IGF-1對血管平滑肌細胞增殖和凋亡的影響
發(fā)布時間：2018-11-08 12:44
【摘要】：目的:探討在炎癥狀態(tài)下胰島素樣生長因子(insulin like growth factor-1,IGF-1)對血管平滑肌細胞增殖和凋亡的影響。方法:1.取雄性SD大鼠,斷頸后分離主動脈,超凈臺內(nèi)去除內(nèi)膜和外膜,按組織貼塊法+酶消化法獲取原代血管平滑肌細胞并傳代,實驗取用第4-8代細胞,α-肌動蛋白陽性表達的細胞鑒定為平滑肌細胞。2.不同濃度的脂多糖(lipopolysaccharide,LPS)誘導RAW264.7細胞制備炎癥模型,ELISA法檢測各組上清液中IL-6的水平,與對照組相比差異有統(tǒng)計學意義為造模成功,即條件培養(yǎng)基(conditioned medium,CM),未加LPS誘導組為對照組即非條件培養(yǎng)基(nonconditioned medium,nCM)。3.平滑肌細胞分別與nCM、CM、CM+IGF-1 60ng/ml、CM+IGF-1 90ng/ml、CM+IGF-1 120ng/ml共培養(yǎng),用MTT法檢測各組OD值,以O(shè)D值反應各組VSMC的數(shù)量,用流式法檢測各組平滑肌細胞的凋亡率。4.統(tǒng)計學方法:以SPSS17.0軟件進行統(tǒng)計,計量資料用均數(shù)±標準差表示,兩組間比較用t檢驗,多組間比較用方差分析,P0.05有統(tǒng)計學差異。結(jié)果:1.使用組織貼塊法+酶消化法可成功獲取實驗所需血管平滑肌細胞。2.一定濃度的LPS誘導RAW264.7細胞后上清液中IL-6的表達量顯著升高。RAW264.7細胞分別在LPS 0ng/ml,10ng/ml,100ng/ml,1ug/ml,10ug/ml濃度刺激下各組上清液中IL-6的濃度分別是(6.75±0.12)pg/ml;(7.82±1.53)pg/ml;(44.09±1.58)pg/ml;(155.71±23.93)pg/ml;(436.59±3.15)pg/ml,與對照組相比,10ng/ml組差異無統(tǒng)計學意義(P=0.916);后三組與對照組相比,差異有統(tǒng)計學意義(P=0.009,P=0.000,P=0.000)。3.CM組能抑制血管平滑肌細胞增殖,加入一定濃度的IGF-1可逆轉(zhuǎn)其對血管平滑肌細胞增殖的抑制。VSMC在五組不同干預下各組OD值分別是(0.53±0.07)、(0.26±0.06)、(0.30±0.10)、(0.62±0.09)、(0.55±0.05)。與nCM組相比,CM組OD值明顯降低(p=0.000),兩組間比較有統(tǒng)計學意義。與CM組相比,CM+IGF-160ng/ml組、CM+IGF-190ng/ml組、CM+IGF-1120ng/ml組OD值升高(p=0.993,p=0.001,p=0.000),后兩組與CM組之間有統(tǒng)計學意義,以90ng/ml組效果最好。4.CM組可促進血管平滑肌細胞凋亡,加入一定濃度的IGF-1可逆轉(zhuǎn)其對血管平滑肌細胞的促凋亡作用。五組干預下VSMC凋亡率分別是(4.89±0.09)%;(10.86±0.15)%;(9.64±0.65)%;(3.85±0.51)%;(4.82±0.08)%。與nCM組相比,CM組凋亡率明顯增加(p=0.000),兩組間差異有統(tǒng)計學意義;與CM組相比,CM+IGF-160ng/ml組、CM+IGF-190ng/ml組、CM+IGF-1120ng/ml組凋亡率顯著降低(p=0.045,p=0.000,p=0.000),差異有統(tǒng)計學意義,以90ng/ml組效果最好。結(jié)論:LPS可誘導RAW264.7細胞極化為M1型巨噬細胞。M1型巨噬細胞所致的炎癥狀態(tài)可促進血管平滑肌細胞凋亡,抑制血管平滑肌細胞增殖,給予一定濃度的IGF-1后可發(fā)現(xiàn)凋亡減少,增殖增加。
[Abstract]:Aim: to investigate the effects of insulin-like growth factor (insulin like growth factor-1,IGF-1) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) in inflammatory condition. Methods: 1. The primary vascular smooth muscle cells (VSMCs) were obtained from male SD rats after cervical dissection, and the intima and adventitia were removed from the ultraclean platform, and the primary vascular smooth muscle cells were obtained by enzyme digestion method, and the 4-8 passage cells were used in the experiment. 偽-actin positive cells were identified as smooth muscle cells. 2. 2. Different concentrations of lipopolysaccharide (lipopolysaccharide,LPS) induced inflammatory model of RAW264.7 cells. ELISA assay was used to detect the level of IL-6 in supernatant of each group. Compared with the control group, the difference was that the model was successful, that is, conditioned medium (conditioned medium,CM). The unconditioned medium (nonconditioned medium,nCM) was the control group without LPS induction. 3. Smooth muscle cells were co-cultured with nCM,CM,CM IGF-1 60ng / ml CM IGF-1 90ng / ml IGF-1 120ng/ml respectively. The OD values of each group were detected by MTT method, and the number of VSMC in each group was measured by OD value. The apoptosis rate of smooth muscle cells in each group was detected by flow cytometry. Statistical methods: SPSS17.0 software was used to analyze the statistical data. The measurement data were expressed as mean 鹵standard deviation. The comparison between the two groups was by t test, and the analysis of variance was used by the analysis of variance between the two groups. There was a statistical difference between the two groups (P0.05). The result is 1: 1. The vascular smooth muscle cells needed in the experiment can be successfully obtained by enzyme digestion with tissue patch method. 2. 2. The expression of IL-6 in the supernatant of RAW264.7 cells was significantly increased by a certain concentration of LPS. The expression of IL-6 in the supernatant of LPS 0ng / ml 10ng / ml 10 ng / ml RAW264.7 cells was 100 ng / ml / ml, respectively. The concentration of IL-6 in supernatant stimulated by 10ug/ml was (6.75 鹵0.12) pg/ml;. (7.82 鹵1.53) pg/ml; (44.09 鹵1.58) pg/ml; (155.71 鹵23.93) pg/ml; (436.59 鹵3.15) pg/ml, there was no significant difference between 10ng/ml group and control group (P0. 916). There was significant difference between the latter three groups and the control group (P0. 009 P0. 000). 3.CM group could inhibit the proliferation of vascular smooth muscle cells. The inhibitory effect of IGF-1 on the proliferation of vascular smooth muscle cells was reversed by adding a certain concentration of IGF-1. The OD value of VSMC in different intervention groups was (0.53 鹵0.07), () 0.26 鹵0.06), (0.30 鹵0.10), (0.62 鹵0.09, respectively. (0.55 鹵0.05). Compared with nCM group, the OD value of CM group was significantly lower than that of nCM group (p0. 000). Compared with CM group, OD value in CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was increased (p0.993P 0.001P 0.000), and there was significant difference between the latter two groups and CM group. The effect of 90ng/ml group was the best. 4.CM group could promote the apoptosis of vascular smooth muscle cells. Adding a certain concentration of IGF-1 could reverse the apoptosis of vascular smooth muscle cells. The apoptotic rates of VSMC were (4.89 鹵0.09)%, (10.86 鹵0.15)%, (9.64 鹵0.65)%, (3.85 鹵0.51)% and (4.82 鹵0.08)%, respectively. Compared with nCM group, the apoptotic rate in CM group was significantly increased (p0. 000), and the difference between the two groups was statistically significant. Compared with CM group, the apoptotic rate of CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was significantly lower (P < 0.05). The difference was statistically significant, especially in 90ng/ml group. Conclusion: LPS can induce RAW264.7 cells to become M1 macrophages. The inflammatory state induced by M1 macrophages can promote the apoptosis of vascular smooth muscle cells and inhibit the proliferation of vascular smooth muscle cells. After given a certain concentration of IGF-1, apoptosis was decreased and proliferation was increased.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54
本文編號：2318541
[Abstract]:Aim: to investigate the effects of insulin-like growth factor (insulin like growth factor-1,IGF-1) on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) in inflammatory condition. Methods: 1. The primary vascular smooth muscle cells (VSMCs) were obtained from male SD rats after cervical dissection, and the intima and adventitia were removed from the ultraclean platform, and the primary vascular smooth muscle cells were obtained by enzyme digestion method, and the 4-8 passage cells were used in the experiment. 偽-actin positive cells were identified as smooth muscle cells. 2. 2. Different concentrations of lipopolysaccharide (lipopolysaccharide,LPS) induced inflammatory model of RAW264.7 cells. ELISA assay was used to detect the level of IL-6 in supernatant of each group. Compared with the control group, the difference was that the model was successful, that is, conditioned medium (conditioned medium,CM). The unconditioned medium (nonconditioned medium,nCM) was the control group without LPS induction. 3. Smooth muscle cells were co-cultured with nCM,CM,CM IGF-1 60ng / ml CM IGF-1 90ng / ml IGF-1 120ng/ml respectively. The OD values of each group were detected by MTT method, and the number of VSMC in each group was measured by OD value. The apoptosis rate of smooth muscle cells in each group was detected by flow cytometry. Statistical methods: SPSS17.0 software was used to analyze the statistical data. The measurement data were expressed as mean 鹵standard deviation. The comparison between the two groups was by t test, and the analysis of variance was used by the analysis of variance between the two groups. There was a statistical difference between the two groups (P0.05). The result is 1: 1. The vascular smooth muscle cells needed in the experiment can be successfully obtained by enzyme digestion with tissue patch method. 2. 2. The expression of IL-6 in the supernatant of RAW264.7 cells was significantly increased by a certain concentration of LPS. The expression of IL-6 in the supernatant of LPS 0ng / ml 10ng / ml 10 ng / ml RAW264.7 cells was 100 ng / ml / ml, respectively. The concentration of IL-6 in supernatant stimulated by 10ug/ml was (6.75 鹵0.12) pg/ml;. (7.82 鹵1.53) pg/ml; (44.09 鹵1.58) pg/ml; (155.71 鹵23.93) pg/ml; (436.59 鹵3.15) pg/ml, there was no significant difference between 10ng/ml group and control group (P0. 916). There was significant difference between the latter three groups and the control group (P0. 009 P0. 000). 3.CM group could inhibit the proliferation of vascular smooth muscle cells. The inhibitory effect of IGF-1 on the proliferation of vascular smooth muscle cells was reversed by adding a certain concentration of IGF-1. The OD value of VSMC in different intervention groups was (0.53 鹵0.07), () 0.26 鹵0.06), (0.30 鹵0.10), (0.62 鹵0.09, respectively. (0.55 鹵0.05). Compared with nCM group, the OD value of CM group was significantly lower than that of nCM group (p0. 000). Compared with CM group, OD value in CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was increased (p0.993P 0.001P 0.000), and there was significant difference between the latter two groups and CM group. The effect of 90ng/ml group was the best. 4.CM group could promote the apoptosis of vascular smooth muscle cells. Adding a certain concentration of IGF-1 could reverse the apoptosis of vascular smooth muscle cells. The apoptotic rates of VSMC were (4.89 鹵0.09)%, (10.86 鹵0.15)%, (9.64 鹵0.65)%, (3.85 鹵0.51)% and (4.82 鹵0.08)%, respectively. Compared with nCM group, the apoptotic rate in CM group was significantly increased (p0. 000), and the difference between the two groups was statistically significant. Compared with CM group, the apoptotic rate of CM IGF-160ng/ml group, CM IGF-190ng/ml group and CM IGF-1120ng/ml group was significantly lower (P < 0.05). The difference was statistically significant, especially in 90ng/ml group. Conclusion: LPS can induce RAW264.7 cells to become M1 macrophages. The inflammatory state induced by M1 macrophages can promote the apoptosis of vascular smooth muscle cells and inhibit the proliferation of vascular smooth muscle cells. After given a certain concentration of IGF-1, apoptosis was decreased and proliferation was increased.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54
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相關(guān)期刊論文 前2條
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