Accelerator for osteogenic differentiation of bone marrow mesenchymal stem cells

A bone marrow mesenchymal and osteogenic differentiation technology, applied in the field of stem cells, can solve problems that have not been reported

Pending Publication Date: 2022-04-29
青岛思拓新源细胞医学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the role of LINC02075 in the adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells

Method used

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  • Accelerator for osteogenic differentiation of bone marrow mesenchymal stem cells
  • Accelerator for osteogenic differentiation of bone marrow mesenchymal stem cells
  • Accelerator for osteogenic differentiation of bone marrow mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Fluorescent quantitative PCR detection of LINC02075 expression level during adipogenic induction

[0036] 1. Adipogenic Induction

[0037] (1) The third-generation human bone marrow mesenchymal stem cells (purchased from Sciencell) in the logarithmic growth phase were selected and inoculated into culture flasks, and cultured by adding complete medium;

[0038] (2) When the cell confluency reached 70%, the medium was replaced with adipogenic induction medium every 3 days, and RNA was extracted after 14 days of adipogenic induction.

[0039] 2. RNA extraction

[0040] RNA was extracted on day 0, day 7 and day 14 respectively, and three controls were set up in each group.

[0041] (1) Remove the medium, add 500 μL Trizol to each well, and repeatedly pipette the cells until a clear and non-viscous liquid is formed;

[0042] (2) Transfer the mixture to a 1.5ml EP tube, add 100μL chloroform, shake vigorously on the shaker for 15 seconds, and let stand at room temperature f...

Embodiment 2

[0061] Fluorescent quantitative PCR detection of the inhibitory effect of sh-LINC02075

[0062](1) The sequence of sh-LINC02075 is as follows:

[0063] Sense strand: 5'-CACCGCTCCTGAAGAAACAGCTTAGCGAACTAAGCTGTTTCTTCAGGAGC-3' (SEQ ID NO.6)

[0064] Antisense strand: 5'-AAAAGCTCCTGAAGAAACAGCTTAGTTCGCTAAGCTGTTTCTTCAGGAGC-3' (SEQ ID NO.7)

[0065] (2) The lentiviral vector was constructed by Jikai Genomics Co., Ltd., and the bone marrow mesenchymal stem cells were transfected according to the transfection instructions. After 48 hours, the RNA was extracted for quantitative fluorescence detection. The steps were the same as in Example 1.

[0066] Experimental results

[0067] Experimental results such as figure 2 As can be seen from the figure, the inhibition rate of sh-LINC02075 was 70.4%.

Embodiment 3

[0069] Effects of LINC02075 inhibition on adipogenic differentiation-related genes PPARγ, C / EBPα and AP2 mRNA levels in bone marrow mesenchymal stem cells

[0070] 1. The primer sequences of PPARγ, C / EBPa and AP2 are as follows:

[0071] gene name Primer sequence PPARγ GCCCAGGTTTGCTGAATGTG (SEQ ID NO. 8) TTGGCAAACAGCTGTGAGGA (SEQ ID NO. 9) C / EBPa AGAACAGCAACGAGTACCGG (SEQ ID NO. 10) GCGGTCATTGTCACTGGTCA (SEQ ID NO. 11) AP2 TGGGCCAGGAATTTGACGAA (SEQ ID NO. 12) CCATCCCATTTCTGCACATGT (SEQ ID NO. 13)

[0072] 2. Bone marrow mesenchymal stem cells transfected with sh-NC and sh-LINC02075 were inoculated on a culture plate for adipogenic induction. After 14 days of adipogenic induction, RNA was extracted, and fluorescent quantitative PCR was used to detect sh-NC group and sh- Expression differences of PPARγ, C / EBPa and AP2 among LINC02075 groups.

[0073] Experimental results

[0074] Experimental results such as image 3 A...

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Abstract

The invention provides an accelerant for osteogenic differentiation of bone marrow mesenchymal stem cells, and belongs to the technical field of stem cells. Experiments prove that mRNA and protein expression of osteogenic differentiation related genes RUNX2, ALP and OCN can be remarkably promoted by inhibiting expression of LINC02075 in the bone marrow mesenchymal stem cells so as to promote osteogenic differentiation of the bone marrow mesenchymal stem cells, so that the LINC02075 can be used as an osteogenic differentiation accelerant of the bone marrow mesenchymal stem cells.

Description

[0001] This case is a divisional application. The invention title of the original application is the role of long-chain non-coding RNA in the osteogenic and adipogenic differentiation of stem cells. The filing date of the original application is: 2020-06-22, and the application number of the original application is: CN202010575929.2. technical field [0002] The invention belongs to the technical field of stem cells, and in particular relates to an accelerator for osteogenic differentiation of bone marrow mesenchymal stem cells. Background technique [0003] Osteoporosis is a systemic, metabolic bone disease. The main pathological features of osteoporosis are decreased bone mass, damaged bone microarchitecture and increased bone fragility, and its main clinical manifestations are pathological fractures and low back pain. With the continuous aggravation of the aging level, osteoporosis has begun to attract more and more people's attention. [0004] Bone marrow mesenchymal s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/713C12N15/113A61P19/10
CPCC12N15/113A61P19/10A61K31/713
Inventor 不公告发明人
Owner 青岛思拓新源细胞医学有限公司
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