CN108410975A - Pelvic prolapse diagnosis marker and its application - Google Patents
Pelvic prolapse diagnosis marker and its application Download PDFInfo
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Abstract
The present invention relates to pelvic prolapse diagnostic flag and its applications, more particularly relate to the application of SALL1 genes and its expression product in pelvic prolapse diagnosis and treatment.Inventor is based on high-flux sequence result and carries out genescreen using bioinformatics method analysis, pick out SALL1 genes, further molecular cytobiology method confirms the apparent high expression in pelvic prolapse tissue of SALL1 genes, it can be used for preparing pelvic prolapse assisting in diagnosis and treatment preparation, there is important clinical value.
Description
Technical field
The present invention relates to biomedicine fields, and in particular to pelvic prolapse diagnostic flag and its application more particularly relate to
The application of SALL1 genes and its expression product in pelvic prolapse diagnosis and treatment.
Background technology
Pelvic organ prolapse (pelvic organ prolapse, POP) be due to basin bottom support structure defect, damage
It is the common disease for influencing middle and aged women quality of life, mainly by basin bottom support structure defect or caused by dysfunction
Or it degenerates, caused by damage and dysfunction.Clinical manifestation is the organs such as uterus, vagina, bladder, urethra, Colon and rectum, small intestine
Prolapsus, the urinary incontinence that often occurs together, urination dysporia, the symptoms such as sex dysfunction.Epidemiological study shows up to 50%
Adult women is perplexed by the disease, and either the incidence of primary POP or recurrent POP are all increased year by year, oneself becomes
The public health problem highly paid close attention to.POP has seriously affected the life and social activities of adult female, studies have pointed out that
The possibility that depressive symptom occurs in POP patient is 5 times of general population.However, since its pathogenesis is still not clear, at present
Most for the treatment of only its symptom is treated, and can not from the cause of disease radical curing of disease.
9 pelvic prolapse case samples of inventor couple and 3 controls carry out high-flux sequence, in conjunction with bioinformatics method
Genescreen is carried out, 6 candidate genes are picked out：CPXM1, SALL1, KIAA1644, RPRM, PCP4, LINC00890.Into one
Step, the present invention have carried out molecular cytobiology method and have confirmed that above-mentioned 6 candidate genes have well with pelvic prolapse
Correlation, candidate gene apparent high expression in pelvic prolapse patient tissue, can be used for preparing pelvic prolapse assisting in diagnosis and treatment preparation,
With important clinical value.
Invention content
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, SALL1, KIAA1644,
RPRM, PCP4, LINC00890.
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, KIAA1644, RPRM, PCP4,
LINC00890。
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, SALL1, KIAA1644,
PCP4, LINC00890.
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, SALL1, KIAA1644,
RPRM, LINC00890.
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, SALL1, KIAA1644,
RPRM, PCP4.
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：SALL1, KIAA1644, RPRM,
PCP4, LINC00890.
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, SALL1, RPRM, PCP4,
LINC00890。
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：SALL1, KIAA1644, RPRM,
LINC00890。
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, KIAA1644, PCP4,
LINC00890。
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, SALL1, KIAA1644,
LINC00890。
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, KIAA1644, RPRM,
LINC00890。
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, KIAA1644, LINC00890.
The purpose of the present invention is to provide application of the molecular marker in preparing pelvic prolapse diagnostic preparation, the molecules
Marker is selected from the expression product of any one or more following gene and/or gene：CPXM1, KIAA1644.
To achieve the above object, the present invention screens candidate base by high-flux sequence combination bioinformatics method first
Cause：CPXM1, SALL1, KIAA1644, RPRM, PCP4, LINC00890.And then pass through molecular cytobiology method validation
The relationship of above-mentioned 6 subsequent genes and pelvic prolapse：Candidate gene has good correlation with pelvic prolapse, can be used for preparing
Pelvic prolapse auxiliary diagnosis preparation has important clinical value.
Further, the molecular marker high expression in pelvic prolapse tissue.CPXM1 genes are in pelvic prolapse group
For the expression knitted higher than control tissue more than 3 times, expression of the SALL1 genes in pelvic prolapse tissue is higher than control group
It knits more than 6 times, expression of the KIAA1644 genes in pelvic prolapse tissue is higher than control tissue more than 3 times, and RPRM genes are in basin
For expression in chamber prolapsed tissue higher than control tissue more than 4 times, expression of the PCP4 genes in pelvic prolapse tissue is high
In control tissue more than 3 times, expression of the LINC00890 genes in pelvic prolapse tissue is higher than control tissue more than 3 times.
Further, the diagnostic preparation of the pelvic prolapse is using PCR kit for fluorescence quantitative, genechip detection pelvic cavity
Above-mentioned candidate gene any one or several expression in prolapsed tissue, it is preferred that contain in the PCR kit for fluorescence quantitative
There is the primer of the above-mentioned candidate gene of a pair of of specific amplification any one or several genes；The genetic chip include with it is upper
State the probe of the nucleic acid array hybridizing of candidate gene any one or several genes.It is furthermore preferred that the specific amplification
The primer of CPXM1, SALL1, KIAA1644, RPRM, PCP4, LINC00890 gene is shown in sequence table SEQ ID NO.15 to SEQ
ID NO.26。
Further, the diagnostic preparation of the pelvic prolapse is using above-mentioned candidate in immunization method detection pelvic prolapse tissue
The expression product of gene any one or several genes, it is preferred that the immunization method is ELISA detections and the detection of/colloidal gold.
Further, the ELISA method of the albumen of any one or several gene expressions of the above-mentioned candidate gene of the detection is to make
Use ELISA detection kit.Commercially available above-mentioned candidate gene any one or several bases can be used in antibody in the kit
The monoclonal antibody of cause.Further, the kit includes：It is coated with the list of any one or several genes of above-mentioned candidate gene
The solid phase carrier of clonal antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme are anti-
Answer terminate liquid etc..
Further, the colloidal gold method of the albumen of any one or several gene expressions of the above-mentioned candidate gene of the detection is to make
With detection kit, the monoclonal that commercially available above-mentioned candidate gene any one or several genes can be used in the antibody is anti-
Body.Further, the gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or colloidal gold percolation.Further,
Detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter have anti-above-mentioned candidate gene any one or
Several gene monoclonal antibodies, quality control region (C) specking have Immunoglobulin IgG.
The present invention also aims to provide a kind of application of molecular marker in preparing pelvic prolapse treatment preparation, institute
State the expression product that molecular marker is selected from any one or more following gene and/or gene：CPXM1, SALL1,
KIAA1644, RPRM, PCP4, LINC00890.
Further, the pelvic prolapse treatment preparation inhibits the transcription and/or expression of molecular marker.
One kind in following methods and/or several usually may be used in the expression of suppressor and its expression product：Pass through
Activate the suppressor of above-mentioned candidate gene any one or several genes, activation inhibit above-mentioned candidate gene any one or it is several
The albumen of a gene expression imports and inhibits on above-mentioned candidate gene any one or the siRNA of several gene expressions, activation promote
State the microRNA of candidate gene any one or several gene mRNAs degradation, import promote above-mentioned candidate gene any one or
The molecule of the protein degradation of several gene expressions, the factor for inhibiting to promote above-mentioned candidate gene any one or several gene expressions
And the expression of albumen.
Further, described to inhibit above-mentioned candidate gene any one or the choosing of the siRNA sequence of several genetic transcriptions or expression
From SEQ ID NO.1 to SEQ ID NO.12 any one and/or it is several.
RNA interference (RNAi) refers to exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo
Selective degradation, the phenomenon that leading to posttranscriptional gene silencing, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal
The expression of certain specific gene, promotes mRNA to degrade, and cells show is made to go out the technology of specific gene missing phenotype.SiRNA is designed
Direct synthesis technique or structure SiRNA expression vector may be used after the completion, the siRNA prepared can be total by calcium phosphate
Mechanical Methods, the cation lipid such as the precipitation method, electroporation, DEAE- glucans and polybrene methods, microinjection or particle gun
The approach transfectional cells such as body reagent method.
The purpose of the present invention is to provide a kind of pelvic prolapse inhibitor, the pelvic prolapse inhibitor inhibits above-mentioned candidate
The transcription or expression of gene any one or several genes, it is preferred that containing inhibiting above-mentioned candidate base in pelvic prolapse inhibitor
Because of the siRNA of transcription or the expression of any one or several genes.It is furthermore preferred that it is described inhibit above-mentioned candidate gene any one
Or the siRNA of transcription or the expression of several genes in sequence table SEQ ID NO.1 to SEQ ID NO.12 any one
And/or it is several.
The purpose of the present invention is to provide a kind of pelvic prolapse diagnostic preparation, the pelvic prolapse diagnostic preparation detection is above-mentioned
The transcription or expression of candidate gene any one or several genes, it is preferred that pelvic prolapse diagnostic preparation uses quantitative fluorescent PCR
Kit, genetic chip, immunization method detection.
Fluorescence quantitative PCR method is that PCR product is marked by the probe of the specificity of fluorescent dye or fluorescent marker
Tracking, real time and on line monitoring reaction process can analyze product in conjunction with corresponding software, calculate sample to be tested template
Initial concentration.
Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types：1) it is fixed on
Nucleic acid probe on polymer matrix film (nylon membrane, nitrocellulose membrane etc.) surface or cDNA segments, usually with isotope labelling
Target gene is hybrid with it, and is detected by radiography technology.2) DNA probe battle array on a glass is fixed with point sample method
Row, are hybridized by the target gene with fluorescent marker and are detected.3) oligonucleotides directly synthesized on the hard surfaces such as glass
Probe array hybridizes with the target gene of fluorescent marker and is detected.
Known antigen or antibody are adsorbed on surface of solid phase carriers by enzyme-linked immunosorbent assay (ELISA), make enzyme mark
The technology that the antigen-antibody reaction of note is carried out in solid phase surface.ELISA detection kit can according to testing goal and operating procedure
It is divided into indirect method, double-antibody method, competition law, double site one-step method, prize law and surveys IgM antibody, using Avidin and biotin
ELISA.Horseradish peroxidase (HRP) or alkaline phosphatase (AP) may be selected in chromogenic substrate in ELISA detection kit.
Common immune colloid gold detection technique：(1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut
Piece can be dyed with the antibody of colloid gold label, can also be enhanced with silver-colored developer solution and marked on the basis of colloid gold label,
So that the silver atoms being reduced is deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(2) it is immunized
Colloidal gold staining method for electron microscopy can use the antibody of colloid gold label or antiantibody to be combined with negative staining Virus Sample or tissue ultra-thin section,
Then negative staining is carried out.It can be used for observation and the viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made
Antigen or antibody point are first added sample to be checked by carrier on film after closing, corresponding with the antibody test of colloid gold label after washing
Antigen or antibody.(4) antigen of specificity or antibody are fixed on ribbon on film by colloidal gold immunity chromatography, colloid
Golden labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be checked is added in the sample pad of test strips one end
Afterwards, it moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad through capillary action, it is fixed when being moved to
When the region of antigen or antibody, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
It is taken in detection, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.
The purpose of the present invention is to provide a kind of gene detecting kit detecting pelvic prolapse, in the kit detection
Candidate gene any one or several genes is stated, using special sense primer and downstream primer.
Further, which is suitable for presently, there are all types fluorescence quantitative gene extender in the market, clever
Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes：Specific primer, internal control primer, quantitative fluorescent PCR
Reaction solution.The internal reference is GAPDH.
The kit also includes RNA extraction agents.
The present invention also has detected this kit sensitivity, as a result shows that this kit detection range is 106-102copies/
μ l, minimum concentrations are 100copies/ μ l.
There is provided a kind of pelvic prolapse protein detection kit, the detection kits to detect above-mentioned time for goal of the invention
Select the albumen of gene any one or several gene expressions.Further, the kit further includes other detection reagents.
It is an object of the present invention to provide it is a kind of detection pelvic prolapse genetic chip, the genetic chip include with it is above-mentioned
The probe of the nucleic acid array hybridizing of candidate gene any one or several genes.
Description of the drawings
Fig. 1 candidate genes relative expression quantity in pelvic prolapse patient group and control group
Each group candidate's mRNA expressions after Fig. 2 RNA interference
The fibroblastic growth curve of Fig. 3 uterosacral ligaments 1
The fibroblastic growth curve of Fig. 4 uterosacral ligaments 2
The fibroblastic growth curve of Fig. 5 uterosacral ligaments 3
The fibroblastic growth curve of Fig. 6 uterosacral ligaments 4
The fibroblastic growth curve of Fig. 7 uterosacral ligaments 5
The fibroblastic growth curve of Fig. 8 uterosacral ligaments 6
Specific implementation mode
Present invention will be further explained below with reference to specific examples, is only used for explaining the present invention, and should not be understood as to this
The limitation of invention.It will be understood by those skilled in the art that：Without departing from the principle and spirit of the present invention may be used
To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent
It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to the item proposed by manufacturer
Part examinations.
The collection of 1 case of embodiment
Take in September, 2012 to the POP patient to go to a doctor in gynemetrics of Concord Hospital during in December, 2013, collection 9, right altogether
According to other gynecological diseases (the removing cancer patient) patient and Check-up crowd being hospitalized from same time gynemetrics, with the age, occupy
Residence is matching factor, collects totally 3.The utero-sacral ligament tissue samples of all research objects are obtained, -80 DEG C of number postposition is low
Temperature refrigerator preserves.
POP group inclusion criterias：A. POP-Q scorings >=II degree after gynecologial examination；
B. sequent occupance Beijing area；
C. history of childbirth at least once.
Exclusion criteria：A. nearly 3 months rows hormone replacement therapy patient；
B. connective tissue disease patient (rheumatic arthritis, systemic loupus erythematosus, polymyositis, Marfan syndrome,
Like Down syndrome)；
C. gestational patients；
D. patient refuses to be added；
E. malignant tumor patient.
Control group inclusion criteria：A.POP-Q scorings≤I degree；
B. history of childbirth at least once；
C. age, residence are matched with case group
D. without POP medical histories.
Exclusion criteria：With POP groups
Pelvic organ prolapse quantifies allotment method (Pelvic Organ Prolapse Quantitation, POP-Q)：It utilizes
2 dissections on vaginal vault, vagina front and rear wall indicate relationship of the point with hymen to define the prolapsus degree of pelvic organ, divide
For 0-IV degree.All patients take lithotomy position when inspection, and do maximum Valsalva actions.
0 degree of expression is not prolapsed；
I degree indicates prolapsus distalmost end in hymen, at hymen ＞ 1cm；
II degree indicates prolapsus distalmost end in the 1cm of virgin's film edge, no matter inside and outside；
III degree indicates that prolapsus distalmost end outside hymen, apart from virgin film edge ＞ 1cm, but is less than (vagina total length-
2cm)；
IV degree indicates that vagina completely or almost completely prolapses, and prolapse distalmost end >=(vagina total length -2cm).
2 high-flux sequence of embodiment and analysis
RNA extractions are carried out to tissue, agarose gel electrophoresis after RNA extractions can be extracted from electrophoresis result with preliminary judgement
RNA sample it is up-to-standard whether, if can be used for further transcriptome analysis.And then pass through NanoDrop1000 points
Light photometer detects the extraction situation of RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2.
Microarray dataset is the 2500 high-flux sequence platforms of HiSeq of Illumina companies, carries out high-throughput transcript profile depth
Sequencing, we use Fast-QC after sequencing
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) software to sequencing
The quality of data carries out total evaluation, includes the quality Distribution value of base, the position distribution of mass value, G/C content, PCR
Duplication contents, the frequency etc. of kmer.In differential genes expression analysis, according to obtained FPKM values, use
Internationally recognized algorithm EBSeq carries out differential screening.Wherein, when screening, LOG2FC>1 or<-1,FDR<0.05.In order to better
Understand the function of difference expression gene, we have carried out Gene Onlogy to difference expression gene and signal path is analyzed, and right
Difference expression gene carries out functional annotation and protein interaction network analysis, in view of data above analysis as a result, in conjunction with
Document we screened 6 difference expression genes：CPXM1, SALL1, KIAA1644, RPRM, PCP4, LINC00890.
The uterosacral ligament tissue candidate gene expression of 3 pelvic prolapse patient of embodiment and control
One, material and method
1, material
Choose 35 pelvic prolapse patient's uterosacral ligament tissues and 5 control uterosacral ligament tissues, it is grouped and
Number.Control group is out that the gynecological benign diseases patient of abdomen or Clinical observation, per vaginam exclude pelvic organ
Prolapsus, no incontinence.In 35 pelvic prolapse patients 17 be II degree, 8 III degree, 10 be IV degree.
2, method
The extraction of the uterosacral ligament total tissue RNA of 2.1 pelvic prolapse patients and control
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation
It is carried out by product description.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into
Row cDNA reverse transcriptions, experimental implementation are carried out by product description.
2.3 Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instruments of ABI, the relative quantitative assay of data is carried out using 2- △ △ CT methods. 2.3.2
Design of primers
Using online primer-design software, gene order is with reference to NCBI:NM_019609.4 (CPXM1), NM_002968.2
(SALL1), NM_001099294.1 (KIAA1644), NM_019845.2 (RPRM), NM_006198.2 (PCP4),
Participate in the election of GAPDH in LINC00890 (LINC00890), is synthesized by invitrogen companies after design of primers.Specific primer sequence is such as
Under：
1 primer sequence of table
Operating process is as follows：
(1) reaction system：Use PowerGreen PCR Master Mix (invitrogen, article No.
4367659) it is expanded, experimental implementation is carried out by product description.Amplification program is：95 ° of 5min, (95 DEG C of 15sec, 60 DEG C
45sec) × 40 cycle.
2 RealTime reaction systems of table
(2) primer screening
After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ l to make mould after dilution
Plate is expanded with target gene primer and reference gene primer respectively, while melt curve analysis analysis, root are carried out at 60-95 DEG C
Primer screening is carried out according to amplification efficiency height and the unimodal principle of solubility curve.
(3) sample RealTimePCR is detected
2 μ l will be taken to make template after 10 times of each sample cDNA dilution, use respectively target gene primer and reference gene primer into
Row amplification.Solubility curve analysis is carried out at 60-95 DEG C simultaneously.
Two, experimental result
Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；According to the relative quantification formula of qRT-PCR：2-
Δ Ct × 100% compares expression of the candidate gene in pelvic prolapse tissue and control tissue.As a result it shows：qRT-PCR
Stable amplification result, expression of the CPXM1 genes in pelvic prolapse tissue are higher than nearly 4 times of control tissue, and SALL1 genes exist
Expression in pelvic prolapse tissue is higher than control tissue more than 6 times, expression of the KIAA1644 genes in pelvic prolapse tissue
For level higher than control tissue more than 3 times, expression of the RPRM genes in pelvic prolapse tissue is higher than control tissue more than 4 times,
Expression of the PCP4 genes in pelvic prolapse tissue is higher than control tissue more than 3 times, and LINC00890 genes are in pelvic prolapse
Expression in tissue (is specifically shown in Fig. 1) higher than control tissue more than 3 times, and result above demonstrates high-throughput transcript profile expression number
According to confluence analysis in candidate gene high expression in pelvic prolapse patient result.
The fibroblastic original cuiture of 4 uterosacral ligament of embodiment, purifying, passage
One, the original cuiture of cell
(1) tissue specimen is laid flat in ware, PBS is rinsed 3 times, and eye scissors shred；
In (2) 37 DEG C of 5% carbon dioxide incubator, the 1ml digestion of 1%I Collagenase Types shreds tissue 2h；
(3) digestive juice containing cell is transferred to sterile 15m1 centrifuge tubes, centrifuged, 40C, 1500rpm, 5min；
(4) after abandoning supernatant, will centrifuge bottom of the tube suspension containing cell with suction pipe blow it is even after be transferred to 25cm2Culture
Bottle, the M199 culture medium 5ml containing 15% fetal calf serum (FBS) are added after static 0.5h, are put into 37 DEG C of 5% carbon dioxide incubator
Middle carry out primitive cell culture, next day replace culture medium；
(5) it is changed the liquid once every 3d：Old culture medium is abandoned, PBS is rinsed 1 time, and the M199 fresh cultureds containing 15%FBS are added
Base 5ml；
(6) form and growing state of cell are observed under conventional inverted microscope；
(7) thin to the uterosacral ligament turned out using adherent method when difference when the cell of growth is paved with bottom of bottle 70%-80%
Born of the same parents purify.
Two, the purifying of cell
(1) on super-clean bench, former culture medium is discarded, is washed twice, is discarded with PBS 2m1,0.25%Trypsin- is added
0.02%EDTA lml digest 1-2 minutes in 37 DEG C of 5% carbon dioxide incubator；
(2) observed under conventional inverted microscope, occur most cells be rounded suspension after, be added 1ml contain 15%
The M199 culture mediums of FBS, are jiggled to neutralize 0.25%Trypsin-0.02%EDTA；
(3) mixed liquor is drawn in sterile 15m1 centrifuge tubes, 1500rpm, 4 DEG C of centrifugation 5min abandon supernatant, addition contains
The M199 culture medium 3m1 of 15%FBS, are gently blown and beaten with suction pipe, after mixing cell, are inoculated in new 25cm2In culture bottle, it is put into
0.5h in 37 DEG C of 5% carbon dioxide incubator, after abandon culture medium, adherent fast cell (fibroblast) stays in bottle wall, adherent
Slow cell (smooth muscle cell) is discarded with culture medium, is added new culture medium and is put into incubator and continues to cultivate；
(4) it is changed the liquid once every 3d：Old culture medium is abandoned, PBS is rinsed 1 time, and the M199 fresh cultureds containing 15%FBS are added
Base 5ml；
(5) when the cell of growth is paved with bottom of bottle 70%-80%, using adherent method when difference according to above-mentioned steps to turning out
Uterosacral ligament cell purified again；
(6) cell for the second time after purification is cultivated with the M199 culture mediums containing 15%FBS, when cell is paved with after purification
When bottom of bottle 70%-80%, secondary culture is carried out.
Three, cell secondary culture
(1) on super-clean bench, former culture medium is discarded, is washed twice, is discarded with PBS 2m1,0.25%Trypsin- is added
0.02%EDTA lml digest 1-2 minutes in 37 DEG C of 5% carbon dioxide incubator；
(2) it is observed under conventional inverted microscope, cytoplasm retraction occurs, space between cells increases, and most cells are rounded change
After suspension, the M199 culture mediums containing 15%FBS of 1ml are added, jiggle to neutralize 0.25%Trypsin-0.02%EDTA；
(3) mixed liquor is drawn in sterile 15ml centrifuge tubes, 1500rpm, 4 DEG C, centrifuges 5min, abandons supernatant, addition contains
The M199 culture medium 2m1 of 15%FBS, are gently blown and beaten with suction pipe, after mixing cell, by 1:2 or 1:3 inoculations and new 25cm2Training
It supports in bottle, is put into 37 DEG C of 5% carbon dioxide incubator, next day replaces culture medium；
(4) after cell covers with bottle wall, cell is passed to new culture bottle with method, the cell in 3-8 generations is used for this experiment.
Embodiment 5 RNAi interference candidate genes are expressed and on the fibroblastic influence of uterosacral ligament
One, material
(1) cell origin
The uterosacral ligament fibroblast that embodiment 4 is cultivated.
(2) siRNA structures and synthesis
According to 2.0 (http of Photographing On-line software siDirect version://design.rnai.jp/), candidate gene
Sequence is with reference to NCBI:NM_019609.4 (CPXM1), NM_002968.2 (SALL1), NM_001099294.1 (KIAA1644),
NM_019845.2 (RPRM), NM_006198.2 (PCP4), LINC00890 (LINC00890) design corresponding siRNA.If
Synesis Company's synthesis is sent to after meter.
Two, experimental method
(1) expression of RNA AF panels uterosacral ligament fibroblast candidate gene
1, the design and synthesis of siRNA
SiRNA expression vector pSIREN-DNR contains neomycin resistance gene and GFP green fluorescent labels, can monitor in real time
Transfection efficiency of the carrier in cell.According to purpose mRNA sequence, each candidate gene, which designs 3 RNA interference target sequences, to be passed through
Testing sieve selects optimum jamming sequence, while designing negative control (being shown in Table 3).For every selected siRNA target sequence, if
SiRNA positive-sense strands and antisense strand are counted, is connected with loop (9nt), referred to as shRNA (shorthairpin RNA).Synthesize every volume
Two single-stranded, single-stranded DNA double chain templates for obtaining shRNA of annealed dna of the DNA profiling of code shRNA.Template strand followed by
RNA PoIyIII polymerase transcriptions stop site, while both ends separately design BamHI and HindIII restriction enzyme sites, can clone
To between BamHI the and HindIII restriction enzyme sites of siRNA carrier multiple cloning sites.SiRNA empty carriers BamHI and HindIII
After double digestion, 1% agarose gel electrophoresis recycles linear carrier.The DNA profiling double-strand of annealing is connected in linear carrier.
Using T4 ligases, the molar ratio of insertion and carrier is about 3:1.Connection product converts DH5 α Escherichia coli, in LB Amp
Coated plate on culture medium, 37 DEG C of overnight incubations.PCR is identified；Sequencing identification.Column extracts positive colony carrier and quantifies.
3 siRNA transcription templates sequences of table
3, cell grouping and transfection
(1) cell is grouped
C groups：Blank control group；C1 groups：Transfect liposome group；C2 groups：Transfect nonspecific siRNA groups；S1, S2, S3
Group：Transfect the siRNA groups of specificity.
(2) it transfects
According to LipofectamineTMThe step of 2000Transfection Reagent are provided carries out.
1. before transfection for 24 hours, the cell pancreatin of logarithmic growth phase is digested and is counted, and adjustment cell concentration is 1 × 105/
Ml takes 2m1 to be inoculated in six orifice plates, is positioned over 37 DEG C, 5%CO2It is cultivated in incubator, when cell is merged up to 80% for turning
Dye.With the DMEM medium cultures 3-4h without serum before transfection.
2. preparing transfection liquid：
A liquid：250u1 serum free mediums dilute 4.0ugDNA, mild mixing；
B liquid：250u1 serum free mediums dilute 10u1Lipofectamine, and mild mixing is placed at room temperature for 5min；
3. transfecting：A liquid is mixed with B liquid, and compound is directly added in every hole by incubation at room temperature 20min, is shaken
Culture plate, gently mixing.In CO2Liquid is changed after 37 DEG C of heat preservations 24-48h, 6h in incubator, the culture medium containing serum is added.
4, the verification of transfection efficiency
(1) cellular morphology and transfected condition are observed under fluorescence inverted microscope
After transfection for 24 hours, culture plate is placed under fluorescence inverted microscope and observes cellular morphology and growth conditions, green fluorescence
Lower observation transfected condition.
(2) variation of the front and back candidate gene expression of Real-time PCR methods detection transfection is applied
1. the structure of standard curve：It is chosen at 1 bottle of the uterosacral ligament fibroblast normally cultivated in 50mI culture bottles,
RNA is extracted, RNA concentration and purity are measured, carries out reverse transcription reaction, ten times of dilutions of DNA templates that reaction is generated obtain phase
When in 104-100The DNA profiling of copies/ul is separately added into candidate gene primer and internal control primer, prepares 25u1 reaction systems,
Using Real-time PCR amplification instruments, pcr amplification reaction is carried out.Obtain candidate and internal reference standard curve.
2. the variation of the front and back candidate gene expression of Real-time PCR methods detection transfection：The RNA of each group cell is extracted,
RNA concentration and purity are measured, reverse transcription reaction is carried out, every group of DNA profiling is carried out at the same time candidate and internal reference Real-time PCR
Reaction, experiment is in triplicate.
3. to PCR product into row agarose gel electrophoresis.
Three, experimental result
As a result show the candidate gene interference carrier of the invention built all to candidate gene in uterosacral ligament fibroblast
Certain inhibiting effect is played in expression, and wherein CPXM1-siRNA, up to 39%, is specifically shown in Fig. 3 to CPXM1 genes inhibiting rate；SALL1-
SiRNA, up to 61%, is specifically shown in Fig. 4 to SALL1 genes inhibiting rate；KIAA1644-siRNA reaches KIAA1644 gene inhibiting rates
42%, it is specifically shown in Fig. 5；RPRM-siRNA, up to 54%, is specifically shown in Fig. 6 to RPRM genes inhibiting rate；PCP4-siRNA is to PCP4 bases
Because inhibiting rate is up to 43%, it is specifically shown in Fig. 7；LINC00890-siRNA, up to 41%, is specifically shown in figure to LINC00890 genes inhibiting rate
8。
6 mtt assay of embodiment detects the fibroblastic growth curve of uterosacral ligament:
One, experiment packet
Control group：To open the gynecological benign diseases patient of abdomen or Clinical observation, per vaginam excludes pelvic cavity
Organ prolapse, no incontinence；Uterosacral ligament is derived from other than ligament and uterine neck junction at 0.5-1.0cm, according to embodiment 4
The method does uterosacral ligament cell culture, secondary culture 4-6 generations；
POP groups：By senior clinician according to POP-Q methods judgement prolapsus degree POP-QII degree patient；Uterosacral ligament takes
From at 0.5-1.0cm, uterosacral ligament cell culture is done according to the method described in embodiment 4 other than ligament and uterine neck junction, pass
It is commissioned to train foster 4-6 generations；
POP interference groups：By senior clinician according to POP-Q methods judgement prolapsus degree POP-QII degree patient；Palace sacrum is tough
Band is derived from ligament at 0.5-1.0cm other than uterine neck junction, and the training of uterosacral ligament cell is done according to the method described in embodiment 4
It supports, secondary culture 4-6 generations, interference carrier is transfected according to the method described in embodiment 5；POP interference groups compare：Faced by senior
Bed doctor according to POP-Q methods judgement prolapsus degree POP-QII degree patient；Uterosacral ligament is derived from other than ligament and uterine neck junction
At 0.5-1.0cm, uterosacral ligament cell culture, secondary culture 4-6 generations, according to implementation are done according to the method described in embodiment 4
Method described in example 5 transfects nonspecific siRNA；
Two, mtt assay experimental procedure
(1) above-mentioned each group cell is taken, cell density is the uterosacral ligament fibroblast of 80%-90%, with 0.25%
Trysin-0.02%EDTA digests, and the M199 culture mediums 1ml containing 10% fetal calf serum (FBS) terminates digestion.After centrifugation, with thin
Born of the same parents' tally counts, and adjustment cell density is 5 × 104/ ml takes the uniform cell suspension of piping and druming, per hole 200u1, kind to 96 holes
Plate is placed in 37 DEG C of 5% carbon dioxide incubator with the M199 culture mediums containing 10%FBS and is cultivated；
(2) second day after kind plate, culture medium is abandoned, 20u1MTT solution (5mg/ml, i.e. 0.5%MTT) is added per hole and continues
Cultivate 4h；
(3) after culture 4h is added in MTT, Formazan crystallizations can be sufficiently formed, and supernatant is gently discarded, and avoid not inciting somebody to action
Formazan crystallizations are removed；
(4) 150u1 dimethyl Asia is added per hole to sough (DMSO), sets low-speed oscillation 10min on shaking table, keeps crystal fully molten
Solution.The light absorption value (A) in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm, and seeks the corresponding average absorbance value of its each group；
(5) second day after kind of plate, survey corresponding A within the 4th day, the 6th day, the 8th day, the tenth day, the 12nd day
Value, and be averaged；
(6) using the time as X-axis, light absorption value is mapped for Y-axis, and it is bent to draw the fibroblastic growth of each group uterosacral ligament respectively
Line.
Three, experimental result
With the fibroblastic growth curve of tetrazolium salts (MTT) colorimetric method for determining, 96 orifice plates are best, and inoculating cell number is 1
×104A/hole.The i.e. A values of the absorbance at 490nm that microplate reader is surveyed are shown in Table 4.As a result it shows：In same time, POP groups
With control group, POP groups and POP interference group, the A value significant differences of POP interference groups control and control group, statistically significant (P<
0.01), it is specifically shown in Fig. 3.The result shows that the growing multiplication activity of POP group cells is less than control group, the growth of POP interference group cells
Proliferation activity is significantly improved compared with POP groups, and difference has statistical significance (P<0.01).
The fibroblastic absorbance A of 4 uterosacral ligament of table (X ± S)
Sequence table
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Claims (10)
1. application of the molecular marker in preparing pelvic prolapse diagnostic preparation, which is characterized in that the molecular marker is
The expression product of SALL1 genes and/or SALL1 genes.
2. application according to claim 1, which is characterized in that the molecular marker high table in pelvic prolapse tissue
It reaches.
3. application according to claim 1, which is characterized in that the pelvic prolapse diagnostic preparation uses fluorescent quantitation
The expression of molecular marker in PCR kit, genechip detection pelvic prolapse tissue, it is preferred that the quantitative fluorescent PCR
Primer containing a pair of of specific amplification molecular marker in kit；
The genetic chip includes the probe with the nucleic acid array hybridizing of molecular marker.
4. application according to claim 1, which is characterized in that the pelvic prolapse diagnostic preparation is examined using immunization method
Survey the expression product of molecular marker in pelvic prolapse tissue, it is preferred that the immunization method is ELISA detections and/colloidal gold
Detection.
5. application of the molecular marker in preparing pelvic prolapse treatment preparation, which is characterized in that the molecular marker is
The expression product of SALL1 genes and/or SALL1 genes.
6. application according to claim 5, which is characterized in that the pelvic prolapse treatment preparation inhibits molecular marker
Transcription and/or expression.
7. application according to claim 6, which is characterized in that use one kind in following methods and/or several inhibition point
The expression of sub- marker：The suppressor of activating molecules marker, activation inhibit the albumen of molecular marker expression, import inhibition
The microRNA of siRNA, activation promotion molecular marker mRNA degradations that molecular marker is transcribed or expressed, promotion molecule is imported
The molecule of marker protein degradation inhibits to promote the factor of molecular marker expression and the expression of albumen.
8. application according to claim 7, which is characterized in that it is described inhibit molecular marker expression siRNA sequence be
SEQ ID NO.3 and SEQ ID NO.4.
9. a kind of pelvic prolapse inhibitor, which is characterized in that the pelvic prolapse inhibitor inhibits transcription or the table of SALL1 genes
It reaches.
10. pelvic prolapse inhibitor according to claim 9, which is characterized in that contain inhibition in pelvic prolapse inhibitor
The siRNA of transcription or the expression of SALL1 genes.
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| EP1337667A2 (en) * | 2000-11-16 | 2003-08-27 | Cedars-Sinai Medical Center | Profiling tumor specific markers for the diagnosis and treatment of neoplastic disease |
| CN101415325A (en) * | 2005-11-29 | 2009-04-22 | 内华达肿瘤研究所 | Targeting of SALL4 for the treatment and diagnosis of proliferative disorders associated with myelodysplastic syndrome (MDS) |
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| ZA200803021B (en) * | 2005-09-12 | 2010-09-29 | Univ Michigan | Recurrent gene fusions in prostate cancer |
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| EP1337667A2 (en) * | 2000-11-16 | 2003-08-27 | Cedars-Sinai Medical Center | Profiling tumor specific markers for the diagnosis and treatment of neoplastic disease |
| CN101415325A (en) * | 2005-11-29 | 2009-04-22 | 内华达肿瘤研究所 | Targeting of SALL4 for the treatment and diagnosis of proliferative disorders associated with myelodysplastic syndrome (MDS) |
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