重组人内抑素对兔耳增生性瘢痕成纤维细胞凋亡的作用及机制研究

发布时间：2018-05-31 22:46

本文选题：重组人Qg抑素 + 增生性瘢痕　；参考：《安徽医科大学》2017年硕士论文

【摘要】：增生性瘢痕(hypertrophic scar,HS)是一种皮肤软组织损伤之后机体过度修复导致的皮肤纤维化疾病,手术后的发病率可达40%-70%,而烧伤后的发病率更高达91%。HS主要的临床表现为损伤局部组织凸于正常皮肤的异常增生,并伴有疼痛、瘙痒等机体感觉不适,同时给患者带来局部组织外观和功能上的改变,如局部关节部位的瘢痕性挛缩等,严重影响了患者的身心健康。其确切的发病原因和机制目前尚未完全明确,临床上也缺乏特异性的治疗措施,而单纯的手术切除会产生新的瘢痕。增生性瘢痕成纤维细胞(hypertrophic scar fibroblasts,HSFs)是HS发生和发展过程中的主要效应细胞,且HSFs增殖和凋亡之间的不平衡也是临床上HS过度增生和持续存在的重要原因,因而抑制HSFs增殖和(或)促进HSFs凋亡可以成为临床上治疗HS的一个重要突破口。内抑素(endostatin)是O’Reilly等人从小鼠血管内皮瘤细胞的培养上清液中分离纯化得到的一种蛋白质,共184个氨基酸残基。先前的研究发现内抑素可特异性抑制血管内皮细胞增殖,通过进一步的研究证实,内抑素可以直接抑制部分肿瘤细胞增殖、迁移并诱导凋亡从而发挥抗肿瘤效应。我们早期的研究证实重组人内抑素(recombinant human endostatin,rh Endostatin)可特异性抑制佐剂性关节炎大鼠成纤维样滑膜细胞增殖并诱导其凋亡;Ren等证明Qg抑素可以通过降低瘢痕组织中bcl-2的表达和抑制瘢痕内新生血管的形成来抑制瘢痕的增生。本课题组前期的研究发现rh Endostatin(6.25,12.5,25,50,100μg/ml)可以直接抑制HSFs增殖,本实验正是在本课题组前期研究的基础上进一步探讨rh Endostatin(100μg/ml)对兔耳HSFs凋亡的作用并探讨其中凋亡相应的分子机制,从而为HS的临床治疗以及寻找药物治疗新靶点提供实验基础和理论依据。目的:观察rh Endostatin对HSFs凋亡的影响,探讨其作用的部分细胞与分子机制。方法:(1)HS模型制备与鉴定:新西兰大耳兔8只,分为正常组(2只)和HS模型组(6只),模型组大耳兔建立兔耳HS模型;术后第28天,瘢痕形成;随机取两只兔耳瘢痕组织和正常兔耳皮肤行组织学观察,鉴定HS。(2)HSFs分离培养和鉴定:采用组织块培养法培养细胞,取第3代(传2代)细胞行波形蛋白免疫细胞化学染色鉴定HSFs。后续实验采用第3代细胞。(3)HSFs凋亡情况检测:将模型组细胞分为未处理组、Qg抑素处理组(100μg/ml)和5-氟尿嘧啶处理组(500μg/ml),流式细胞仪(FCM)对细胞凋亡情况进行检测。(4)HSFs凋亡相关基因与蛋白检测:实时荧光定量PCR法检测c-fos,c-jun,NF-κB,fas,caspase-3,bcl-2和p53 m RNA;Western Blot法检测相应蛋白表达。(5)HSFs胞质内的Ca~(2+)检测:Fluo-4/AM作为Ca~(2+)指示剂,其终浓度为100mg/ml rh Endostatin灌流HSFs,同时用激光共聚焦显微镜(confocal laser scanning microscope,CLSM)动态观察和记录HSFs在有无Ca~(2+)液时胞质中的Ca~(2+)荧光强度(fluorescence intensity,FI)变化,检测rh Endostatin对HSFs胞质Ca~(2+)浓度(cytosolic free calcium concentration)[Ca~(2+)]i的影响。结果:(1)术后28天,HS即形成。HE染色结果显示瘢痕组织较周围正常皮肤明显增厚,同时瘢痕组织内的胶原纤维也明显变粗变大,呈现出旋涡或结节状,形成胶原结节;同时伴有大量的成纤维细胞增殖以及小血管增生。(2)波形蛋白免疫细胞化学染色对第3代(传2代)细胞进行鉴定的结果显示:细胞胞质内含有大量的棕黄色颗粒,呈现成纤维细胞特性。(3)FCM分析结果显示,rh Endostatin(100μg/ml)可促进HSFs早期及晚期凋亡,且以早期凋亡更为显著,分别为(10.78±0.06)%、(2.12±0.04)%,差异较未处理对照组HSFs均具有统计学意义(P0.01)。(4)实时荧光定量PCR及Western Blot分析结果显示,与未处理对照组HSFs相比较,rh Endostatin(100μg/ml)能明显降低HSFs c-jun,c-fos,NF-κB,fas,p53,caspase-3和bcl-2 m RNA和蛋白的表达水平(P0.01)。(5)CLSM检测结果显示,当细胞处于无Ca~(2+)液(D-Hanks缓冲液)中时,rh Endostatin(100mg/ml)不能引起HSFs胞质内Ca~(2+)FI的改变。当细胞外缓冲液为有Ca~(2+)液(Hanks缓冲液)时,终浓度为100mg/ml rh Endostatin灌流HSFs可使胞质Ca~(2+)FI急剧增加达峰值,随即荧光开始减弱,FI随时间而缓慢下降。结论:(1)rh Endostatin可促进HSFs凋亡。(2)rh Endostatin促进HSFs凋亡与其降低c-fos,c-jun,NF-κB和bcl-2基因的表达与活化有关,同时该凋亡过程不依赖于fas,p53表达增加,也不被caspase-3活性降低所抑制。(3)rh Endostatin可引起HSFs胞外Ca~(2+)内流,细胞内钙超载可能作为HSFs凋亡的一个重要始动环节。
[Abstract]:Hypertrophic scar (HS) is a kind of skin fibrosis disease caused by excessive repair of soft tissue after skin soft tissue injury. The incidence of after operation is up to 40%-70%. The incidence of post burn is higher than that of 91%.HS. The main clinical manifestation of 91%.HS is the abnormal hyperplasia of local tissue, accompanied by pain, itching, and so on. The body feel discomfort, and bring the changes of the local tissue appearance and function, such as the scar contracture of the local joint, which seriously affect the physical and mental health of the patients. The exact cause and mechanism of the disease are not completely clear at present, and the specific treatment measures are lacking in clinical. The scar. Hypertrophic scar fibroblasts (hypertrophic scar fibroblasts, HSFs) are the main effector cells in the development and development of HS, and the imbalance between HSFs proliferation and apoptosis is also an important reason for the clinical HS hyperproliferation and persistence, thus inhibiting the proliferation of HSFs and / or promoting HSFs apoptosis can be a clinical treatment. An important breakthrough for the treatment of HS. Endostatin is a protein isolated and purified by O 'Reilly and others from the culture supernatant of mouse hemangioendothelioma cells. A total of 184 amino acid residues were found. Previous studies have found that endostatin inhibits vascular endothelial cell proliferation. Further studies have confirmed that endostatin is possible. Our early study confirmed that recombinant human endostatin (recombinant human endostatin, Rh Endostatin) can specifically inhibit the proliferation of fibroblast like synovial cells in adjuvant arthritis rats and induce apoptosis, and Ren etc. prove that Qg inhibits can be used. To reduce the expression of Bcl-2 in scar tissue and inhibit the formation of neovascularization in scar tissue to inhibit the proliferation of scar. The previous study in our group found that RH Endostatin (6.25,12.5,25,50100 mu g/ml) could directly inhibit the proliferation of HSFs. This experiment is the further study of RH Endostatin (100 micron g/ml) on the basis of previous research in this group. The effect of HSFs apoptosis in rabbit ear and the molecular mechanism of apoptosis, which can provide experimental basis and theoretical basis for the clinical treatment of HS and the new target of drug treatment. Objective: To observe the effect of RH Endostatin on the apoptosis of HSFs and to explore the mechanism of partial cell and subdivision of its action. Method: (1) HS model preparation and identification: New West 8 rabbits were divided into normal group (2) and HS model group (6 rats). The rabbit model of model group was established by HS model of rabbit ear. The scar formation was formed at twenty-eighth days after operation. Two rabbit ears scar tissue and normal rabbit ear skin were randomly selected to identify HS. (2) HSFs isolation and culture and identification: tissue culture method was used to culture cells and third generation (2 generation) cells were taken. Third generations of HSFs. cells were used in the subsequent experiment of vimentin immunocytochemical staining. (3) detection of HSFs apoptosis: the model group cells were divided into untreated group, Qg statin treatment group (100 mu g/ml) and 5- fluorouracil treatment group (500 mu g/ml), and flow cytometry (FCM) was used to detect the cell apoptosis. (4) apoptosis related genes and proteins of HSFs Detection: real-time fluorescence quantitative PCR method was used to detect c-fos, c-jun, NF- kappa B, Fas, Caspase-3, Bcl-2 and p53 m RNA. (5) detection of the corresponding protein in the cytoplasm. Microscope, CLSM) dynamically observed and recorded the changes of the Ca~ (2+) fluorescence intensity (fluorescence intensity, FI) in the cytoplasm of the Ca~ (2+) liquid when there was no Ca~ (2+). Results: (1) 28 days after the operation, the staining results showed scar tissue. The thickness of the normal skin was thicker than the surrounding normal skin, and the collagen fibers in the scar tissue became thicker and larger, showing a whirlpool or nodular form, forming a collagen nodule, and accompanied by a large number of fibroblast proliferation and small vascular proliferation. (2) the results of the identification of the third generation (2 generation) cells by vimentin immunocytochemical staining showed that: Cytoplasm contained a large number of brown and yellow granules and showed fibroblast properties. (3) FCM analysis showed that RH Endostatin (100 g/ml) could promote the early and late apoptosis of HSFs, and the early apoptosis was more significant, (10.78 + 0.06)%, (2.12 + 0.04)% respectively. The difference was statistically significant (P0.01). (4) real-time fluorescence (4). The results of PCR and Western Blot analysis showed that compared with the untreated control group HSFs, Rh Endostatin (100 mu g/ml) could obviously reduce HSFs c-Jun, c-fos, NF- kappa B. (100mg/ml) can not cause the change of Ca~ (2+) FI in the cytoplasm of HSFs. When the extracellular buffer is Ca~ (2+) solution (Hanks buffer), the final concentration of 100mg/ml RH Endostatin perfusion HSFs can increase the peak value rapidly, then the fluorescence begins to weaken and decreases with time. Conclusion: (1) (2) Endostatin promotes HSFs apoptosis and reduces the expression and activation of c-fos, c-jun, NF- kappa B and Bcl-2 genes, and the apoptosis process is not dependent on Fas, p53 expression is increased, and caspase-3 activity is not inhibited. (3) RH Endostatin can cause extracellular flow. Intracellular calcium overload may be an important initiation of apoptosis. Link.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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