Effect Of Cell Transcription Effector YAP On Differentiation Of Mesenchymal Stem Cells Into Tendons

Background:Injuries of tendons and ligaments constitute a large part of injuries in the musculoskeletal system-related diseases.The tendon has a special anatomical structure,which has a small supply of blood vessels,poor self-healing ability,long healing time after injury,and scar-based healing,so the treatment of tendon injury has brought considerable challenges to clinicians.At present,tendon injuries include two treatments.One of which is non-surgical treatment,which includes non-steroidal anti-inflammatory drugs,corticosteroids,extracorporeal shock wave therapy,ultrasound treatment,hyaluronic acid,platelet-rich plasma,etc.The other is surgical technique has also made rapid progress,but no matter what treatment method is selected,it is currently unable to achieve satisfactory results.The recovery is slow,the treatment course is long,and the cost is high.Even after healing,the tendon is difficult to reach the initial state.It may cause scarring,calcification,atrophy,etc.,leading to significant dysfunction.The fundamental reason is that the tendon cells have low proliferation ability and high degree of differentiation,and it is difficult to form after injury.For the past few years,the research of tissue engineering and regenerative medicine based on stem cells has become a hotspot in the medical community.The field of tendon and ligament restore and regeneration is extensively put forward with great expectations.MSCs stand out in the field of tendon and ligament repair and regeneration because of their relatively low level of differentiation and their potential for multi-directional differentiation.Some studies have confirmed that YAP is an important protein that regulates the differentiation of MSCs into fat,bone,and cartilage.However,the role of YAP in the differentiation of MSCs into tendons has not yet been reported.Objective:To investigate whether BMP-12 can induce differentiation of mouse C3H/10T1/2 and the optimal conditions for differentiation into tendon cells.By interfering with the expression of YAP protein in the Hippo signaling pathway,the effect of YAP protein on the differentiation of MSCs into tendons induced by bmp-12 was further investigated.To investigate the role of YAP,a downstream factor of Hippo signaling pathway,in the differentiation of stem cells into tendons.Method:1.Design different BMP-12 induction concentration gradients and action time to induce C3H/10T1/2 cells to differentiate,and use Immunofluorescence method to measure the fluorescence signal intensity of the tendon cell marker protein Tenomodulin to screen the best BMP-12 induction concentration and the most Best time.2.After C3H/10T1/2 cells were induced by BMP-12 at 10 ng/ml,the changes of tendon cell marker proteins Tenascin C(TNC),Tenomodulin(TNMD),and Scleraxis(SCX)were investigated by Immunofluorescence and Western-Blot methods,respectively.3.Immunofluorescence was used to determine the change of fluorescence signal of YAP protein after BMP-12 induced tendon differentiation.4.Western-Blot to screen plasmids with the best effect on YAP protein expression.5.Western-Blot and Immunofluorescence were used to detect the effects of interference with YAP expression on expression of tendon marker protein when BMP-12 induced C3H/10T1/2 differentiation.Results:1.The fluorescence signal of Tenomodulin was significantly enhanced after 10ng/ml BMP-12 induction.The fluorescence signal of Tenomodulin was significantly enhanced after 48 h and 72 h induction with 10 ng/ml BMP-12.2.Immunofluorescence showed that:C3H/10T1/2 cells were induced by BMP-12 at 10 ng/ml for 48 hours.and the immunofluorescence signal intensity of tendon cell marker proteins Scleraxis,Tenascin C and Tenomodulin all increased significantly.Western-Blot results showed that Scleraxis,Tenascin C and Tenomodulin induced 48 h protein expression higher than the normal group(P<0.05).3.Immunofluorescence showed that the signal intensity of YAP protein was significantly enhanced and transferred to the nucleus after 48 h induction with10 ng/ml BMP-12.4.After transfection of C3H/10 T 1/2 with the plasmid YAP-MUS-1283 for 48 h,the YAP protein expression was significantly decreased compared with the normal group(P < 0.05).5.The results of western-Blot showed that the plasmid YAP-MUS-1283 was transfected into C3H/10T1/2 cells and then induced by BMP-12 for 48 h,the tendon markers Scleraxis,Tenascin C and Tenomodulin were significantly higher than those induced by BMP-12 for 48 hours Downward trend(P<0.05).Immunofluorescence results:C3H/10T1/2 cells were induced with BMP-12 for48 hours after YAP was down-regulated by plasmid transfection method,the fluorescence expression intensity of tendon marker proteins Scleraxis,Tenascin C and Tenomodulin was significantly lower than that induced by BMP-12 only for 48 hours.Conclusion:1.In vitro experiments,BMP-12 can obviously induce tendon differentiation of C3H/10T1/2 cells.BMP-12 at 10ng/ml induces tendon differentiation of C3H/10T1/2 cells for 48 h.2.YAP protein is transferred to the nucleus during BMP-12-induced tendon differentiation.YAP may be a transcription factor related to tendon differentiation to affect stem cell differentiation into tendon system.3.YAP protein regulates the differentiation of MSCs induced by BMP-12 into tendons,and YAP expression is required during differentiation.