Application of ginsenoside Rg1 in promoting growth and osteogenic differentiation of dental pulp stem cells

Abstract

The application provides application of neomussaenoside in promoting growth and osteogenic differentiation of dental pulp stem cells, and belongs to the technical field of dental pulp stem cells. The application finds through experiments that neomussaenoside can promote proliferation of dental pulp stem cells and can promote osteogenic differentiation of the dental pulp stem cells; further research finds that the neomussaenoside can effectively regulate mRNA expression of cell cycle genes and protein expression of osteogenic differentiation genes.

Description

Technical Field

[0001] This invention belongs to the field of dental pulp stem cell technology, and particularly relates to the application of lingxianxin glycoside in promoting the proliferation and osteogenic differentiation of dental pulp stem cells. Background Technology

[0002] Dental pulp stem cells are a type of stem cell isolated from dental pulp tissue. They possess an immune phenotype similar to bone marrow mesenchymal stem cells and have the potential for self-renewal and multi-lineage differentiation. Under the induction of different growth factors, dental pulp stem cells can differentiate into various cell lineages, including bone, dentin, and nerve cells.

[0003] Currently, regenerative medicine research, including dental pulp regeneration and reconstruction, has received widespread attention, and dental pulp stem cells have become a research hotspot. However, the specific regulatory mechanisms of dental pulp stem cell proliferation and differentiation are not yet fully understood. Therefore, how to better promote the proliferation and differentiation of dental pulp stem cells is of great significance.

[0004] Clematis glycoside, also known as clematisin, is an active ingredient extracted from the traditional Chinese medicine Clematis chinensis. In in vitro studies, clematisin has been shown to reduce Ox-LDL-induced foam cell formation and inflammatory responses in RAW264.7 cells by activating autophagy, thus potentially useful in the treatment of atherosclerosis. However, the role of clematisin in dental pulp stem cells remains unclear. Therefore, this invention explores the application of clematisin in the proliferation and osteogenic differentiation of dental pulp stem cells. Summary of the Invention

[0005] The purpose of this invention is to provide a new application of lecithin in promoting the growth and osteogenic differentiation of dental pulp stem cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of lecithin in the preparation of formulations that promote the growth of dental pulp stem cells.

[0008] Preferably, the lecithin promotes the proliferation of dental pulp stem cells.

[0009] Preferably, the lindane promotes the mRNA expression of the cell cycle promoting gene Cyclin-D1, while effectively inhibiting the mRNA expression of the cell cycle repressor gene P21.

[0010] Preferably, the formulation comprises lecithin and pharmaceutically acceptable excipients and / or carriers.

[0011] Secondly, this invention provides the application of lecithin in the preparation of formulations that promote osteogenic differentiation of dental pulp stem cells.

[0012] Preferably, the lecithin promotes the calcification of dental pulp stem cells.

[0013] Preferably, the lindane promotes the protein expression of the osteogenic differentiation genes RUNX2 and ALP in dental pulp stem cells.

[0014] Preferably, the formulation comprises lecithin and pharmaceutically acceptable excipients and / or carriers.

[0015] The beneficial effects of this invention are:

[0016] 1. This invention provides a novel use of lingxianxin glycoside in promoting the proliferation of dental pulp stem cells, and discovers that lingxianxin glycoside can effectively promote the mRNA expression of the cell cycle promoting gene Cyclin-D1, while effectively inhibiting the mRNA expression of the cell cycle repressor gene P21.

[0017] 2. This invention provides a novel use of linexinoside in promoting osteogenic differentiation of dental pulp stem cells, and finds that linexinoside can effectively promote calcification of dental pulp stem cells and promote the expression of RUNX2 and ALP by dental pulp stem cells during osteogenic differentiation. Attached Figure Description

[0018] Figure 1 The effect of clematisin on the proliferation of dental pulp stem cells;

[0019] Figure 2 The effect of cyclinopeptide on the mRNA expression of Cyclin-D1 and P21 in dental pulp stem cells;

[0020] Figure 3 The effect of clematisin on cell calcification of dental pulp stem cells;

[0021] Figure 4 The effect of lindane on the expression of RUNX2 and ALP proteins in dental pulp stem cells. Detailed Implementation

[0022] The illustrated embodiments are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the illustrated embodiments. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description of the invention still fall within the protection scope of the present invention.

[0023] Example 1

[0024] Effects of clematisin on dental pulp stem cell proliferation

[0025] 1. P3 generation dental pulp stem cells, prepared as cell suspensions, were mixed at a ratio of 1×10⁻⁶. 4 Seeds were planted per well into 96-well plates and incubated in a cell culture incubator for 12 hours;

[0026] 2. Change the culture medium. The control group is not treated. Experimental group 1 is given 50 μmol / L lingxianxin glycoside, and experimental group 2 is given 100 μmol / L lingxianxin glycoside. After the addition is completed, the cells are placed in a cell culture incubator and cultured for 72 h.

[0027] 3. After the culture is complete, add 10 μL of CCK-8 to each well, gently tap the culture plate to mix it thoroughly, place the 96-well plate in a cell culture incubator and continue to culture for 3 hours, and then measure the absorbance at 450 nm.

[0028] The results obtained from the experiment are shown in Figure 1 In the study, the OD value of the control group was 0.514±0.039, the OD value of experimental group 1 was 0.681±0.043, and the OD value of experimental group 2 was 0.779±0.066. Compared with the control group, the OD values ​​of experimental groups 1 and 2 were increased, indicating that lingxianxin glycoside has a promoting effect on the proliferation of dental pulp stem cells.

[0029] Example 2

[0030] Effects of clematisin on cyclin in dental pulp stem cells

[0031] 1. P3 generation dental pulp stem cells, which were prepared into cell suspension, were seeded into 6-well plates. When the cell density reached about 70%, the culture medium was changed. The control group was not treated. Experimental group 1 was supplemented with 50 μmol / L lingxianxin glycoside, and experimental group 2 was supplemented with 100 μmol / L lingxianxin glycoside. After the addition was completed, the cells were placed in a cell culture incubator and cultured for 72 h.

[0032] 2. After the culture is complete, gently wash the cells three times with PBS, add 1 ml of Trizol reagent to each well, repeatedly pipette the cells, and then collect the mixture into a centrifuge tube.

[0033] 3. After standing at room temperature for 5 minutes, add 200 μL of chloroform, mix well, and let stand at room temperature for 5 minutes.

[0034] 4. Adjust the centrifuge parameters to 4℃, 12000rpm, 15min, and centrifuge after placing the centrifuge tubes in the centrifuge.

[0035] 5. After centrifugation, the liquid separates into 3 layers. Carefully transfer the upper aqueous phase to a new enzyme-free centrifuge tube.

[0036] 6. Add an equal volume of isopropanol, mix well, let stand at room temperature for 10 minutes, adjust the centrifuge parameters to 4℃, 12000rpm, 15 minutes, put it into a centrifuge tube and centrifuge.

[0037] 7. After centrifugation, discard the supernatant, add 75% ethanol to the precipitate to wash the precipitate, adjust the centrifuge parameters to 4℃, 7500rpm, 5min, and then centrifuge in a centrifuge tube.

[0038] 8. After centrifugation, discard the supernatant, place in a clean bench to dry for 5-10 minutes until the alcohol has completely evaporated, add RNase-free water to dissolve the precipitate;

[0039] 9. Perform the experiment according to the instructions of the Takara PrimeScript RT reagent Kit to reverse transcribe RNA into cDNA;

[0040] 10. Design primers for PCR reactions of the cell cycle promoting gene Cyclin-D1 and the cell cycle repressor gene P21. The primer sequences are as follows:

[0041]

[0042] The results obtained from the experiment are shown in Figure 2 Compared to the control group, the mRNA expression levels of Cyclin-D1 were significantly increased in experimental groups 1 and 2, while the mRNA expression levels of P21 were significantly decreased in both groups. These results indicate that treatment of dental pulp stem cells with cyclin-D1 can effectively promote the expression of the cell cycle promoting gene Cyclin-D1 and effectively inhibit the expression of the cell cycle repressor gene P21.

[0043] Example 3

[0044] Effects of clematis treatment on osteogenic differentiation of dental pulp stem cells

[0045] 1. P3 generation dental pulp stem cells with good growth were seeded into 6-well plates. When the cell density reached more than 85%, the medium was replaced with osteogenic induction medium. The control group was not treated. Experimental group 1 was supplemented with 50 μmol / L ligustrazine and experimental group 2 was supplemented with 100 μmol / L ligustrazine. After the addition was completed, the cells were placed in a cell culture incubator and cultured for 14 days. The medium was changed every 2-3 days, and the composition was the same as the initial addition.

[0046] 2. After culturing, remove the culture medium, gently wash the cells with PBS, and fix the cells with 4% paraformaldehyde for 30 minutes;

[0047] 3. After fixation, the paraformaldehyde was discarded, the cells were washed with PBS, and then incubated with alizarin red staining solution at room temperature in the dark for 30 min.

[0048] 4. After staining, remove the staining solution, wash the cells twice with PBS, and then photograph the cells.

[0049] The results obtained from the experiment are shown in Figure 3 As shown in the figure, compared with the control group, the number of calcified nodules was significantly increased in experimental groups 1 and 2. Therefore, the alizarin red staining experiment demonstrates that treatment of dental pulp stem cells with linalool can effectively improve the osteogenic differentiation of dental pulp stem cells.

[0050] Example 4

[0051] Effects of ginsenosides on the expression of osteogenic differentiation-specific transcription factors RUNX2 and ALP proteins in dental pulp stem cells

[0052] 1. P3 generation dental pulp stem cells with good growth were seeded into 6-well plates. When the cell density reached more than 85%, the medium was replaced with osteogenic induction medium. The control group was not treated. Experimental group 1 was supplemented with 50 μmol / L ligustrazine and experimental group 2 was supplemented with 100 μmol / L ligustrazine. After the addition was completed, the cells were placed in a cell culture incubator and cultured for 14 days.

[0053] 2. After the culture is complete, add 100 μl of cell lysis buffer to each well. After scraping the cells off with a cell scraper, collect the lysis buffer into a centrifuge tube and place the centrifuge tube on ice for lysis for 30 min, vortexing once every 10 min.

[0054] 3. Adjust the centrifuge parameters to 4℃, 12000rpm, 18min, and centrifuge after placing the centrifuge tubes in the centrifuge.

[0055] 4. After centrifugation, transfer the supernatant to a new centrifuge tube, take 2 μl of the supernatant to measure the protein concentration, add loading buffer, and boil in water for 5 min to obtain the protein sample.

[0056] 5. After preparing the 5% stacking gel and 12% separating gel, assemble the electrophoresis rack and start loading the samples. After loading the samples, turn on the power to start electrophoresis.

[0057] 6. When the bromophenol blue reaches the bottom of the separating gel, stop electrophoresis, assemble the electrotransfer clamp, add electrotransfer buffer, and perform electrotransfer.

[0058] 7. After the electroporation is complete, remove the PVDF membrane and place it in 5% skim milk powder, then seal it at room temperature for 1 hour;

[0059] 8. After sealing, remove the membrane and incubate it with ALP, RUNX2 and GAPDH primary antibodies in a 4°C freezer overnight.

[0060] 9. On the second day, the primary antibody was recovered, and the membrane was washed three times with TBST for 10 minutes each time, followed by incubation with the corresponding secondary antibody.

[0061] 10. After incubating at room temperature in the dark for 1 hour, wash the membrane with TBST and develop it in a dark room.

[0062] Experimental results

[0063] The results obtained from the experiment are shown in Figure 4 As can be seen from the figure, compared with the control group, the expression levels of ALP and RUNX2 were significantly increased in experimental groups 1 and 2, indicating that treatment of dental pulp stem cells with lindane can effectively improve the protein expression of specific transcription factors RUNX2 and ALP in osteogenic differentiation.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of lecithin in the preparation of formulations that promote the growth of dental pulp stem cells.

2. The application according to claim 1, characterized in that, The ligusticin promotes the proliferation of dental pulp stem cells.

3. The application according to claim 1, characterized in that, The ligustilide promotes the mRNA expression of the cell cycle promoter gene Cyclin-D1, while inhibiting the mRNA expression of the cell cycle repressor gene P21.

4. The application according to claim 1, characterized in that, The formulation includes lecithin and pharmaceutically acceptable excipients and / or carriers.

5. Application of lingxianxin glycoside in the preparation of formulations that promote osteogenic differentiation of dental pulp stem cells.

6. The application according to claim 5, characterized in that, The ligusticin promotes the calcification of dental pulp stem cells.

7. The application according to claim 5, characterized in that, The ligusticin promotes the protein expression of the osteogenic differentiation genes RUNX2 and ALP in dental pulp stem cells.

8. The application according to claim 5, characterized in that, The formulation includes lecithin and pharmaceutically acceptable excipients and / or carriers.

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