EtNBSe-PDT对成纤维细胞增殖和TGF-β1分泌的影响及信号通路研究

发布时间：2018-03-06 21:02

本文选题：光动力疗法　切入点：成纤维细胞　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的 1.观察EtNBSe-PDT对体外培养的成纤维细胞增殖活性及TGF-β1分泌的影响。 2.观察EtNBSe-PDT对体外培养成纤维细胞内MAPK家族成员ERK、JNK、P38活性影响和信号蛋白的时相性变化,以及抑制剂干预中磷酸化表达水平的影响。方法 1.分离培养正常人包皮成纤维细胞,采用角蛋白和Ⅰ、Ⅲ型胶原染色进行成纤维细胞的鉴定。 2.采用梯度红光照射、梯度EtNBSe浓度和EtNBSe联合红光照射处理成纤维细胞；在抑制剂干预研究中,应用ERK、JNK、P38信号通路特异性抑制剂(PD98059、SP600125、SB203580)分别处理成纤维细胞,四甲基偶氮唑盐(MTT)法检测各组成纤维细胞增殖和抑制的变化。 3. EtNBSe联合红光照射处理成纤维细胞,酶联免疫吸附(ELISA)法检测TGF-β1分泌变化。 4.蛋白质免疫印迹(western blot)法测定磷酸化ERK、JNK、P38的活性及时相性变化；同时观察在抑制剂干预中,磷酸化ERK、JNK、P38的变化。结果 1.体外培养的细胞经Ⅰ、Ⅲ型胶原染色阳性,角蛋白染色阴性,细胞具有成纤维细胞形态。 2.成纤维细胞经梯度红光照射(10、30、60、90min)后,与对照组比较,细胞不产生明显的细胞增殖及细胞毒效应(P0.05)。在梯度浓度EtNBSe (30、60、120、240pM)分别作用培养1、2h后,各组细胞间未观察到明显的增殖活性和细胞毒反应(P0.05)。在梯度EtNBSe (30、60、120、240pM)+红光照射组,梯度EtNBSe处理1h后照光,与对照组比较,对细胞不产生明显的细胞毒反应及细胞增殖活性(P0.05)；在梯度EtNBSe处理2h后照光,EtNBSe (30、60、120pM)对成纤维细胞有增殖活性,以EtNBSe (60、120pM)增殖活性最明显(P0.05)。在抑制剂干预组中,与对照组比较,抑制剂中(PD98059、SB203580)+照光组,细胞活性受到明显抑制(P＜0.05); SP600125细胞活性无明显变化。 3.与对照组比较,成纤维细胞在经60、120pM EtNBSe-PDT处理后,TGF-β1分泌均增加(P＜0.05),以120pM更明显。 4.浓度组：与对照组比较,30、60、120pM EtNBSe-PDT可促进磷酸化ERK蛋白的活化；30、60pM可促进磷酸化JNK和P38蛋白的活化,随着剂量的增加逐渐抑制两者的活性。时间组：ERK、JNK、P38均有不同程度的活化,与对照组比较,ERK在60分钟时达到峰值； JNK和P38在90分钟时达到峰值。干预组：与对照组比较,PD98059、SB203580可显著抑制磷酸化ERK、 P38的活性；SP600125对磷酸化JNK抑制作用不明显。结论 1. EtNBSe-PDT可促进体外培养的成纤维细胞的增殖,诱导TGF-β1分泌的增加。 2. EtNBSe-PDT可影响体外培养的成纤维细胞内MAPK家族成员(ERK, JNK、p38)的活性,其中对JNK、p38具有双向性作用。
[Abstract]:objective
1. the effects of EtNBSe-PDT on the proliferation of fibroblasts and the secretion of TGF- beta 1 in cultured fibroblasts were observed.
2., we observed the effects of EtNBSe-PDT on the MAPK family members ERK, JNK, P38 activity and signal protein phase changes in vitro, as well as the effect of inhibitor intervention on phosphorylation level.
Method
1. the normal human foreskin fibroblasts were isolated and cultured. Keratin and type I and type III collagen were used to identify the fibroblasts.
2. using gradient red light irradiation, gradient concentration of EtNBSe and EtNBSe combined with red light irradiation treated fibroblasts; inhibitors in the intervention study, application of ERK, JNK, a specific inhibitor of P38 pathway (PD98059, SP600125, SB203580) were treated fibroblasts, four methyl thiazolyl tetrazolium (MTT) assay the fibroblast proliferation and the inhibition of change.
3. EtNBSe combined with red light irradiation treatment of fibroblasts and enzyme linked immunosorbent assay (ELISA) to detect the changes of TGF- beta 1 secretion.
4. protein immunoblot (Western blot) method was used to detect the phosphorylation of ERK, JNK, P38 activity and phase change. Meanwhile, we observed the changes of ERK, JNK and P38 in inhibitor intervention.
Result
1. the cells cultured in vitro were stained positive by type I, type III collagen and negative in keratin, and the cells had fibroblast morphology.
2. fibroblast cells by gradient red light irradiation (10,30,60,90min), compared with the control group, there were no obvious cell proliferation and cytotoxicity (P0.05). The concentrations of EtNBSe (30,60120240pM) respectively. After cultured 1,2h cells were not observed between proliferation and cytotoxicity (P0.05). In the gradient EtNBSe (30,60120240pM) + irradiation group, gradient EtNBSe after the treatment with 1H light, compared with the control group, no cytotoxicity and cell proliferation activity in cell (P0.05); light gradient in EtNBSe after 2H treatment, EtNBSe (30,60120pM) on fibroblast proliferation activity by EtNBSe (60120pM) the most obvious proliferation (P0.05) inhibitors. In the intervention group, compared with control group (PD98059, SB203580) inhibitor + irradiation group, the cell activity was inhibited (P < 0.05); the activity of SP600125 cells had no obvious change.
3. compared with the control group, after the treatment of 60120pM EtNBSe-PDT, the secretion of TGF- beta 1 increased (P < 0.05), and 120pM was more obvious.
The concentration of 4. groups: compared with the control group, 30,60120pM EtNBSe-PDT can promote the activation of phosphorylated ERK protein; 30,60pM can promote the activation of phosphorylated JNK and P38 protein, with the increase of the dose gradually suppress the activity of them. The time group: ERK, JNK, activation of P38 was different, compared with the control group, ERK peak in 60 minutes; JNK and P38 reached the peak at 90 minutes. The intervention group: compared with the control group PD98059, SB203580 significantly inhibited the phosphorylation of ERK, the activity of P38; SP600125 on the phosphorylation of JNK inhibition is not obvious.
conclusion
1. EtNBSe-PDT can promote the proliferation of cultured fibroblasts in vitro, and induce the increase of TGF- beta 1 secretion.
2. EtNBSe-PDT can affect the activity of MAPK family members (ERK, JNK, p38) in cultured fibroblasts in vitro, which has a two-way effect on JNK and p38.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R751

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