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CircRNA-FTO for regulating and controlling ADSCs osteogenic differentiation and tissue regeneration and application of circRNA-FTO

A technology for bone tissue regeneration and osteogenic differentiation, applied in DNA/RNA fragments, recombinant DNA technology, bone/connective tissue cells, etc., can solve problems that have not been reported

Pending Publication Date: 2020-08-07
辽宁省肿瘤医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there is no report on the role of Circ RNA-FTO in adipose-derived stem cells and its relationship with adipose-derived stem cell osteogenic differentiation and tissue regeneration.

Method used

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  • CircRNA-FTO for regulating and controlling ADSCs osteogenic differentiation and tissue regeneration and application of circRNA-FTO
  • CircRNA-FTO for regulating and controlling ADSCs osteogenic differentiation and tissue regeneration and application of circRNA-FTO
  • CircRNA-FTO for regulating and controlling ADSCs osteogenic differentiation and tissue regeneration and application of circRNA-FTO

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Adipose stem cell biological characteristics

[0063] 1. Isolation and culture of human adipose-derived stem cells

[0064] Abdominal adipose tissue was collected during liposuction and stored in a 20mL sterile syringe for later use, and stored in ice packs. Adipose tissue was centrifuged in a 50mL centrifuge tube at 1000rpm / 5min to remove the lower liquid. Adipose tissue was digested with 0.2% type I collagenase at a ratio of 1:1 for 40 minutes on a shaker at 37°C. After standing still, it could be seen that it was divided into three layers. Remove the upper grease layer, fully shake and filter (200 mesh filter). Then centrifuge at 1000rmp / 5min for three times to remove the supernatant liquid, and the cell sediment therein can be seen. Pipette the cells evenly with cell culture medium. Cells were then seeded in culture dishes at a density of 5×105 / mL. The cells were then placed at 37°C, 5% CO 2 , 95% humidity incubator. Subcultured to the third generat...

Embodiment 2

[0069] Example 2 Circ RNA-FTO was screened out, and its cell localization and expression changes in osteogenesis induction

[0070] 1. The adipose-derived stem cells were induced into osteogenesis for 0d, 3d, 5d, and 7d, and RNA was extracted, and the expression trend of CircRNA-FTO at different times was detected by RT-q PCR.

[0071] Experimental results such as Figure 4 As shown, the expression of Circ RNA-FTO increased over time during osteogenic differentiation.

[0072] 2. Design and transfection of small interfering RNA sequences

[0073] Circ RNA-FTO sequence (circbase ID: hsa-circ-0005941) designed primers, primers were synthesized and purified by Shanghai Gemma Biotechnology Co., Ltd., designed small interfering RNA sequences to inhibit the expression of Circ RNA-FTO (because the FTO sequence is too large, It is extremely difficult to successfully construct an overexpression vector, so the present invention conducts a reverse research demonstration by inhibiting t...

Embodiment 3C

[0085] Example 3 Circ RNA-FTO regulates tissue regeneration of adipose stem cells in vivo

[0086] The human adipose stem cell complex was subcutaneously implanted into the back of nude mice, and its tissue formation ability was observed.

[0087] 1. Transfer siRNA and negative control (Negative control, NC) into hADSCs by lipofection method

[0088] Human adipose stem cells were cultivated by the tissue block method and enzyme digestion method, and the vigorously growing third-generation adipose stem cells were inoculated on a 60mm culture dish, and the culture components were α-MEM medium (containing 10% fetal bovine serum, 2mmol / L glutamine Amide, 100 U / ml penicillin, 100 μg / ml streptomycin). siRNA and negative control (Negative control, NC) were transferred into adipose-derived stem cells by lipofection method, and the experiment was divided into two groups: (1) siRNA-NC transfected adipose-derived stem cells group (2) siRNA-FTO transfected adipose-derived stem cells ste...

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Abstract

The invention discloses circRNA-FTO for regulating and controlling ADSCs osteogenic differentiation and tissue regeneration and application of the circRNA-FTO and belongs to the technical field of bone tissue regeneration. The circRNA-FTO circbase ID is has-circ-0005941, the cDNA sequence is as shown in SEQ ID NO: 1, and the sequence is a cyclic structure formed by connecting the sequences as shown in the sequence end to end. In-vivo and in-vitro experiments of regulating ADSCs osteogenic differentiation and tissue regeneration by the circRNA-FTO prove that the circRNA-FTO plays an important regulating role in the adipose-derived stem cell osteogenic differentiation and / or tissue regeneration process; the circRNA-FTO can be used as a potential target for tissue regeneration treatment, newcircRNA-FTO related drugs or factors are promoted to be developed, and finally the circRNA-FTO related drugs or factors are applied to the field of regenerative medicine, so that real clinical precision medical treatment is achieved.

Description

Technical field: [0001] The invention belongs to the technical field of bone tissue regeneration, and specifically relates to CircRNA-FTO for regulating ADSCs osteogenic differentiation and tissue regeneration and application thereof. Background technique: [0002] Bone defects caused by trauma, tumors and infections are common clinical diseases. Autologous bone transplantation has always been the first choice for the treatment of bone defects, but it is greatly limited due to its limited source, poor plasticity, and damage to the donor site. Immune rejection exists in allogeneic bone transplantation. Decellularization of allogeneic bone in order to reduce its immunogenicity results in a loss of osteogenic activity. Its sources are limited and its infection rate is high. Immune rejection of xenografts is more severe, and may transmit zoonotic diseases. They cause a heavy social and economic burden and seriously affect the quality of life of patients. Adipose Derived Stem...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N5/077A61K45/00A61K48/00A61K31/713A61P19/08
CPCC12N15/113C12N5/0654A61K45/00A61K31/713A61P19/08C12N2310/14C12N2501/01C12N2506/1384
Inventor 康悦赵峰
Owner 辽宁省肿瘤医院
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