RNA interfering target of osteoclast proton pump sub-gene a3 and application thereof

An RNA interference and osteoclast technology, applied in the field of molecular biology and biomedicine, can solve the problems of inability to demineralize bone, lack of bone marrow cavity, and lack of extracellular acidification of osteoclasts.

Inactive Publication Date: 2010-02-03
ZHEJIANG TIANHUANG MEDICINAL PLANT PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results showed that all a3 mutant homozygous (- / -) mice had growth retardation, abnormal long bone growth, osteosclerosis with loss of bone building and remodeling, tooth disintegration, and died at about 4 weeks after birth; a3- / - Mice lack bone marrow cavity, bone growth surface is enlarged, and calcified cartilage area is extended; a3- / - mice have normal number of osteoclasts attached to the bone, but cannot form bone resorption lacunae
A3- / - mouse osteoclasts cultured in vitro lack extracellular acidification and fail to demineralize bone

Method used

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  • RNA interfering target of osteoclast proton pump sub-gene a3 and application thereof
  • RNA interfering target of osteoclast proton pump sub-gene a3 and application thereof
  • RNA interfering target of osteoclast proton pump sub-gene a3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Primary osteoclast culture

[0042] Referring to Yang, S. and Li, Y.P. (2007) J. Bone Miner. Res., 22, 45-54, primary cultured bone marrow mononuclear cells were induced to differentiate into mature osteoclasts. C57BL6 mice aged 6-8 weeks were sacrificed by pulling their necks. After routine disinfection, the femurs and tibias of their hind limbs were placed in ice-cold RIMP-1640 culture medium containing antibiotics. Use scissors and tweezers to separate the vascular connective tissue on the bone surface, and rinse with ice-cold RIMP-1640 culture solution containing antibiotics. The two ends of the femur and tibia were cut off with scissors, and the bone marrow cells were blown into ice-cold α-MEM medium containing 10% FBS with a sterile syringe (1 ml). The syringe needle was aspirated repeatedly to form a single cell suspension. Centrifuge at 4°C (1100rpm×6min), and resuspend the cells in α-MEM+10%FBS medium containing M-CSF (10ng / ml) and RANKL (10ng / ml) ...

Embodiment 2

[0043] Example 2: Antibody preparation

[0044] synthetic peptide a3 816 CFYSGTGYKLSPFTFTVDSD 834 Coupled to KLH, immunized male New Zealand white rabbits to obtain polyclonal antibody against a3, and monoclonal antibody against GAPDH was purchased from cellsignaling company.

Embodiment 3

[0045] Example 3: Screening for effective siRNA sequences

[0046] Use Dharmacon siDESIGN center (http: / / www.dharmacon.com) to design small interfering siRNA, and select three specific target sequences against Atp6v0a3 mRNA

[0047] a3siRNA1: 5'-AGATGAAGGCAGTGTACCT-3' (SEQ ID NO.1);

[0048] a3siRNA2: 5'-CTCGGCGTTTCATCTGTGG-3' (SEQ ID NO.2);

[0049] a3siRNA3: 5'-ACGGACTGCTCATGTTTCT-3' (SEQ ID NO. 3).

[0050] The negative control is the target sequence si-LacZ specific for LacZ gene mRNA: 5'-CTCGGCGTTTCATCTGTGG-3'. The target sequence of the chemically synthesized short hairpin structure was ligated between the BglII / HindIII sites on the pSUPER vector (OligoEngine) after annealing and renaturation. The efficiency of knocking down a3 expression by short hairpin RNA (shRNA) was tested in human embryonic kidney cell line 293T (HEK293T, ATCC No.CRL-11268 TM ) to co-express plasmid pFlag-CMV-4-a3 (Flag-a3) and plasmid pSUPER-siRNA. That is: use lipofectamine reagent (Invitrog...

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Abstract

The invention belongs to the technical field of molecule biology and biomedical science, and relates to targets in an a3 sequence for RNA interference, siRNA obtained in various ways for the targets and application thereof in preparing new medicines for treating osteoporosis. A slow virus is used for infecting osteoclast and expressing shRNA, and a method of osteoclast function detection after invitro gene is knocked down is also used; thus, three targets for RNA interference in an a3 gene are screened, and the No.2 target is preferred. ShRNA for the No.2 target can obviously inhibit the expression of a3 protein, stably inhibit the capabilities of extracellular acidification and bone resorption of the osteoclast, and does not affect the formation of filiform actin rings of the mature osteoclast. Based the target sequence, biological medicines for treating osteoporosis can be prepared.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and biomedicine, and relates to interference targets used for RNA interference on the sequence of osteoclast proton pump subunit a3, and small interfering RNA molecules obtained in various ways for these targets and their Application in preparation of medicine for treating osteoporosis. Background technique [0002] Osteoporosis is the most common bone metabolic disease with high morbidity, high mortality and high cost of health care. It is estimated that more than half of women and approximately one third of men will suffer an osteoporotic fracture during their lifetime. Fractures bring great misery to people's lives, the light ones make people's quality of life decline, and severe ones will cause paralysis (as femoral fractures) and cause many complications and even death. The occurrence of these diseases is related to the overactivity of osteoclasts. Osteoclasts and osteoblasts (Os...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N15/867A61K48/00A61K31/713A61P19/10C12N15/113
Inventor 李亦平
Owner ZHEJIANG TIANHUANG MEDICINAL PLANT PHARMA
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