本文選題：重組人Qg抑素 + 增生性瘢痕　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：增生性瘢痕(hypertrophic scar,HS)是一種皮膚軟組織損傷之后機(jī)體過(guò)度修復(fù)導(dǎo)致的皮膚纖維化疾病,手術(shù)后的發(fā)病率可達(dá)40%-70%,而燒傷后的發(fā)病率更高達(dá)91%。HS主要的臨床表現(xiàn)為損傷局部組織凸于正常皮膚的異常增生,并伴有疼痛、瘙癢等機(jī)體感覺不適,同時(shí)給患者帶來(lái)局部組織外觀和功能上的改變,如局部關(guān)節(jié)部位的瘢痕性攣縮等,嚴(yán)重影響了患者的身心健康。其確切的發(fā)病原因和機(jī)制目前尚未完全明確,臨床上也缺乏特異性的治療措施,而單純的手術(shù)切除會(huì)產(chǎn)生新的瘢痕。增生性瘢痕成纖維細(xì)胞(hypertrophic scar fibroblasts,HSFs)是HS發(fā)生和發(fā)展過(guò)程中的主要效應(yīng)細(xì)胞,且HSFs增殖和凋亡之間的不平衡也是臨床上HS過(guò)度增生和持續(xù)存在的重要原因,因而抑制HSFs增殖和(或)促進(jìn)HSFs凋亡可以成為臨床上治療HS的一個(gè)重要突破口。內(nèi)抑素(endostatin)是O’Reilly等人從小鼠血管內(nèi)皮瘤細(xì)胞的培養(yǎng)上清液中分離純化得到的一種蛋白質(zhì),共184個(gè)氨基酸殘基。先前的研究發(fā)現(xiàn)內(nèi)抑素可特異性抑制血管內(nèi)皮細(xì)胞增殖,通過(guò)進(jìn)一步的研究證實(shí),內(nèi)抑素可以直接抑制部分腫瘤細(xì)胞增殖、遷移并誘導(dǎo)凋亡從而發(fā)揮抗腫瘤效應(yīng)。我們?cè)缙诘难芯孔C實(shí)重組人內(nèi)抑素(recombinant human endostatin,rh Endostatin)可特異性抑制佐劑性關(guān)節(jié)炎大鼠成纖維樣滑膜細(xì)胞增殖并誘導(dǎo)其凋亡;Ren等證明Qg抑素可以通過(guò)降低瘢痕組織中bcl-2的表達(dá)和抑制瘢痕內(nèi)新生血管的形成來(lái)抑制瘢痕的增生。本課題組前期的研究發(fā)現(xiàn)rh Endostatin(6.25,12.5,25,50,100μg/ml)可以直接抑制HSFs增殖,本實(shí)驗(yàn)正是在本課題組前期研究的基礎(chǔ)上進(jìn)一步探討rh Endostatin(100μg/ml)對(duì)兔耳HSFs凋亡的作用并探討其中凋亡相應(yīng)的分子機(jī)制,從而為HS的臨床治療以及尋找藥物治療新靶點(diǎn)提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。目的:觀察rh Endostatin對(duì)HSFs凋亡的影響,探討其作用的部分細(xì)胞與分子機(jī)制。方法:(1)HS模型制備與鑒定:新西蘭大耳兔8只,分為正常組(2只)和HS模型組(6只),模型組大耳兔建立兔耳HS模型;術(shù)后第28天,瘢痕形成;隨機(jī)取兩只兔耳瘢痕組織和正常兔耳皮膚行組織學(xué)觀察,鑒定HS。(2)HSFs分離培養(yǎng)和鑒定:采用組織塊培養(yǎng)法培養(yǎng)細(xì)胞,取第3代(傳2代)細(xì)胞行波形蛋白免疫細(xì)胞化學(xué)染色鑒定HSFs。后續(xù)實(shí)驗(yàn)采用第3代細(xì)胞。(3)HSFs凋亡情況檢測(cè):將模型組細(xì)胞分為未處理組、Qg抑素處理組(100μg/ml)和5-氟尿嘧啶處理組(500μg/ml),流式細(xì)胞儀(FCM)對(duì)細(xì)胞凋亡情況進(jìn)行檢測(cè)。(4)HSFs凋亡相關(guān)基因與蛋白檢測(cè):實(shí)時(shí)熒光定量PCR法檢測(cè)c-fos,c-jun,NF-κB,fas,caspase-3,bcl-2和p53 m RNA;Western Blot法檢測(cè)相應(yīng)蛋白表達(dá)。(5)HSFs胞質(zhì)內(nèi)的Ca~(2+)檢測(cè):Fluo-4/AM作為Ca~(2+)指示劑,其終濃度為100mg/ml rh Endostatin灌流HSFs,同時(shí)用激光共聚焦顯微鏡(confocal laser scanning microscope,CLSM)動(dòng)態(tài)觀察和記錄HSFs在有無(wú)Ca~(2+)液時(shí)胞質(zhì)中的Ca~(2+)熒光強(qiáng)度(fluorescence intensity,FI)變化,檢測(cè)rh Endostatin對(duì)HSFs胞質(zhì)Ca~(2+)濃度(cytosolic free calcium concentration)[Ca~(2+)]i的影響。結(jié)果:(1)術(shù)后28天,HS即形成。HE染色結(jié)果顯示瘢痕組織較周圍正常皮膚明顯增厚,同時(shí)瘢痕組織內(nèi)的膠原纖維也明顯變粗變大,呈現(xiàn)出旋渦或結(jié)節(jié)狀,形成膠原結(jié)節(jié);同時(shí)伴有大量的成纖維細(xì)胞增殖以及小血管增生。(2)波形蛋白免疫細(xì)胞化學(xué)染色對(duì)第3代(傳2代)細(xì)胞進(jìn)行鑒定的結(jié)果顯示:細(xì)胞胞質(zhì)內(nèi)含有大量的棕黃色顆粒,呈現(xiàn)成纖維細(xì)胞特性。(3)FCM分析結(jié)果顯示,rh Endostatin(100μg/ml)可促進(jìn)HSFs早期及晚期凋亡,且以早期凋亡更為顯著,分別為(10.78±0.06)%、(2.12±0.04)%,差異較未處理對(duì)照組HSFs均具有統(tǒng)計(jì)學(xué)意義(P0.01)。(4)實(shí)時(shí)熒光定量PCR及Western Blot分析結(jié)果顯示,與未處理對(duì)照組HSFs相比較,rh Endostatin(100μg/ml)能明顯降低HSFs c-jun,c-fos,NF-κB,fas,p53,caspase-3和bcl-2 m RNA和蛋白的表達(dá)水平(P0.01)。(5)CLSM檢測(cè)結(jié)果顯示,當(dāng)細(xì)胞處于無(wú)Ca~(2+)液(D-Hanks緩沖液)中時(shí),rh Endostatin(100mg/ml)不能引起HSFs胞質(zhì)內(nèi)Ca~(2+)FI的改變。當(dāng)細(xì)胞外緩沖液為有Ca~(2+)液(Hanks緩沖液)時(shí),終濃度為100mg/ml rh Endostatin灌流HSFs可使胞質(zhì)Ca~(2+)FI急劇增加達(dá)峰值,隨即熒光開始減弱,FI隨時(shí)間而緩慢下降。結(jié)論:(1)rh Endostatin可促進(jìn)HSFs凋亡。(2)rh Endostatin促進(jìn)HSFs凋亡與其降低c-fos,c-jun,NF-κB和bcl-2基因的表達(dá)與活化有關(guān),同時(shí)該凋亡過(guò)程不依賴于fas,p53表達(dá)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+)內(nèi)流,細(xì)胞內(nèi)鈣超載可能作為HSFs凋亡的一個(gè)重要始動(dòng)環(huán)節(jié)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R622

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