| OBJECTIVEOur objective is to investigate the high expression and correlation of urotensin Ⅱ and its receptor. GPR14, in the aorta of diabetic, and to compare the different expression of urotensin Ⅱ and GPR14 between diabetic artery smooth muscle cells and non-diabetic artery smooth muscle cells. The aim is to explore new target spots and new approaches to prevent and treat diabetic artery lesions.METHODSThe artery tissue was obtained from coronary artery bypass grafting (CABG) and aortic dissection (AD), common and classical operation of cardiovascular surgery. Human primary artery smooth muscle cells were cultured by tissue - piece inoculation method and identified with morphology and immunofluorescence method. Cells under 3rd to 6th generation were selected for our experiment. They were divided into diabetic group and non-diabetic group according to whether the patients had been suffering from diabetes or not. The patients had suffered from diabetes more than one year with irregular blood glucose, and the average blood glucose was above 11.1mmol/L before operation. While, the average blood glucose of the patients without diabetes was at normal level (3.9-6. lmmol/L). Diabetic and non-diabetic group were respectively divided into UⅡ group, UT antagonist group and the control group according to different disposal measures and dealt respectively with U 11,SB-657510,control deal for 24 hours. It was detected that the different effect of urotensin Ⅱ and its receptor GPR14 on migration rate of diabetes and non-diabetes aortic smooth muscle cell by wound healing assay. Compared the degree of cell proliferation by CCK-8 test to demonstrate the promoting proliferation effect of urotensin Ⅱ and its receptor GPR14 in diabetes group and non-diabetes group. Total protein and total mRNA of cells in every group were extracted and compared respectively to demonstrate the different expression of urotensin Ⅱ and its receptor GPR14 in normal and diabetes aortic smooth muscle cells by western blot and RT-PCR method. Data was sent to SPSS19.0, a common application for statistical processing.RESULTS1. Culture and identification of smooth muscle cellsThe human primary culture cells of aortic smooth muscle were cultured successfully by tissue adherent method. Morphological observation was adopted to identify the human aortic smooth muscle cells. Then we could see that cells were spindle or long spindle. They could grow overlap to multilayer, to lie in undulation, in a special form of "peak-valley", which is the typical grow form of vascular smooth muscle cells (just like the attached figure 4). The immunofluorescence identification was based on the anti-alpha actin of smooth muscle cells. We can see green fluorescence in the cell cytoplasm, and the purity of smooth muscle cells is above 90% (just like the attached figure 5).2. CCK-8 proliferation test①As time went on, the OD value of every group increased within a certain range;②In the diabetic group(DM for short), the cell proliferation activity of diabetic U Ⅱ treated group (UⅡ+DM group for short) was markedly higher than that of diabetic UⅡreceptor antagonism group (UT+DM for short) (P<0.01) and diabetic control group (DM control group for short) (P<0.01) at every point in time, at the same time, the cell proliferation activity of UT+DM group was lower than that of DM control group(P<0.05);③In normal group, the cell proliferation activity of diabetic UⅡ treated group(U Ⅱ+normal group for short) was higher than that of normal UⅡ receptor antagonism group (UT+ normal for short) (P<0.05) and normal control group (P<0.05), but there was no remarkable difference between UT+ normal group and the normal control group (P>0.05);④At every point in time, the cell proliferation activity of UⅡ+DM group was higher than that of UⅡ+normal group (P<0.05).3. Cell wound scratch assay①In diabetic group, the cells’ migration distance of UⅡ+DM group was remarkably farther than that of UT+DM group (P<0.01) and DM control group (P<0.01), then the distance of UT+DM was nearer than that of DM control group (P<0.05);②In the normal group, the cells’ migration distance of UⅡ+normal group was also farther than that of UT+ normal group (P<0.05) and normal control group (P<0.05), while, the difference between UT+ normal group and normal control was no statistical significance (P>0.05);③Between the diabetic group and the normal group, the cells’ migration distance of UⅡ+DM group was farther than that of UⅡ+normal group (P<0.05).4. Western blot and RT-PCR④In diabetic group, the protein and mRNA expression quantity of UⅡ and its receptor GPR14 in UⅡ+DM group was remarkably higher than that of UT+DM group (P<0.01) and the DM control group (P<0.01), then the protein and mRNA expression quantity of UⅡ and its receptor GPR14 in UT+DM group was lower than that of normal control group (P<0.05);②In the normal group, the protein and mRNA expression quantity of UⅡ and its receptor GPR14 in UⅡ+normal group was higher than that of UT+ normal group (P<0.05) and normal control group (P<0.05), however, there was no significant difference between UT+ normal and normal control group (P>0.05);③The protein and mRNA expression quantity of UⅡ and its receptor GPR14 in UⅡ+DM group was higher than that of UⅡ+normal group (P<0.05).CONCLUSIONS1. It confirmed the high expression of UⅡ and its receptor GPR14 is closely related to the proliferation and migration of human aortic smooth muscle cells from a new perspective.2. The aortic smooth muscle cells of diabetic was more sensitive to UⅡ than that of normal one, which made proliferation and migration more easily to occur, thus leading to suffering atherosclerosis more easily. In addition, the atherosclerosis of diabetic was more serious and destructive. The above process played a number of complex reactions in cells through second messenger pathway which was mediated by GPR14.3. In addition to "UⅡ and its receptor GPR14", there may be some other pathways to promote the proliferation and migration of vascular smooth muscle cell, which may accelerate the occurrence and development of atherosclerosis.4. SB-657510, as a kind of sulfa UⅡ receptor antagonist, can significantly inhibit the proliferation and migration of vascular smooth muscle cell and can put off the progress of atherosclerosis. As a result, it may play an important role in prevent and treatment of atherosclerosis, which will has a vast potential prospect for future development. |
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