CN107828885A - 11 β HSD2 and the epigenetic early sign thing susceptible as articular cartilage depauperation and osteoarthritis purposes - Google Patents
11 β HSD2 and the epigenetic early sign thing susceptible as articular cartilage depauperation and osteoarthritis purposes Download PDFInfo
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Abstract
The invention discloses the purposes of early sign thing 11 β HSD2 and epigenetic susceptible as articular cartilage depauperation and osteoarthritis.Result of study shows, compared with Normal group, 11 β HSD2 promoter regions acetylation of histone and expression in IUGR neonatal umbilical cords mescenchymal stem cell or overall umbilical cord reduce；IUGR neonatal umbilical cord mescenchymal stem cell cartilage differentiation abilities are lower, are easier the similar cartilage cell's regression performance of osteoarthritis occur under glucocorticoid effect.Therefore, the risk of the osteochondrodysplasia of subject and future trouble osteoarthritis of growing up is judged by detecting expression and the epigenetic level of the β HSD2 genes of umbilical cord 11.Early warning technology for articular cartilage depauperation and tire source property adult osteoarthritis provides research foundation, and the also integrated control for tire source property adult osteoarthritis provides new approaches.
Description
Technical field
The invention belongs to technical field of clinical medicine, be related to a kind of beta-hydroxysteroid dehydrogenase of 2 type 11 (11 β-
Hydroxysteroid dehydrogenase type-2,11 β-HSD2) gene and its epigenetic develop as articular cartilage
Bad and adult osteoarthritis early warning biomarker.
Background technology
Osteoarthritis is a kind of chronic joint diseases using articular cartilage retrogression pathological changes as major pathologic features.Tradition is seen
Point thinks that osteoarthritis is neurodegenerative disease.Because its incidence of disease is high, treatment difficulty is big, the diagnosis and treatment to osteoarthritis are entered
Row Research Significance is great.Intrauterine growth retardation (intrauterine growth retardation, IUGR) refers to that term infant goes out
Raw body is less than 2.5kg again, or fetal weight is less than two standard deviations of its pregnant age average weight.Large sample epidemiological study is sent out
Existing, the multi-joint osteoarthritis such as low birth weight infant adult defensive position, hip is susceptible [1-3].This room early-stage Study discovery, a variety of pregnancy periods
Allogene exposure (such as caffeine, nicotine, ethanol, dexamethasone) can cause Offspring rat IUGR, and IUGR individuals in utero occur
Osteochondrodysplasia, shows as that staining cartilaginous matrix shoals, cell viability declines, and it is susceptible existing osteoarthritis to be present after adult
As [4,5].Prompting, osteoarthritis, which has, in utero to originate from.However, early warning and the Diagnosis Technique of tire source property adult osteoarthritis
Scarcity, susceptible molecular target is not yet clearly relevant with tire source property adult osteoarthritis.
Glucocorticoid (glucocorticoid, GC) is to determine in utero tire tissue morphology, the key factor of function maturation,
Disease in Infants GC take part in the growth and development of fetus early stage, and participate in the development of multiple internal organs (such as articular cartilage). 11β-HSD2
By metabolic inactivation Disease in Infants GC, can effectively adjust tire tissue local active GC is horizontal and function [6,7].We study
Show, the in utero neuroendocrine metabolism caused by a variety of allogenes (such as caffeine, nicotine, ethanol) of pregnancy period exposure changes (bag
HPAA functional development is included to suppress and the change of periphery glucose-lipid metabolism), with Disease in Infants GC exposures and tissue excessively
Related β-HSD2 the low expressions of important gene 11 of interior development are relevant；And 11 β-HSD2 expression or activity reduce, Disease in Infants can be amplified
Inhibitory action of the GC to embryo growth and development.Prompting, 11 β-HSD2 may also regulate and control cartilage part GC in cartilage local expression
Played an important role in effect.Separately studies have found that, articular cartilage matrix can be caused to subtract while treating osteoarthritis with GC
Less, articular chondrocyte apoptosis increase [8,9].Prompting, 11 β-HSD2 can be used as articular cartilage depauperation and adult osteoarthritis easy
The molecular target of sense.However, have not yet to see any relevant report.
People's huatong plastic mescenchymal stem cell (human Wharton`s jelly-derived mesenchymal stem
Cells, WJ-MSCs) it is a kind of pluripotent stem cell, cartilage cell [10-12] can be divided into vitro.It is soft in WJ-MSCs
External evoked using IL-1 β (interleukin-1 β, IL-1 β) on bone cell differentiation model, the bone that can establish cellular level closes
Save scorching original mold type.In ontogenetic process, what mescenchymal stem cell (including WJ-MSCs) can influence as in utero poor environment
It target tissue, can be programmed, i.e., " remember " pessimal stimulation [13] for influenceing function after adult received during primordial growth traits.
Studies have reported that the recognizable epigenetic marking [14,15] is remained with the WJ-MSCs in small for gestational age infant source.It is worth note
Meaning, current each laboratory is not yet reached common understanding in terms of WJ-MSCs separation and amplification in vitro, and there is no a kind of method energy
Rapidly and efficiently isolate homogeneous and meet the WJ-MSCs that clearly defines, this is obviously clinical to be carried out in the future based on high flux screening
The implementation of early warning provides inconvenience.Have been reported that prompting, the epigenetic analysis in overall umbilical cord is possibly used for assessing
Fat and metabolic syndrome neurological susceptibility [16] after individual birth.Research prompting above, it is bad that WJ-MSCs can turn into checking fetus
The preliminary screening system of potential molecular mechanism is developed, it programs the marking can also embody in overall umbilical cord, based on overall umbilical cord
The epigenetic marking is detected as providing new think of based on extensive neonatal high flux screening and predictive disease risk in the future
Road.
Bibliography：
1.Clynes,M.A.,et al.,Further evidence of the developmental origins of
osteoarthritis:results from the Hertfordshire Cohort Study.J Dev Orig Health
Dis,2014.5(6):p.453-8.
2.Sayer,A.A.,et al.,Weight from birth to 53years:a longitudinal study
of the influence on clinical hand osteoarthritis.Arthritis Rheum,2003.48(4):
p.1030-3.
3.Poole,J.,et al.,Birth weight,osteoarthritis of the hand,and
cardiovascular disease in men.Ann Rheum Dis,2003.62(10):p.1029；author reply
1029.
4.Tan,Y.,et al.,Caffeine-induced fetal rat over-exposure to maternal
glucocorticoid and histone methylation of liver IGF-1might cause skeletal
growth retardation.Toxicol Lett,2012.214(3):p. 279-87.
5.Deng,Y.,et al.,Nicotine-induced retardation of chondrogenesis
through down-regulation of IGF-1 signaling pathway to inhibit matrix
synthesis of growth plate chondrocytes in fetal rats.Toxicol Appl Pharmacol,
2013.269(1):p.25-33.
6.Wyrwoll,C.S.,M.C.Holmes,and J.R.Seckl,11beta-hydroxysteroid
dehydrogenases and the brain: from zero to hero,a decade of progress.Front
Neuroendocrinol,2011.32(3):p.265-86.
7.Tsugita,M.,et al.,Differential regulation of 11beta-hydroxysteroid
dehydrogenase type-1and-2gene transcription by proinflammatory cytokines in
vascular smooth muscle cells.Life Sci,2008.83(11-12): p.426-32.
8.Kou,H.,et al.,Maternal glucocorticoid elevation and associated
blood metabonome changes might be involved in metabolic programming of
intrauterine growth retardation in rats exposed to caffeine
prenatally.Toxicol Appl Pharmacol,2014.275(2):p.79-87.
9.Conde,J.,et al.,Corticoids synergize with IL-1in the induction of
LCN2.Osteoarthritis Cartilage,2017. 25(7):p.1172-1178.
10.Tanthaisong,P.,et al.,Enhanced Chondrogenic Differentiation of
Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stem Cells by GSK-
3Inhibitors.PLoS One,2017.12(1):p.e0168059.
11.Sukarieh,R.,et al.,Molecular pathways reflecting poor intrauterine
growth are found in Wharton's jelly-derived mesenchymal stem cells.Hum
Reprod,2014.29(10):p.2287-301.
12.Batsali,A.K.,et al.,Mesenchymal stem cells derived from Wharton's
Jelly of the umbilical cord: biological properties and emerging clinical
applications.Curr Stem Cell Res Ther,2013.8(2):p. 144-55.
13.Broholm,C.,et al.,Epigenetic programming of adipose-derived stem
cells in low birthweight individuals.Diabetologia,2016.59(12):p.2664-2673.
14.Sukarieh,R.,et al.,Molecular pathways reflecting poor intrauterine
growth are found in Wharton's jelly-derived mesenchymal stem cells.Human
Reproduction,2014.29(10):p.2287-2301.
15.Tan,P.Y.,et al.,E2F1Orchestrates Transcriptomics and Oxidative
Metabolism in Wharton's Jelly-Derived Mesenchymal Stem Cells from Growth-
Restricted Infants.PLoS One,2016.11(9):p. e0163035.
16.Godfrey,K.M.,et al.,Epigenetic gene promoter methylation at birth
is associated with child's later adiposity.Diabetes,2011.60(5):p.1528-34.
The content of the invention
In order to overcome the shortcomings of the prior art, the purpose of the present invention is dry thin by detecting neonatal umbilical cord mesenchyma
The level of 11 β-HSD2 gene expressions and epigenetic in born of the same parents WJ-MSCs and overall umbilical cord, is in utero articular cartilage depauperation
And the early warning offer research foundation that property adult osteoarthritis in tire source is susceptible.
The purpose of the present invention is achieved through the following technical solutions：
First aspect present invention provides the β-HSD2 genes of neonatal umbilical cord mescenchymal stem cell WJ-MSCs or overall umbilical cords 11
Expression and promoter region H3K9ac are easy for detecting early warning osteochondrodysplasia and tire source property adult osteoarthritis in preparation
Application in the kit of sense.
Preferably, the 11 β-HSD2 gene expressions and promoter region H3K9ac are in neonatal umbilical cord mescenchymal stem cell
Horizontal decline in WJ-MSCs or overall umbilical cords, then there are osteochondrodysplasia and the susceptible risk of tire source property adult osteoarthritis.
Preferably, the reagent comprising 11 β-HSD2 gene expressions of detection in described kit detects 11 β-HSD2 genes and opened
Reagent horizontal mover area H3K9ac.
Preferably, the primer pair comprising 11 β-HSD2 of amplification, detection in the reagent of 11 β-HSD2 gene expressions of the detection
Primer pair horizontal 11 β-HSD2 gene promoter area H3K9ac.
Preferably, 11 β-HSD2 of amplification primer pair sequence is as described in Seq ID No.1 and Seq ID No.2.
Preferably, primer pair such as Seq ID No.3 horizontal 11 β-HSD2 gene promoter area H3K9ac of the detection and
Described in Seq ID No.4.
Second aspect of the present invention provides a kind of early warning osteochondrodysplasia and property adult osteoarthritis in tire source is susceptible
Kit, the primer pair comprising 11 β-HSD2 of amplification, 11 β-HSD2 gene promoter area H3K9ac of detection horizontal primer pair.
Preferably, 11 β-HSD2 of amplification primer pair sequence is as described in Seq ID No.1 and Seq ID No.2.
Preferably, primer pair such as Seq ID No.3 horizontal 11 β-HSD2 gene promoter area H3K9ac of the detection and
Described in Seq ID No.4.
The present invention is confirmed by studying, compared with control group, pregnancy period caffeine exposure (prenatal caffeine
Exposure, PCE) organize the β-HSD2 of Offspring rat tire cartilage 11 and cartilage marker gene COL2A1 and proteoglycans
The mRNA expressions of (aggrecan, AGAN) reduce (P<0.01)；Compared with control group, the β-HSD2 of PCE tires cartilage 11 start
The horizontal obvious reduction (P of sub-district H3K9ac<0.01), above change is from utero to after birth.Further confirm, chronic stress
Afterwards, compared with control group, PCE groups cartilage cell arrangement is more disorderly, and cartilage cell's number significantly reduces, and Mankin ' s scorings are notable
Rise, cartilage marker gene COL2A1 expression significantly reduces, and becomes apparent (P than being reduced before chronic stress<0.01).More than
Prompting, the local 11 β-HSD2 genes low expressions of PCE group Offspring rats cartilage and promoter region H3K9ac low-levels, and from utero
After extending to birth, cause cartilage local endogenous activity GC to increase, cause cartilage phenotype gene persistently to suppress and cartilage development
Bad, presentation osteoarthritis is susceptible under poor environment-chronic stress induction.
Then, the WJ-MSCs cartilage directed differentiations in inventor discovery IUGR sources are bad and under IL-1 β processing
Cartilage matrix degraded becomes apparent from.Further, 11 β-HSD2 and downstream in IUGR and normal newborn umbilical cord WJ-MSCs are compared
Mrna expression, compared with control group, the β-HSD2 of IUGR umbilical cords WJ-MSCs 11 and downstream gene COL2A1 and AGAN
MRNA expression is obvious to reduce (P<0.01), while the β-HSD2 promoter regions H3K9ac of IUGR umbilical cords WJ-MSCs 11 levels substantially drop
Low (P<0.01).Prompting, there is 11 β-HSD2 genes low expressions and the low-level WJ-MSCs of promoter region H3K9ac cartilage be present
Poorly differentiated, and the like cell reaction of presentation osteoarthritis is more easy under inflammatory stimulus.
Further, inventor compares the gene of 11 β-HSD2 in IUGR infants and normal newborn entirety umbilical cord
Expression and the horizontal changes of promoter region H3K9ac.It was found that compared with normal umbilical cord, 11 β-HSD2 gene expression in IUGR umbilical cords
Significantly reduced with promoter region H3K9ac levels.Prompting, IUGR entirety umbilical cord exist identical with the WJ-MSCs in IUGR sources
Molecular changes, i.e. 11 β-HSD2 gene low expression and promoter region H3K9ac low-levels.
To sum up, the β-HSD2 gene expressions of neonatal umbilical cord 11 and promoter region H3K9ac levels join together to can be used for closing
Save osteochondrodysplasia and the susceptible early warning of tire source property adult osteoarthritis.
Brief description of the drawings
Fig. 1, control group and the β-HSD2 genes of PCE group tires cartilage 11 and its promoter region H3K9ac, cartilage marker gene two
Collagen Type VI (collagen type II alpha 1, COL2A1) and proteoglycans (aggrecan, AGAN) mRNA expressions.
Compared with control group,*P<0.05,**P<0.01。
12 weeks β-HSD2 genes of cartilage 11 and its promoter region H3K9ac and cartilage mark after Fig. 2, control group and the birth of PCE groups
Will mrna expression.Compared with control group,*P<0.05,**P<0.01。
2 weeks articular cartilage of immobilized color substrates of chronic stress, Mankin ' s are given within 10 weeks after Fig. 3, control group and the birth of PCE groups
Scoring and COL2A1 mRNA expressions.Compared with control group,**P<0.01。
Fig. 4, WJ-MSCs cartilage directed differentiation and the reaction of osteoarthritis like cell；
11 β-HSD2 genes and its promoter region H3K9ac and cartilage mark in Fig. 5, IUGR and normal newborn WJ-MSCs
Mrna expression.Compared with control group,**P<0.01。
11 β-HSD2 genes and its promoter region H3K9ac and cartilage mark base in Fig. 6, IUGR and normal newborn umbilical cord
Because of mRNA expressions.Compared with control group,**P<0.01。
Embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the inventive method, remaining content without limiting the invention in any way announcement.
SPSS 17 and Prism 5.0 is used for data analysis.Quantitative data be represented as mean value ± standard error (mean ±
S.E.M.) and obtain qRT-PCR independent samples t tests analysis.Mankin ' s scorings and COL2A1 mRNA after chronic stress
Expression data use two-way analysis of variance.
【Embodiment 1】
After control group and PCE group tires cartilage, birth 12 weeks β-HSD2 promoter regions H3K9ac of cartilage 11,11 β-HSD2 and
Cartilage marker gene Type Ⅱ collagen (collagen type II alpha 1, COL2A1) and proteoglycans (aggrecan,
AGAN) mRNA expressions.
1st, experimental animal
SPF level health Wistar rats, purchased from Disease Prevention Control Center, Hubei Prov, animal credit number：SCXK (Hubei Province)
2008-2010.This research obtains the approval of medical board Ethics Committee of Wuhan University, and protects certification in strict accordance with International Laboratory Animal
Appraisal agency's relevant treatment criterion performs.
Experimental animal feeding is in barrier environment, and 22~25 DEG C of temperature, humidity 50%, day alternates with night within 12 hours.
2nd, animal is handled
The Wistar rats of health and adult are bought from Disease Control and Prevention Center of Hubei Province, Female Body Weight is 209 ± 12g, male body
Weight is 258 ± 17g.Every night 6 when according to male and female 2:1 is mated, secondary morning check cloudy bolt and sperm smear determine female mice whether into
Work(is become pregnant, and is designated as pregnant 0 day (gestation day 0, GD0).Pregnant rats are randomly divided into 2 groups：Control group, PCE groups.In
GD9 starts, and during every morning 8-10, PCE groups give caffeine (120mg/kg.d) gavage, the isometric physiology of control group gavage
Salt solution.In GD20, a part of pregnant mouse by tire mouse lower limb detachment, retains complete knee through etherization and Caesarean mouse (n=8)
Joint, left knee, which freezes, is stored in that -80 DEG C of refrigerators are stand-by, and right knee is placed in neutral formalin and fixed, and embeds, section, row hematoxylin-eosin
(hematoxylin-eosin, HE) and Toluidine blue staining.The pregnant mouse of another part (including control group, PCE groups) spontaneous labor.
After using produces day as birth 0 day (postnatal day 0, PD0), 2 male newborn mouses are selected per nest to after being born：Wherein 1
Only put to death at 12 weeks (postnatal week 12, PW12)；Give after frozen water swimming in 2 weeks stimulates for other 1 and put to death in PW10,
And sample (n=8) is preserved in the manner described above.3rd, genetic test
In utero tire mouse cartilage, PW12 and the PW12 cartilage total serum IgEs for giving frozen water swimming stimulation in 2 weeks, reverse transcription are for extraction
CDNA, quantitative analysis related gene expression situation is carried out by qRT-PCR.Each gene primer sequence is as shown in table 1：
Design of primers：Utilize Primer Premier 5.0 and the design of NCBI Blast databases and checking primer.Design
After good primer sequence, Sangon Biotech (Shanghai) Co., Ltd. is transferred to synthesize, and PAGE is purified.New close will be housed
EP pipes 7500g into primer is centrifuged 10 minutes, adds the molecular biology ultra-pure water of tube wall subscript injection body product, and concussion mixes standby
With.
Table 1：RT-PCR primer sequence
4th, chromatin immune is co-precipitated
Cartilaginous tissue after homogenate washs the formaldehyde of addition 37% in lower floor's cell precipitation acquired in 3 times with 1ml PBS,
Room temperature places 10min and carries out chromatin crosslinking, and the glycine room temperature for then adding 2.5mM places 5min termination crosslinkings.PBS is clear
Wash after collecting cell and split with Ultrasonic Cell Disruptor (condition of work be 30% power, 2s on, 1s off), progress ultrasonication 5min
Solve DNA.12,000rpm centrifuges 10min under 4 DEG C of environment, takes supernatant to collect DNA fragmentation.DNA supernatants are dispensed, are respectively
IgG, Input, H3K9ac tri- is managed.The BSA closing Protein G that 1mg/ml is added in DNA supernatants are respectively dispensed in addition to Input
Beads is closed, and adds the corresponding antibody (abcam, USA) that total amount is 1 μ g, 4 DEG C of overnight incubations.3000rpm centrifuges 1min, receives
Collect immunoprecipitate.Clean and centrifuge 3 immunoprecipitates, 65 DEG C of water-baths solve crosslinking overnight.Using PCR purification kits
(Tiangen Co., Beijing, China) purifies DNA, and DNA after purification is dissolved with 50 μ L water, in -20 DEG C of preservations.Each
Specimen needle after purification carries out qRT-PCR to 11 β-HSD2 promoter regions, and primer sequence is as shown in table 2, acetylation of histone knot
Fruit is using Input as reference.
Table 2.RT-PCR primer sequences
5th, result
Control group, the β-HSD2 of PCE group tires cartilage 11 and cartilage marker gene mRNA expression are as shown in Figure 1A.With control group phase
Than the β-HSD2 of PCE tires cartilage 11 and cartilage marker gene COL2A1 and AGAN mRNA expression reduce (P<0.05,P<0.01).
11 β-HSD2 promoter regions H3K9ac are horizontal to be changed as shown in Figure 1B.Compared with control group, the β-HSD2 promoters of PCE tires cartilage 11
The horizontal obvious reduction (P of area H3K9ac<0.01).
Control group, PCE groups birth after 12 weeks β-HSD2 of cartilage 11 and cartilage marker gene mRNA expressions such as Fig. 2A,
Shown in 2B.Compared with control group, the β-HSD2 of PCE groups cartilage 11 and cartilage marker gene COL2A1 and AGAN mRNA expression drops
Low (P<0.05,P<0.01), while 11 β-HSD2 promoter region H3K9ac levels also reduce (P<0.01).
Toluidine blue staining (Fig. 3 A) after 2 weeks chronic stresses is given within 10 weeks after control group, the birth of PCE groups, Mankin ' s are commented
Divide (Fig. 3 B) and COL2A1 mRNA expression (Fig. 3 C) as shown in Figure 3.Compared with control group, do not receive the filial generation of chronic stress,
PCE group articular cartilage matrix dyssynthesis, cartilage cell's arrangement disorder, cartilage marker gene COL2A1 are reduced.Chronic stress
Afterwards, PCE groups cartilage cell arrangement is more disorderly, and cartilage cell's number significantly reduces, Mankin ' s scorings significantly rise, cartilage mark
Gene C OL2A1 is significantly reduced, and becomes apparent from (P than being reduced before chronic stress<0.01).
The present embodiment result is prompted, and PCE group Offspring rat cartilages locally lie in 11 β-HSD2 genes low expressions and promoter
Area's H3K9ac low-levels, and after in utero birth is extended to, cause cartilage local endogenous activity GC to increase, cause cartilage phenotype
Gene persistently suppresses and osteochondrodysplasia, and it is susceptible that osteoarthritis finally is presented under poor environment (such as chronic stress) induction.
【Embodiment 2】
Normal group and IUGR group neonatal umbilical cord mescenchymal stem cells (Wharton`s jelly-derived
Mesenchymal stem cells, WJ-MSCs) 11 β-HSD2 promoter regions H3K9ac, 11 β-HSD2 and downstream gene mRNA
Expression.
1st, umbilical cord mesenchymal stem cells WJ-MSCs cultures carry out Total RNAs extraction
From Wuhan University, Central-South obstetrics and gynecology hospital collects neonatal umbilical cord sample.It is divided into 2 groups：Normal group and IUGR
Group (n≤8), total RN A are extracted after cultivating mescenchymal stem cell.1ml Trizol are added in homogenizer, are fully homogenized on ice
Afterwards, go in no RNase 1.5ml EP pipes；200 μ l chloroforms are added, 15s is acutely shaken, stands 10min on ice；4℃
12,000 × rpm centrifuges 15min；Careful 500 μ l upper strata aqueous phases of drawing move to one newly without the body such as in RNase 1.5ml EP pipes, adding
Long-pending isopropanol, overturn and mix precipitation RNA, 20~30 DEG C of standing 10min；4 DEG C 12,000 × rpm centrifugation 10min, supernatant is abandoned,
Add the ethanol of 1ml 75% washing precipitation, overturn and mix；4 DEG C of 9500 × rpm centrifuge 5min, abandon ethanol (being repeated twice), room temperature is put
10min is put to dry；Add the DEPC processing water 20 μ l of 60 DEG C of incubations；Ultraviolet specrophotometer surveys RNA concentration and purity, -80 DEG C of guarantors
Deposit.
2nd, umbilical cord WJ-MSCs is to osteoarthritis like cell modeling after Chondrocyte Differentiation
Osteoarthritis permissive cell model after umbilical cord WJ-MSCs cartilage directed differentiations.By the 4th generation WJ-MSCs with common complete
Full medium culture 24 hours, then adds cartilage differentiation culture medium, and it is small to contain DMEM/F12 95%, Gibco in culture medium
100 × ITS, 100nM dexamethasone, 10ng/mL TGF β 1,40 μ g/mL proline, the 50 μ g/mL of cow's serum 5%, 1% resist
Bad hematic acid, 100mg/L pyruvic acid, 10% mycillin etc., it is placed in 37 DEG C of constant incubators, maintains CO2Concentration be 5%, it is wet
Spend and broken up for 95%, in induction 21 days, directed differentiation was cartilage cell.Cell osteoarthritis after the beta induced differentiation of IL-1
Modeling.By differential medium be changed to containing rh Interleukin 1β (recombinant human interleukin-1 β,
RhIL-1 β) 10ng/mL DMEM/F12 culture medium, cultivate 24 hours carry out osteoarthritis modeling.
3rd, 11 β-HSD2 and downstream gene expression detection in neonatal umbilical cord WJ-MSCs
Cell culture propagation WJ-MSCs 5 days, reverse transcription is cDNA after extracting cell total rna, and passes through qRT-PCR
Carry out quantitative analysis related gene expression situation.Each gene primer sequence is as shown in table 1.
4th, the horizontal detections of the β-HSD2 promoter regions H3K9ac of neonatal umbilical cord WJ-MSCs 11
Normal group and IUGR infants are obtained into umbilical cord mesenchymal stem cells cell suspension, it is coprecipitated to carry out chromatin immune
Form sediment and test, finally obtain the different IP products of Input, H3K9ac, IgG, and pass through DNA Purification Kit samples, qRT-
PCR detection index of correlation changes.Primer sequence is as shown in table 2.
5th, experimental result
After cartilage directed differentiation and after osteoarthritis like cell modeling, the similar performance of osteoarthritis chondrocytes is such as Fig. 4 institutes
Show.After orienting cartilage differentiation, compared with Normal group, the WJ-MSCs color substrates in IUGR sources significantly shoal；Further exist
After IL-1 β processing, compared with Normal group, worse state is presented in the cellular matrix dyeing after IUGR components.
11 β-HSD2 and downstream gene mRNA expression (Fig. 5 A) and startup in IUGR and normal newborn umbilical cord WJ-MSCs
Sub-district H3K9ac levels (Fig. 5 B) are as shown in Figure 5.Compared with Normal group, the β-HSD2 of IUGR umbilical cords WJ-MSCs 11 and downstream
Gene C OL2A1 and AGAN mRNA expression are obvious to reduce (P<, while the β-HSD2 promoters of IUGR umbilical cords WJ-MSCs 11 0.01)
The horizontal obvious reduction (P of area H3K9ac<0.01).
The present embodiment result is prompted, 11 β-HSD2 genes low expressions and promoter region in the WJ-MSCs in IUGR sources
H3K9ac low-levels, be characterized in WJ-MSCs cartilage directed differentiations it is bad and break up after cell be more easy under inflammatory stimulus be in
Existing arthritis like cell reaction.
【Embodiment 3】
The Normal group and β-HSD2 promoter regions H3K9ac of IUGR groups neonatal umbilical cord 11,11 β-HSD2 and cartilage mark
Gene Type Ⅱ collagen (collagen type II alpha 1, COL2A1) and proteoglycans (aggrecan, AGAN) mRNA tables
Change up to level.
1st, 11 β-HSD2 expression and cartilage marker gene detection of expression in neonatal umbilical cord tissue
From Wuhan University, Central-South obstetrics and gynecology hospital collects neonatal umbilical cord sample.It is divided into 2 groups：Normal group and IUGR
Group (n≤8).10cm umbilical cords are taken to shred addition culture medium, (Invitrogen is public containing (1mg/mL) hyaluronidase clostridiopetidase A I
Department).37 DEG C are at the uniform velocity swayed 12 hours, add PBS liquid to blow and beat dilution repeatedly, collect dilution (containing tissue), 3500rpm centrifugations
20min, supernatant and the adipose tissue of floating are then abandoned, collect tissue.1ml Trizol are added in homogenizer, on ice fully
After homogenate, go in no RNase 1.5ml EP pipes；200 μ l chloroforms are added, 15s is acutely shaken, stands 10min on ice；4
DEG C 12,000 × rpm centrifugation 15min；Careful 500 μ l upper strata aqueous phases of drawing move to one newly without in RNase 1.5ml EP pipes, add
Isometric isopropanol, overturn and mix precipitation RNA, 20~30 DEG C of standing 10min；4 DEG C 12,000 × rpm centrifugation 10min, abandon
Supernatant, add the ethanol of 1ml 75% washing precipitation, overturn and mix；4 DEG C of 9500 × rpm centrifuge 5min, abandon ethanol (being repeated twice),
Room temperature is placed 10min and dried；Add the DEPC processing water 20 μ l of 60 DEG C of incubations；Ultraviolet specrophotometer surveys RNA concentration and purity ,-
80 DEG C of preservations.Reverse transcription is cDNA after extracting total tissue RNA, and carries out quantitative analysis related gene expression by qRT-PCR
Situation.Each gene primer sequence is as shown in table 1.
2nd, the horizontal detections of the β-HSD2 promoter regions H3K9ac of neonatal umbilical cord WJ-MSCs 11
Will normally and IUGR infants obtain umbilical cord tissue, carry out chromatin immune co-precipitation experiment, finally acquisition Input,
H3K9ac, IgG different IP products, and pass through DNA Purification Kit samples, RT-PCR detection index of correlation changes.Draw
Thing sequence is as shown in table 2.
3rd, result
11 β-HSD2 and cartilage marker gene mRNA expression (Fig. 6 A), promoter region in IUGR and normal newborn umbilical cord
H3K9ac levels (Fig. 6 B) are as shown in Figure 6.Compared with Normal group, 11 β-HSD2 and cartilage marker gene in IUGR umbilical cords
COL2A1, AGAN mRNA expression are obvious to reduce (P<0.01), while the β-HSD2 promoter regions H3K9ac of IUGR umbilical cords 11 is horizontal
It is obvious to reduce (P<0.01).
The present embodiment result is prompted, and 11 β-HSD2 genes low expressions and promoter region H3K9ac low-levels exist in
In the overall umbilical cord in IUGR sources.
Sequence table
<110>Wuhan University
<120>11 β-HSD2 and the epigenetic early sign thing susceptible as articular cartilage depauperation and osteoarthritis
Purposes
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gacatgccat atccgtgctt 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gctggatgat gctgaccttg 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccgggagaga agtgaaggaa 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gacactcgct ttctctgctc 20
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gaaatcccat caccatcttc cag 23
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gagtccttcc acgataccaa ag 22
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gctcccagaa catcacctac ca 22
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
acagtcttgc cccacttacc g 21
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
aagggcgagt ggaatgatgt 20
<210> 10
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
cgcttctgta gtctgcgttt gt 22
Claims (9)
1.11 β-HSD2 gene expressions and promoter region H3K9ac are being prepared for detecting early warning osteochondrodysplasia and tire
Application in the susceptible kit of source property adult osteoarthritis, it is characterised in that the 11 β-HSD2 genes and promoter region
H3K9ac derives from neonatal umbilical cord mescenchymal stem cell WJ-MSCs or overall umbilical cords.
2. application according to claim 1, it is characterised in that the 11 β-HSD2 gene expressions and promoter region H3K9ac
Horizontal decline in neonatal umbilical cord mescenchymal stem cell WJ-MSCs or overall umbilical cords, then have osteochondrodysplasia and tire source
Property the susceptible risk of adult osteoarthritis.
3. application according to claim 1 or 2, it is characterised in that 11 β-HSD2 bases of detection are included in described kit
Because of the horizontal reagent of the reagent and 11 β-HSD2 gene promoter area H3K9ac of detection of expression.
4. application according to claim 3, it is characterised in that included in the reagent of 11 β-HSD2 gene expressions of the detection
Expand 11 β-HSD2 primer pair, the primer pair of detection 11 β-HSD2 gene promoter area H3K9ac levels.
5. application according to claim 4, it is characterised in that 11 β-HSD2 of amplification primer pair sequence such as Seq ID
Described in No.1 and Seq ID No.2.
6. application according to claim 4, it is characterised in that 11 β-HSD2 gene promoter areas H3K9ac water of the detection
Flat primer pair is as described in Seq ID No.3 and Seq ID No.4.
A kind of susceptible kit of osteoarthritis 7. early warning osteochondrodysplasia and tire source property are grown up, it is characterised in that bag
The primer pair of the 11 β-HSD2 containing amplification, 11 β-HSD2 gene promoter area H3K9ac of detection horizontal primer pair.
8. kit according to claim 7, it is characterised in that 11 β-HSD2 of amplification primer pair sequence such as Seq
Described in ID No.1 and Seq ID No.2.
9. kit according to claim 7, it is characterised in that 11 β-HSD2 gene promoter area H3K9ac of the detection
Horizontal primer pair is as described in Seq ID No.3 and Seq ID No.4.
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| CN201711378099.9A CN107828885A (en) | 2017-12-19 | 2017-12-19 | 11 β HSD2 and the epigenetic early sign thing susceptible as articular cartilage depauperation and osteoarthritis purposes |
| CN202110775991.0A CN113481292A (en) | 2017-12-19 | 2017-12-19 | Application of 11 beta-HSD 2 and epigenetic marker as early markers of joint cartilage dysplasia and osteoarthritis susceptibility |
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| CN202110775991.0A Pending CN113481292A (en) | 2017-12-19 | 2017-12-19 | Application of 11 beta-HSD 2 and epigenetic marker as early markers of joint cartilage dysplasia and osteoarthritis susceptibility |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904047A (en) * | 2019-11-25 | 2020-03-24 | 武汉大学 | Cell model for screening developmental toxic exogenous compounds by taking 11 β -HSD2 as target spot, construction method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904047A (en) * | 2019-11-25 | 2020-03-24 | 武汉大学 | Cell model for screening developmental toxic exogenous compounds by taking 11 β -HSD2 as target spot, construction method and application thereof |
| CN110904047B (en) * | 2019-11-25 | 2022-01-04 | 武汉大学 | Cell model for screening developmental toxicity exogenous compound by taking 11 beta-HSD 2 as target spot, construction method and application thereof |
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