CN103820438A - RNA (Ribonucleic Acid) molecule for improving osteogenic capability of mesenchymal stem cell and effecting target point and application thereof - Google Patents
RNA (Ribonucleic Acid) molecule for improving osteogenic capability of mesenchymal stem cell and effecting target point and application thereof Download PDFInfo
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- CN103820438A CN103820438A CN201410050371.0A CN201410050371A CN103820438A CN 103820438 A CN103820438 A CN 103820438A CN 201410050371 A CN201410050371 A CN 201410050371A CN 103820438 A CN103820438 A CN 103820438A
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Abstract
The invention discloses an RNA (Ribonucleic Acid) molecule for improving the osteogenic capability of a mesenchymal stem cell, and an effecting target point and application of the RNA molecule, and belongs to the technical field of biology. The nucleotide sequence of the RNA molecule disclosed by the invention is as shown in SEQ ID NO.1; the RNA molecule can specially inhabit expression of ICAM-1 in the mesenchymal stem cell, can remarkably relieve the inhibition function of ICAM-1 for bone formation and improve the mesenchymal stem cell to osteoblast differentiation, is beneficial for improving the osteogenic capability of the mesenchymal stem cell in an inflammatory micro environment and prompting bone regeneration, and is beneficial for bone repair. The invention further discloses application of the small RNA molecule in preparing a medicament for improving the osteogenic capability of the mesenchymal stem cell and treating osteoporosis, bone destruction and knitting slowness caused by inflammation factors.
Description
Technical field
The invention belongs to biological technical field, be specifically related to improve RNA molecule and action target spot and the application of mescenchymal stem cell osteogenic ability.
Background technology
Osseous tissue that the reason such as wound and disease causes is damaged is clinical common and more unmanageable problem, how to promote that the reparation that osseous tissue is damaged is association area scholar's research emphasis always.In recent years, by the application of tissue engineering technique, associated problem have obtained solution to a certain degree.Fill the biomaterial of analog cell epimatrix by giving damaged part, implant and there is the seed cell that is divided into cambium ability, realize the reparation of damaged tissue, proved a comparatively effectively therapeutic strategy by fundamental research and clinical application.
Mescenchymal stem cell is a kind of adult stem cell with multi-lineage potential, can break up mature cells such as becoming scleroblast, lipoblast and chondroblast under suitable condition.The characteristic of this Multidirectional Differentiation of mescenchymal stem cell, makes it likely become the seed cell of repairing Various Tissues damage.Mescenchymal stem cell also has the highly ability of propagation, can be expanded to clinical treatment desired number from lesser amt in the short period of time, meets the needs of clinical treatment.Because mescenchymal stem cell is convenient to amplification, can be divided in vivo and in vitro Various Tissues cell, at present, it has become the study hotspot in regenerative medicine field.Researchist uses mescenchymal stem cell treatment osteogenesis imperfecta congenita; bone is damaged; the diseases such as osteochondrodysplasia; and obtain satisfied effect (Chiu LH, Lai WF, Chang SF; Wong CC; Fan CY, Fang CL, Tsai YH.The effect of type II collagen on MSC osteogenic differentiation and bone defect repair.Biomaterials.2014; 35 (9): 2680-2691.McCoy RJ; Widaa A; Watters KM; Wuerstle M; Stallings RL; Duffy GP, O'Brien FJ.Orchestrating osteogenic differentiation of mesenchymal stem cells--identification of placental growth factor as a mechanosensitive gene with a pro-osteogenic role.Stem Cells.2013; 31 (11): 2420-2431.Pereira RF; O'Hara MD; Laptev AV; Halford KW; Pollard MD, Class R, Simon D; Livezey K, Prockop DJ.Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis imperfecta.Proc Natl Acad Sci U S is A.1998; 95 (3): 1142-1147.).But along with going deep into that mescenchymal stem cell is studied, increasing tera incognita shows, and waits for further investigation.
The bone repair of mescenchymal stem cell is usually subject to the adjusting of local microenvironment.The damaged osseous tissue that usually causes of bone due to wound and infection infects, and the damaged part of bone is usually accompanied by inflammatory cytokine and infiltrates.And also increasing with inflammatory cytokine in local bone tissue usually of the destruction of bone that autoimmune disorder causes.Inflammatory cytokine, as tumour necrosis factor and interleukin 1 etc. usually suppress mescenchymal stem cell to osteoblast differentiation, affects osteanagenesis reparation.Therefore, find new action target spot, promotion mescenchymal stem cell to osteoblast differentiation, improves the effect that osteanagenesis is repaired in inflammatory microenvironment, is the major issue of association area scientific research and clinical application.
Find by research recently: ICAM-1 (intercellular cell adhesion molecule, ICAM-1) is the target spot of a regulation and control mescenchymal stem cell to osteoblast differentiation.ICAM-1 belongs to adhesion molecule immunoglobulin superfamily (IgSF), can adhere to and migration by mediated lymphocytes, in immunity of organism process and Inflammatory response, plays an important role.Recently there are some researches show that ICAM-1 is the key molecule of mescenchymal stem cell performance immunosuppressive action.But, whether there is regulating effect about ICAM-1 for the biological characteristics of mescenchymal stem cell self especially differentiation capability and mechanism of action but has no report.
Summary of the invention
The object of the present invention is to provide a kind of small RNA molecular that improves mescenchymal stem cell osteogenic ability.
Second object of the present invention is to provide the action target spot of above-mentioned small RNA molecular.
The 3rd object of the present invention is to provide a kind of mescenchymal stem cell.
The 4th object of the present invention is to provide above-mentioned small RNA molecular to improve the application in mescenchymal stem cell osteogenic ability medicine in preparation.
The osteoporosis that provides small RNA molecular to cause at preparation treatment inflammatory factor, destruction of bone, the application in the slow medicine of knitting are provided the 5th object of the present invention.
The 6th object of the present invention is that the osteoporosis, destruction of bone, the knitting that provide above-mentioned mescenchymal stem cell to cause at preparation treatment inflammatory factor are slow, nature osteogenesis imperfecta, and bone is damaged, the application of the application in osteochondrodysplasia medicine.
Realize technical scheme of the present invention as follows:
Improve a small RNA molecular for mescenchymal stem cell osteogenic ability, described small RNA molecular is the antisense nucleic acid of the mRNA of target ICAM-1, and its sequence is
5’-UUUGACAGACUUCACCACCTT-3’（SEQ?ID?NO.1）。
The action target spot of above-mentioned small RNA molecular, in the mRNA sequence that described action target spot is ICAM-1 one section, its nucleotide sequence is as shown in SEQ ID NO.2.
The sequence of action target spot is 5 '-GGUGGUGAAGUCUGUCAAA-3 ' (SEQ ID NO.2).
A kind of mescenchymal stem cell, it contains above-mentioned small RNA molecular.
Osteoporosis, destruction of bone, knitting that above-mentioned mescenchymal stem cell causes at preparation treatment inflammatory factor are slow, nature osteogenesis imperfecta, and bone is damaged, the application in osteochondrodysplasia medicine.
Above-mentioned small RNA molecular improves the application in mescenchymal stem cell osteogenic ability medicine in preparation.
The osteoporosis that above-mentioned small RNA molecular causes at preparation treatment inflammatory factor, destruction of bone, the application in the slow medicine of knitting.
Small RNA molecular of the present invention can be by the method preparation of RNA synthetic.
Small RNA molecular of the present invention can specificity suppresses the expression of ICAM-1 on mescenchymal stem cell, and significantly remove ICAM-1 for osteoplastic restraining effect, promote mescenchymal stem cell to osteoblast differentiation.Small RNA molecular of the present invention contributes to improve the osteogenic ability of mescenchymal stem cell in inflammatory microenvironment, promotes osteanagenesis, is conducive to bone reparation.Method in the present invention is simple to operate, target is clear and definite, convenient and practical, can significantly promote mescenchymal stem cell to osteoblast differentiation, there is positive effect for the bone reparation improving in inflammatory microenvironment.
Accompanying drawing explanation
Fig. 1 is early stage skeletonization (alkaline phosphatase staining) and skeletonization in late period (Von kossa) ability that rheumatoid arthrosis chamber liquid suppresses mescenchymal stem cell; In figure, scale all represents 100 microns.
Fig. 2 is that rheumatoid arthrosis chamber liquid suppresses important albumen Runx2 (Fig. 2 A) in osteogenetic process and the expression of OCN (Fig. 2 B).（*,P＜0.05，**,P＜0.01）。
Fig. 3 is the mrna expression of the ICAM-1 in the liquid inducing mesenchymal stem cell of rheumatoid arthrosis chamber; Fig. 3 A is the mrna expression of ICAM-1 and the concentration relationship of rheumatoid arthrosis chamber liquid; Fig. 3 B be the ICAM-1 in the liquid inducing mesenchymal stem cell of rheumatoid arthrosis chamber mrna expression over time; Fig. 3 C is the protein expression (*, P < 0.05, * *, P < 0.01) of the ICAM-1 on rheumatoid arthritis arthral fluid inducing mesenchymal stem cell surface.
Fig. 4 is the impact that gives antisense sequences nucleic acid and the osteogenic ability of nonsense sequencing nucleic acid on mescenchymal stem cell of target ICAM-1, and in figure, scale all represents 100 microns.
Fig. 5 is the expression (*, P < 0.05) that the antisense sequences nucleic acid that gives target ICAM-1 can improve Runx2 (Fig. 5 A) and OCN (Fig. 5 B) in mescenchymal stem cell.
Fig. 6 adopts genetically engineered to import early stage skeletonization (alkaline phosphatase staining) and skeletonization in late period (Von kossa) ability that ICAM-1 can suppress mescenchymal stem cell, and in figure, scale all represents 100 microns.
Fig. 7 adopts genetically engineered to import ICAM-1 can suppress important albumen Runx2 (Fig. 7 A) in osteogenetic process and the expression of OCN (Fig. 7 B).（*,P＜0.05，**,P＜0.01）。
Fig. 8 is the osteogenic ability that the antisense sequences nucleic acid of target ICAM-1 improves mescenchymal stem cell, and in figure, scale all represents 100 microns.
Fig. 9 is the expression (*, P < 0.05, * *, P < 0.01) that the antisense sequences nucleic acid of target ICAM-1 improves the interior Runx2 (Fig. 9 A) of mescenchymal stem cell and OCN (Fig. 9 B).
Embodiment
Below will by specific embodiment, the present invention will be further described, as do not specialize, biochemical reagents used in embodiment are commercial reagent, and in embodiment, technique means used is the conventional means that art technology people knows.
The preparation of embodiment 1 small RNA molecular of the present invention
Show that through contriver's result of study ICAM-1 can suppress the Osteoblast Differentiation of mescenchymal stem cell.Stimulate mescenchymal stem cell with Inflammatory substances (arthral fluid of Patients With Rheumatoid Arthritis), then mescenchymal stem cell is broken up to scleroblast induction, simulate the osteogenetic process in inflammatory microenvironment with this.Albumen and the mrna expression level of finding after deliberation the ICAM-1 in mescenchymal stem cell all significantly raise, and in this simultaneously, the Osteoblast Differentiation ability of mescenchymal stem cell significantly reduces.For further checking ICAM-1 is for the restraining effect of Osteoblast Differentiation, employing gene manipulation techniques is transfected into ICAM-1 in mescenchymal stem cell, makes ICAM-1 high expression level in mescenchymal stem cell.Found that high expression level ICAM-1 suppresses the osteogenic ability of mescenchymal stem cell.Protein kinase (MAPK) path of mitogen activation and the propagation of cell and break up closely related, suppress the molecular mechanism of skeletonization activity in order to disclose ICAM-1, for further confirmation, the molecular mechanism of having studied ICAM-1 inhibition mescenchymal stem cell osteogenic ability, result shows that ICAM-1 suppresses skeletonization activity by activation ERK/MAPK path.
The RNA molecule that improves mescenchymal stem cell osteogenic ability of the present invention is the antisense nucleic acid for the mRNA of ICAM-1.Its action target spot is one section of sequence special in the mRNA sequence of ICAM-1
5 '-GGUGGUGAAGUCUGUCAAA-3 ', for shot design antisense sequences, the sequence of small RNA molecular of the present invention is 5 '-GGUGGUGAAGUCUGUCAAATT-3 ' (SEQ ID NO.1).
Above-mentioned small RNA molecular entrusts Guangzhou Rui Bo company directly synthetic, carries out drying treatment and become dry powder after synthesizing.
The preparation of the mescenchymal stem cell that embodiment 2 contains small RNA molecular of the present invention
The mescenchymal stem cell that contains above-mentioned small RNA molecular of the present invention, it can be by obtaining small RNA molecular together with the common transfection mescenchymal stem cell of transfection reagent Lipofectamine2000.
Wherein, the transfection final concentration of described small RNA molecular can be 10-100nM, is preferably 50nM, and the transfection final concentration of Lipofectamine2000 can be 10-100 μ L/mL, is preferably 50 μ L/mL.The transfection time is 24-96hours, is preferably 48hours.
The mescenchymal stem cell substratum using in transfection process is to add foetal calf serum in α-MEM substratum, and the final concentration that makes foetal calf serum is 5-20%(volumn concentration) substratum that obtains; Medium optimization for to add foetal calf serum in α-MEM substratum, and the final concentration of foetal calf serum is 10%(volumn concentration) substratum that obtains.
The composition of α-MEM substratum: HEPES buffer and 2.4g NaHCO that the L-glutaminate (L-glutamine) that 1L α-MEM substratum is 2mmol/L containing 12.1g α-MEM powder, final concentration and final concentration are 25mmol/L
3.
The condition of described cultivation can be 35-38 ℃, CO for temperature
2concentration is 5-15%(volumn concentration), time of saturated humidity, cultivation is 3-4 days; Described temperature is preferably 37 ℃, described CO
2concentration is preferably 5%.
Described in aforesaid method, mescenchymal stem cell is specifically as follows people, mouse, rat, rabbit, dog and monkey mescenchymal stem cell.
Embodiment 3 improves the Osteoblast Differentiation ability of mescenchymal stem cell in inflammatory microenvironment for the anti sense nucleotide sequence of ICAM-1
1, prepare acellular arthral fluid
Rheumatoid arthritis is a class autoimmune disorder, to invade extremities joint and finally to cause joint destruction of bone as principal character.In patient's joint cavity, be usually full of inflammatory arthral fluid, there are some researches show and wherein include a large amount of inflammatory cytokines as interleukin 1 and tumor necrosis factor alpha etc., powerful (the Rivollier A of bone metabolism in simulation inflammatory microenvironment, Mazzorana M, Tebib J, Piperno M, Aitsiselmi T, Rabourdin-Combe C, Jurdic P, Servet-Delprat C.Immature dendritic cell transdifferentiation into osteoclasts:a novel pathway sustained by the rheumatoid arthritis microenvironment.Blood.2004, 104 (13): 4029-4037.), therefore, the present invention has adopted arthral fluid and the mescenchymal stem cell co-cultivation of Patients With Rheumatoid Arthritis according to an international practice, observe mescenchymal stem cell in inflammatory microenvironment skeletonization situation and and ICAM-1 between relation.The volunteer of 20 informed consents, all meets rheumatism association of U.S. Case definition in 1987, without anti-inflammatory treatment.The mode of getting arthral fluid sample is that under aseptic condition, puncture is extracted to anticoagulant tube.
By the arthral fluid sample of anti-freezing with 6000G centrifugal 20 minutes, get supernatant liquor, abandon cell, after supernatant is filtered, frozen for subsequent use in-70 ℃ of refrigerators.
2, stimulate mescenchymal stem cell with acellular arthral fluid
Mouse mesenchymal cell C3H10T1/2 (buying from ATCC company of the U.S.), by 2 × 10
5/ hole is inoculated in 6 well culture plates; mescenchymal stem cell substratum (is made up of α-MEM substratum and foetal calf serum; all, purchased from GIBCO company of the U.S., the final concentration of foetal calf serum is 5-20%(volumn concentration)) and acellular arthral fluid mix (100:0 according to different volume ratio; 95:5; 90:10; 80:20), each culture hole adds 3ml, stimulates 24 hours.
3, the directional induction of Osteoblast Differentiation and evaluation
Every hole adds osteogenic induction system (dexamethasone 0.1 μ mol/L, sodium β-glycerophosphate salt 10mmol/L, ascorbyl phosphate 50 μ mol/L) 3ml, within every 2 days, full dose is changed liquid, cultivate and carry out ALP dyeing by alkaline phosphatase (ALP) detection kit operation steps on the 2nd week, cultivate and within 3 weeks, give Von kossa dyeing.ALP dyeing is the early stage detection index of skeletonization, and Von kossa dyeing reflection be skeletonization late period time calcium deposition situation.As shown in Figure 1, when the articulat cavity fluid concentration of Patients With Rheumatoid Arthritis rises to 5% from 0,10% and 20% time, the ALP dyeing of mescenchymal stem cell is thin out gradually, the movable decline of early stage skeletonization is described, and the result of Von kossa dyeing also shows, along with the lifting of articulat cavity fluid concentration, late period, skeletonization activity was also subject to inhibition.In figure, micro-scale all represents 100 μ m.
4, detect the important adjusting albumen of Osteoblast Differentiation
Runx2 and OCN are the important adjusting albumen that promotes Osteoblast Differentiation, and the expression that detects them can judge from molecule aspect the Osteoblast Differentiation situation of mescenchymal stem cell.Induce after 10 days, full dose sucking-off induced liquid, adds 3mlPBS, jiggles culture plate, washing residue induced liquid.Then each culture hole adds the Trizol of 1ml, extract RNA, according to PrimeScript reverse transcription test kit specification sheets, be cDNA by total RNA reverse transcription, utilize SYBR Green JumpStartTM Taq ReadyMixTM test kit and carry out real-time PCR take 60 ℃ as annealing temperature.Primer sequence refers to table 1.Result represents with mean number ± standard deviation, relatively adopts Student ' s t test between group.What Fig. 2 A and Fig. 2 B showed is that the expression level of Runx2 and OCN declines gradually along with the articulat cavity fluid concentration of Patients With Rheumatoid Arthritis constantly promotes.Arthral fluid stimulates each group compared with stimulating group not, and fall all has statistical significance.(*,P＜0.05;**,P＜0.01)。
Table 1 is primer and the condition of gene real-time PCR
5, arthral fluid raises the expression of ICAM-1 gene and the expression of cell surface in mescenchymal stem cell
ICAM-1 is a kind of intercellular adhesion molecule, it is generally acknowledged, only has and in the time that it expresses at cell surface, just may bring into play regulating effect.This part is by the genetic expression of quantitative PCR detection ICAM-1, the expression by Flow cytometry ICAM-1 at cell surface.
(1) mouse mesenchymal cell C3H10T1/2 (buying from ATCC company of the U.S.), by 2 × 10
5/ hole is inoculated in 6 well culture plates; mescenchymal stem cell substratum (is made up of α-MEM substratum and foetal calf serum; the final concentration of foetal calf serum is 10%, all purchased from GIBCO company of the U.S.) and acellular arthral fluid mix (100:0 according to different volume ratio; 95:5; 90:10; 80:20), each culture hole adds 3ml, stimulates 24 or 72 hours.
(2) full dose sucking-off stimulation fluid, adds 3mlPBS, jiggles culture plate, washing residue induced liquid.Then each culture hole adds the Trizol of 1ml, extract RNA, according to PrimeScript reverse transcription test kit specification sheets, be cDNA by total RNA reverse transcription, utilize SYBR Green JumpStartTM Taq ReadyMixTM test kit and carry out real-time PCR take 60 ℃ as annealing temperature.ICAM-1 primer sequence refers to table 1.Result represents with mean number ± standard deviation, relatively adopts Student ' s t test between group.As shown in Figure 3A, along with articulat cavity fluid concentration constantly promotes, the expression level of ICAM-1 rises result gradually.Arthral fluid stimulates each group compared with stimulating group not, and ascensional range all has statistical significance.(*,P＜0.05;**,P＜0.01)。As shown in Figure 3 B, along with the prolongation of arthral fluid stimulation time, the expression level of ICAM-1 rises gradually.Arthral fluid stimulates each group compared with stimulating group not, and ascensional range all has statistical significance.(*,P＜0.05;**,P＜0.01)。
(3) collect 2 × 10
5cell/EP manages (1.5ml), and requirement to specifications adds anti-mouse ICAM-1 (anti-mouse ICAM-1) and the corresponding homotype control antibodies (IgG2a PE) of PE mark, and 4 ℃ of lucifuges are hatched 30 minutes.Wash twice, 4 ℃ of whizzer 300g rotating speed of cell, machine analysis on flow cytometry with PBS.
As shown in Figure 3 C, along with articulat cavity fluid concentration constantly promotes, ICAM-1's result gradually rises at the expression level of cell surface.Arthral fluid stimulates each group compared with stimulating group not, and ascensional range all has statistical significance.(*,P＜0.05;**,P＜0.01)。
7, adopt the antisense sequences nucleic acid transfection of ICAM-1, detect the recovery of skeletonization activity
(1) mouse mesenchymal cell C3H10T1/2 (buying from ATCC company of the U.S.), by 2 × 10
5/ hole is inoculated in 6 well culture plates; mescenchymal stem cell substratum (is made up of α-MEM substratum and foetal calf serum; the final concentration of foetal calf serum is 10%, all purchased from GIBCO company of the U.S.) and acellular arthral fluid mix (100:0 according to different volume ratio; 95:5; 90:10; 80:20), each culture hole adds 3ml, stimulates 24 hours.
(2) by centrifugal the antisense sequences nucleic acid for ICAM-1 (sequence is as shown in SEQ ID NO.1 for ICAM-1SiRNA, small RNA molecular of the present invention) dry powder synthetic commercialization, add the deionized water dissolving without RNA enzyme, storage concentration is formulated as to 50 μ M.Adopt one section of irregular nonsense sequencing nucleic acid (NC) in contrast.
(3) preferably antisense nucleic acid final concentration is 50nM.Add 3ml substratum according to 6 well culture plate single holes and calculate, get antisense sequences nucleic acid solution and the 3 μ l that 3 μ l storage concentration are 50 μ M and store the Lipofectamine2000 mixing that concentration is 50 μ M, incubated at room 15 minutes.
(4) mixed solution is added in 6 well culture plates, mix gently.
(5) Osteoinductive differentiation is carried out in transfection after 48 hours.
(6) induction is carried out ALP dyeing by ALP detection kit operation steps on the 2nd week, induces and within 3 weeks, gives Von kossa dyeing.
(7) induction, after 10 days, is extracted RNA reverse transcription, by the expression of quantitative PCR detection RUNX2 and OCN.
Skeletonization action result as shown in Figure 4, with transfection compared with the mescenchymal stem cell of NC, transfection in the mescenchymal stem cell of ICAM-1SiRNA ALP and Von kossa dyeing all strengthen to some extent, after showing to have suppressed ICAM-1, early stage and all recoveries to some extent of osteogenic ability in late period of mescenchymal stem cell.
The expression of RUNX2 and OCN, as shown in Fig. 5 A and Fig. 5 B, is compared with NC transfection group with arthral fluid stimulating group, and in the mescenchymal stem cell of ICAM-1SiRNA group, the expression of RUNX2 and OCN all recovers to some extent, and difference has statistical significance.(*,P＜0.05)。After showing to have suppressed ICAM-1, can promote the movable recovery of skeletonization in molecule aspect.
Embodiment 4 can improve the Osteoblast Differentiation ability of the mescenchymal stem cell of the high expression level ICAM-1 of genetically engineered foundation for the anti sense nucleotide sequence of ICAM-1
Mouse mesenchymal cell (the ICAM-1 of high expression level ICAM-1
highmSC) and empty carrier contrast mescenchymal stem cell (empty vector MSC) according to the method for having delivered build (Xu Fenfen, Zhu Heng, Chen Jide, etc.ICAM-1 gene transfection becomes the impact of fat differentiation function on mouse mesenchymal cell.Chinese Journal of Hematology.2013;34(5):435-438.）。Also can obtain by purchasing channel.
1, the directional induction of Osteoblast Differentiation and evaluation
Mouse mesenchymal cell C3H10T1/2, empty vector MSC and ICAM-1
highmSC, by 2 × 10
5/ hole is inoculated in 6 well culture plates, every hole adds osteogenic induction system (dexamethasone 0.1 μ mol/L, sodium β-glycerophosphate salt 10mmol/L, ascorbyl phosphate 50 μ mol/L) 3ml, within every 2 days, full dose is changed liquid, cultivate and carry out ALP dyeing by alkaline phosphatase (ALP) detection kit operation steps on the 2nd week, cultivate and within 3 weeks, give Von kossa dyeing.As shown in Figure 6, compare ICAM-1 with empty vector MSC with mescenchymal stem cell
highthe ALP dyeing of MSC is thin out, the movable decline of early stage skeletonization is described, and the result of Von kossa dyeing also shows, ICAM-1
highthe activity of MSC skeletonization in late period has also been subject to inhibition.In figure, micro-scale all represents 100 μ m.
2, detect the important adjusting albumen of Osteoblast Differentiation
Induce after 10 days, full dose sucking-off induced liquid, adds 3mlPBS, jiggles culture plate, washing residue induced liquid.Then each culture hole adds the Trizol of 1ml, extract RNA, according to PrimeScript reverse transcription test kit specification sheets, be cDNA by total RNA reverse transcription, utilize SYBR Green JumpStartTM Taq ReadyMixTM test kit and carry out real-time PCR take 60 ℃ as annealing temperature.RUNX2 and OCN primer sequence refer to table 1.Result represents with mean number ± standard deviation, relatively adopts Student ' s t test between group.
Result, as shown in Fig. 7 A and 7B, is compared ICAM-1 with empty vector MSC with mescenchymal stem cell
highthe Runx2 of MSC and OCN express all and lower, and this species diversity has statistical significance.(*,P＜0.05;**,P＜0.01)。
3, adopt the antisense sequences nucleic acid transfection of ICAM-1, detect the recovery of skeletonization activity
(1) mouse mesenchymal cell C3H10T1/2, empty vector MSC and ICAM-1
highmSC, by 2 × 10
5/ hole is inoculated in 6 well culture plates.
(2) by centrifugal the antisense sequences nucleic acid for ICAM-1 (sequence is as shown in SEQ ID NO.1 for ICAM-1SiRNA, small RNA molecular of the present invention) dry powder synthetic commercialization, add the deionized water dissolving without RNA enzyme, storage concentration is formulated as to 50 μ M.Adopt one section of irregular nonsense sequencing nucleic acid (NC) in contrast.
(3) preferably antisense nucleic acid final concentration is 50nM.Add 3ml substratum according to 6 well culture plate single holes and calculate, get antisense sequences nucleic acid solution and the 3 μ l that 3 μ l storage concentration are 50 μ M and store the Lipofectamine2000 mixing that concentration is 50 μ M, incubated at room 15 minutes.
(4) mixed solution is added in 6 well culture plates, mix gently.
(5) Osteoinductive differentiation is carried out in transfection after 48 hours.
(6) induction is carried out ALP dyeing by ALP detection kit operation steps on the 2nd week, induces and within 3 weeks, gives Von kossa dyeing.
(7) induction, after 10 days, is extracted RNA reverse transcription, by the expression of quantitative PCR detection RUNX2 and OCN.
Skeletonization action result as shown in Figure 8, with transfection the ICAM-1 of NC
highmSC compares, transfection the ICAM-1 of ICAM-1SiRNA
highin MSC, ALP and Von kossa dyeing all strengthen to some extent, after showing that SiRNA has suppressed ICAM-1, and early stage and all recoveries to some extent of osteogenic ability in late period of mescenchymal stem cell.
The expression of RUNX2 and OCN is as shown in Fig. 9 A and Fig. 9 B, with ICAM-1
highmSC and transfection the ICAM-1 of NC
highmSC compares, transfection the ICAM-1 of ICAM-1SiRNA group
highin MSC, the expression of RUNX2 and OCN all recovers to some extent, and difference has statistical significance.(*,P＜0.05;**,P＜0.01)。After showing to have suppressed ICAM-1, can promote the movable recovery of skeletonization in molecule aspect.
Claims (6)
1. a small RNA molecular that improves mescenchymal stem cell osteogenic ability, is characterized in that, described small RNA molecular is the antisense nucleic acid of the mRNA of target ICAM-1, and its nucleotide sequence is as shown in SEQ ID NO.1.
2. the action target spot of small RNA molecular claimed in claim 1, is characterized in that, in the mRNA sequence that described action target spot is ICAM-1 one section, its nucleotide sequence is as shown in SEQ ID NO.2.
3. a mescenchymal stem cell, it contains small RNA molecular claimed in claim 1.
4. the small RNA molecular shown in claim 1 improves the application in mescenchymal stem cell osteogenic ability medicine in preparation.
5. the osteoporosis that the small RNA molecular shown in claim 1 causes at preparation treatment inflammatory factor, destruction of bone, the application in the slow medicine of knitting.
6. mescenchymal stem cell claimed in claim 3 causes at preparation treatment inflammatory factor osteoporosis, destruction of bone, knitting are slow, nature osteogenesis imperfecta, and bone is damaged, the application in osteochondrodysplasia medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513571A (en) * | 2017-09-30 | 2017-12-26 | 首都医科大学附属北京口腔医院 | MiRNA application |
| CN107513571B (en) * | 2017-09-30 | 2020-07-07 | 首都医科大学附属北京口腔医院 | Application of miRNA |
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