【作者】 蔡宏；

【导师】 顾瑛；

【作者基本信息】 中国人民解放军军医进修学院 ， 康复医学与理疗学， 2008， 博士

【摘要】 目的:增生性瘢痕（HS）是成纤维细胞异常增殖和细胞外基质过度沉积的纤维化疾病,是医学界亟待解决的难题。本文研究以血卟啉单甲醚（HMME）为光敏剂的光动力学疗法（PDT）对瘢痕成纤维细胞（HSF）增殖与功能的影响并初步探讨其作用机制,在动物瘢痕模型上观察HMME-PDT的生物学效应,为临床应用HMME-PDT防治增生性瘢痕提供实验依据。方法:1.HSF对HMME的吸收特性及HMME-PDT对其增殖效应的研究:取原代培养的第4～6代HSF,经流式细胞仪（FCM）检测孵育浓度（0～40μg/ml）和孵育时间（0～120min）对HSF细胞吸收HMME的影响;MTT法检测HMME-PDT对HSF细胞存活率的影响;银染法检测HSF中核仁组成区相关嗜银蛋白（AgNORs）的表达情况;FCM分析细胞周期的变化。2.HMME-PDT对HSF中TGF-β₁/Smad信号通路的影响:用酶联免疫吸附法（ELISA）检测HMME-PDT后HSF分泌至上清液的TGF-β₁蛋白表达量;用Smad3-FITC标记后在荧光显微镜上观察细胞内Smad3的荧光强度;将细胞分为对照组、光敏剂组、照光组和HMME-PDT组,经Western-blot方法检测各组细胞浆内TGF-β₁、磷酸化和非磷酸化Smad3、Smad7蛋白的表达量。3.HMME-PDT诱导HSF凋亡效应的研究:Hoechst33258染色后在荧光显微镜上观察HMME-PDT后HSF细胞形态学变化;Annexin V-FITC-PI双染后经FCM检测HMME-PDT后HSF细胞凋亡率和坏死率;将HSF爬片后分为对照组、HMME-PDT组和Z-DEVD-FMK抑制剂组,Caspase3-FITC-PI染色后在荧光显微镜上观察各组细胞内Caspase3的荧光强度;收集各组细胞在active-Caspase3-FITC单染后经FCM检测active-Caspase3阳性细胞百分率;收集各组细胞在PI单染后经FCM检测细胞凋亡率。4.HMME-PDT对兔耳瘢痕的效应研究:建立兔耳瘢痕模型后,将瘢痕块分为不同组别,照光功率密度为20mW/cm²,光敏剂剂量为10mg/kg,分别在给药后即刻、0.5、1、3h给予2.5、5、10、20J/cm²的能量密度照光,治疗后第10天用游标卡尺测量并计算瘢痕增生指数（HI）;HE染色后观察瘢痕厚度、细胞及胶原分布等形态学变化;观察HMME-PDT后瘢痕中微血管数量、形态变化,采用Weidner计数方法,计算平均微血管密度（MVD）;运用透射电镜观察兔耳瘢痕增生块中成纤维细胞超微结构的变化。结果:1.在一定孵育浓度范围内（0～40μg/ml）,HSF细胞对HMME的吸收量随孵育浓度的增高和孵育时间的增加而增大。实验范围内的HMME-PDT对HSF细胞的杀伤效应与孵育浓度和激光能量密度正相关;HMME-PDT能够减少HSF细胞中AgNORs的含量,使I.S%值降低;HMME-PDT降低HSF增殖指数,改变各期细胞的分布比例,抑制S期DNA合成,使细胞滞留在G₀/G₁期。2.HSF分泌至细胞上清液的TGF-β₁蛋白含量较高,而HMME-PDT后降低;HSF合成的TGF-β₁蛋白水平高,HMME-PDT后HSF中的TGF-β₁和磷酸化Smad3蛋白含量减少,Smad7蛋白含量增加,光敏剂组和照光组中上述蛋白的表达无显著改变;PDT不能改变HSF内Smad3蛋白表达量,但其在细胞内的分布发生变化,进入细胞核的Smad3蛋白减少。3.HMME-PDT治疗后细胞核染色质高度凝聚,呈团块颗粒状,或呈波纹状、折缝样改变;AnnexinV-FITC-PI双染结果表明PDT后凋亡率显著升高,并伴有细胞坏死的发生,但组内坏死率低于凋亡率;对照组和Z-DEVD-FMK抑制剂组HSF的细胞浆中Caspase3-FITC荧光微弱,PDT组荧光显著增强;对照组active-Caspase-3阳性细胞百分率低,HMME-PDT组上升,Z-DEVD-FMK组降低;PI染色分析表明对照组凋亡率低,PDT组的凋亡率显著升高,Z-DEVD-FMK组的凋亡率低于PDT组,但仍然显著高于对照组。4.在本研究的兔耳增生性瘢痕块中,静脉注射HMME10mg/kg后30min,给予功率密度为20mW/cm²,能量密度为5J/cm²的630nm红光照射能对瘢痕块产生较理想的抑制效应;对照组兔耳瘢痕块真皮层显著增厚,血管分布丰富,HMME-PDT组瘢痕块厚度下降,MVD显著减少;在透射电镜下观察到对照组中有大量HSF,胞浆内粗面内质网丰富,高尔基体发达,线粒体增加,PDT组胞浆内粗面内质网萎缩且数量减少,核蛋白体有不同程度的脱颗粒和解聚,胞质中见较多游离核糖体,线粒体肿胀,嵴溶解。结论:1.HSF对HMME的吸收随孵育浓度和孵育时间的增加而增加,HMME-PDT处理HSF时,光敏剂孵育浓度和激光能量密度是影响细胞存活率的重要因素,PDT改变各期细胞的分布比例,抑制细胞增殖。2.HMME-PDT能够阻止TGF-β₁的信号经由Smad蛋白传向细胞核,改变TGF-β₁、磷酸化Smad3和Smad7在HSF细胞水平的表达,使细胞增殖及胶原合成有关的靶基因得不到激活,从而抑制HSF增殖和胶原合成。3.HMME-PDT诱导HSF发生的凋亡效应与Caspase-3的激活密切相关,但该凋亡效应并不依赖于Caspase-3,可能与AIF等因子的释放或其它信号途径的激活有关。4.HMME-PDT对兔耳增生性瘢痕的生物学效应是一个光动力综合作用的结果,与光敏剂的剂量和激光照光时间、给药后至照光的时间间隔及能量密度密切相关。在瘢痕形成早期,HMME-PDT能够抑制兔耳HSF的增殖,破坏蛋白合成场所,并能诱导部分细胞进入凋亡途径,HMME-PDT有可能成为瘢痕防治的有效方法。更多还原

【Abstract】 Objective:Photodynamic therapy （PDT） is a promising treatment for various kinds of dermatosis. Hypertrophic scar （HS） is a pathological process characterized by fibroblastic hyperproliferation. In this study, we investigated the cellular response to PDT which induced by hematoporphyrin monomerthyl ether （HMME） in human fibroblasts from hypertrophic scar （HSF）, and explored the cell signal mechanisms initially. Furthermore, we observed the biological effects of HMME-PDT on hypertrophic scarring in a rabbit ear model.Methods:1. The absorption characteristics of HSF on HMME and the inhibitory effect of HMME-PDT: Fibroblasts were cultured from nontreated burn hypertrophic scars, and the 4-6 passage cells were used in the experiments. HSF was incubated with HMME at different concentrations （0～40μg/ml） and different incubation time （0-120min）. The absorption characteristics of HSF on HMME were detected by flow cytometry （FCM）. Cell viability after HMME-PDT was detected by MTT assay. The AgNORs expression was determined with the standard silver-staining method and the cell cycle was calculated by FCM.2. TGF-β₁/Smad signal mechanisms in HSF mediated by HMME-PDT: The expression of TGF-β₁ in supernatant of HSF was detected by enzyme linked immunosorbent assay （ELISA）. Fluorescence intensity of Smad3 was observed with immunofluorescence staining. The expression of TGF-β₁ Smad3, Phospho-Smad3, and Smad7 was analyzed by Western blot.3. The apoptotic effects of HSF induced by HMME-PDT: The morphological changes in HSF were observed with Hoechst 33258 staining. The rate of apoptotic or necrotic cells was respectively detected by FCM through double staining of Annexin V-FITC and popodium iodide （PI）. Caspase-3 activity assay and immunofluorescence staining method were applied to the investigate Caspase-3 expression in HSF by FCM and fluorescence microscope.4. Effect of HMME-PDT on Hypertrophic Scarring in Rabbit Ear Model: After the acute model of dermal hypertrophic scar in the rabbit ear was established, scar wounds randomly received HMME-PDT with different treatment parameters. Hypertrophic index （HI） was measured by slide caliper. Scar histomorphology and thickness were observed with haematoxylin-eosin staining. The microvessel density （MVD） was calculated under microscope. HSF was observed under transmission electron microscope （TEM）.Results:1. The absorption of HMME by HSF was increased with increased concentration and incubation time. HMME-PDT inhibited the proliferation of HSF significantly. The growth curve was declined following the increased HMME concentration and light dose. HMME-PDT decreased the expression of AgNORs and led to cell cycle arrest in G₀/G₁ phase.2. HMME-PDT down-regulated the protein level of TGF-β₁ both in supernatant of HSF and in HSF. It prevented the activation of Smad3 while increased the expression of Smad7 protein.3. Marked morphological features of cell apoptosis were viewed under the fluorescent microscope through Hoechst 33258 staining. The analysis of FCM indicated that the apoptotic rate was significantly increased after HMME-PDT, accompanied by the presence of active-caspase-3. The apoptotic rate was still high after used Z-DEVD-FMK which could inactivate caspase-3.4. Compared with the control group, HMME-PDT in the experimental group （photosensitizer dose 10mg/kg, power density 20mw/cm² , energy fluence 5J/cm² ） reduced scar formation, and the HI. Mitochondria, rough ER and Golgi of HSF were abundant in the control group, while decreased and destroyed by PDT. Some fibroblasts underwent marked apoptosis after HMME-PDT.Conclusions:1. Incubation concentration and time are two important factors for the absorption of HMME by HSF. HMME-PDT inhibits the proliferation of HSF which depending on HMME concentration and laser energy density, and also blocks the cell cycle.2. HMME-PDT induces HSF apoptosis, and stimulates caspase-3 activation, but caspase-3 is dispensable for apoptosis in this process.3. HMME-PDT can inhibit the TGF-β₁/Smad signal transduction pathway and prevent HSF’s proliferation and collagen’s synthesis.4. HMME-PDT can inhibit hypertrophic scarring in rabbit ear. The biological effect is determined by photosensitizer, interval injection of photosensitizer and irradiation, power density and energy fluence. HMME-PDT may have good potent effects on the formation of hypertrophic scar.更多还原