CN107028971A - Applications of the miR 141 in preparing anti-osteoporosis, suppressing bone information preparation - Google Patents

Abstract

The invention belongs to field of biomedical materials, applications of the miR 141 in preparing anti-osteoporosis, suppressing bone information preparation is disclosed.It is verified by experiments, miR 141 is by adjusting the expression of osteoclast differentiation associated gene, so as to suppress the differentiation process of osteoclast.MiR 141 can suppress the generation of the loss and osteoporosis of bone amount, therefore can be used to prepare anti-osteoporosis by miR 141, suppress the different kind organism pharmaceutical preparation of bone loss.

Description

Applications of the miR-141 in preparing anti-osteoporosis, suppressing bone information preparation

Technical field

The invention belongs to technical field of biomedical materials, dredged more particularly, to a kind of miR-141 preparing anti-sclerotin Pine, the application suppressed in bone information preparation.

Background technology

Osteoporosis is a kind of because of bone amount reduction, and bone tissue Microstructure Fracture, bone fragility increase easily occurs fracture and is characterized Systemic osteopathy.As world population ages' trend is increasingly serious, the incidence of disease more and more higher of osteoporosis.According to the 6th time Census shows that the elderly of China's over-65s has accounted for the 7.5% of total population, it is contemplated that suffer to the year two thousand twenty China osteoporosis Person will account for more than half of whole world sufferers of osteoporosis face more than 2.5 hundred million.The World Health Organization is by osteoporosis and glycosuria Disease, cardiovascular disease are classified as the three big killers of harm senior health and fitness jointly.

Osteoclast is a kind of cell type in medullary system source, plays important during skeleton development and bone information Effect.The regulation mechanism for deeply understanding osteoclast function is to resist the precondition of osteoporosis.Osteoclast is sclerotin Important target spot in terms of loose and fracture prevention and treatment, and diphosphonate is the most popular medicine of this respect.However, Research shows that the medicine and atypia femoral fracture, which is used for a long time, has Close relation, and clinical vein injects the trouble of diphosphonate Easily there is jawbone necrosis in person.Therefore, the research and development of the anti-bone information medicine with brand-new target spot have urgent realistic meaning.

MicroRNA (abbreviation miRNA) is that a class is endogenic, and length is the non-coding tiny RNA of 18 to 22 nucleotides. Expression of the miRNA by the complementary pairing with said target mrna to gene on post-transcriptional level carries out negative regulation, causes mRNA's Degraded suppresses translation.Up to the present, it has been reported that thousands of kinds of miRNA are present in a variety of organisms such as animal, plant, fungi It is interior.Each miRNA can have multiple target genes, and several miRNAs can also adjust same gene.This complicated regulation Network both can regulate and control the expression of multiple genes by a miRNA, can also be by several miRNAs combination come fine Regulate and control the expression of some gene.Progressively going deep into for the research expressed with miRNA controlling genes, will be helped us understand high true The complexity and complicated gene expression regulation network of the biological genome of core.

MiR-141 is less to its function report at present as one of member of miR-200 families, only finds that some swell Expression occurs abnormal in knurl, such as the miR-141 of unconventionality expression take part in the propagation and kidney cancer cell of cholangiocarcinoma cell Invasion and attack activity.MiR-141 can be combined with ZEB2 3 ' UTR, intracellular ZEB2 level be reduced, so as to suppress liver cancer The effect of development.But have not yet to see any report during anti-osteoporosis, bone information etc. on miR-141.

The content of the invention

Preparing anti-osteoporosis it is an object of the invention to provide miR-141 new application, i.e. miR-141, suppressing bone Application in absorbable preparation.

Above-mentioned purpose of the present invention is achieved by the following technical programs：

Applications of the miR-141 in preparing anti-osteoporosis, suppressing bone information preparation

In order to be better understood from the essence of the present invention, illustrate it in pharmaceutical field with reference to pharmacological evaluation and result New application.

Research finds macaque miR-141 (5 '-aacacugucugguaaagaugg-3 ') (SEQ ID NO:I it is) thin in macaque Intracellular, which is overexpressed, can significantly reduce myelomonocyte (BMM) cell synthesis Tartrate resistant acid phosphatase (TRAP), while real When PCR result show：The intracellular TRAPmRNA of BMM level is significantly reduced.RANKL induction BMM cells it is osteoclastic to point During change, miR-141 level is decreased obviously.After BMM cell transfectings miR-141, the BMM cells of RANKL inductions are osteoclastic Substantially reduce, compared with control group to the ability of differentiation, cell expression TRAP ability is substantially reduced.Illustrate miR-141 broken Bone plays important regulating and controlling effect into induction atomization.

MiR-141 expression declines in the bone tissue of female old age macaque and osteoporosis patient.Passed using nucleic acid Send after miR-141 is delivered in female old Yokogawa monkey body by system again, by detecting that osteoclast division guideline judges miR- 141 can suppress the differentiation of osteoclast.And detected by Micro CT, find delivering miR-141 experimental group macaque Bone density, the quantity of bone trabecula and thickness increase than control group macaque.Illustrate miR-141 to female old macaque osteoporosis Model has certain adjustment effect.

Compared with prior art, the invention has the advantages that：

1. the present invention has excavated new medical application to known miR-141, a new application field has been opened up.

2. miR-141 of the present invention inhibitor can lower miR-141 gene expression abundance, promote point of osteoclast differentiation Change, strengthen bone information, so as to cause osteoporosis.MiR-141 can suppress osteoclast differentiation, further suppress bone information and make With, can by miR-141 be used for prepare suppress bone loss, all kinds of pharmaceutical preparations of anti-osteoporosis.

Brief description of the drawings

Fig. 1 is the expression that real-time fluorescence quantitative PCR detects the marker gene in macaque osteoclast atomization.

Fig. 2 is that real-time fluorescence quantitative PCR detects expressed in abundance degree of the miR-141 in macaque osteoclast atomization.

Fig. 3 suppresses osteoclast differentiation index of correlation to transiently transfect miR-141.

Fig. 4 is that miR-141 suppresses osteoclast differentiation TRAP coloration result figures.

Fig. 5 is control group and experimental group macaque femur CT scan figure.

Fig. 6 is the changes of weight of control group and experimental group macaque.

Fig. 7 is control group and the histotomy of experimental group major organs.

Fig. 8 is control group and experimental group serum of macaque physiology and bio-chemical changes.

Embodiment

Present disclosure is further illustrated with reference to specific embodiment, but be should not be construed as limiting the invention. Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.Except non-specifically Illustrate, the reagent of the invention used, method and apparatus is the art conventional reagent, methods and apparatus.

MiR-141 is purchased from Shanghai JiMa pharmacy Technology Co., Ltd.

Using the old female macaque of health in embodiment, purchased from Nanning Ling Kangsainuoke bio tech ltd (Wincon Thera Cells Biotechnologies Co Ltd, Wincon).Participate in experiment macaque age 19~ Between 23 years old, body weight is between 5.8~6.5kG.Every equal single cage of experiment monkey is raised, and daily feeding feed twice, is aided with appropriate Fruit, free water.The control of receptacle environment temperature is in 24~30 DEG C, illumination 12 hours, same point automatically of whole day.Experimental animal Using and nursing apply and permitted with the Wincon animal welfares committee in advance.Before experiment simultaneously anaesthetized into Row Bone mineral density (lumbar vertebrae and hipbone), finally extracts 3mL venous blood.And NTx, CTx and TRAP of its serum are detected.

For participating in the macaque that ovary is won, anaesthetized first, extract 3mL blood, centrifugation obtains serum.Then collect Sample bone, CT detection bone densities.Extract after ovary every one and a half months collection serum, sample bone and progress CT detections.

The macaque BMM cell inductions of embodiment 1

The marrow of 2 monthly age macaques is gone out, with the α-MEM nutrient solutions containing 10%FBS, in 5%CO₂37 DEG C it is incubated Cultivated 24 hours in case.By cell with 1 × 10 during induction⁴/ hole is inoculated into 24 orifice plates, adds 50ng/ml M-CSF inductions 3 My god, 50ng/ml M-CSF and 100ng/ml RANKL inducing cells are added after 3 days, liquid, cell culture 5 days are changed to cell daily Follow-up Molecular Detection and TRAP dyeing are carried out afterwards.Osteoclast differentiation specific gene primer sequence is as shown in table 1：(F, on Swim primer；R, anti-sense primer)

The osteoclast of table 1 breaks up specific gene primer sequence

The cell total rna of embodiment 2 is extracted

Treat that cell covers with the 80%-90% of each aperture of 24 porocyte culture plates, add 1ml Trizol cell lysis simultaneously Blown and beaten repeatedly with pipette tips, room temperature is acted on 5 minutes.Lysate is moved into 1.5ml centrifuge tubes, 200ul chloroforms is added, covers tightly pipe Lid, firmly vibrates centrifuge tube, is stored at room temperature 5 minutes up and down, under the conditions of 4 DEG C, and 12000g/min is centrifuged 15 minutes.On colourless Layer aqueous phase liquid is transferred in new 1.5ml centrifuge tubes, adds 500 μ L isopropanols, and mixing of turning upside down is stored at room temperature 10 points Clock, under the conditions of 4 DEG C, 12000g/min is centrifuged 10 minutes.Supernatant is drawn with pipettor, abandoned, retains bottom white precipitate. The ethanol of 1ml 75% (preparation of DEPC water) is added, gently white precipitate is upspring with hand, then under the conditions of 4 DEG C, 7500g/min Centrifugation 5 minutes.Supernatant is abandoned, drying at room temperature 10-15 minutes adds 30-40 μ lLDEPC water and dissolved again, is placed in -80 DEG C of refrigerators Preserve or directly use.

The osteoclastic detection to differentiation marker gene expression of the macaque BMM cells of embodiment 3

Use the reverse transcription reagent box (PrimeScript of TaKaRa companies^TMRT reagent Kit with gDNA Eraser, Code No.:RR047A), according to the operating process of kit, by the total serum IgE reverse transcription of extraction into cDNA.Use reality When fluorescent quantitative PCR technique detection macaque BMM atomizations in marker gene expression.Fig. 1 is real-time fluorescence quantitative PCR Detect the expression of the marker gene in macaque osteoclast atomization.From fig. 1, it can be seen that TRAP relative expression levels and CTSK relative expression levels increase with the increase of differentiation number of days.The molecular indexes of osteoclast differentiation are with the differentiation of cell Content increases, and as a result illustrates that osteoclast breaks up successfully.

The detection of the macaque myelomonocyte miR-141 gene expression abundances into osteoclast atomization of embodiment 4

The total serum IgE of different differentiation period macaque myelomonocytes is extracted, the Mir- of TaKaRa companies is used X^TMMiRNAFirst-Strand Synthesis Kit synthesize cDNA, use the PCR primer for miR-141 and TaKaRa Mir-X^TM miRNAqRT-PCRKit carries out quantitative fluorescent PCR, as shown in table 2.Using U6 genes as internal reference to miR- 141 testing results are standardized correction.Fig. 2 is that real-time fluorescence quantitative PCR detects miR-141 in macaque BMM cell differentiation mistakes Expressed in abundance degree in journey.As a result show, with the differentiation of osteoclast, miR-141 content is gradually reduced.

Table 2 is used for the primer sequence for the miR-141 that real-time fluorescence quantitative PCR is analyzed

Gene Name	Primer sequence
		Mml-miR-141	5’-AACACTGTCTGGTAAAGATGG-3’
U6	5’-CGACTGCATAATTTGTGGTAGTGG-3

The cell transfection assays of embodiment 5

By cell with 1 × 10⁴Individual/hole is inoculated in 24 orifice plates, cell induction transfection in 2 days once, when cell induction 4 days again Transfection is once.Appropriate miR-141 is dissolved in serum-free Optimal Medium during transfection, while the P3000 in Lip3000 is mixed with it Close, finally Lip3000 is added in the mixture, is stored at room temperature 5 minutes.Archaeocyte nutrient solution is sucked, fresh medium is changed, Transfection liquid is added in fresh medium, 37 DEG C are incubated 24 hours.The culture medium containing transfection liquid is finally discarded, training completely is added Base is supported to continue to cultivate.Fig. 3 suppresses osteoclast differentiation index of correlation to transiently transfect miR-141.As a result show, osteoclast turns Contaminate after miR-141, it is suppressed that it further breaks up.

The osteoclast trap of embodiment 6 is dyed

After osteoclast breaks up 6 days, supernatant is abandoned, PBS cell is used, then adds fixative.Room temperature fixes cell 30 Second, then with deionized water rinsing 3 times.By 0.5mL Fast Garnet GBC Base solution and 0.5mL Sodium Nitrite solution are mixed, and are overturned and are mixed, are stored at room temperature 2 minutes.Above-mentioned mixed liquor and 45mL are preheated to 37 DEG C Ionized water, 0.5mL Naphthol AS-BI phosphate solution, 2mL Acetate solution and 1mL Tartrate solution are mixed, the water-bath in 37 DEG C of water-baths.Above-mentioned solution is added to 37 DEG C of incubations in Tissue Culture Dish 1 hour.Fig. 4 is that miR-141 suppresses osteoclast differentiation TRAP coloration result figures.Wherein, before (a) is transfection miR-141, (b) After transfection miR-141, as can be known from Fig. 4, experimental group osteoclast is decreased obviously, and is as a result shown, osteoclastic after transfection miR-141 The quantity of cell declines, and illustrates that miR-141 breaks up inhibited to osteoclast.

The macaque femur CT of embodiment 7 is detected

It is euthanized in the 12nd week macaque by experimental group and control group, and Henghe is fixed with 4% paraformaldehyde cardiac perfusion Monkey.Take out the leg bone of rhesus macaque, lumbar vertebrae and hipbone.4% paraformaldehyde fixes 1 week, is then cleaned with 10% sucrose solution.Make The bone sample of macaque is carried out with microcomputer layer scanning technology (micro computed tomography, Micro-CT) Scanning.After the completion of scanning, metaphysis bone trabecula is analyzed.Volume ratio (BV/TV) including bone trabecula, the thickness of bone trabecula (Tb.Th) and bone trabecula quantity (Tb.N).Fig. 5 is that control group and experimental group macaque femur CT scheme.Wherein, (a) is control group, (b) it is experimental group, as can be known from Fig. 5, the bone density of experimental group substantially increases.As a result show, experimental group macaque bone amount is than control Group is high, illustrates to give the bone loss that old macaque is alleviated after miR-141.

The miR-141 safety evaluations of embodiment 8

Experimental group and control group macaque body weight are recorded weekly and blood is extracted, and are pacified experimental group and control group macaque within the 12nd week It is happy dead, take its major organs (heart, liver,spleen,kidney) to carry out histological stain.And the physiological and biochemical index of its serum is examined Survey.Fig. 5 is the changes of weight of control group and experimental group macaque.Experimental result shows that experimental group and control group macaque body weight do not have Notable difference.Fig. 6 is control group and the histotomy of experimental group major organs.As a result show, experimental group and the main device of control group Official does not have notable difference.Fig. 7 is experimental group and control group serum of macaque physiology and bio-chemical changes, as shown in Figure 7, miR-141 Obvious tissue damage is not caused to the heart, liver, spleen, lung tissue.As a result show, experimental group and control group do not have notable difference. To sum up tell and give macaque miR-141 and do not produce obvious side effect.

Fig. 8 is control group and experimental group serum of macaque physiology and bio-chemical changes.Macaque is given it can be seen from Fig. 8 After miR-141, compared with control group, glutamic-pyruvic transaminase and internal glucose content, and amyotrophic lateral sclerosis journey Degree illustrates that give miR-141 does not produce apparent side effect to macaque all without significant change.

The studies above shows that miR-141 can suppress osteoclast differentiation, further suppresses bone resorption, therefore can be by MiR-141, which is used to prepare, suppresses bone loss, all kinds of pharmaceutical preparations of anti-osteoporosis.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, is combined and simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (1)

1. Applications of the 1.miR-141 in preparing anti-osteoporosis, suppressing bone information preparation.