Preliminary Study Of Mechanical Stretch On The Proliferation And Apoptosis Of Vascular Smooth Muscle Cells

BackgroundThis study started from the thinking of a large number of clinical phenomena. Mechanical stress is an important factor in tissue repair, and to reduce the mechanical stress can relieve excessive tissue repair. In the skin repair process, the scar area of the joint activity area is larger than other areas. The callus in the unstable fracture ends appears much more hyperplasia than other stable area. These clinical phenomena show that absolute braking can reduce excessive tissue repair and the mechanical stress is one of the important factors that affect organizational excessive repair. Our preliminary research results show that add a-cyanoacrylate glue can reduce excessive movement of blood vessels and inhibit the excessive movement of the blood vessels can inhibit the excessive neointimal hyperplasia. Many scholars have widely studied the effect of mechanical stimulation on cells in many experiments. This study is on the cellular and molecular biological level to discuss mechanical stretch on the proliferation and apoptosis of vascular smooth muscle cells. Apply mechanical stretch to VSMCs and observe the the changes at the aspects of the proliferation and apoptosis and explore the related genes, proteins, and factor involved.PurposeIn order to further study mechanical stretch on the proliferation and apoptosis of VSMCs and confirm mechanical stress can relieve excessive tissue repair on the cell biology level. Our study used Flexcell-5000T cell stretching device to apply mechanical stretch to VSMCs and by regulating the tensile force size and stretch time. Observe the the changes at the aspects of the proliferation and apoptosis and explore the related genes, proteins, and factor involved.Method(1) The fitst step is the primary cell extraction and subculture. Draw the thoracic aorta materials from male SD-rats weight 200-220g. To get the generation of smooth muscle cells with the method of tissue block until 3-8 generation for follow-up study.(2) Then to apply mechanical stretch on VSMCs with the use of Flexcell-5000T cell stretching device. Putting the VSMCs at the density of 3*105/ml inoculation on Bioflex 6 holes culture plate, and adding 2～3 ml of 20% FBS DMEM culture in each hole. Accept the FX-5000T mechanical tensile force and divide into groups according to the mechanical tensile force. Experimental group, Group A 5% tensile force, the observation time 0 min,15 min,30 min,60 min,120 min,1 HZ; GroupB,C,D,E,F are respectively divide from tensile force of 10%,15%,20%,25%,30% elongation with the same observation time and frequency. The control group is of 0% elongation tensile force with the same observation time and frequency.(3) And observe the changes of various indicators. To get the results of cell cycle with flow cytometry instrument analysis. To observe the cell morphology with microscope. Count cells with the cell count meter calculation. Analyse cell proliferation with the method of MTT and analyse cell apoptosis with flow cytometry. Analyse cell proliferation related genes with RT-PCR analysis such as a-SMA, ERK, MMP-2, NF-kB, PCNA and detect the level of these proteins related to cell proliferation with Western blot analysis. Detect the level of these factors such as MCP-1、PDGF、TGF-β、VEGF related to cell proliferation with ELISA analysis. Detect apoptosis index of hydrogen peroxide content.Results(1) Successfully putting the generation 3-8 of VSMCs of mechanical stretch with the use of Flexcell-5000T cell stretching device.(2) Compare each cell cycle and the results show that group B has the highest percentage of S phase with 54.65% at the time of 15 min and group F has the lowest percentage of S phase with 8.17% at the time of 60 min. Compared with the control group, P<0.05, the difference was statistically significant and the influence of tensile force and time to cell apoptosis is different.(3) Compare each cell count and the results show that group B has the most number of cells with 5675* 106 at the time of 15 min and group F has the least number of cells with 2636* 106 at the time of 60 min. Compared with the control group, P<0.05, the difference was statistically significant and the influence of tensile force and time to number of cells is different.(4) Compare each 570nm absorbance value and the results show that group B has the highest number with 1.01 and group F has the lowest number with 0.6. Compared with the control group, P<0.05, the difference was statistically significant and the influence of tensile force and time to absorbance value is different.(5) Compare each apoptosis ratio and the results show that group E has the most ratio with 25.64% at the time of 60 min and group B has the lowst ratio with 4.34% at the time of 0 min. Compared with the control group, P<0.05, the difference was statistically significant and the influence of tensile force and time to apoptosis ratio is different.(6) Detect the proliferation of the related genes with RT-PCR technique and the tensile force size was 10%, the tensile time 15 min. The a-SMA related mRNA level was 1.94, ERK related mRNA level was 2.23, MMP-2 related mRNA level was 2.11, NF-kB related mRNA level was 1.33 and PCNA mRNA level was 1.56.(7) Detect the proliferation related to the proteins with Western blot technique in the tensile force size was 10% and the tensile time 15 min. The a-SMA related protein level was 8.73, ERK protein levels related was 3.68, MMP-2 related protein was 4.79, NF-κB related protein level was 8.52 and PCNA related protein level was 8.52.(8) Detect the contents of each proliferation related factors with ELISA. The tensile force size was 10% and tensile time 15 min. The content of MCP-1 was 482.47 pg/ml;The content of PDGF was 345.31 pg/ml;The content of TGF-β was 4563.19 pg/ml;The content of VEGF was 50.03 pg/ml.(9) Detect apoptosis index of hydrogen peroxide content. The tensile force size 10% stretch time 15 min showed the lowest level of hydrogen peroxide with 4.20 uM.ConclusionModerate size and time of mechanical tensile can promote the cell cycle S period and promote cell proliferation and increase cell numbers. To change the size of the mechanical tensile and time can affect S period cell cycle, cell proliferation and cell count. Moderate size and time of mechanical tensile can promote cell apoptosis, and change the size of mechanical tensile and time affect cell apoptosis.Mechanical tensile force stimulates cells by signaling to the cell mechanics with the corresponding cytokine content changes. The corresponding cell protein and gene level change, these changes affect cell proliferation and apoptosis, specific influence mechanism is yet to be studied.