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Application of Flot2 inhibitor to inhibition of osteoclast formation and/or osteoclastic activity

A technology for inhibiting osteoclasts and inhibitors, applied in the direction of organic active ingredients, anti-inflammatory agents, bone diseases, etc., can solve the problems of unreported effects on bone cells

Active Publication Date: 2020-08-25
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of Flot2 on the destruction of bone cells has not been reported

Method used

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  • Application of Flot2 inhibitor to inhibition of osteoclast formation and/or osteoclastic activity
  • Application of Flot2 inhibitor to inhibition of osteoclast formation and/or osteoclastic activity
  • Application of Flot2 inhibitor to inhibition of osteoclast formation and/or osteoclastic activity

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Experimental program
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Effect test

Embodiment 1

[0037] Our previous studies found that the expression of Flot2 gene was significantly increased in RANKL, LPS, and tumor supernatant-activated osteoclast precursor cells. Therefore, it is speculated that the targeted inhibition of Flot2 gene expression may have the effect of inhibiting osteoclast formation. For the Flot2 gene, three specific siRNA sequences are involved, and the specific sequences are shown in Table 1, which were synthesized by Gemma Gene Company.

[0038] Table 1 RNA oligo sequence list

[0039]

[0040] 1. Cell culture:

[0041] Cell source: RAW264.7 mouse mononuclear macrophage leukemia cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0042] Culture method: select RAW264.7 cells in good growth state and press 2×10 4 / well density seeded in a 24-well plate, at 37 ° C, 5% CO 2 Incubator incubation.

[0043] 2. Cell transfection:

[0044] After the cells were stably cultured for 4-6 hours, press Instructions for RNAiMAX T...

Embodiment 2

[0077] Example 2 Effect of Flot2-siRNA on Inducing Osteoclast Precursor Cells to Differentiate into Osteoclasts

[0078] Select RAW264.7 cells in good growth state and press 2×10 4 / well density seeded in 24-well plate, at 37°C, 5% CO 2 Incubator incubation. After the cells were stably cultured for 4-6 hours, press Instructions for RNAiMAX TransfectionReagent Add the first siRNA (-260) Flot2-siRNA (Gimma gene) and the control NC-siRNA (Gemma gene) with the highest knockout efficiency obtained through screening to the cells, transfect for 24 hours, and then add RANKL ( The final concentration is 50ng / ml), TRAP staining was carried out on the 4th to 5th day of induction, observation was taken under a microscope, and osteoclasts were counted as TRAP-positive cells with more than 3 nuclei.

[0079] The result is as image 3 As shown, it can be seen from the results that Flot2-siRNA can significantly inhibit the formation of TRAP-positive osteoclasts in RAW264.7 cells induced b...

Embodiment 3

[0080] Example 3 Effect of Flot2-siRNA on osteoclast activity of induced osteoclasts

[0081] Inoculate RAW264.7 cells on the artificial bone-coated Osteo Assay was cultured in 96-well plates, and the cells were washed away on the 7th day of induction, and photographed under a 40×Nikon inverted optical microscope. The results are as follows: Figure 4 As shown in A, the percentage of bone resorption area in each hole was calculated by Image-Pro Plus software, and the results are shown in the attached Figure 4 Shown in B.

[0082] As can be seen from the results: compared with the NC control group, the area of ​​white bone depressions in the Flot2-siRNA group was significantly reduced, indicating that Flot2-siRNA can significantly reduce the area of ​​bone depressions formed by osteoclasts on bone slices, that is, Flot2-siRNA can significantly reduce the area of ​​bone depressions formed by osteoclasts. Inhibits the bone resorption activity of osteoclasts.

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Abstract

The invention discloses application of a Flot2 inhibitor to inhibition of osteoclast formation and / or osteoclastic activity for the first time. The osteoclast generation and bone resorption induced byRANKL can be obviously reduced after the Flot2 expression is inhibited by Flot2-siRNA specific interference. A study team of the inventor discovers that the Flot2 directly participates in the osteoclast generation and bone resorption functions, and is possible to become a novel target of an osteoclast differentiation inhibitor. On the basis of the inhibition effect of Flot2-siRNA on osteoclasts and the application prospects of RNA interference medicine, the application of the Flot2 inhibitor as a medicine of inhibiting the osteoclast generation is provided.

Description

technical field [0001] The invention relates to the field of biomedicine, more specifically, the invention relates to the application of Flot2 gene inhibitors in inhibiting osteoclast formation and / or osteoclast activity. Background technique [0002] The stabilization of bone metabolism requires a dynamic balance and coupling between bone resorption mediated by osteoclasts (OCs) and bone formation mediated by osteoblasts (OBs), which maintain function in healthy individuals Balance, and once the balance is broken, it may lead to the occurrence of bone diseases. [0003] Osteoclasts are the only cells in the body responsible for bone resorption. It can differentiate into functional mature osteoclasts from osteoclast precursor cells after activation. Because differentiated and mature osteoclasts can absorb bone, their excessive activation can cause bone loss, osteoporosis, fracture pain, secondary reactive bone hyperplasia, and even disability. There are many diseases rela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/06A61K31/713A61K48/00A61P19/08A61P19/10A61P35/04A61P19/02A61P29/00A61P31/04A61P17/06A61P1/02
CPCA61K31/713A61K45/06A61P1/02A61P17/06A61P19/02A61P19/08A61P19/10A61P29/00A61P31/04A61P35/04
Inventor 李晓娟邹斌华郑洁煌
Owner SOUTHERN MEDICAL UNIVERSITY
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