【作者】 杨力；

【导师】 李荟元；

【作者基本信息】 第四军医大学 ， 外科学， 2002， 博士

【摘要】 瘢痕疙瘩是多种生长因子刺激成纤维细胞、血管内皮细胞等修复细胞增殖并合成大量细胞外基质的结果。TGF-β在瘢痕疙瘩的形成过程中发挥着关键作用。 TGF-β与细胞膜上的TGF-β受体结合后，经过其在细胞内的信号介导子--SMADs蛋白家族，将TGF-β的刺激信号传入核内调控基因转录。这就是TGF-β／SMADs信号转导通路。但TGF-β如何影响SMADs、以何种方式影响哪些靶基因等问题尚远未研究清楚。特别是，在TGF-β／SMADs信号传导通路与瘢痕疙瘩的形成的关系研究方面，有关的报道少见，作者在研究过程中仅检索到一篇文献(Chin GS，et al．P．R．S．2001；108；423)。因此，有必要对此进行研究。 本文对KFB胞膜TGF-βⅠ、Ⅱ受体和TGF-β／SMADs信号传导通路中的SMAD3、7信号介导子的表达及其调控机制进行了研究。并利用反义寡 第四军医大学傅士学位论文 核苦酸掺入技术，对 TGF－5／SMADS信号通路阻断后的 KFB的增殖与胶原 合成的影响进行了初步研究。 首先，利用免疫组织化学（SABC法）和图像分析的方法，观察了 TGF． PI、11型受体在体外培养的正常皮肤成纤维细胞（NFB）、KFB中的表达。 结果发现，两种受体在KFB中染色的阳性着色（棕黄色）均深于NFB细胞 （P＜0．05）；当受到 TGF－日；刺激后，KFB阳性着色深于未受刺激的 K厂以P＜0．05）。提示吓B中的 TGF－6I、11型受体高表达，而且这种表达 可因配体的刺激而上调。 其次，采用逆转录PCR、Western蛋白印迹杂交、免疫荧光等技术， 对Smad3、7mRNA及其蛋白在KFB中的表达和调控机制进行了研究。结果 发现：Smad3mRNA及其蛋白在NFB和KFB中的表达水平并无明显差别， 但SMAD3蛋白在KFB核内的荧光强度强于NFB。TGF－pl刺激KFB后，Smad3 ITutN A表达随K卜日 剂量增加（O，5，n，500＊m。1几）和时间的延续 （1，2，4，24，d卜而降低，呈剂量依赖性和时间依赖性。枷d7m趴A 在 5 pmol／L TGF－P 作用 90。in就上调到对照组的近 3倍，并延续到 24h 才恢复至大约正常水平。SWhD3表达在兀F－p 作用24h后下降了70％， 而SAfD7在TGF－日；刺激2小时就达到最高值，说明Smad7 aRNA表达对TGF－ 日；的刺激更为敏感而反应迅速。用蛋白合成阻断剂CHX阻断KFB的蛋白 合成后，Smad3 mRNA表达下调明显，说明Smad3 mRNA表达具有蛋白合成 依赖性，而Smad7 mRNA表达不受影响，呈非蛋白合成依赖性，即 Smad7 可能是SWDS的直接靶基因。 再次，对SMAD3、7蛋白在KFB中的空间分布进行的研究发现：当KFB 受到TGF－p 刺激后，SMAD3由胞浆向核内转位，具有剂量和时间信赖性。 SMAD7在KFB未受刺激时表达很弱，受TGF－日；刺激后，迅速从在胞浆 聚集。 4 第四军医大学博士学位论文 在研究 fNfN－Y对 TGF－e／SM－AM－Ak信号通路的影响时，我们发现， 500U／ml I刚－v旨使Sm叨7 InRNA在作用30ruin 后以非蛋白合成信赖性 方式上调到刺激前水平的8倍多，并持续达sh之久。IFN－Y还能抑制TGF－ e；引起的SMAD3向核内转位。说明Iry－Y通过直接促进SMAD7表达， 抑制TGF－日／ShaD信号转导过程。 最后，我们将 Smad3、Smad4反义寡核苦酸（AS03，AS04）掺入 KFB 中，以直接阻断TGF－p；’SMADS信号转导通路，结果发现，AS03、AS04均 能使 MTT反应 A值下降（P＜0．01），并使们－脯氨酸的掺入量降低（P＜0．05）， 说明 AS03或 AS04能阻断 TGF日／SMAD信号通路，并抑制 KF增殖与胶原 合成。 综上所述，本研究得出如下结论： 1．TGF－pl、11型受体在 KFB高表达并具有配体依赖性。 2．yB内可能存在较高水平的磷酸化SMAD3。 3．KFB存在着与其它细胞类似的 TGF－日／SMDS信号传递过程和调 控机制。 4．在KFB中，IFN－Y以非蛋白合成信赖的方式迅速上调SMAD7的 表达而抑制TG卜p／刁MDS通路信号转导。 5．Smad3、Smad4反义寡核苦酸能阻断TGF－D／SMAD信号通路并抑制 KFB的增殖和胶原合成。 在本研究的基础上，结合目前有关细胞信号转导的进展更多还原

【Abstract】 It is generally agreed that keloid is characterized by excessive deposition of extracellular matrix and over-proliferated fibroblast. TGF- 3 plays an crucial role in keliod development.TGF- 3 initiates signaling through their interaction wiJi TGF- 3 receptor complexes. The SMAD complex, which has been activated by TGF-3 receptors, translocates from the cytoplasm into the nucleus and stimulates gene transcription. But its mechanism ,such as how TGF- 3 regulates the expression of SMADs ,what kinds of cellular gene expression may be activated and how this activation occurs, is still incompletely characterized, especially in the fields of keloid researches.The expression and regulation of TGF- 3 I / II receptor and SMAD3 and SMAD7 of keloid-derived fibroblasts(KFB) has been studied. Theeffects of the Smad3 and Smad4 antisense oligonucleotides, which were used to inhibit endogenous Smad expression, on the proliferation and synthesis of collagen of KFB also have been observed.Firstly, the expression of type I and II TGF- P receptor of fibroblasts in vitro has been observed using immunohistochemistry and imaging analysis techniques. The results show that the expression of type I and II TGF- P receptor of KFB is higher than that of NFB(p<0.05).The same results can be obtained between treated and untreated KFB with TGF- P i.Secondly, the expression and regulation of SMAD3 , SMAD7 and their rnRNA of KFB have been studied by applying RT-PCR and Western blot and cellular immunofluorescence. The results show that the expression of SMAD3 and its mRNA between KFB and NFB has no difference, But SMAD3-associated fluorescence in nuclear of KFB shows a high degree compared with that of NFB. The Smad3 mRNA expression was reduced in a dose- and time-dependent manner and the levels of SMAD3 were reduced by 70% in KFB exposed to TGF- P ,.The induction of Smad? mRNA were markedly up-regulated by 3 fold of control exposed to 5 pmol/L TGF- P i for 90 min, and By 24 h, it returned to control levels. The expression of SMAD7 were increased maximal value upon incubation with TGF- PI for 2 h. Inhibition of de novo protein synthesis with CHX caused down-regulated Smad3 mRNA levels by TGF- P i In contrast, TGF- P i induction of SmadTmRNA was unaffected by pretreatment with CFDC These results suggest that SmadV gene is a direct target of receptor-activated SMAD signals. Thirdly, the localization of SMAD3 and SMAD7 has been detected. The results indicate that TGF- P i induces a translocation of SMAD3 from thecytoplasm into the nucleus in a dose- and time- dependent manner and a accumulation of SMAD7-specific immunofluorescence in the cytoplasm of KFB.Fourthly, the opposite effects of IFN- y on TGF- P /SMAD signal transduction pathway of KFB also has been investigated. We found that IFN-Y could increase Smad? mRNA levels at least 8 fold over the basal level . This increase was maximal 30 min after IFN- Y addition and still present after 8h. It was unaffected by the protein-synthesis inhibitor CHX. IFN- Y also inhibited the translocation of SMAD3 caused by TGF- 3 i.It indicates IFN-Y may cause the inhibition of TGF- P /SMAD signaling by increasing the expression of Smad7 mRNA directly.Finally, the SmadS mRNA antisense oligonucleotide were added directly to the monolayer cultures of KFB, both of the MTT reaction A values and 3H-proline incorporation were decreased. The similar effects were found in the Smad4 mRNA antisense oligonucleotide. These results suggest the disruption of TGF- P /SMAD signaling by the SmadS or Sad4 mRNA antisense oligonucleotide can inhibit the proliferation and the synthesis of collagen of KFB. Conclusion:1. The levels of type I> II TGF- P receptors expression is higher in KFB.2. The activated-SMAD3 may be in a higher level in KFB than in NFB.3. The similar mechanism of TGF- P /SMDA is present in KFB , NFB and others.4. The inhibition of TGF- P /SMAD signaling can be caused by IFN- Y , which can increase the expression of Smad? mRNA directly.5. The disruption of T更多还原