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二氢丹参酮Ⅰ作用于婴幼儿血管瘤的机制研究
中文摘要

目的:婴幼儿血管瘤(infantile hemangiomas,IH),为婴幼儿最常见的良性肿瘤之一,发病率约5%-10%。IH的发病机制至今模糊,约50%以上的患儿可自行消退,部分患儿在治疗后易出现色素沉着,同时伴有浅瘢痕、皮肤萎缩下垂等体征,严重者可能导致毁容,从而给患儿及家庭造成沉重的心理负担。激素和普萘洛尔是目前口服治疗IH的首要选择,两者在疗效显著的同时,也面临诸多副作用,因而更多有效的治疗手段仍需进一步探索。中医中药作为我国独有的医疗特色,具有巨大的潜在治疗价值,中药的有效成分研究为疾病的治疗提供了更多的可能性。传统医学中IH归属外科之“血瘤”,长于肌肤分肉之间,多因着经络阻滞,营卫不和、气血凝滞所致,当以活血化瘀为主,本研究拟从中医学理论出发结合现代药物化学,筛选具有活性的中药单体,为IH提供一种高效安全的治疗选择。我们在前期研究中选取萜类化合物进行筛查,发现丹参提取的化合物抑制血管瘤作用明显,随后对丹参中主要的化合物进行系统的分析,结果提示二氢丹参酮I(DHTS)在丹参提取的化合物中疗效最佳,体外细胞增殖实验(CCK8)提示DHTS抑制血管瘤细胞的能力为普萘洛尔的近二十倍,故本研究旨在进一步探索DHTS治疗婴幼儿血管瘤的作用机制,为临床的药物研发提供理论基础。 方法:利用流式细胞术结合Hochest33258染色观察并探索DHTS对血管瘤细胞凋亡的影响,Western blot检测其对血管瘤细胞凋亡通路中经典蛋白的变化;采用血管成管实验,在相差显微镜下观察DHTS对血管瘤细胞血管生成的影响,并利用western blot检测是否对血管生成过程的关键蛋白VEGFR2及MMP9的表达有影响;进行裸鼠皮下移植瘤实验,成瘤后,对裸鼠进行腹腔药物注射,观察DHTS是否对体内血管瘤具有抑制增殖的作用,同时设立普萘洛尔对照组,比较两者的治疗效果。 结果:体外实验结果提示,在DHTS作用于血管瘤细胞后,流式细胞技术提示其能显著引起血管瘤细胞早期凋亡,western blot提示DHTS在低浓度时更能诱发线粒体凋亡路径,高浓度时诱发Fas/Fasl凋亡途径;血管成管实验表明,DHTS能显著抑制血管瘤细胞血管生成,同时抑制血管生成过程中的关键蛋白—VEGFR2及MMP9的生成;体内裸鼠移植瘤的实验初步证明,DHTS能抑制体内血管瘤的增殖,免疫组化提示其抑制作用可能与诱导血管瘤细胞凋亡及抑制血管生成相关。 结论:DHTS对婴幼儿血管瘤具有潜在的治疗作用,体外及体内实验初步表明, DHTS作用效果显著优于普萘洛尔,其作用机制可能是通过促进血管瘤细胞凋亡,抑制血管瘤血管生成的过程发挥作用。 关键词:二氢丹参酮Ⅰ;婴幼儿血管瘤;普萘洛尔;细胞增殖;凋亡;血管生成

英文摘要

Background: Infantile hemangiomas (IH) is a common and benign vascular neoplasms with an estimated prevalence of 5-10%. The etiopathogenesis of IH have not been fully elucidated. Although majority of the IHs can spontaneous regress, some patients may be associated with complications resulting in pain, functional impairment, or permanent disfigurement. Treatment options of IH include corticosteroids, surgery, vincristine, interferon or cyclophosphamide, while none of therapeutic modalities are ideal due to restrictions or potential serious side effects. There is thus a need to explore novel treatments for IH. Plant-derived phytochemicals have been widely used as therapeutic agents for thousands of years in China. Herbal extracts are used commonly around the world for treating illness and improving health. Hemangiomas generally belong to “RouLiu” in traditional Chinese medicine, which are caused by obstruction of meridian and collateral, and are required to “activate blood circulation”. The dry root of S.miltiorrhiza, also called Tanshen, is widely used in traditional Chinese medicine for the effect of “active blood circulation” . In this study, we tested the effects of lipophilic tanshinones on EOMA cell function, and we identified DHTS as the most potently cytotoxic of the tanshinones examined. The results of our studies showed DHTS was the most potent cytotoxity of all tanshinones, which had ever been reported to have cytotoxicity and anti-angiogenesis to tumor cells. The same effect was also observed in the cells treated by propranolol, but with almost 20-fold of drug concentration as compared with DHTS. Our research may provide a foundation for the clinical development of DHTS as an alternative treatment for infantile hemangiomas. Methods: Cell viability, apoptosis, protein expression and anti-angiogenesis were analyzed by CCK8, Annexin V staining, Western blot and tube formation, respectively. The anti-tumor activity of DHTS in vivo was evaluated using a mouse xenograft model. We set propranolol as positive control group for the following experiment. Results: Fourteen major compounds extracting from tanshinones were screened for the inhibition of hemangiomas cells. Of these compounds, DHTS showed the most potent inhibition effects on hemangioma cells. DHTS could significantly decrease EOMA cells proliferation by inducing cell apoptosis, which is much more efficient than propranolol in vitro. Apoptosis relevant proteins including PARP, AIF, Caspase9, Bax, Cyst3, FADD and Caspase8 were significantly regulated after DHTS treatment in EOMA cells. In addition, DHTS significantly inhibited tube formation in vitro by downregulating vascular endothelial cell growth factor receptor 2 ( VEGFR2 ) and matrix metalloproteinase 9 (MMP9 ) expression. In nude mice xenograft experiment, DHTS(10㎎/㎏) could significantly inhibited the tumor growth of EOMA cells as well as propranolol(40㎎/㎏). Conclusion: Our findings reveal for the first time that in vivo and in vitro, the anti-angiogenic and apoptosis-inducible activity of DHTS occur at significantly lower doses than propranolol, which could be potentially become a new and high effective compound for the treatment of infantile hemangiomas. Keywords: Dihydrotanshinone I; Infantile hemangiomas; Propranolol: Proliferation; Apoptosis; Angiogenesis.

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