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Application of long chain non-coded RNA-HOXA-AS2 in bone tissue damage repair

An RNA-HOXA-AS2, HOXA-AS2 technology, applied in the application field of long-chain non-coding RNA-HOXA-AS2 in the repair of bone tissue damage, can solve the problems of immune rejection, many complications, few sources, etc. The effect of enhancing alkaline phosphatase activity and promoting calcium ion deposition

Active Publication Date: 2019-01-04
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For sports-induced bone injuries, traditional methods such as autologous bone grafting, allogeneic bone grafting, blood-supplied free bone grafting, and biomaterial replacement are often used in the medical field. Rejection and iatrogenic infection and many other problems, the treatment effect is not ideal

Method used

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  • Application of long chain non-coded RNA-HOXA-AS2 in bone tissue damage repair
  • Application of long chain non-coded RNA-HOXA-AS2 in bone tissue damage repair
  • Application of long chain non-coded RNA-HOXA-AS2 in bone tissue damage repair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Acquisition of HOXA-AS2 gene fragments

[0031] According to the RNA nucleotide sequence (RefSeq sequence number: NR_122069.1, as shown in SEQ ID NO: 1 in the sequence listing) of the long-chain non-coding RNA-HOXA-AS2 in the NCBI database and BamH I on the lentiviral vector pLV and Xba I-specific enzyme cutting site, using DNA chem192 synthesizer, according to the principle of solid-phase phosphoramidite triester method, double-strand synthesis of HOXA-AS2 gene fragment, and adding two Specific enzyme cutting sites BamH I (GGATCC) and Xba I (TCTAGA) were obtained to obtain a HOXA-AS2 gene fragment containing specific enzyme cutting sites, the nucleotide sequence of which is shown in SEQ ID NO: 2 in the sequence listing.

Embodiment 2

[0032] Example 2: Construction of lentiviral expression vector pLV-HOXA-AS2

[0033] Using restriction endonucleases BamH I and Xba I, the lentiviral empty vector pLV (its nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing) and the HOXA-AS2 gene fragment obtained in Example 1 were respectively carried out Double digestion, use T4 DNA ligase system to ligate the digested HOXA-AS2 gene fragment and linear lentiviral empty vector pLV, then transform competent cells, screen positive colonies, extract plasmids of positive colonies, and obtain lentiviral expression Vector pLV-HOXA-AS2.

[0034] 1. Enzyme digestion of lentiviral empty vector pLV

[0035]The enzyme digestion reaction system is as follows (20 μL):

[0036]

[0037] Reaction conditions for enzyme cleavage: react at 37°C for 4 hours.

[0038] 2. Enzyme digestion of HOXA-AS2 gene fragment

[0039] The enzyme digestion reaction system is as follows (20 μL):

[0040]

[0041] Reaction conditions...

Embodiment 3

[0054] Example 3: Lentiviral Packaging

[0055] HEK293T cells were seeded in six-well culture plates for culture, and cultured in a cell culture incubator at 37°C with DMEM complete medium containing 10% fetal bovine serum (FBS). When the cell density reached about 70%, the Lipofectamine 3000 liposome transfection reagent (purchased from Thermo Fisher, model L3000015), respectively transfected lentiviral expression vector pLV-HOXA-AS2 and lentiviral empty vector pLV into HEK293T cells for lentiviral packaging. Specific steps are as follows:

[0056] 1) Take two 1.5mL EP tubes and add 150μL serum-free DMEM medium to each;

[0057] 2) Add 1 μg lentiviral expression vector pLV-HOXA-AS2, 0.5 μg lentiviral packaging plasmid pSPAX2, and 0.5 μg lentiviral packaging plasmid pMD2G into one of the EP tubes, and mix well;

[0058] 3) Add Lipofectamine 3000 liposome transfection reagent into another EP tube and mix well;

[0059] 4) Quickly add the plasmid solution obtained in step 2) ...

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Abstract

The invention provides application of long chain non-coded RNA-HOXA-AS2 in bone tissue damage repair. Experiments verify that overexpressed HOXA-AS2can promote calcium ion deposition in a mesenchymalstem cell obviously, enhance the activity of alkaline phosphatase in the mesenchymal stem cell obviously, improve expression quantities of osteogenic differentiation markers RUNX2, SP7 and SPP1 genesin the mesenchymal stem cell obviously and inhibit the activity of an NF-kappaB signal obviously.

Description

technical field [0001] The invention belongs to the technical field of stem cell bioengineering, and more specifically relates to the application of long-chain non-coding RNA-HOXA-AS2 in repairing bone tissue damage. Background technique [0002] Bone injury repair and regeneration has always been a major problem in the medical field. For sports-induced bone injuries, traditional methods such as autologous bone grafting, allogeneic bone grafting, blood-supplied free bone grafting, and biomaterial replacement are often used in the medical field. Rejection and iatrogenic infection and many other problems, the treatment effect is not satisfactory. With the continuous development of bone tissue engineering technology, people gradually began to use tissue engineering methods to solve the problem of bone tissue damage repair. As a kind of seed cells with good value-proliferating ability and multi-directional differentiation potential, mesenchymal stem cells have come into people...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N5/10C12Q1/6883
CPCC12N15/113C12Q1/6883C12Q2600/158C12Q2600/178
Inventor 朱鑫星林俊堂于金金钟根深郭睿杜江杨芬
Owner XINXIANG MEDICAL UNIV
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