CN103966348A - Application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA)or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and kit thereof - Google Patents

Application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA)or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and kit thereof Download PDF

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CN103966348A
CN103966348A CN201410233905.3A CN201410233905A CN103966348A CN 103966348 A CN103966348 A CN 103966348A CN 201410233905 A CN201410233905 A CN 201410233905A CN 103966348 A CN103966348 A CN 103966348A
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cystatin
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cst4mrna
cst1mrna
bladder cancer
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CN103966348B (en
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王弢
渠香云
何林富
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Jiangsu is the real biopharmaceutical technology Limited by Share Ltd
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SHANGHAI DUWEI MEDICAL TECHNOLOGY Co Ltd
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Abstract

The invention discloses an application of CST1mRNA (Cystatin 1 Messenger RNA) and CST4mRNA (Cystatin 4 Messenger RNA) or proteins coded by CST1mRNA and CST4mRNA in preparation of bladder cancer markers and a kit thereof. When the CST1mRNA and the CST4mRNA or the proteins coded by the CST1mRNA and the CST4mRNA are combined and used for diagnosing and predicting bladder cancer, the specificity and sensitivity are higher than those of one marker therein. The invention further discloses the kit for detecting the markers. The kit is convenient in use, can be used for directly collecting urine for detecting without collecting serum so as to reduce the sampling pain of patients, has the characteristics of good specificity and high sensitivity, and can be used for bladder cancer diagnosis, curative effect assessment in the treatment process and metastasis recurrence monitoring after treatment so as to provide a guidance for advanced intervention of doctors.

Description

Application and the test kit thereof of the protein of CST1mRNA and CST4mRNA or its coding in preparation bladder cancer mark
Technical field
The invention belongs to diagnostic field, be specifically related to Cystatin SN and the Cystatin S application in preparation bladder cancer mark, also relate to the test kit of diagnosing bladder cancer.
Background technology
Bladder cancer is the modal malignant tumour of urinary system, is also one of whole body ten large kinds of tumor.Account for first of China's urogenital neoplasm sickness rate, and its sickness rate is only second to prostate cancer in west, occupies the 2nd.It is 6.61/10 ten thousand that national tumour in 2012 is registered the sickness rate of regional bladder cancer, the 9th of row Cancer Mortality.Bladder cancer can betide any age, even children.Its sickness rate increases with age growth, 50～70 years old age occurred frequently.Along with China's aging process is accelerated, bladder cancer sickness rate presents the trend rising year by year.In domestic big city as Beijing, Shanghai, Tianjin, the sickness rate of bladder cancer has ranked the 6th of male sex's common cancer, and mortality ratio is ranked the 7th.
Cysteine proteinase inhibitor C ystatin SN and Cystatin S are mankind Cystatin family members, respectively by CST1 gene and CST4 genes encoding.Cystatin SN and Cystatin S are typical secretory protein, are distributed in Fluids and secretions, as tears, saliva, serum, blood plasma etc., can be used as markers for breast cancer.And CST1 and the CST4 expression amount in gastrointestinal tumor tissue is higher than healthy tissues, and the expression of CST1 and CST4 is relevant to invasive depth, distant metastasis and neoplasm staging (TNM analysis); Survival analysis shows, 5 years survival rates of CST1 and CST4 high expression level patient are significantly higher than without expression group; Cox regression analysis shows, CST1 and CST4 are independent prognostic factors; Thereby prompting CST1 and CST4 may bring into play the effect of similar tumor-inhibiting factor in the generation of gastrointestinal tumor, evolution.But have no up to now, the report of CST1 and CST4 and bladder cancer dependency.
Summary of the invention
In view of this, one of object of the present invention is to provide CST1mRNA and the combined utilization of CST4mRNA in preparation diagnosis and indication bladder cancer mark, by CST1mRNA and CST4mRNA joint-detection, improves the specificity of diagnosis and indication bladder cancer; The advantages such as two of object of the present invention is to provide CST1mRNA and CST4mRNA combined detection kit, and it is high that this test kit has specificity, easy to use; Three of object of the present invention is to provide Cystatin SN and the combined utilization of Cystatin S in preparation diagnosis and indication bladder cancer mark.
For achieving the above object, the invention provides following technical scheme:
1.CST1mRNA and the CST4mRNA combined utilization in preparation diagnosis and indication bladder cancer mark, the nucleotide sequence of described CST1mRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of described CST4mRNA is as shown in SEQ ID NO.2.
Preferably, the judgment formula of CST1mRNA and CST4mRNA combined utilization is P=exp (1.341+0.005a-0.001b)/[1+exp (1.341+0.005a-0.001b)], wherein a=CST1 copy number, b=CST4 copy number.
Preferably, described diagnosis and be shown in advance diagnosis, curative effect evaluation or transfer and relapse monitoring.
2, CST1mRNA and CST4mRNA combined detection kit, comprises the quantitative detecting reagent of CST1mRNA and CST4mRNA; Described CST1mRNA quantitative detecting reagent comprises detection primer and the TaqMAN probe of nucleotide sequence as shown in SEQ ID NO.20 as shown in SEQ ID NO.7 and SEQ ID NO.8; Described CST4mRNA quantitative detecting reagent comprises detection primer and the TaqMAN probe of nucleotide sequence as shown in SEQ ID NO.22 as shown in SEQ ID NO.9 and SEQ ID NO.10.
Preferably, described test kit also comprises SDHA mRNA quantitative detecting reagent, and described SDHA mRNA quantitative detecting reagent comprises detection primer and the TaqMAN probe of nucleotide sequence as shown in SEQ ID NO.24 as shown in SEQ ID NO.11 and SEQ ID NO.12.
Preferably, described test kit also comprises the magnetic bead of the special CST1mRNA of catching, CST4mRNA and SDHA mRNA, the magnetic bead of the described special CST1mRNA of catching is combined with the probe as shown in SEQ ID NO.4, the magnetic bead of the described special CST4mRNA of catching is combined with the probe as shown in SEQ ID NO.5, and the magnetic bead of the described special SDHA of catching mRNA is combined with the probe as shown in SEQ ID NO.6.
Preferred, the concentration that target sequence is caught magnetic bead in liquid is 500 μ g/mL, and the concentration of special capture probe is 2 μ M.
Preferably, described test kit also comprises 10 × Buffer, dNTP, MgCl 2, DMSO, DTT, Taq enzyme, UDG, reversed transcriptive enzyme and RNA enzyme inhibitors.
3, Cystatin SN and the Cystatin S combined utilization in preparation diagnosis and indication bladder cancer mark, the nucleotide sequence of coding Cystatin SN is as shown in SEQ ID NO.1, and the nucleotide sequence of coding Cystatin S is as shown in SEQ ID NO.2.
Preferably, the judgment formula of Cystatin SN and Cystatin S combined utilization is P=exp (43.523+0.272a+0.027b)/[1+exp (43.523+0.272a+0.027b)], wherein a=Cystatin SN concentration, b=Cystatin S concentration.
Preferably, described diagnosis and be shown in advance diagnosis, curative effect evaluation or transfer and relapse monitoring.
Beneficial effect of the present invention is: the new mark that the invention discloses diagnosis and indication bladder cancer, be CST1mRNA and CST4mRNA or its coded protein, utilize two markers in detecting specificity and highly sensitive in use one of them mark; The invention also discloses the test kit of diagnosis and indication bladder cancer mark, this test kit is easy to use, can directly collect urine detects, do not need to gather serum, reduce patient's sampling misery, and have the good He highly sensitive feature of specificity, its diagnostic result is consistent with clinical diagnosis result, in monitor procedure, can also find early transfer and relapse situation, provide guidance for doctor intervenes in advance.
Brief description of the drawings
In order to make object of the present invention, technical scheme and beneficial effect clearer, the invention provides following accompanying drawing:
Fig. 1 is that paramagnetic particle method is from Urinary Specific enrichment mRNA schematic diagram.
Fig. 2 is specific probe and non-specific probe enrichment CST1 genetic comparison result.
Fig. 3 is specific probe and non-specific probe enrichment CST4 genetic comparison result.
Fig. 4 is specific probe and non-specific probe enrichment SDHA genetic comparison result.
Fig. 5 is the ratio result figure of Bladder Cancer and the CST1 of cancer beside organism relative expression quantity.
Fig. 6 is the ratio result figure of Bladder Cancer and the CST4 of cancer beside organism relative expression quantity.
Fig. 7 is CST1 absolute quantitation typical curve.
Fig. 8 is CST4 absolute quantitation typical curve.
Fig. 9 is the CST1 gene content result figure that detects 97 routine samples.
Figure 10 is the CST4 gene content result figure that detects 97 routine samples.
Figure 11 is experimenter CST1 gene ROC curve.
Figure 12 is experimenter CST4 gene ROC curve.
Figure 13 is experimenter CST1 gene and CST4 gene joint-detection ROC curve.
Figure 14 is that ELISA detects Cystatin SN at Bladder Cancer and cancer beside organism's expression (1-T and 2-T represent Bladder Cancer, and 1-N and 2-N represent cancer beside organism).
Figure 15 is that ELISA detects Cystatin S at Bladder Cancer and cancer beside organism's expression (1-T and 2-T represent Bladder Cancer, and 1-N and 2-N represent cancer beside organism).
Figure 16 is Cystatin SN protein standard curve.
Figure 17 is Cystatin S protein standard curve.
Figure 18 is the ROC curve of the independent detection of Cystatin SN and Cystatin S mark and joint-detection.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition, for example, condition described in molecular cloning experiment guide (third edition, the work such as J. Pehanorm Brooker), or the condition of advising according to manufacturer.
Embodiment 1, paramagnetic particle method are from the mRNA of Urinary Specific enrichment CST1, CST4 and SDHA gene
According to CST1, the mRNA sequences Design of CST4 and SDHA is caught CST1, (nucleotide sequence of CST1mRNA is as shown in SEQ ID NO.1 for the specific probe of CST4 and SDHA gene mRNA, the nucleotide sequence of CST4mRNA is as SEQ ID NO.2, the nucleotide sequence of SDHA mRNA is as SEQ ID NO.3), specific as follows: the probe of catching CST1mRNA is 5 '-aaagagcacaactgtttcttctgca (dA) 30-3 ' (SEQ ID NO.4), the probe of catching CST4mRNA is 5 '-taccaggtctattagaagca (dA) 30-3 ' (SEQ ID NO.5), the probe of catching reference gene SDHA (ubiquinone reductase) mRNA is 5 '-ggagcgaatggctggcgggacg (dA) 30-3 ' (SEQ ID NO.6), above-mentioned specific probe can with magnetic bead (GE, article No. is 3815-2103-010150) the complementary combination of olig (dT), must be in conjunction with the magnetic bead of specific probe.
The mRNA of enrichment CST1, CST4 and RPN1 gene, concrete steps are as follows: freshly voided urine is preserved to the ratio that liquid is 2:1 according to volume ratio with sample transport and mix, obtain urine specimen after treatment, under 4 DEG C of conditions of this urine specimen after treatment, can preserve one week, under-20 DEG C of conditions, can preserve 1 year; Then the target sequence that respectively 200 μ L is contained to magnetic bead is caught liquid and is joined in 200 μ L urine after treatment, under 75 DEG C of conditions, processes 5 minutes; After vortex mixes, room temperature leaves standstill 15 minutes; Then sample is placed on magnetic separator, after 5 minutes, inhales and abandon supernatant, add 1mL rinsing liquid, vortex mixes, and upper magnetic separator, after 5 minutes, is inhaled and abandoned supernatant; Last room temperature (18～25 DEG C) leaves standstill 5 minutes, adds elutriant 20 μ L, and the piping and druming of rifle head mixes, and upper magnetic frame 5 minutes, is transferred to supernatant in new EP pipe, and enrichment principle as shown in Figure 1.
In enrichment process, in sample transport preservation liquid, each concentration of component is as follows: 110mM LiDS (lithium dodecyl sulfate), 10mM NaH 2pO 4, 10mM Na 2hPO 4, 5mM EDTA, 7mM EGTA, pH7.5; It is as follows that target sequence is caught in liquid each concentration of component: 135mM HEPES, 1.25M LiCl, 110mM LiOH, 10mM EDTA, pH7.0,500 μ g/mL magnetic beads, 2 μ M capture probes; In rinsing liquid, each concentration of component is as follows: 100mM HEPES, 350mM NaCl, 10mM NaOH, 2mM EDTA, 3% ethanol, 0.2% hydroxy methyl, 0.1% hydroxy propyl ester, 0.1%SDS, pH7.5; In elutriant, each concentration of component is as follows: 20mM Tris-HCl, pH7.5,1mM EDTA.
Press above-mentioned identical method unspecific enrichment mRNA with non-specific probe oligo (dT) simultaneously.
The mRNA that above-mentioned enrichment is obtained detects respectively the relative content of CST1mRNA, CST4mRNA and SDHAmRNA by quantitative PCR, detect the primer using as follows:
CST1 detects primer: upstream primer: 5 '-agagccaggcaacagacc-3 ' (SEQ ID NO.7)
Downstream primer: 5 '-gttcatggaaggcacagg-3 ' (SEQ ID NO.8)
CST4 detects primer: upstream primer: 5 '-atgaacagccagaactgca-3 ' (SEQ ID NO.9)
Downstream primer: 5 '-caagaaggaaggagggag-3 ' (SEQ ID NO.10)
SDHA detects primer: upstream primer: 5 '-attactccaagcccatcc-3 ' (SEQ ID NO.11)
Downstream primer: 5 '-gcacagtcagcctcgttc-3 ' (SEQ ID NO.12)
Then build following detection system: SYBR Green2 × Mix10 μ L (be purchased from Japan and spin (Shanghai) bio tech ltd), final concentration is respectively upstream primer and the downstream primer of 250nM, and template 2 μ L, add ddH 2volume is supplemented to 20 μ L by O.
And detect by following condition: first denaturation 5 minutes, then, in 95 DEG C of sex change 10 seconds, 15 seconds, 72 DEG C extensions of 60 DEG C of annealing 20 seconds, carry out 45 circulations; Finally melt: 95 DEG C, 1 minute; 40 DEG C, 1 minute; 65 DEG C, 1 second; 95 DEG C, 1 minute, cooling 50 DEG C, 30sec.
Simultaneously taking the mRNA of oligo (dT) unspecific enrichment for contrasting, detecting primer, system and testing conditions carries out as stated above, then compare CST1, CST4 and SDHA gene mRNA CP value (Cross Point), result as in Figure 2-4.
From Fig. 2-4, use the CP value of the specific probe CST1mRNA, the CST4mRNA that are enriched to and SDHA mRNA less, show that the concentration of its enrichment is higher, there is background low, the feature that signal to noise ratio is high, is better than using the mRNA of non-specific probe enrichment.
Embodiment 2, detection CST1 and CST4 expression in Bladder Cancer
From Shanghai, the 5th the People's Hospital Urology Surgery is collected bladder cancer, the other pairing of cancer tissue samples 30 examples, is switched to grain of rice size, puts to RNAlater and preserves-80 DEG C of storages in liquid, uses forward horizontal stand to room temperature.Then according to the method for embodiment 1 enrichment bladder cancer and other CST1mRNA, CST4mRNA and the SDHA mRNA organizing that match of cancer respectively, detect primer, CST4 detection primer and reference gene SDHA detection primer with CST1 respectively again and detect CST1 gene and CST4 gene relative expression quantity in 30 routine sample bladder cancer and the other pairing tissue of cancer, detection system and testing conditions are identical with embodiment 1.Detected result adopts 2 -Δ Δ CPmethod is calculated relative expression quantity, then adds up other pairing of bladder cancer and cancer and organizes the ratio (C/N) of relative expression quantity, and statistics as shown in Figure 5 and Figure 6.From Fig. 5 and Fig. 6, CST1 gene and CST4 gene obviously raise in Bladder Cancer, and apparently higher than the expression amount of cancer beside organism, the above results indication detects CST1mRNA and CST4mRNA content can be for diagnosing bladder cancer.
Embodiment 3, structure bladder cancer detection kit
1, build CST1 recombinant plasmid
Taking bladder cancer cell line T24 as the total mRNA of material extraction bladder cancer cell line T24, then taking the mRNA that extracts as the synthetic cDNA of template, and according to the primer of CST1 gene order design construction CST1 recombinant plasmid, upstream primer is 5 '-ctggagccccaaggagga-3 ' (SEQ ID NO.13), downstream primer is 5 '-accagtccaggggtggga-3 ' (SEQ ID NO.14), taking Nucleotide shown in SEQ ID NO.13 and SEQ ID NO.14 as primer, synthetic cDNA is that template is carried out pcr amplification, and amplification condition is: 94 DEG C of sex change 5 minutes; 30 seconds, 72 DEG C extensions of 30 seconds, 60 DEG C annealing of 94 DEG C of sex change 1 minute, carry out 45 circulations; 72 DEG C are extended 5 minutes, and 4 DEG C cooling.Amplified production is connected with pTZ57R carrier, builds CST1 recombinant plasmid, and send the order-checking of order-checking mechanism by recombinant plasmid, the nucleotide sequence that order-checking shows amplified production is as shown in SEQ IDNO.15.This sequence is identical with theoretical amplicon sequence, then by CST1 recombinant plasmid glycerine conservation, as the standard substance that detect CST1 gene.
2, build CST4 recombinant plasmid
According to the primer of CST4 gene order design construction CST4 recombinant plasmid, upstream primer is 5 '-tctgaggagaccatggcc-3 ' (SEQ ID NO.16), downstream primer is 5 '-tgtaccaggtctattagaagcaag-3 ' (SEQ ID NO.17), then taking the Nucleotide of SEQ ID NO.16 and SEQ ID NO.17 as primer, synthetic cDNA is that template is carried out pcr amplification, and amplification condition is: 94 DEG C of sex change 5 minutes; 30 seconds, 72 DEG C extensions of 30 seconds, 60 DEG C annealing of 94 DEG C of sex change 1 minute, carry out 45 circulations; 72 DEG C are extended 5 minutes, and 4 DEG C cooling.Amplified production is connected with pTZ57R carrier, builds CST4 recombinant plasmid, and send the order-checking of order-checking mechanism by recombinant plasmid, the nucleotide sequence that order-checking shows amplified production is as shown in SEQ ID NO.18.This sequence is identical with theoretical amplicon sequence, then by CST4 recombinant plasmid glycerine conservation, as the standard substance of CST4 gene.
By CST1 recombinant plasmid and CST4 recombinant plasmid, by double digestion (EcoR I, BamH I) linearizing target sequence, rubber tapping is reclaimed and is obtained pure linearizing fragment respectively.Use in-vitro transcription test kit, taking linearizing sequence as template, under the effect of t7 rna polymerase, prepare RNA, after RNA purification kit purifying, obtain RNA standard substance storage liquid, measure concentration.Make RNA integrity analysis through Agilent2100, electrophoretogram shows except target sequence significantly mixes and is with and RNA degraded band without other, can be used as RNA standard substance storage liquid, prepares against downstream use.
The typical curve of embodiment 4, drafting CST1 and CST4
The mRNA standard substance that embodiment 3 is obtained are done as the gradient dilution of table 1 and table 2, specific as follows:
Table 1, CST1RNA standard substance dilution gradient
Standard substance numbering Concentration (copy/ μ L)
STD1 100000
STD2 10000
STD3 1000
STD4 100
Table 2, CST4RNA standard substance dilution gradient
Standard substance numbering Concentration (copy/ μ L)
STD1 100000
STD2 10000
STD3 1000
STD4 100
[0068]primer and the probe of design detection by quantitative CST1 gene and CST4 gene, specific as follows:
CST1 detection by quantitative primer is as shown in SEQ ID NO.7 and SEQ ID NO.8, and amplicon sequence is as shown in SEQ ID NO.19, and probe is: FAM-5 '-tacttcttcgacgtagaggtgggcc-3 '-TAMRA (SEQ ID NO.20);
CST4 detection by quantitative primer is as shown in SEQ ID NO.9 and SEQ ID NO.10, and amplicon sequence is as shown in SEQ ID NO.21, and probe is FAM-5 '-aacagttgtgctctttcgagatcta-3 '-TAMRA (SEQ ID NO.22).
SDHA detection by quantitative primer is as shown in SEQ ID NO.11 and SEQ ID NO.12, and amplicon sequence is as shown in SEQ ID NO.23, and probe is FAM-5 '-caacagaagaagccctttgagg-3 '-TAMRA (SEQ ID NO.24).
Then adopt the single stage method RT-PCR concentration of CST1RNA and CST4RNA in table 1 and table 2 respectively, detection system is 2 μ L10 × Buffer, 3 μ L2.5mM dNTP, 2 μ L25mM MgCl 2, 0.75 μ L concentration is the CST1 of 10 μ m or the upstream and downstream primer of CST4,0.5 μ L concentration is SEQ ID NO.20 or the SEQ ID NO.22 probe of 10 μ m, DMSO analytical pure 1 μ L; 10mMDTT1 μ L; 0.1 μ LRoche HS TAQ (article No. 12032953001), 0.1 μ L UDG (purchased from NEB company, article No. EN0362); 0.1 μ L reversed transcriptive enzyme and 0.1 μ L RNA enzyme inhibitors (its composition uses Thermo reverse transcription test kit, article No. K1622), sterilizing purified water 8.6 μ L, reaction conditions is: 37 DEG C, 5 minutes; 50 DEG C, 15 minutes; 94 DEG C, 5 minutes; 94 DEG C, 10 seconds, 60 DEG C, 30 seconds, 45 circulations, last 50 DEG C cooling 30 seconds, then, according to detected result drawing standard curve, result is as shown in Figure 7 and Figure 8.
Embodiment 5, CST1 are or/and the specificity of CST4 gene test and sensitivity
Collect urine specimen 100 examples, wherein transitional cell bladder carcinoma 50 examples, benign lesion Urine in Patients 50 examples such as bladder inflammation from BJ Union Hospital's urology department.According to the method for embodiment 1 enrichment CST1mRNA, CST4mRNA and SDHA mRNA respectively, then by a step TRAP of embodiment 4, the sample of enrichment SDHA mRNA is detected, present " S " type with SDHA amplification curve, and CP value is less than 35 sample for interpretation of result.Analytical results shows in 100 routine samples wherein usable samples 97 examples, bladder cancer 49 examples, benign lesion 48 examples.The CST1mRNA and the CST4mRNA content that continue to detect by a step TRAP of embodiment 4 again 97 routine usable samples, its detection method is identical with embodiment 4.Detected result respectively as shown in Figure 9 and Figure 10.Result demonstration, in 49 routine bladder cancer samples, the median of CST1 gene copy number is that the median of 602, CST4 gene copy number is 305; In 48 routine benign lesion samples, the median of CST1 gene copy number is that the median of 134, CST4 gene copy number is 101.Draw experimenter's performance curve (ROC curve) according to detected result, as shown in Figure 11 and Figure 12.。Result demonstration, when CST1 gene and CST4 gene detect separately, area under curve is respectively 0.803 and 0.653, cutoff value is got 167copy/ μ L, when 159.5copy/ μ L, diagnostic sensitivity is respectively 83.7% and 71.4%, and specificity is respectively 77.1% and 60.4%.According to detecting gained CST1mRNA and CST4mRNA copy number, in the situation that specificity reaches 75%, application Logistic regression and statistical method draws the judgment formula of CST1mRNA and CST4mRNA joint-detection: P=exp (1.341+0.005a-0.001b)/[1+exp (1.341+0.005a-0.001b)] (a=CST1 copy number, b=CST4 copy number), when P is more than or equal to 0.75, positive; When P is less than 0.75, negative.Then draw joint-detection performance curve (ROC curve), as shown in figure 13.Result demonstration, joint-detection area under curve is 0.846, and diagnostic sensitivity is 70.9%, and specificity is 97.9%.Therefore, adopt the specificity of CST1 and CST4 joint-detection bladder cancer higher.In order to obtain higher specificity, the mark using CST1mRNA and CST4mRNA as diagnosing bladder cancer, and build CST1 and CST4 combined detection kit with this, it comprises following component:
CST1mRNA detection by quantitative primer and TaqMAN probe, its primer is respectively as shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of TaqMAN probe is as shown in SEQ ID NO.17;
CST4mRNA detection by quantitative primer and TaqMAN probe, its primer sequence is respectively as shown in SEQ ID NO.9 and SEQ ID NO.10, and the nucleotide sequence of TaqMAN probe is as shown in SEQ ID NO.19;
SDHA mRNA detection by quantitative primer and TaqMAN probe, its primer sequence is respectively as shown in SEQ ID NO.11 and SEQ ID NO.12, and the nucleotide sequence of TaqMAN probe is as shown in SEQ ID NO.21;
Other conventional reagent that also comprise detection by quantitative, comprising: 10 × Buffer, dNTP, MgCl 2, DMSO, DTT, the reagent of Taq enzyme, UDG, reversed transcriptive enzyme and RNA enzyme inhibitors and paramagnetic particle method enrichment CST1mRNA, CST4mRNA and SDHAmRNA.
Embodiment 6, CST1 and CST4 combined detection kit diagnosing bladder cancer
Collect carcinoma of urinary bladder fluid samples 30 examples, benign lesion sample 30 examples from Shanghai No. 5 People's Hospital.Urine is adopted to method enrichment CST1mRNA, CST4mRNA and the SDHA mRNA of embodiment 1, present " S " type with SDHA amplification curve, and sample employing CST1 and CST4 combined detection kit detection CST1mRNA and CST4mRNA relative expression quantity that CP value is less than 35, and positive and negative according to the judgment formula judgement of CST1 and CST4 joint-detection, according to clinical analysis diagnosis, result is as shown in table 3 simultaneously.
Table 3, CST1 and CST4 combined detection kit diagnosing bladder cancer result
Add up detected result and clinical diagnosis results relevance with χ 2, result shows, P < 0.05, show that CST1 and CST4 joint-detection bladder cancer and clinical diagnosis result have dependency, and consistence is better, and Kappa value is 86.7%.
Embodiment 7, CST1 and CST4 combined detection kit assessment bladder cancer curative effect
Urine from 10 routine bladder cancer patients treatments are got by Jiangsu Prov. Tumour Hospital, with method enrichment CST1mRNA, CST4mRNA and the SDHA mRNA of embodiment 1, present " S " type with SDHA amplification curve, and CP value is less than 35 the sample concentration for detection of CST1mRNA in urine and CST4mRNA, treatment is got Urine in Patients after finishing again, with detecting CST1mRNA and CST4mRNA concentration after method enrichment CST1mRNA, the CST4mRNA of embodiment 1 and SDHA mRNA.Then utilize CST1 and CST4 joint-detection judgment formula P=exp (1.341+0.005a-0.001b)/[1+exp (1.341+0.005a-0.001b)] (a represents CST1 concentration, b represents CST4 concentration) calculating P value, then according to changing assessment curative effect with the rear P value for the treatment of before treatment, judging criterion is: P value declines and is less than 30% compared with before treatment, is judged as and fails to respond to any medical treatment; P value declines and is more than or equal to 30% compared with before treatment, is judged as the state of an illness and improves; P value declines and is more than or equal to 70% compared with before treatment, is judged as result for the treatment of remarkable, and its assessment result is as shown in table 4, and according to clinical symptom assessment curative effect, result is as shown in table 4 simultaneously.
Table 4, CST1 and CST4 combined detection kit assessment bladder cancer efficacy result
Patient's numbering Change in concentration per-cent before and after treatment Clinical evaluation
1 Decline 76% Evident in efficacy
2 Raise 10% Invalid
3 Reduce by 59% Improve
4 Reduce by 37% Improve
5 Raise 12% Invalid
6 Reduce by 27% Invalid
7 Reduce by 48% Improve
8 Reduce by 80% Evident in efficacy
9 Reduce by 38% Invalid
10 Reduce by 49% Improve
As shown in Table 4, using CST1 and CST4 combined detection kit is to have 2 examples evident in efficacy to 10 routine bladder cancer patients curative effect evaluation results, and wherein after 4 example treatments, the state of an illness improves, all the other 4 routine inefficacies.And clinical diagnosis result is that 2 examples are evident in efficacy, after 4 example treatments, the state of an illness improves, all the other 4 routine inefficacies.Therefore adopt the judged result of CST1 and CST4 joint-detection consistent with clinical judgment result.
Embodiment 8, CST1 and CST4 combined detection kit monitoring bladder cancer transfer and relapse
Bladder cancer early stage patient after finishing for the 6 routine courses for the treatment of is followed the tracks of and followed up a case by regular visits to, after treatment, within 6 weeks, get first urine, method enrichment CST1mRNA, CST4mRNA and SDHA mRNA by urine with embodiment 1, present " S " type with SDHA amplification curve, and CP value is less than 35 sample for detection of CST1 in urine and CST4mRNA concentration, every three months detects once later, follows the tracks of nine months, detects altogether four times.According to detected result, utilize joint-detection judgment formula P=exp (1.341+0.005a-0.001b)/[1+exp (1.341+0.005a-0.001b)] (a represents CST1 concentration, b represents CST4 concentration) calculating P value, in the time that P is more than or equal to 0.75, it is transfer and relapse; In the time that P is less than 0.75, show not occur transfer and relapse, be Progression free survival, its result is as shown in table 5; According to clinical symptom monitoring bladder cancer transfer and relapse situation, result is as shown in table 5 simultaneously.
Table 5, CST1 and CST4 combined detection kit monitoring bladder cancer transfer recurrence
Patient's numbering 6 weeks 3 months 6 months 9 months Clinical evaluation
1 0.13 0.33 0.58 0.80 Transfer and relapse
2 0.26 0.42 0.38 0.44 Progression free survival
3 0.58 0.61 0.56 0.60 Progression free survival
4 0.39 0.43 0.51 0.56 Progression free survival
5 0.42 0.34 0.52 0.63 Progression free survival
6 0.67 0.71 0.79 0.85 Transfer and relapse
As shown in Table 5, in the time following the tracks of 9th month, in 6 routine patients, there are 2 examples to occur transfer and relapse, there is not transfer and relapse in all the other 4 examples, for Progression free survival, consistent with clinical evaluation result, but in monitor procedure, CST1 and CST4 joint-detection can be predicted the trend of bladder cancer transfer recurrence, can provide guidance for doctor intervenes in advance.
In sum, CST1mRNA and CST4mRNA can combined utilization, as the mark of diagnosis and indication bladder cancer, and detect the sensitivity of CST1mRNA and CST4mRNA2 mark and specificity higher than detection unique identification thing simultaneously, can improve the accuracy of diagnosis.
The ELISA detection kit of embodiment 9, structure Cystatin SN and Cystatin S
For probing into the expression of Cystatin SN and Cystatin S in Bladder Cancer, build the ELISA detection kit that detects Cystatin SN and Cystatin S.In test kit, anti-Cystatin SN monoclonal antibody is purchased from R & D company of the U.S. (article No. is: MAB1285); The anti-human Cystatin SN of rabbit Anti-TNF-α is purchased from Sino Biological Inc.; Anti-Cystatin S monoclonal antibody is purchased from R & D company of the U.S. (article No. is: MAB1296); The anti-human Cystatin S of rabbit Anti-TNF-α (article No. is: 11542-RP02), purchased from Sino Biological Inc., its each component and concentration thereof are as shown in table 6.
The ELISA detection kit of table 6, detection Cystatin SN and Cystatin S
Embodiment 10, Cystatin SN and the expression of Cystatin S in Bladder Cancer
Get cancer beside organism's sample of 2 routine bladder cancer and pairing, carry out 15% SDS-PAGE.After electrophoresis, by albumen electrotransfer to nitrocellulose filter, be that 5% skim-milk and the massfraction PBS that is 0.1%Tween-20 was in the lower sealing of room temperature (18-25 DEG C) 2 hours with containing massfraction, add respectively anti-Cystatin SN monoclonal antibody and anti-Cystatin S monoclonal antibody to be incubated overnight in 4 DEG C, with the PBS washing that contains 0.1%Tween-20 3 times, the goat anti-rabbit igg that adds again horseradish peroxidase (HRP) mark was in 37 DEG C of incubations 1 hour, with the PBST washing that contains 0.15%Tween-20 4 times, again with PBST washing 1 time, use afterwards TMB peroxidase substrate color developing detection.Taking β-actin as internal reference albumen, detect according to the method described above, result respectively as shown in Figure 14 and Figure 15 simultaneously.Result shows, in Bladder Cancer, Cystatin SN and Cystatin S expression amount are higher than the expression amount in cancer beside organism.
The specificity of the ELISA detection kit of embodiment 11, Cystatin SN and Cystatin S and sensitivity evaluation
By the ELISA detection kit of the Cystatin SN of structure and Cystatin S respectively detectable level be 0pg/mL, 50pg/mL, 100pg/mL, 250pg/mL, the Cystatin SN albumen of 500pg/mL and 1000pg/mL and Cystatin S albumen, and under 450nm condition, detect OD value, then according to detected result drawing standard curve, as shown in figure 16, Cystatin S protein standard curve as shown in figure 17 for Cystatin SN protein standard curve.From Figure 16 and Figure 17, the linearity range of the ELISA detection kit of Cystatin SN and Cystatin SN is 50～1000pg, correlation coefficient r >=0.990 in linearity range.
Utilize the ELISA detection kit of Cystatin SN and Cystatin S to collect urine specimen 100 examples, wherein transitional cell bladder carcinoma 50 examples, benign lesion Urine in Patients 50 examples such as bladder inflammation to BJ Union Hospital's urology department.Application Cystatin SN and Cystatin S ELISA detection kit detect, carry out Logistic regression statistical analysis according to detected result, provide the judgment formula of joint-detection: be specially: P=exp (43.523+0.272a+0.027b)/[1+exp (43.523+0.272a+0.027b)], (a=Cystatin SN concentration, b=Cystatin S concentration), and judge whether according to P value ill, when P is more than or equal to 0.75 positive; When P is less than 0.75 negative.Draw experimenter's performance curve (ROC curve) according to detected result, result as shown in figure 18.Below result demonstration Cystatin SN and the independent detection curve of Cystatin S mark, amass and be respectively 0.933 and 0.836, and under the detection curve of joint-detection, area is 0.987.Hence one can see that, utilizes the effect that Cystatin SN and Cystatin S are markers in detecting more excellent.
The ELISA detection kit diagnosing bladder cancer of embodiment 12, Cystatin SN and Cystatin S
Collect carcinoma of urinary bladder fluid samples 30 examples, benign lesion sample 30 examples from Shanghai No. 5 People's Hospital.Application Cystatin SN and Cystatin S ELISA detection kit detect above-mentioned 60 routine samples, then according to the judgment formula P=exp (43.523+0.272a+0.027b) of Cystatin SN and Cystatin S joint-detection/[1+exp (43.523+0.272a+0.027b)] (a=Cystatin SN concentration, b=Cystatin S concentration) calculating P value, positive and negative according to the judgement of P value, according to clinical diagnosis, result is as shown in table 7 simultaneously.
Table 7, Cystatin SN and Cystatin S associating diagnosing bladder cancer result
Utilize χ 2 to add up the dependency of Cystatin SN and Cystatin S combined detection kit diagnostic result and clinical diagnosis result, result shows P < 0.05, show that Cystatin SN and Cystatin S associating diagnosing bladder cancer and clinical diagnosis result have dependency, and consistence is better, Kappa value is 93.3%.
Embodiment 13, Cystatin SN and Cystatin S combined detection kit are for bladder cancer curative effect evaluation
Urine from 10 routine bladder cancer patients treatments are got by Jiangsu Prov. Tumour Hospital, detects Cystatin SN albumen and Cystatin S protein concentration in urine, gets Urine in Patients again and detect CystatinSN albumen and Cystatin S protein concentration after treatment finishes.Utilize judgment formula P=exp (43.523+0.272a+0.027b)/[1+exp (43.523+0.272a+0.027b)] (a=Cystatin SN concentration of Cystatin SN and Cystatin S joint-detection, b=Cystatin S concentration) calculating P value, then and treatment rear P value assessment curative effect front according to treatment, judging criterion is: P value declines and is less than 30% compared with before treatment, is judged as and fails to respond to any medical treatment; P value declines and is more than or equal to 30% compared with before treatment, is judged as the state of an illness and improves; P value declines and is more than or equal to 70% compared with before treatment, is judged as result for the treatment of remarkable, and its assessment result is as shown in table 8; Assess its curative effect according to clinical symptom, result is as shown in table 8 simultaneously.
Table 8, CystatinSN and Cystatin S joint assessment bladder cancer curative effect
Patient's numbering Change in concentration per-cent before and after treatment Clinical evaluation
1 Decline 36% Improve
2 Raise 5% Invalid
3 Raise 19% Invalid
4 Reduce by 77% Evident in efficacy
5 Raise 10% Invalid
6 Reduce by 37% Improve
7 Reduce by 44% Improve
8 Reduce by 58% Improve
9 Reduce by 45% Invalid
10 Reduce by 73% Evident in efficacy
As shown in Table 8, using Cystatin SN and Cystatin S combined detection kit is in 10 routine patients to bladder cancer curative effect evaluation result, there are 2 examples evident in efficacy, wherein after 5 example treatments, the state of an illness improves, all the other 3 routine inefficacies, and clinical diagnosis result is in 10 routine patients, has 2 examples evident in efficacy, have 4 routine conditions of patients to improve, all the other 4 examples are without result for the treatment of.Therefore, use Cystatin SN/Cystatin S albumen combined detection kit its coincidence rate compared with clinical judgment result to reach 90%.
Embodiment 14, Cystatin SN and Cystatin S combined detection kit are for the monitoring of bladder cancer transfer and relapse
Bladder cancer early stage patient after finishing for the 6 routine courses for the treatment of is followed the tracks of and followed up a case by regular visits to, within 6 weeks, gets first urine, and detect Cystatin SN and Cystatin S protein concentration in urine after treatment, later every three months detects once, follows the tracks of nine months, detects altogether four times.According to detected result, calculation formula P=exp (43.523+0.272a+0.027b)/[1+exp (43.523+0.272a+0.027b)] (a=Cystatin SN concentration of Cystatin SN and Cystatin S joint-detection, b=Cystatin S concentration) calculating P value, in the time that P is more than or equal to 0.75, it is transfer and relapse; In the time that P is less than 0.75, show not occur transfer and relapse, be Progression free survival, its result is as shown in table 9, and monitors its transfer and relapse situation according to clinical symptom in the time of 9 months, and its result is as shown in table 9.
Table 9, Cystatin SN and Cystatin S combined detection kit monitoring bladder cancer transfer recurrence result
Patient's numbering 6 weeks 3 months 6 months 9 months Clinical evaluation
1 0.13 0.33 0.38 0.40 Progression free survival
2 0.36 0.42 0.38 0.44 Progression free survival
3 0.58 0.69 0.66 0.77 Transfer and relapse
4 0.40 0.43 0.39 0.56 Progression free survival
5 0.29 0.34 0.62 0.80 Transfer and relapse
6 0.39 0.41 0.49 0.46 Progression free survival
As shown in Table 9, Cystatin SN and Cystatin S combined detection kit monitored results are to have 2 examples to occur transfer and relapse in 6 routine patients, and transfer and relapse does not occur all the other 4 examples, is Progression free survival, consistent with clinical judgment result.But in monitor procedure, Cystatin SN and Cystatin S joint-detection can be predicted the trend of bladder cancer transfer recurrence, can provide guidance for doctor intervenes in advance.
In sum, Cystatin SN and Cystatin S combine the mark that can be used as diagnosis and indication bladder cancer, detect the sensitivity of 2 marks and specificity higher than detecting unique identification thing simultaneously, can improve the accuracy of diagnosis.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can make various changes to it in the form and details, and not depart from the claims in the present invention book limited range.

Claims (10)

1.CST1mRNA and the CST4mRNA combined utilization in preparation diagnosis and indication bladder cancer mark, the nucleotide sequence of described CST1mRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of described CST4mRNA is as shown in SEQ ID NO.2.
2. application according to claim 1, it is characterized in that: the judgment formula of CST1mRNA and CST4mRNA combined utilization is P=exp (1.341+0.005a-0.001b)/[1+exp (1.341+0.005a-0.001b)], wherein a=CST1 copy number, b=CST4 copy number.
3. application according to claim 1 and 2, is characterized in that: described diagnosis and be shown in advance diagnosis, curative effect evaluation or transfer and relapse monitoring.
4.CST1mRNA and CST4mRNA combined detection kit, is characterized in that, comprises the quantitative detecting reagent of CST1mRNA and CST4mRNA; Described CST1mRNA quantitative detecting reagent comprises detection primer and the TaqMAN probe of nucleotide sequence as shown in SEQ ID NO.20 as shown in SEQ ID NO.7 and SEQ ID NO.8; Described CST4mRNA quantitative detecting reagent comprises detection primer and the TaqMAN probe of nucleotide sequence as shown in SEQ IDNO.22 as shown in SEQ ID NO.9 and SEQ ID NO.10.
5. test kit according to claim 4, it is characterized in that: described test kit also comprises SDHA mRNA quantitative detecting reagent, described SDHA mRNA quantitative detecting reagent comprises detection primer and the TaqMAN probe of nucleotide sequence as shown in SEQ ID NO.24 as shown in SEQ ID NO.11 and SEQ ID NO.12.
6. test kit according to claim 5, it is characterized in that: described test kit also comprises the magnetic bead of the special CST1mRNA of catching, CST4mRNA and SDHA mRNA, the magnetic bead of the described special CST1mRNA of catching is combined with the probe as shown in SEQ IDNO.4, the magnetic bead of the described special CST4mRNA of catching is combined with the probe as shown in SEQ ID NO.5, and the magnetic bead of the described special SDHA of catching mRNA is combined with the probe as shown in SEQ ID NO.6.
7. according to the test kit described in claim 4-6 any one, it is characterized in that: described test kit also comprises 10 × Buffer, dNTP, MgCl 2, DMSO, DTT, Taq enzyme, UDG, reversed transcriptive enzyme and RNA enzyme inhibitors.
8.Cystatin SN and the Cystatin S combined utilization in preparation diagnosis and indication bladder cancer mark, it is characterized in that: the nucleotide sequence of coding Cystatin SN is as shown in SEQ ID NO.1, and the nucleotide sequence of coding Cystatin S is as shown in SEQ IDNO.2.
9. application according to claim 8, it is characterized in that: the judgment formula of Cystatin SN and Cystatin S combined utilization is P=exp (43.523+0.272a+0.027b)/[1+exp (43.523+0.272a+0.027b)], wherein a=Cystatin SN concentration, b=Cystatin S concentration.
10. application according to claim 8 or claim 9, is characterized in that: described diagnosis and be shown in advance diagnosis, curative effect evaluation or transfer and relapse monitoring.
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Address after: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 BioBAY No. 218 building, Room 301, C4 201

Patentee after: Jiangsu is the real biopharmaceutical technology Limited by Share Ltd

Address before: Suzhou City, Jiangsu Province, Suzhou Industrial Park, 215123 Street No. 218 Nano Technology Park building C4 Room 301

Patentee before: Suzhou Micro Diag Biomedicine Co., Ltd.