利用RNA干扰技术下调血管瘤中VEGF基因的表达对血管瘤影响的实验研究

发布时间：2018-07-10 03:55

本文选题：血管瘤 + 动物模型　；参考：《第四军医大学》2010年硕士论文

【摘要】： 血管瘤是婴幼儿最常见的皮肤肿瘤,好发于头面部和四肢等体表部位,是以血管内皮细胞增生为主的良性肿瘤。其特点是在婴幼儿出生后第1年迅速增长,在随后的7-9年缓慢消退。一般将其分为增殖期、消退期和消退后期3个阶段。其中相当一部分血管瘤可以自然消退,不留下任何后遗症;还有部分血管瘤可能破溃感染留下瘢痕,大约20%血管瘤会破坏正常组织甚至危及生命。但是其发病机理和消退机制到目前还不清楚。因此血管瘤的治疗方法有很多,但是疗效却不确切。研究其发病机制不仅能够预防血管瘤的发生,同时也有助于临床的治疗。目前研究发现,有助于血管生成的因子有:血管内皮细胞生长因子(VEGF)、雌二醇(oestradiol)、成纤维生长因子(FGF)、血管生长素(angiogenin)、转化生长因子(TGF)、α-肿瘤坏死因子(TNF-α)、血小板衍生生长因子(PDGF)、白介素-8(IL-8)等。其中VEGF被认为与血管瘤的生成、发展和消退有着重要的联系。VEGF具有促进血管化、维持内皮细胞的活性、促进内皮细胞的迁移、促进内皮细胞的增殖、增加微血管通透性等特性。它是内皮细胞(Endothelial cell,ECs)的特异性有丝分裂原,能选择性的作用于EC,促进其增殖、迁移,有利于血管生成。在肿瘤、缺血缺氧等情况下,VEGF及其受体呈高表达。而VEGF不足时会导致血管管腔闭合、血管退化。目前利用基因干扰技术下调VEGF的表达在研究与治疗肿瘤方面取得了极大的进展。同时有学者发现增殖性血管瘤(婴幼儿血管瘤,IH)不同于其它类型的血管畸形,其早期迅速的扩张是血管内皮细胞的非控性增生,这些增生是血管形成刺激因子或抑制因子水平异常引起的。其中VEGF被认为在这种血管瘤的形成中起十分重要的作用。将VEGF作为一种新的治疗靶点,已部分应用到血管瘤的临床诊断和治疗中。本课题拟利用基因干扰技术,通过体内和体外实验研究,下调血管瘤动物模型和婴幼儿血管瘤血管内皮细胞中VEGF基因的表达,观察血管瘤和血管瘤内皮细胞的发展变化,同时探讨血管瘤的增殖和消退机制。为进一步临床应用研究奠定理论基础。 1.裸鼠血管瘤移植模型的建立目的:建立人毛细血管瘤裸鼠移植模型,探讨血管瘤裸鼠模型建立的最佳条件。方法:将手术切除的雌激素受体阳性的儿童增生期血管瘤组织制成组织块,植入20只裸鼠(BALB/c nude mice)皮下,每只4处,将20只裸鼠分为4个实验组。实验1组在移植后给予普通鼠食喂养;实验2组在1组基础上每周肌注雌二醇0.01 mg;实验3组在1组基础上每周肌注雌二醇0.1 mg;实验4组在1组基础上每周肌注雌二醇1mg,于移植后第30、60、90天切取移植瘤。移植瘤标本进行病理学光镜检查,用血管内皮细胞单克隆抗体CD31、CD34、Ki-67行免疫组化染色。结果:移植后早期各组标本内皮细胞大量变性、坏死,30d后,单纯喂养的实验1组及实验2组部分移植瘤开始吸收或形成脓肿及纤维化。实验3、4组移植瘤开始缓慢生长。90d后实验1组实验2组移植瘤均未成活,实验4组移植瘤部分成活,而实验3组移植瘤全部成活。光镜下成活的移植瘤与原血管瘤组织生物学特点相似。结论:不同剂量的雌激素对血管瘤裸鼠移植模型的建立有一定影响,适量的应用雌激素可建立稳定的人血管瘤裸鼠移植动物模型。该模型可以应用到基础和临床的血管瘤研究。 2.增殖期血管瘤内皮细胞的培养和鉴定目的:体外培养和鉴定血管瘤内皮细胞(Hemangioma endothelial cell,HemEC)。方法:无菌条件下手术切取增生期血管瘤组织标本,利用组织块法进行培养。用含20%胎牛血清的M199培养基,加入内皮细胞生长支持物(Endothelial cell growth supplement,ECGS,浓度为100μg/ml)在含5%CO2、37℃培养箱中培养,每隔两到三天换液,细胞铺满瓶底后传代。用光学显微镜观察细胞形态和生长情况,并用免疫组织化学检测细胞表达Ⅷ因子相关抗原和透射电镜检测鉴定细胞。结果:组织块接种3天后有细胞开始从边缘移出,细胞成多角形,1周左右混合成片,部分汇合成岛状。大约3周可以铺满瓶底,随后消化传代。传代细胞成典型的“鹅卵石”样排列铺满瓶底。细胞Ⅷ因子染色为阳性,透射电镜显示细胞质内含有ECs特征性的Weibel-Palade小体(W-P小体,呈点状或卵形)。结论:利用组织块法可以获得纯度较高的血管瘤内皮细胞,该方法简单易用。 3.VEGF基因干扰质粒对血管瘤内皮细胞的影响目的:应用人血管内皮生长因子(VEGF)干扰质粒,作用于体外培养的人增生期血管瘤内皮细胞,观察其对VEGF分泌及对血管瘤内皮细胞的影响。方法:利用脂质体法将真核表达质粒pGCsi-U6NeoRFP-shVEGF转染到HemECs。采用倒置荧光显微镜观察质粒转染效果;用MTT法和酶联免疫吸附(ELISA)法分析观察shVEGF对HemECs的影响。结果:转染组的HemECs与对照组相比, VEGF表达明显减少,转染组培养上清中的VEGF表达量在转染的第1、3、5、7天分别为141.3±7.82、92.34±5.71、68.07±5.95及40.65±6.71pg/ml于其它组比较P0.05,且VEGF质粒的转染使VEGF的表达下降也促进了细胞的凋亡。结论:转染VEGF干扰质粒可以明显减少血管瘤内皮细胞VEGF的表达,同时也促进了细胞的凋亡。 4.VEGF干扰慢病毒对裸鼠血管瘤移植模型的影响目的:制作VEGF慢病毒干扰载体Lenti-siRNA-VEGF,将其作用于裸鼠血管瘤移植模型,观察血管瘤的生长和凋亡的变化,探讨新的血管瘤防治方法。方法:将VEGF干扰质粒包装成慢病毒载体的Lenti-siRNA-VEGF。制作裸鼠血管瘤移植模型,将Lenti-siRNA-VEGF作用于移植模型,大体观察瘤体变化,利用HE、免疫组化和TUNEL等方法观察瘤体切片。再用Western-blot方法从蛋白质水平探讨VEGF被沉默后血管瘤的生长和消退情况。结果:实验组瘤体在注射后1周瘤体体积开始变小,1个月后瘤体缩小变硬;对照组无明显变化。HE检测可见管腔闭塞,瘤体纤维化。CD31、CD34免疫组化检测发现阳性细胞数分别为7.68±0.83、10.25±1.29(P0.05)。TUNEL检测发现细胞凋亡较对照组明显增多。Western-blot发现实验组VEGF蛋白表达明显下降。结论:Lenti-siRNA-VEGF作用于移植模型可以明显降低瘤体VEGF的表达,促进瘤体内皮细胞凋亡。该方法为血管瘤进一步实验和临床研究提供一个新的方法。
[Abstract]:Hemangioma is the most common skin tumor in infants and infants. It is well distributed in the surface of the head, face and limbs. It is a benign tumor with vascular endothelial cell proliferation, which is characterized by a rapid growth of first years after birth and a slow decline in the next 7-9 years. It is generally divided into 3 stages of proliferation, regression and retreat. When a part of the hemangioma can naturally fade away without leaving any sequelae, some hemangiomas may break the infection and leave the scar, about 20% of the tumor will destroy the normal tissue or even life. But the mechanism and mechanism of the disease are still unclear. Therefore, there are many treatments for the hemangioma, but the curative effect is not accurate. Studying its pathogenesis can not only prevent the occurrence of hemangioma, but also help clinical treatment.
Current studies have found that the factors contributing to angiogenesis are vascular endothelial growth factor (VEGF), estradiol (oestradiol), fibroblast growth factor (FGF), angiogenin (angiogenin), transforming growth factor (TGF), alpha tumor necrosis factor (TNF- alpha), platelet derived growth factor (PDGF), and interleukins -8 (IL-8). VEGF is considered to be the same. The formation, development and regression of hemangioma have important connections with.VEGF, which can promote vascularization, maintain endothelial cell activity, promote endothelial cell migration, promote endothelial cell proliferation, and increase microvascular permeability. It is a specific mitogen for Endothelial cell (ECs), which can selectively act on EC and promote it. The proliferation and migration of the VEGF and its receptors are highly expressed in the cases of tumor, ischemia and hypoxia, and the insufficiency of VEGF will lead to the closure of the vascular lumen and the degeneration of blood vessels.
At present, the use of gene interference to reduce the expression of VEGF has made great progress in the research and treatment of tumor. At the same time, some scholars have found that proliferative hemangioma (infant hemangioma, IH) is different from other types of vascular malformation, its early rapid expansion is the non controlled proliferation of vascular endothelial cells, which are vascular formation spines. VEGF is considered to play an important role in the formation of this kind of hemangioma. VEGF, as a new therapeutic target, has been applied to the clinical diagnosis and treatment of hemangioma.
In this study, we use gene interference technique to reduce the expression of VEGF gene in the animal model of hemangioma and the vascular endothelial cells of infantile hemangioma through the study of in vivo and in vitro, to observe the development and changes of the endothelial cells of hemangioma and hemangioma, and to explore the mechanism of proliferation and regression of hemangioma, and lay the foundation for further clinical application. Theoretical basis.
Establishment of 1. model of hemangioma transplantation in nude mice
Objective: to establish a nude mouse model of human capillary hemangioma and to explore the optimum conditions for the establishment of a nude mouse model of hemangioma. Methods: the tissue block was made by the surgical resection of the oestrogen receptor positive hyperplastic hemangioma in children, and 20 nude mice (BALB/c nude mice) were implanted subcutaneously, each of the 20 nude mice was divided into 4 experimental groups. The 1 groups were moved to the experimental group. The 2 groups in the experimental group were injected estradiol 0.01 mg per week on the basis of 1 groups, and the 3 groups were injected estradiol 0.1 mg per week on the basis of the 1 groups, and the 4 groups were injected with estradiol 1mg per week on the basis of 1 groups. The transplanted tumor markers were examined by pathological light microscopy, and vascular endothelial cells were used for vascular endothelial cells. The monoclonal antibodies CD31, CD34, and Ki-67 were immunohistochemical staining. Results: after transplantation, the endothelial cells were denatured and necrotic at the early stage of the transplantation. After 30d, the 1 groups of experimental group and 2 groups of experimental groups began to absorb or form abscess and fibrosis. In the experimental 3,4 group, after the slow growth of.90d, the 1 groups of transplanted tumors were not transplanted. Survival, the experimental 4 groups of transplanted tumors survived, and all of the experimental 3 groups survived. The survival of the transplanted tumor was similar to that of the primary hemangioma. Conclusion: different doses of estrogen have a certain influence on the establishment of the transplanted model of hemangioma in nude mice. Proper use of estrogen can establish a stable transplantation of human hemangioma in nude mice. The model can be applied to basic and clinical studies of hemangioma.
Culture and identification of endothelial cells of 2. proliferative hemangioma
Objective: to cultivate and identify Hemangioma endothelial cell (HemEC) in vitro (HemEC). Methods: the tissue specimens of hyperplastic hemangioma were harvested under aseptic conditions and cultured with tissue block method. The M199 culture medium containing 20% fetal bovine serum was used to add Endothelial cell growth supplement, ECGS, and concentration. 100 g/ml) was cultured in a incubator containing 5%CO2,37. The cells were replaced every two to three days and the cells were spread through the bottom of the bottle. The cell morphology and growth were observed by optical microscopy. The cells expressed VIII related antigens and transmission electron microscopy were detected and identified by the immunohistochemical staining. The results were: the cells began to start from 3 days after inoculation. The edges are moved out and the cells are polygonal and mixed into slices for about 1 weeks. Some converts into an island shape. About 3 weeks can be filled with a bottle bottom and then digested. The passages are filled with a typical "cobblestone" arrangement. Cell VIII staining is positive. Transmission electron microscopy shows the cytoplasm of the cytoplasm containing ECs Weibel-Palade corpuscles (W-P corpuscles, Conclusion: tissue mass method can be used to obtain high purity hemangioma endothelial cells. This method is simple and easy to use.
Effect of 3.VEGF gene interference plasmid on hemangioma endothelial cells
Objective: To observe the effect of human vascular endothelial growth factor (VEGF) on the endothelial cells of human hyperplastic hemangioma in vitro, and to observe its effect on the secretion of VEGF and the endothelial cells of hemangioma. Method: transfection of eukaryotic expression plasmid pGCsi-U6NeoRFP-shVEGF to HemECs. by liposome method to observe plasmids by inverted fluorescence microscope. Transfection effect, the effect of shVEGF on HemECs was observed by MTT method and enzyme linked immunosorbent assay (ELISA). Results: the expression of VEGF in the transfected group decreased significantly compared with the control group, and the expression of VEGF in the transfected group was 141.3 + 7.82,92.34 + 5.71,68.07 + 5.95 and 40.65 + 6.71pg/ml in the other groups. Compared with P0.05, the transfection of VEGF plasmid reduced the expression of VEGF and promoted the apoptosis of the cells. Conclusion: transfection of VEGF interfering plasmids can obviously reduce the expression of VEGF in the endothelial cells of hemangioma, and also promote the apoptosis of the cells.
Effect of 4.VEGF interference Lentivirus on nude mice model of hemangioma transplantation
Objective: to make the VEGF lentivirus interference carrier, Lenti-siRNA-VEGF, to function in the model of hemangioma transplantation in nude mice, observe the changes of the growth and apoptosis of hemangioma, and discuss the new method of prevention and cure of hemangioma. Method: the VEGF interference plasmid is packaged into the Lenti-siRNA-VEGF. of the lentivirus vector to make the nude mouse hemangioma transplantation model, and Lenti-siRNA-VEGF The tumor body changes were observed and the tumor bodies were observed by HE, immunohistochemistry and TUNEL. The growth and decline of the tumor after the silence of VEGF were investigated by the Western-blot method. Results: the tumor volume began to become smaller after 1 weeks after the injection, and the tumor was narrowed and hardened after 1 months. There was no obvious change in the group with.HE detection. The occlusion of the lumen, the fibrotic.CD31 of the tumor, the number of positive cells detected by CD34 immunohistochemical detection were 7.68 + 0.83,10.25 + 1.29 (P0.05).TUNEL detection, respectively, and found that the apoptosis of the cells increased obviously than the control group.Western-blot found that the VEGF protein table of the experimental group decreased obviously. Conclusion: Lenti-siRNA-VEGF's effect on migration The implant model can significantly reduce the expression of VEGF and promote the apoptosis of the endothelial cells of the tumor. This method provides a new method for further experimental and clinical study of hemangioma.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5

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