本文選題：光動(dòng)力療法　切入點(diǎn)：成纖維細(xì)胞　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 1.觀察EtNBSe-PDT對(duì)體外培養(yǎng)的成纖維細(xì)胞增殖活性及TGF-β1分泌的影響。 2.觀察EtNBSe-PDT對(duì)體外培養(yǎng)成纖維細(xì)胞內(nèi)MAPK家族成員ERK、JNK、P38活性影響和信號(hào)蛋白的時(shí)相性變化,以及抑制劑干預(yù)中磷酸化表達(dá)水平的影響。 方法 1.分離培養(yǎng)正常人包皮成纖維細(xì)胞,采用角蛋白和Ⅰ、Ⅲ型膠原染色進(jìn)行成纖維細(xì)胞的鑒定。 2.采用梯度紅光照射、梯度EtNBSe濃度和EtNBSe聯(lián)合紅光照射處理成纖維細(xì)胞；在抑制劑干預(yù)研究中,應(yīng)用ERK、JNK、P38信號(hào)通路特異性抑制劑(PD98059、SP600125、SB203580)分別處理成纖維細(xì)胞,四甲基偶氮唑鹽(MTT)法檢測(cè)各組成纖維細(xì)胞增殖和抑制的變化。 3. EtNBSe聯(lián)合紅光照射處理成纖維細(xì)胞,酶聯(lián)免疫吸附(ELISA)法檢測(cè)TGF-β1分泌變化。 4.蛋白質(zhì)免疫印跡(western blot)法測(cè)定磷酸化ERK、JNK、P38的活性及時(shí)相性變化；同時(shí)觀察在抑制劑干預(yù)中,磷酸化ERK、JNK、P38的變化。 結(jié)果 1.體外培養(yǎng)的細(xì)胞經(jīng)Ⅰ、Ⅲ型膠原染色陽性,角蛋白染色陰性,細(xì)胞具有成纖維細(xì)胞形態(tài)。 2.成纖維細(xì)胞經(jīng)梯度紅光照射(10、30、60、90min)后,與對(duì)照組比較,細(xì)胞不產(chǎn)生明顯的細(xì)胞增殖及細(xì)胞毒效應(yīng)(P0.05)。在梯度濃度EtNBSe (30、60、120、240pM)分別作用培養(yǎng)1、2h后,各組細(xì)胞間未觀察到明顯的增殖活性和細(xì)胞毒反應(yīng)(P0.05)。在梯度EtNBSe (30、60、120、240pM)+紅光照射組,梯度EtNBSe處理1h后照光,與對(duì)照組比較,對(duì)細(xì)胞不產(chǎn)生明顯的細(xì)胞毒反應(yīng)及細(xì)胞增殖活性(P0.05)；在梯度EtNBSe處理2h后照光,EtNBSe (30、60、120pM)對(duì)成纖維細(xì)胞有增殖活性,以EtNBSe (60、120pM)增殖活性最明顯(P0.05)。在抑制劑干預(yù)組中,與對(duì)照組比較,抑制劑中(PD98059、SB203580)+照光組,細(xì)胞活性受到明顯抑制(P＜0.05); SP600125細(xì)胞活性無明顯變化。 3.與對(duì)照組比較,成纖維細(xì)胞在經(jīng)60、120pM EtNBSe-PDT處理后,TGF-β1分泌均增加(P＜0.05),以120pM更明顯。 4.濃度組：與對(duì)照組比較,30、60、120pM EtNBSe-PDT可促進(jìn)磷酸化ERK蛋白的活化；30、60pM可促進(jìn)磷酸化JNK和P38蛋白的活化,隨著劑量的增加逐漸抑制兩者的活性。時(shí)間組：ERK、JNK、P38均有不同程度的活化,與對(duì)照組比較,ERK在60分鐘時(shí)達(dá)到峰值； JNK和P38在90分鐘時(shí)達(dá)到峰值。干預(yù)組：與對(duì)照組比較,PD98059、SB203580可顯著抑制磷酸化ERK、 P38的活性；SP600125對(duì)磷酸化JNK抑制作用不明顯。 結(jié)論 1. EtNBSe-PDT可促進(jìn)體外培養(yǎng)的成纖維細(xì)胞的增殖,誘導(dǎo)TGF-β1分泌的增加。 2. EtNBSe-PDT可影響體外培養(yǎng)的成纖維細(xì)胞內(nèi)MAPK家族成員(ERK, JNK、p38)的活性,其中對(duì)JNK、p38具有雙向性作用。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R751

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 魏駿;;皮膚老化研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(老年醫(yī)學(xué)分冊(cè));1998年05期

2 吳和巖,周華;光老化的預(yù)防與治療[J];國(guó)外醫(yī)學(xué)(衛(wèi)生學(xué)分冊(cè));2003年03期

3 魯建云;于小峰;康健;歐陽澤輝;耿雪瑞;向亞平;黃進(jìn)華;;強(qiáng)脈沖光對(duì)成纖維細(xì)胞分泌TGF-β_1的影響及JNK抑制劑的干預(yù)作用(英文)[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年05期

4 王繼華;劉垠;何永靜;杜永貴;趙亞南;;TGF-β_1對(duì)UVA照射皮膚成纖維細(xì)胞Ⅰ型,Ⅲ型膠原合成和表達(dá)的影響[J];中國(guó)美容醫(yī)學(xué);2009年03期

5 楊紅華;A型肉毒毒素治療上半面部皺紋1000例[J];實(shí)用美容整形外科雜志;2001年04期

6 蔡澤浩,劉偉,李卿,武曉莉,錢云良,曹誼林;阻斷轉(zhuǎn)化生長(zhǎng)因子-β信號(hào)轉(zhuǎn)導(dǎo)抑制成纖維細(xì)胞增殖和Ⅰ型膠原合成[J];中華實(shí)驗(yàn)外科雜志;2003年02期

7 魯建云;于小峰;黃進(jìn)華;歐陽澤輝;耿雪瑞;陳靜;左成忻;向亞平;楊盛波;譚麗娜;康健;;強(qiáng)脈沖光照射對(duì)成纖維細(xì)胞分泌TGF-β_1的影響及信號(hào)機(jī)制研究[J];中國(guó)現(xiàn)代醫(yī)學(xué)雜志;2010年16期

，

本文編號(hào)：1576473