本文關(guān)鍵詞： 增生性瘢痕 siRNA 結(jié)締組織生長因子 基因載體 殼聚糖 納米粒　出處：《第二軍醫(yī)大學》2017年博士論文　論文類型：學位論文

【摘要】：研究背景增生性瘢痕往往是皮膚在遭受創(chuàng)傷或重度燒傷后難以避免的愈合結(jié)果。瘢痕繼續(xù)發(fā)展還會導致皮膚瘙癢、活動受限、外觀畸形以及攣縮等諸多問題,給患者生理和心理兩方面均造成不良影響。據(jù)統(tǒng)計,每年有數(shù)百萬人遭受瘢痕帶來的身心傷害,生活質(zhì)量很差。由于傳統(tǒng)治療手段難以產(chǎn)生滿意的效果,因此迫切需要尋找一種新的治療方式來防治增生性瘢痕。1991年Bradham等首先在人臍靜脈內(nèi)皮細胞培養(yǎng)基中發(fā)現(xiàn)人類CTGF。CTGF作為一類基質(zhì)細胞蛋白,主要表達于成纖維細胞、肝星狀細胞、軟骨細胞等間質(zhì)細胞,在機體生長發(fā)育及損傷時表達相應(yīng)增加。CTGF病理生理功能主要是刺激細胞有絲分裂、粘附、凋亡、ECM合成分泌以及促進其他類型細胞的遷移,同時它還能夠改變其他分子的活性。最近,越來越多文獻證實CTGF在病理性瘢痕真皮層中過量表達,但是其參與瘢痕形成的具體機制仍不十分清楚。干擾(interference RNA)RNA是一種短雙鏈RNA,作為一種基因沉默技術(shù),其可以與m RNA互補配對并促使其降解,最終發(fā)揮特異性基因阻斷作用。si RNA作為RNAi技術(shù)的一種,相比反義核酸技術(shù),具備基因抑制率高、特異性強以及濃度低等優(yōu)勢,在基因功能研究領(lǐng)域已得到廣泛應(yīng)用。然而,由于體內(nèi)外環(huán)境的復(fù)雜性,諸如核酸酶等酶類的存在,本身就不穩(wěn)定的裸si RNA在進入細胞和胞核前極易被降解,因此總體轉(zhuǎn)染效率不高。之后雖然化學修飾或病毒載體等一些改進措施可以延緩其降解,但又帶來細胞毒性大、效果差等缺點。因而,急需尋找一種更理想的轉(zhuǎn)染載體來介導si RNA進入細胞并發(fā)揮基因阻斷效應(yīng)。殼聚糖是一種非病毒載體,具有細胞毒性低、免疫原性低、降解性好和生物相容性良好等優(yōu)點。此外,作為唯一一種天然陽離子材料,殼聚糖還可以與帶負電荷的核酸如DNA、si RNA以及mi RNA等結(jié)合形成納米復(fù)合物。殼聚糖納米載體主要通過增進與帶負電細胞膜的粘附和避免si RNA被內(nèi)源性核酸酶降解兩個方面來提高si RNA的基因干擾效率。近年來,殼聚糖納米粒作為一種安全、經(jīng)濟的非病毒載體,已經(jīng)獲得了越來越多的關(guān)注�；谝陨媳尘�,本研究通過將載siCTGF殼聚糖納米粒應(yīng)用于新愈合兔耳皮膚創(chuàng)面,通過靶向干擾CTGF的表達及其生物學功能,進而抑制成纖維細胞過度增殖、分化以及減少膠原蛋白合成沉積,最終發(fā)揮減輕瘢痕形成的作用。載siCTGF殼聚糖納米粒有望成為一種安全、有效的瘢痕防治技術(shù)。第一部分構(gòu)建siCTGF以及篩選最佳干擾序列研究目的:分離培養(yǎng)及鑒定原代瘢痕成纖維細胞;確認構(gòu)建序列的有效性并篩選最佳干擾序列。研究方法:采用組織塊貼壁和胰蛋白酶聯(lián)合消化的方法,分離培養(yǎng)瘢痕成纖維細胞,并采用流式細胞術(shù)鑒定瘢痕成纖維細胞。參照si RNA設(shè)計原則,應(yīng)用RNA在線設(shè)計軟件,設(shè)計3段RNA干擾序列,分別轉(zhuǎn)染瘢痕成纖維細胞,并以無同源性的亂碼si RNA作為對照組,應(yīng)用RT-PCR、蛋白印跡等方法檢測各組細胞CTGF m RNA及蛋白的表達,比較分析后選出沉默效率最大的一組siCTGF。研究結(jié)果:該改進的方法能夠快速分離出高活性的成纖維細胞,流式細胞儀得到FSP陽性率可達94.37±1.31%。該合成方法所構(gòu)建的si RNA能成功轉(zhuǎn)染成纖維細胞。與未轉(zhuǎn)染組和亂碼組相比,候選序列3的基因抑制效率最高,CTGF m RNA表達降低到0.294±0.093倍,CTGF蛋白表達降低到0.33±0.065倍,均有統(tǒng)計學差異(P0.05)。研究結(jié)論:干擾序列3的成纖維細胞CTGF基因沉默效率最高。第二部分載siCTGF殼聚糖納米粒的制備及相關(guān)特性的研究研究目的:探討載siCTGF殼聚糖納米粒制備方法及其相關(guān)物理特征。研究方法:采用經(jīng)改進的離子膠凝法,通過對分子量、氮磷比等條件進行摸索來制備載siCTGF殼聚糖納米粒。粒徑分析儀和透射電鏡分別檢測納米粒平均粒徑和電位,紫外分光光度計計算載藥率,同時對其在體外的控釋周期以及細胞毒性實驗進行測定,最后完成納米粒在細胞內(nèi)的示蹤。研究結(jié)果:投射電鏡圖像顯示殼聚糖納米粒呈圓形顆粒,大小一致,分布均勻,平均粒徑在98.3±2.7nm左右。粒徑分析儀測得zeta電位為15.3±4.2m V。siCTGF的包封率為96.5±2.4%。在PBS溶液中體外控釋周期最長可達6天。CCK8細胞毒性實驗提示空白對照組與陰性對照組相比,各濃度殼聚糖納米粒組未見明顯細胞毒性,無統(tǒng)計學差異(P0.05)。研究結(jié)論:本方法制備的殼聚糖納米粒具備粒徑小、細胞毒性低以及體外控釋周期長等特點,是一種較好的基因轉(zhuǎn)染載體。第三部分體外實驗評價載siCTGF殼聚糖納米粒對瘢痕成纖維細胞的作用研究目的:評價體外應(yīng)用載siCTGF殼聚糖納米粒對成纖維細胞纖維化基因表達的抑制效果。研究方法:實驗按照空白對照組、載亂碼RNA殼聚糖納米粒組、三種濃度載siCTGF殼聚糖納米粒組(50nmol/ml,100nmol/ml,200nmol/ml)分成5個組。選用CCK8試驗方法比較各組對瘢痕成纖維細胞增殖效率的影響;RT-PCR、Western-blot等技術(shù)測定目標基因CTGF、細胞外基質(zhì)成分Ⅰ型膠原以及細胞分化標志α-SMA的表達。研究結(jié)果:CCK8法結(jié)果顯示相比空白組和陰性對照組,各濃度載siCTGF殼聚糖納米粒組細胞增殖得到不同程度抑制,均存在統(tǒng)計學差異(P0.05)。RT-PCR、Western-blot等技術(shù)測定目標基因CTGF、細胞外基質(zhì)成分Ⅰ型膠原以及細胞分化標志α-SMA表達,結(jié)果顯示載siCTGF殼聚糖納米粒組3種基因的表達水平均明顯低于空白組和陰性對照組(P0.05),又以200nmol/ml載siCTGF殼聚糖納米粒組下降程度最高。研究結(jié)論:體外應(yīng)用載siCTGF殼聚糖納米粒能有效抑制成纖維細胞增殖,Ⅰ型膠原蛋白合成以及向肌成纖維細胞轉(zhuǎn)分化。第四部分局部應(yīng)用載siCTGF殼聚糖納米粒對兔耳瘢痕增生的影響研究目的:評價局部應(yīng)用載siCTGF殼聚糖納米粒對瘢痕增生的抑制效果。研究方法:實驗動物按照空白對照組、載亂碼RNA殼聚糖納米粒組,三種濃度載siCTGF殼聚糖納米粒組分成5組。采用活體成像儀監(jiān)測納米粒在裸鼠體內(nèi)的控釋周期;建立兔耳瘢痕模型,局部注射不同濃度載siCTGF殼聚糖納米粒,肉眼觀察局部創(chuàng)面瘢痕增生的大體圖像,并超聲探測儀動態(tài)監(jiān)測治療過程中瘢痕厚度的變化。采用免疫組織化學染色法檢測局部真皮內(nèi)CTGF的表達分布,膠原合成(Ⅰ型膠原)以及向肌成纖維細胞轉(zhuǎn)分化(α-SMA)的情況差異,并采用Masson三色染色法觀察真皮內(nèi)膠原的堆積及排列情況。同時,提取皮膚組織蛋白,采用Western-blot技術(shù)檢測各組皮膚內(nèi)CTGF、Ⅰ型膠原、α-SMA三種蛋白的表達差異。研究結(jié)果:納米粒組裸鼠創(chuàng)面可觀察到紅色熒光并持續(xù)到第5天。大體圖像可見第2周創(chuàng)面基本愈合,之后開始向外增生突起,到第6周增生達到頂峰,B型超聲結(jié)果顯示相比空白對照組和載亂碼RNA殼聚糖納米粒組,三種濃度載siCTGF殼聚糖納米粒組超聲測量值(瘢痕厚度)呈現(xiàn)不同程度下降,有統(tǒng)計學差異(P0.05)。組織學結(jié)果顯示治療組真皮內(nèi)CTGF蛋白表達減少,而其他纖維化相關(guān)因子表達也有不同程度下降,且Masson染色顯示載siCTGF殼聚糖納米粒組膠原堆積減少且排列較規(guī)則。研究結(jié)論:載siCTGF殼聚糖納米粒能有效減輕兔耳瘢痕增生,在增生性瘢痕防治中具有較大的應(yīng)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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R622

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