【作者】 冉霞；

【导师】 秦路平； 张宏；

【作者基本信息】 华东师范大学 ， 植物学， 2012， 硕士

【摘要】 增生性瘢痕是皮肤创面愈合后瘢痕持续增生的一种病理现象,主要特征是真皮层成纤维细胞的增殖异常和以胶原为主的细胞外基质过度沉积。瘢痕病人会遭受瘙痒、红肿、疼痛等痛苦,还伴随身体的功能障碍,影响美观,这些都给瘢痕病人带来身体和心理上的双重压力。增生性瘢痕的发病机制目前还不清楚,缺乏疗效持久副作用小的有效治疗手段。我们前期的研究显示川芎挥发油能抑制瘢痕成纤维细胞的增殖,诱导其凋亡,在兔耳瘢痕模型实验中能预防瘢痕的发生和发展,然而很好的预防作用不意味着一定具备较好的治疗作用。为此,本课题将展开川芎挥发油对已经形成的瘢痕组织是否具有较好的治疗作用的相关研究。本研究采用组织块培养法培养瘢痕成纤维细胞,选择四川省4个产地,新都,彭山,敖平和都江堰的川芎分别进行石油醚萃取和水蒸气蒸馏来提取其中的挥发油,用MTT筛选出最佳产地和最佳提取方法,即比较它们对成纤维细胞的活性的影响,用最佳提取方法提取最佳产地的川芎挥发油用于接下来的实验研究。结果发现最佳产地是新都,最佳提取方法是水蒸气蒸馏,最佳给药时间是24h,最佳给药浓度是200μ g/ml。于是我们用水蒸气蒸馏法大量提取新都产地的川芎挥发油用于下面的探究。用新西兰大耳兔建立兔耳瘢痕模型,造模28天后,将兔子分为空白组(未造模),模型组,阳性组和川芎挥发油(LEO)剂量组,空白组不给任何药物,模型组给基质,阳性组给康瑞宝,川芎挥发油组分别给2.5%,5%和10%LEO,每天一次,给药28天后,取兔耳瘢痕组织进行切片,HE染色,Masson染色,TUNEL染色检测凋亡率,EL ISA检测I、III型胶原,PCR检测MMP1、TGF-β1、Caspase-3和Caspase-9的mRNA的表达水平。结果发现,阳性组和LEO剂量组相对于模型组瘢痕指数明显降低,胶原束明显减少,且排列更规律,Ⅰ、Ⅲ型胶原的含量显著下降,MMP1和TGF-β1的mRNA表达显著降低,Caspase-3和Caspase-9的mRNA表达显著上升,凋亡率也显著上升。表明川芎挥发油对已经形成的瘢痕有很好的治疗作用。采用气相色谱-质谱联用技术(GC-MS)对挥发油成分进行分离鉴定；小鼠100只,随机分为10组,观察川芎挥发油对小鼠灌胃和腹腔给药的急性毒性反应；家兔20只,分为破损组和完整组,每组又分别有空白组,12.5%EO剂量组,25%EO剂量组,50%EO剂量组,100%EO剂量组,观察川芎挥发油对家兔皮肤的刺激性反应；主动过敏性实验(ACA),豚鼠60只,随即分为6组,12.5%EO剂量组,25%EO剂量组,50%EO剂量组,100%EO剂量组,阴性和阳性对照组,观察川芎挥发油对豚鼠皮肤的过敏性反应。GC-MS共检出74个色谱峰,含量达到1%以上的有12种化合物,化合物藁本内酯含量最大,为54.98%；小鼠急性毒性灌胃给药的LDso为7.23g/kg,腹腔注射的LDso为2.52g/kg,分别相当于川芎原药材临床最大用量的14606和5091倍；家兔完整及破损组给药组中25%E0,50%E0和100%EO剂量组均有轻度刺激性反应；豚鼠皮肤未见过敏性反应。这些毒理学研究数据显示其在临床用量范围内安全。综上,川芎挥发油对已经形成的瘢痕组织有很好的治疗作用,所用剂量在临床用量范围内安全无毒,故川芎挥发油有望成为治疗增生性瘢痕的一剂良药。更多还原

【Abstract】 Hypertrophic scar is a pathological phenomenon, which results from skin wound healing. It’s characterized by proliferative disorder of dermal fibroblasts and overproduction of collagen and excessive deposition of extracellular matrix.Patients with hypertrophic scars often report itching and pain, and experience serious functional and cosmetic problems. The pathogenesis is still unclear, so there is no effective treatment without side effects. Our previous study showed that essential oil of Ligusticum chuanxiong Hort.(LEO) can inhibited fibroblasts proliferation and induced apoptosis, and had a good preventive effect in rabbit ear model with hypertrophic scars. But the good preventive effect of a drug does not mean its favorable therapeutic action.Therefore we investigate the therapeutic effect on hypertrophic scar.Hypertrophic fibroblasts (FB) were cultured from HS tissue. Chuanxiong rhizome collecting from Xindu, Pengshan, Aoping and Du jiangyan were extracted by petroleum ether and hydrodistillation respectively. We picked up the best habit and extract way by comparing their inhibitory effect on FB proliferation. Then extract essential oil through the best extract way from Chuanxiong rhizome which is collected from the best habit. The LEO would be used in the next study. The results showed that the best habit is Xindu, best way of extraction is hydrodistillation, and the best concentration is200μg/ml when the best inhibitory effect. So we extract LEO from Chuanxiong rhizome produced in Xindu through hydrodistillation.EO was prepared as a liposomal formulation (liposome-enveloped essential oil, LEO) and a rabbit ear model with hypertrophic scars was established. On postoperative day29and afterwards, LEO (2.5,5, and10%), contractubex and liposomes without EO were applied once daily to the scars for28days. One further unwounded rabbit with full-thickness skin on its ears received neither liposomes (with or without EO) nor contractubex. On postoperative day56, the scar tissue was excised for HE and masson’s trichrome staining, detection of fibroblast apoptosis, assays of the levels of collagens I and III, and analysis of the mRNA expression of matrix metalloproteinase-1(MMP-1), caspase-3and-9, and transforming growth factor P1(TGF-β1). In addition, the scar elevation index (SEI) was also determined. As a result, LEO treatment significantly alleviated formed hypertrophic scars on rabbit ears. The levels of caspase-3and-9levels and apoptosis cells were markedly increased, and TGF-β1, MMP-1, collagen Ⅰ, and collagen Ⅲ were evidently decreased in the scar tissue. SEI was also significantly reduced. Histological findings exhibited significant amelioration of the collagen tissue. These results suggest that LEO possesses the favorable therapeutic effects on formed hypertrophic scars in the rabbit ear model and may be an effective cure for human hypertrophic scars.The essential oil was extracted by hydrodistillation and analyzed by GC-MS;100mice were divided into10groups, the first five groups were intragastric administrated EO, the last five groups were intraperitoneal EO;20rabbits were divided into2groups, normal skin group and injured skin group. There are six subgroups, normal group,12.5%EO,25%EO,50%EO and100%EO group. Observe the irritative reaction after using EO by external application;60guinea pigs were divided into6groups,12.5%EO,25%EO,50%EO and100%EO group, positive and negative group. Observe the allergic reaction of EO. Fingerprint was established and74common peaks were found, of which12components were listed. LD50of EO by intragastric administration and intraperitoneal to mice is7.23g/kg and2.52g/kg, which is respectively equal to14606times and5091times of60kg people’s daily dried medicinal herb expenses; rabbits skin test shows that there were slight erythema and edema response after the intact skin and the damaged skin had contact with25%EO,50%EO and100%EO, but the stimulus index were all below2.0and the doses is equal to101,202and404times of60kg people’s daily dried medicinal herb expenses; EO and basic ointment did not cause anaphylaxis such as erythema and edema etc. on the guinea pigs skin. The findings above showed that EO belongs to the safe preparation.In conclusion, EO had a good therapeutic effect on hypertrophic scar, and the dose is safe in clinic. It has the potential to be a new therapeutic medicine on hypertrophic scar.更多还原