载siCTGF壳聚糖纳米粒在兔耳增生性瘢痕中的应用研究

发布时间：2018-01-21 11:13

本文关键词： 增生性瘢痕 siRNA 结缔组织生长因子基因载体壳聚糖纳米粒　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景增生性瘢痕往往是皮肤在遭受创伤或重度烧伤后难以避免的愈合结果。瘢痕继续发展还会导致皮肤瘙痒、活动受限、外观畸形以及挛缩等诸多问题,给患者生理和心理两方面均造成不良影响。据统计,每年有数百万人遭受瘢痕带来的身心伤害,生活质量很差。由于传统治疗手段难以产生满意的效果,因此迫切需要寻找一种新的治疗方式来防治增生性瘢痕。1991年Bradham等首先在人脐静脉内皮细胞培养基中发现人类CTGF。CTGF作为一类基质细胞蛋白,主要表达于成纤维细胞、肝星状细胞、软骨细胞等间质细胞,在机体生长发育及损伤时表达相应增加。CTGF病理生理功能主要是刺激细胞有丝分裂、粘附、凋亡、ECM合成分泌以及促进其他类型细胞的迁移,同时它还能够改变其他分子的活性。最近,越来越多文献证实CTGF在病理性瘢痕真皮层中过量表达,但是其参与瘢痕形成的具体机制仍不十分清楚。干扰(interference RNA)RNA是一种短双链RNA,作为一种基因沉默技术,其可以与m RNA互补配对并促使其降解,最终发挥特异性基因阻断作用。si RNA作为RNAi技术的一种,相比反义核酸技术,具备基因抑制率高、特异性强以及浓度低等优势,在基因功能研究领域已得到广泛应用。然而,由于体内外环境的复杂性,诸如核酸酶等酶类的存在,本身就不稳定的裸si RNA在进入细胞和胞核前极易被降解,因此总体转染效率不高。之后虽然化学修饰或病毒载体等一些改进措施可以延缓其降解,但又带来细胞毒性大、效果差等缺点。因而,急需寻找一种更理想的转染载体来介导si RNA进入细胞并发挥基因阻断效应。壳聚糖是一种非病毒载体,具有细胞毒性低、免疫原性低、降解性好和生物相容性良好等优点。此外,作为唯一一种天然阳离子材料,壳聚糖还可以与带负电荷的核酸如DNA、si RNA以及mi RNA等结合形成纳米复合物。壳聚糖纳米载体主要通过增进与带负电细胞膜的粘附和避免si RNA被内源性核酸酶降解两个方面来提高si RNA的基因干扰效率。近年来,壳聚糖纳米粒作为一种安全、经济的非病毒载体,已经获得了越来越多的关注。基于以上背景,本研究通过将载siCTGF壳聚糖纳米粒应用于新愈合兔耳皮肤创面,通过靶向干扰CTGF的表达及其生物学功能,进而抑制成纤维细胞过度增殖、分化以及减少胶原蛋白合成沉积,最终发挥减轻瘢痕形成的作用。载siCTGF壳聚糖纳米粒有望成为一种安全、有效的瘢痕防治技术。第一部分构建siCTGF以及筛选最佳干扰序列研究目的:分离培养及鉴定原代瘢痕成纤维细胞;确认构建序列的有效性并筛选最佳干扰序列。研究方法:采用组织块贴壁和胰蛋白酶联合消化的方法,分离培养瘢痕成纤维细胞,并采用流式细胞术鉴定瘢痕成纤维细胞。参照si RNA设计原则,应用RNA在线设计软件,设计3段RNA干扰序列,分别转染瘢痕成纤维细胞,并以无同源性的乱码si RNA作为对照组,应用RT-PCR、蛋白印迹等方法检测各组细胞CTGF m RNA及蛋白的表达,比较分析后选出沉默效率最大的一组siCTGF。研究结果:该改进的方法能够快速分离出高活性的成纤维细胞,流式细胞仪得到FSP阳性率可达94.37±1.31%。该合成方法所构建的si RNA能成功转染成纤维细胞。与未转染组和乱码组相比,候选序列3的基因抑制效率最高,CTGF m RNA表达降低到0.294±0.093倍,CTGF蛋白表达降低到0.33±0.065倍,均有统计学差异(P0.05)。研究结论:干扰序列3的成纤维细胞CTGF基因沉默效率最高。第二部分载siCTGF壳聚糖纳米粒的制备及相关特性的研究研究目的:探讨载siCTGF壳聚糖纳米粒制备方法及其相关物理特征。研究方法:采用经改进的离子胶凝法,通过对分子量、氮磷比等条件进行摸索来制备载siCTGF壳聚糖纳米粒。粒径分析仪和透射电镜分别检测纳米粒平均粒径和电位,紫外分光光度计计算载药率,同时对其在体外的控释周期以及细胞毒性实验进行测定,最后完成纳米粒在细胞内的示踪。研究结果:投射电镜图像显示壳聚糖纳米粒呈圆形颗粒,大小一致,分布均匀,平均粒径在98.3±2.7nm左右。粒径分析仪测得zeta电位为15.3±4.2m V。siCTGF的包封率为96.5±2.4%。在PBS溶液中体外控释周期最长可达6天。CCK8细胞毒性实验提示空白对照组与阴性对照组相比,各浓度壳聚糖纳米粒组未见明显细胞毒性,无统计学差异(P0.05)。研究结论:本方法制备的壳聚糖纳米粒具备粒径小、细胞毒性低以及体外控释周期长等特点,是一种较好的基因转染载体。第三部分体外实验评价载siCTGF壳聚糖纳米粒对瘢痕成纤维细胞的作用研究目的:评价体外应用载siCTGF壳聚糖纳米粒对成纤维细胞纤维化基因表达的抑制效果。研究方法:实验按照空白对照组、载乱码RNA壳聚糖纳米粒组、三种浓度载siCTGF壳聚糖纳米粒组(50nmol/ml,100nmol/ml,200nmol/ml)分成5个组。选用CCK8试验方法比较各组对瘢痕成纤维细胞增殖效率的影响;RT-PCR、Western-blot等技术测定目标基因CTGF、细胞外基质成分Ⅰ型胶原以及细胞分化标志α-SMA的表达。研究结果:CCK8法结果显示相比空白组和阴性对照组,各浓度载siCTGF壳聚糖纳米粒组细胞增殖得到不同程度抑制,均存在统计学差异(P0.05)。RT-PCR、Western-blot等技术测定目标基因CTGF、细胞外基质成分Ⅰ型胶原以及细胞分化标志α-SMA表达,结果显示载siCTGF壳聚糖纳米粒组3种基因的表达水平均明显低于空白组和阴性对照组(P0.05),又以200nmol/ml载siCTGF壳聚糖纳米粒组下降程度最高。研究结论:体外应用载siCTGF壳聚糖纳米粒能有效抑制成纤维细胞增殖,Ⅰ型胶原蛋白合成以及向肌成纤维细胞转分化。第四部分局部应用载siCTGF壳聚糖纳米粒对兔耳瘢痕增生的影响研究目的:评价局部应用载siCTGF壳聚糖纳米粒对瘢痕增生的抑制效果。研究方法:实验动物按照空白对照组、载乱码RNA壳聚糖纳米粒组,三种浓度载siCTGF壳聚糖纳米粒组分成5组。采用活体成像仪监测纳米粒在裸鼠体内的控释周期;建立兔耳瘢痕模型,局部注射不同浓度载siCTGF壳聚糖纳米粒,肉眼观察局部创面瘢痕增生的大体图像,并超声探测仪动态监测治疗过程中瘢痕厚度的变化。采用免疫组织化学染色法检测局部真皮内CTGF的表达分布,胶原合成(Ⅰ型胶原)以及向肌成纤维细胞转分化(α-SMA)的情况差异,并采用Masson三色染色法观察真皮内胶原的堆积及排列情况。同时,提取皮肤组织蛋白,采用Western-blot技术检测各组皮肤内CTGF、Ⅰ型胶原、α-SMA三种蛋白的表达差异。研究结果:纳米粒组裸鼠创面可观察到红色荧光并持续到第5天。大体图像可见第2周创面基本愈合,之后开始向外增生突起,到第6周增生达到顶峰,B型超声结果显示相比空白对照组和载乱码RNA壳聚糖纳米粒组,三种浓度载siCTGF壳聚糖纳米粒组超声测量值(瘢痕厚度)呈现不同程度下降,有统计学差异(P0.05)。组织学结果显示治疗组真皮内CTGF蛋白表达减少,而其他纤维化相关因子表达也有不同程度下降,且Masson染色显示载siCTGF壳聚糖纳米粒组胶原堆积减少且排列较规则。研究结论:载siCTGF壳聚糖纳米粒能有效减轻兔耳瘢痕增生,在增生性瘢痕防治中具有较大的应用前景。
[Abstract]:The research background of hypertrophic scar is often difficult to avoid the skin healing in trauma or severe burn scar. Continue to develop will cause skin itching, activity limitation, many problems and contracture deformity, two patients with physiological and psychological adverse effects. According to statistics, there are millions of people suffering from physical and psychological harm caused by scar every year, the quality of life is poor. The traditional treatment is difficult to produce satisfactory results, so there is an urgent need to find a new way to prevention and treatment of hypertrophic scar.1991 Bradham culture medium firstly found in human CTGF.CTGF stromal cells as a kind of protein in human umbilical vein endothelial cells, mainly expressed in fibroblasts, liver stellate cells, cartilage cells and stromal cells, the corresponding increase in the pathophysiology of.CTGF function in body growth and injury is mainly to stimulate cell expression Mitosis, cell adhesion, apoptosis, ECM synthesis and secretion and promote the migration of other cell types, at the same time it can also change other molecular activity. Recently, more and more literatures demonstrate that overexpression of CTGF in pathological scar in the dermis, but its involvement in the specific mechanism of scar formation is still unclear. Interference (interference RNA) RNA is a short double stranded RNA as a gene silencing technique, which can be m and RNA complement and promote its degradation, eventually play specific gene blocking.Si RNA as a RNAi technology, compared with the antisense nucleic acid technique, gene inhibiting rate, specificity and concentration has advantages. Widely used in the field of gene function research. However, due to the complexity of the in vivo environment, such as nuclease enzymes, itself is not stable in RNA bare Si into the cell nucleus before and can easily be reduced The solution, so the overall transfection efficiency is not high. Although after chemical modification or viral vectors and some improvement measures can delay the degradation, but also bring cell toxicity, disadvantages of poor effect. Therefore, the urgent need to find a more ideal transfection vector mediated Si RNA gene into cells and play blocking effect of chitosan is. A non viral vector with low cytotoxicity, low immunogenicity, good compatibility with the advantages of good biodegradability and biology. In addition, only as a natural cationic material, chitosan can also negatively charged nucleic acids such as DNA, RNA and Mi combined with Si RNA to form a nanocomposite chitosan. The nano carrier mainly through the promotion and adhesion of negatively charged membrane and avoid Si RNA by two aspects of endogenous nuclease degradation to improve the efficiency of Si RNA gene interference. In recent years, chitosan nanoparticles as a safe and economical Non viral vectors, has gained more and more attention. Based on the above background, this study of the siCTGF loaded chitosan nanoparticles used in the new ear skin wound healing of rabbits, by targeting CTGF expression and its biological function, and inhibit the proliferation of fibroblasts, differentiation and reduced synthesis of collagen deposition, eventually reduce scar play the role of the formation of. SiCTGF loaded chitosan nanoparticles is expected to become a safe and effective technique for treating scars. The first part constructs siCTGF and Study on the selection of the best interference Objective: isolation culture and identification of primary fibroblasts; confirm the effectiveness of the construction sequence and select the optimum interference sequence. Methods: using the method of tissue explants the wall and trypsin digestion, scar fibroblasts were isolated and cultured by flow cytometry, and identification of scar fibroblasts by Si. RNA design principles, application of RNA online design software, design 3 RNA interference sequences were transfected into fibroblasts, and no homologous garbled Si of RNA as control group, using RT-PCR, Western blotting was used to detect the expression of CTGF cells m RNA and protein, comparative analysis to choose a set of siCTGF. results silence: the maximum efficiency of the improved method can quickly isolate fibroblasts with high activity, Si RNA positive rate of FSP was 94.37 + 1.31%. the synthesis method constructed by flow cytometry can be successfully transfected into fibroblasts. Compared with untransfected group and garbled group, gene candidate sequences 3 of the highest inhibition efficiency the expression of CTGF, m RNA decreased to 0.294 + 0.093 times, the expression of CTGF protein decreased to 0.33 + 0.065 times, were statistically significant (P0.05). Conclusion: 3 the fibroblast interference sequence CTGF gene silencing efficiency is the highest. The two part of the study of preparation and related properties of siCTGF loaded chitosan nanoparticles: To investigate siCTGF loaded chitosan nanoparticles preparation method and its related physical characteristics. Methods: using the ion gelation method improved, the molecular weight, ratio of nitrogen to phosphorus conditions were groping to the preparation of siCTGF loaded chitosan nanoparticles. The particle size analyzer and transmission electron microscopy were used to detect the average particle size of nanoparticles and the calculation of potential, drug loading rate, UV spectrophotometry, and the in vitro release cycle and cell toxicity test were determined, finally completed nanoparticles in cells. Tracer results: transmission electron microscopy images show that chitosan nanoparticles were round particles, uniform size, uniform distribution, the average particle size of 98.3 + 2.7nm. The entrapment efficiency of particle size analyzer measured zeta potential was 15.3 + 4.2m V.siCTGF 96.5 + 2.4%. in PBS solution in vitro release The longest period of up to 6 days of.CCK8 cell toxicity test showed that compared with the blank control group and negative control group, the concentration of no shell chitosan nanoparticles group obvious cytotoxicity, no significant difference (P0.05). Conclusion: the method for preparation of chitosan nanoparticles with small particle size, low cytotoxicity and in vitro release characteristics of long cycle control that is a good gene transfection vector. The third part is to evaluate the in vitro siCTGF loaded chitosan nanoparticles to the effect of fiber cells on scar: To evaluate the application of siCTGF loaded chitosan nanoparticles in vitro inhibitory effect on expression of fibroblast fibrosis gene. Methods: according to the experimental control group, RNA loaded chitosan garbled three kinds of nanoparticles group, the concentration of siCTGF loaded chitosan nanoparticles group (50nmol/ml, 100nmol/ml, 200nmol/ml) are divided into 5 groups. Compared to the scar fibroblasts by CCK8 test method Effect of cell proliferation efficiency; RT-PCR, determination of target gene CTGF Western-blot, extracellular matrix collagen and cell differentiation marker expression of alpha -SMA. Results: CCK8 results showed that compared with the blank control group and negative control group, the concentration of siCTGF loaded chitosan nanoparticles group cell proliferation inhibited both there was significant difference (P0.05.RT-PCR), determination of target gene of CTGF Western-blot, extracellular matrix collagen and cell differentiation markers alpha -SMA expression revealed that the expression level of siCTGF loaded chitosan nanoparticles group of 3 genes were significantly lower than that of control group and negative control group (P0.05), and siCTGF loaded with 200nmol/ml the highest degree of chitosan nanoparticles was decreased. Conclusion: in vitro applications siCTGF loaded chitosan nanoparticles can effectively inhibit the proliferation of fibroblasts, collagen and eggs to Bai Hecheng Myofibroblast transdifferentiation. The fourth part of the topical application of siCTGF loaded chitosan nanoparticles on rabbit ear hypertrophic scar effect Objective: the inhibitory effect of topical application of siCTGF loaded chitosan nanoparticles for evaluation of scar proliferation. Methods: the experimental animal in the blank control group, RNA group of chitosan nanoparticles loaded garbage, three concentration of airborne siCTGF chitosan nanoparticles were divided into 5 groups. Using in vivo imaging to monitor the nanoparticles in vivo release cycle; establish rabbit ear scar model, local injection of different concentrations of siCTGF loaded chitosan nanoparticles, a general picture of hypertrophic scar wound eye, and the scar thickness of ultrasonic detector dynamic monitoring in the treatment of the distribution and expression changes. Immunohistochemical staining was used to detect the local dermal CTGF, collagen (collagen) and myofibroblast transdifferentiation (alpha -SMA). The difference, and the Masson staining method was used to observe the accumulation of collagen in the dermis and arrangement. At the same time, the extraction of skin tissue protein, using Western-blot technology to detect CTGF in the skin, collagen expression of alpha -SMA three proteins. Results: nanoparticles group can be observed in nude mice wound red fluorescence and continued up to fifth days the general image visible for second weeks. The wound healed, then began to proliferation processes, sixth weeks of proliferation peak, ultrasonic B display results compared with the blank control group and RNA group of chitosan nanoparticles loaded garbage, three concentrations of siCTGF loaded chitosan nanoparticles group ultrasonic measurements (scar thickness) showed varying degrees of decline, there was the difference (P0.05). The histological results showed that the treatment group in the dermis decreased CTGF protein expression and other fibrosis related gene expression decreased with different degree, and Masson staining showed that siC Conclusion: siCTGF chitosan nanoparticles can effectively reduce the scar formation of rabbit ears, and have great application prospects in the prevention and treatment of hypertrophic scars. Conclusion: chitosan nanoparticles can reduce the accumulation of collagen in TGF chitosan nanoparticles.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R622

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