immunodetection Diagnosis of osteoporosis comprises: the antibody be combined with PIGK protein-specific; The product of described in situ hybridization Diagnosis of osteoporosis comprises: with the probe of the nucleic acid array hybridizing of PIGK gene; The product of described chip Diagnosis of osteoporosis comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with PIGK protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of PIGK gene.
In specific embodiment of the invention scheme, the product of described real-time quantitative PCR Diagnosis of osteoporosis at least comprises the sequence of the primer of a pair specific amplified PIGK gene as shown in SEQIDNO.3 and SEQIDNO.4.
Preferably, described diagnostic tool comprises chip, test kit, test paper or high-flux sequence platform.Wherein, high-flux sequence platform is a kind of special diagnostic tool, and the product detecting PIGK genetic expression can be applied to the detection of this platform realization to the expression of PIGK gene.Along with the development of high throughput sequencing technologies, will become the structure of the gene expression profile of a people and work very easily.By contrasting the gene expression profile of Disease and normal population, easily analyze exception and the disease-related of which gene.Therefore, in high-flux sequence, the exception of the PIGK gene purposes that also belong to PIGK gene relevant to osteoporosis is known, equally within protection scope of the present invention.
Present invention also offers a kind of instrument of Diagnosis of osteoporosis, described diagnostic tool comprises chip, test kit, test paper or high-flux sequence platform.
Wherein, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for PIGK gene for detecting PIGK gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of PIGK albumen of solid phase carrier; Described gene chip can be used for detecting the expression level of the multiple genes (such as, relevant to osteoporosis multiple genes) comprising PIGK gene.Described protein chip can be used for detecting the expression level of the multiple protein (such as relevant to osteoporosis multiple protein) comprising PIGK albumen.By being detected by multiple mark with osteoporosis simultaneously, the accuracy rate of diagnosis of osteoporosis greatly can be improved.
Wherein, described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting PIGK gene transcription level; Described protein immunization detection kit comprises the specific antibody of PIGK albumen.Further, described reagent comprises the reagent used needed in RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip method detection PIGK gene expression dose process.Preference, described reagent comprises primer for PIGK gene and/or probe.The primer and probe that may be used for detecting PIGK gene expression dose is easily designed according to the nucleotide sequence information of PIGK gene.
Can be DNA, RNA, DNA-RNA mosaic, PNA or other derivative with the probe of the nucleic acid array hybridizing of PIGK gene.The length of described probe does not limit, if complete specific hybrid, with object nucleotide sequence specific binding, any length can.The length of described probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150,300 base pairs or longer, even whole gene.Because different probe length has different impacts to hybridization efficiency, signal specificity, the length of described probe is at least 14 base pairs usually, the longlyest generally be no more than 30 base pairs, best with 15-25 base pair with the length of object nucleotide sequence complementary.Described probe self-complementary sequences most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
Described high-flux sequence platform comprises the reagent detecting PIGK gene expression dose.
Described test paper comprises test paper carrier and is fixed on the oligonucleotide on test paper carrier, and described oligonucleotide can detect the transcriptional level of PIGK gene.
Further, the specific antibody of described PIGK albumen comprises monoclonal antibody, polyclonal antibody.The specific antibody of described PIGK albumen comprise complete antibody molecule, any fragment of antibody or modification (such as, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.As long as described fragment can retain the binding ability with PIGK albumen.Well known to a person skilled in the art during preparation for the antibody of protein level, and the present invention can use any method to prepare described antibody.
In specific embodiment of the invention scheme, the described primer sequence for PIGK gene is as follows: forward primer sequence is as shown in SEQIDNO.3, and reverse primer is as shown in SEQIDNO.4.
Include but not limited to that blood, tissue juice, urine, saliva, spinal fluid etc. can obtain the body fluid of genomic dna for the PIGK gene of Diagnosis of osteoporosis and the source of expression product thereof.In specific embodiment of the invention scheme, be blood for the PIGK gene of Diagnosis of osteoporosis and the source of expression product thereof.
In the context of the present invention, " PIGK gene " comprises the polynucleotide of any function equivalent of PIGK gene and PIGK gene.PIGK gene comprises and has more than 70% homology with PIGK gene (NC_000001.11) DNA sequence dna in current international common core sequence databank GeneBank, and coding identical function protein DNA sequence;
Preferably, the encoding sequence of PIGK gene comprises any DNA molecular following:
(1) DNA sequence dna shown in SEQ ID NO.1;
(2) under strict conditions with 1) DNA sequence dna that limits hybridizes and identical function protein DNA sequence of encoding;
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, more than 90% homology, and coding identical function protein DNA molecule.
In specific embodiment of the invention scheme, the encoding sequence of described PIGK gene is the DNA sequence dna shown in SEQIDNO.1.
In the context of the present invention, PIGK gene expression product comprises the partial peptide of PIGK albumen and PIGK albumen.The partial peptide of described PIGK albumen contains the functional domain relevant to osteoporosis.
" PIGK albumen " comprises any function equivalent of PIGK albumen and PIGK albumen.Described function equivalent comprises PIGK albumen conservative variation's protein or its active fragments, or its reactive derivative, allelic variant, natural mutation, induced mutants, can with the protein coded by the DNA of the DNA hybridization of PIGK under high or low stringent condition.
Preferably, PIGK albumen is the protein with following amino acid sequences:
(1) protein be made up of the aminoacid sequence shown in SEQ ID NO.2;
(2) aminoacid sequence shown in SEQIDNO.2 had the protein derivative by the aminoacid sequence shown in SEQIDNO.2 of identical function with the aminoacid sequence shown in SEQIDNO.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.The amino acid whose number replacing, lack or add is generally 1-50, preferably 1-30, and more preferably 1-20,1-10 is individual best.
(3) with the aminoacid sequence shown in SEQIDNO.2, there is at least 80% homology (being also called sequence iden), more preferably, with the aminoacid sequence shown in SEQIDNO.2 at least about 90% to 95% homology, be often 96%, 97%, 98%, 99% homology aminoacid sequence form polypeptide.
In specific embodiment of the invention scheme, described PIGK albumen is the protein with the aminoacid sequence shown in SEQIDNO.2.
Usually, it is known that in a protein one or more amino acid whose modification can not affect the function of protein.Those skilled in the art can approve the amino acid that changes single amino acids or little per-cent or be conservative modifications to indivedual interpolations of aminoacid sequence, disappearance, insertion, replacement, and wherein the change of protein produces the protein with identity function.Intimate amino acid whose Conservative substitution tables is provided to be well known in the art.
By adding the fusion rotein that the example of the protein of an amino acid or multiple Modification of amino acid residues is PIGK albumen.Peptide or protein with PIGK protein fusion are not limited, as long as the fusion rotein of gained retains the biologic activity of PIGK albumen.
PIGK albumen of the present invention also comprises the non-conservative modification to the aminoacid sequence shown in SEQIDNO.2, as long as the protein through modifying still can retain the biologic activity of PIGK albumen.The amino acid number suddenlyd change in this type of modifying protein normally 10 or less, such as 6 or less, such as 3 or less.
In the context of the present invention, " Diagnosis of osteoporosis " had both comprised and had judged whether experimenter has suffered from osteoporosis, also comprised and judge whether experimenter exists the risk suffering from osteoporosis.
Advantage of the present invention and beneficial effect:
Late Cambrian of the present invention PIGK genetic expression is relevant to osteoporosis, by detecting the expression of PIGK in experimenter, can judge whether experimenter suffers from osteoporosis or judge whether experimenter exists the risk suffering from osteoporosis, thus instruct clinicist to provide prevention scheme or treatment plan to experimenter.
Present invention finds a kind of new molecular marked compound-PIGK gene, compare traditional detection means, gene diagnosis more in time, more special, sensitiveer, the early diagnosis of osteoporosis can be realized, thus reduce the mortality ratio of osteoporosis.
Accompanying drawing explanation
Fig. 1 display utilizes the differential expression of genechip detection PIGK gene in patients with osteoporosis and normal people;
Fig. 2 display utilizes QPCR to detect the differential expression of PIGK gene in patients with osteoporosis and normal people.
Concrete embodiment
Below in conjunction with drawings and Examples, the present invention is further detailed explanation.Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1 screens the gene of differential expression in patients with osteoporosis and normal people
1, research object:
Osteoporosis group: randomly draw the 10 routine Osteoporosis that hospital orthopedics is accepted for medical treatment, man, each 5 examples of female, minimum 50 years old of age, maximum 75 years old.Inclusive criteria: meet " Chinese's osteoporosis suggestion Case definition " (the second original text).Cause the various endocrinopathys of secondary osteoporosis without the obvious heart, liver, kidney, pulmonary insufficiency, nothing, get rid of other serious diseases such as tumour, diabetes interference bone metabolism person.
Normal group: choose age 50-75 year healthy volunteer 10 example, men and women each 5 example.
Between two groups, age, gender difference not statistically significant (P>0.10), have comparability.
All research objects are all known the inside story to this research and be endorsed Informed Consent Form.
2, the extraction of total serum IgE in blood
(1) homogenized (Homogenization)
Directly get fresh blood, add 3 times of volume erythrocyte cracked liquids, after mixing, room temperature places 10 minutes, centrifugal 1 minute of 10,000rpm.Supernatant is abandoned in thorough suction, collects leukocyte cell pellet.The leukocyte cell pellet of every 100-200 μ l blood collecting adds 1mlTRIzol.
(2) layering (PhaseSeparation)
A., after sample adds TRIzol, room temperature places 5min, makes the abundant cracking of sample.
B. every 1mlTRIzol adds 200 μ l chloroforms, and after thermal agitation mixing, room temperature is placed 3-5min and made its natural phase-splitting.
(3) RNA precipitation (RNAPrecipitation)
A.4 DEG C 12,000rpm centrifugal 10-15min.Sample can be divided into three layers: yellow organic phase, middle layer and colourless aqueous phase, RNA, mainly in aqueous phase, transfers to aqueous phase (usually can draw 550 μ l) in new pipe.
B. in supernatant, add the ice-cold Virahol of equal-volume, room temperature places 10-20min.4 DEG C of 12,000rpm centrifugal 10min, abandon supernatant, RNA is deposited at the bottom of pipe.
(4) RNA rinsing (RNAWash)
Add 1ml75% ethanol (with the preparation of RNase-free water) in a.RNA precipitation, gentle vibration centrifuge tube, suspend precipitation.Every 1mlTRIzol adds 1ml75% ethanol.
B.4 DEG C 5,000-8,000rpm centrifugal 1-2min, abandon supernatant; Of short duration centrifugal fast, carefully inhale with pipettor and abandon supernatant, room temperature is placed and is dried precipitation in 1-2 minute.
(5) RNA (RedissolvingtheRNA) is dissolved
Add 50-100 μ lRNase-free water in precipitation, flick tube wall, fully to dissolve RNA ,-70 DEG C of preservations.
3, RNA quality and purity detecting
RNA quality: represented by RNA integrity, available plain agar sugar gel electrophoresis (deposition condition: 1.2% glue; 0.5 × TBE electrophoretic buffer; 150v, 15 minutes) detect integrity.
RNA purity: OD260/OD280 ratio is the index weighing protein contamination degree in RNA sample.High-quality RNA sample, OD260/OD280 value (10mMTris, pH7.5) is about 2.0.
4, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP marks, and the cRNAs after fluorescent mark adopts RNEASYMiniKit purifying, carries out fragmentation process with the RNAFragmentationReagents of Amhion to the cRNAs marked.Adopt people's full genome chip of expression spectrum (4x44K gene) of Agilent company of the U.S., in chip hybridization stove, 65 DEG C of hybridization 17h, then wash-out, dyeing, finally use AgilentDNAMicroarrayScanner scanner scanning.
5, chip data treatment and analyses
Chip after hybridization after chip scanner reading of data point, by data importing analysis software, the natural logarithm absolute value for two groups of ratios be greater than 2.0 or be less than 0.5 gene as difference expression gene.
6, statistical procedures
Adopt SPSS13.0 statistical software to carry out data analysis, group difference compares employing one-way analysis of variance method, and P<0.05 difference has significant.
7, result
Result display (as shown in Figure 1), compared with normal people, in patients with osteoporosis blood, the mRNA level in-site of PIGK gene significantly raises, and difference has statistical significance (P<0.05).
The gene of differential expression in embodiment 2QPCR experimental verification patients with osteoporosis and normal people
1, research object:
Osteoporosis group: randomly draw the 50 routine Osteoporosis that hospital orthopedics is accepted for medical treatment, man, each 25 examples of female, minimum 50 years old of age, maximum 75 years old.Inclusive criteria: meet " Chinese's osteoporosis suggestion Case definition " (the second original text).Cause the various endocrinopathys of secondary osteoporosis without the obvious heart, liver, kidney, pulmonary insufficiency, nothing, get rid of other serious diseases such as tumour, diabetes interference bone metabolism person.
Normal group: choose age 50-75 year healthy volunteer 46 example, men and women each 23 example.
Between two groups, age, gender difference not statistically significant (P>0.10), have comparability.
All research objects are all known the inside story to this research and be endorsed Informed Consent Form.
2, the extraction of total serum IgE in blood
Step is with embodiment 1.
3, reverse transcription
With RT Buffer, reverse transcription synthesis cDNA is carried out to l μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, adds following component respectively: DEPC water in PCR pipe, 5 × RT Buffer, 10mmol/LdNTP, 0.1mmol/lDTT, 30 μm of mol/lOligodT, 200U/ μ lM-MLV, template ribonucleic acid.Hatch 1h for 42 DEG C, 72 DEG C of 10min, of short duration centrifugal.
4、QPCR
(1) design of primers
According to the encoding sequence design QPCR amplimer of PIGK gene and GAPDH gene in Genbank, synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
PIGK gene:
Forward primer is 5 '-CAGTCACATTGTCCTAAT-3 ' (SEQIDNO.3);
Reverse primer is 5 '-CTCGTAACTTCTATAATCCA-3 ' (SEQIDNO.4),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQIDNO.5);
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQIDNO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBRGreen polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction conditions: 95 DEG C of 10min, (95 DEG C of 10s, 60 DEG C of 40s) * 42 circulation.Using SYBRGreen as fluorescent marker, in the enterprising performing PCR reaction of LightCycler quantitative real time PCR Instrument, by melt curve analysis analysis and electrophoresis determination object band, Δ Δ CT method carries out relative quantification.
5, statistical method
Result data is all represent in the mode of mean+SD, adopts SPSS13.0 statistical software to carry out statistical study, and difference between the two adopts t inspection, thinks to have statistical significance as P<0.05.
6, result
As shown in Figure 2, compared with normal people, in patients with osteoporosis blood, the mRNA level in-site of PIGK gene significantly increases result, and difference has statistical significance (P<0.05), result is homogenic array experiment.
The explanation of above-described embodiment is just for understanding method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also will fall in the protection domain of the claims in the present invention.
Claims (10)
1. detect the application of product in the instrument preparing Diagnosis of osteoporosis of PIGK genetic expression.
2. application according to claim 1, it is characterized in that, described product comprises: by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, chip or high-flux sequence detection of platform PIGK gene expression dose with the product of Diagnosis of osteoporosis.
3. application according to claim 2, is characterized in that, the product of described RT-PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified PIGK gene; The product of described real-time quantitative PCR Diagnosis of osteoporosis at least comprises the primer of a pair specific amplified PIGK gene; The product of described immunodetection Diagnosis of osteoporosis comprises: the antibody be combined with PIGK protein-specific; The product of described in situ hybridization Diagnosis of osteoporosis comprises: with the probe of the nucleic acid array hybridizing of PIGK gene; The product of described chip Diagnosis of osteoporosis comprises: protein chip and gene chip; Wherein, protein chip comprises the antibody be combined with PIGK protein-specific, and gene chip comprises the probe with the nucleic acid array hybridizing of PIGK gene.
4. application according to claim 3, is characterized in that, the primer of a pair specific amplified PIGK gene that the product of described real-time quantitative PCR Diagnosis of osteoporosis at least comprises is as shown in SEQIDNO.3 and SEQIDNO.4.
5. for an instrument for Diagnosis of osteoporosis, it is characterized in that, described instrument can carry out Diagnosis of osteoporosis by the expression detecting PIGK gene in sample.
6. instrument according to claim 5, is characterized in that, described instrument comprises chip, test kit, test paper or high-flux sequence platform.
7. instrument according to claim 6, is characterized in that, described chip comprises gene chip, protein chip; Described gene chip comprises solid phase carrier and is fixed on the oligonucleotide probe of solid phase carrier, and described oligonucleotide probe comprises the oligonucleotide probe for PIGK gene for detecting PIGK gene transcription level; Described protein chip comprises solid phase carrier and is fixed on the specific antibody of PIGK albumen of solid phase carrier; Described test kit comprises gene detecting kit and protein immunization detection kit; Described gene detecting kit comprises the reagent for detecting PIGK gene transcription level; Described protein immunization detection kit comprises the specific antibody of PIGK albumen; Described test paper comprises the reagent for detecting PIGK gene transcription level; Described high-flux sequence platform comprises the reagent for detecting PIGK gene transcription level.
8. instrument according to claim 7, is characterized in that, the reagent of described detection PIGK gene transcription level comprises primer for PIGK gene and/or probe.
9. instrument according to claim 8, its spy is characterised in that, the described primer sequence for PIGK gene is as follows: forward primer sequence is as shown in SEQIDNO.3, and reverse primer is as shown in SEQIDNO.4.
10. the instrument according to any one of claim 5-9, is characterized in that, described sample is blood.
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