發(fā)布時(shí)間：2018-02-14 21:34

  本文關(guān)鍵詞： 增生性瘢痕 色甘酸鈉 肥大細(xì)胞 類胰蛋白酶　出處：《西南醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:本實(shí)驗(yàn)擬建立大鼠深度創(chuàng)面模型,使用肥大細(xì)胞膜穩(wěn)定劑色甘酸鈉干預(yù)創(chuàng)面愈合過(guò)程中肥大細(xì)胞的作用,通過(guò)觀察愈合后瘢痕增生情況并檢測(cè)肥大細(xì)胞數(shù)量、類胰蛋白酶等相關(guān)指標(biāo),探究以肥大細(xì)胞作為瘢痕預(yù)防和治療靶點(diǎn)的可行性。方法:選取40只雄性SD大鼠,隨機(jī)將其等分為A、B、C、D四組,每組10只,A、B、C組為實(shí)驗(yàn)組,其中A組為大劑量色甘酸鈉組(40mg/kg),B組為中劑量色甘酸鈉組(20mg/kg),C組為小劑量色甘酸鈉組(10mg/kg),D組為陰性對(duì)照組。使用脫毛劑對(duì)所有實(shí)驗(yàn)對(duì)象進(jìn)行背部脫毛后,麻醉?xiàng)l件下于背部脊柱兩側(cè)用手術(shù)刀形成兩個(gè)對(duì)稱的面積約15mm*10mm*2mm的切割傷深度創(chuàng)面。創(chuàng)面以2%稀釋碘伏溶液消毒后予以暴露,每日觀察大鼠背部創(chuàng)面情況(是否有滲血和異常滲液)。分別于術(shù)前30min;術(shù)后第24、第48、第72小時(shí),對(duì)A、B、C組所有實(shí)驗(yàn)對(duì)象腹腔注射對(duì)應(yīng)劑量色甘酸鈉溶液,D組注射相等體積生理鹽水。記錄創(chuàng)面初始面積,定期觀察創(chuàng)面面積大小,并在術(shù)后第3d取背部左側(cè)創(chuàng)面組織,對(duì)組織進(jìn)行固定脫水及常規(guī)HE染色,行甲苯胺藍(lán)染色(肥大細(xì)胞計(jì)數(shù))、免疫組化染色(類胰蛋白酶)等處理。記錄全部實(shí)驗(yàn)對(duì)象創(chuàng)面愈合時(shí)間,組間取其均數(shù)進(jìn)行比較。術(shù)后第42d處死所有實(shí)驗(yàn)對(duì)象,并收集背部右側(cè)瘢痕組織,對(duì)其進(jìn)行常規(guī)HE染色。肥大細(xì)胞計(jì)數(shù):組織切片后使用甲苯胺藍(lán)染色法,用Image-pro6.0圖像分析軟件系統(tǒng),在雙盲高倍鏡下(×400)連續(xù)計(jì)數(shù)單位面積(個(gè)/mm2),每張隨機(jī)挑選5個(gè)視野,計(jì)數(shù)其中肥大細(xì)胞的總數(shù)后,取其平均值作為該實(shí)驗(yàn)對(duì)象最終數(shù)據(jù)。并對(duì)脫顆粒和未脫顆粒進(jìn)行定義。脫顆粒后肥大細(xì)胞胞膜不完整,邊界模糊,且周圍可見(jiàn)散在顆粒物質(zhì)分布。而完整肥大細(xì)胞形態(tài)相對(duì)完整規(guī)則,與周邊組織交界分明。類胰蛋白酶分析方法:免疫組化染色后,用image-pro6.0圖像分析軟件系統(tǒng),雙盲高倍鏡下(×400)連續(xù)計(jì)數(shù)單位面積(個(gè)/mm2),同樣每張切片隨機(jī)選取5個(gè)視野,記錄其中胰蛋白酶陽(yáng)性肥大細(xì)胞數(shù),并取其平均值作為最后結(jié)果。結(jié)果:1、創(chuàng)面初始面積:通過(guò)方差分析進(jìn)行組間比較后再兩兩比較均可得p0.05,提示所有實(shí)驗(yàn)對(duì)象創(chuàng)面初始面積無(wú)統(tǒng)計(jì)學(xué)差異。2、創(chuàng)面愈合時(shí)間:通過(guò)方差分析進(jìn)行組間比較后再兩兩比較均可得p0.05,提示所有實(shí)驗(yàn)對(duì)象創(chuàng)面愈合時(shí)間無(wú)統(tǒng)計(jì)學(xué)差異。3、瘢痕面積及硬度:術(shù)后第42天,見(jiàn)所有實(shí)驗(yàn)對(duì)象背部創(chuàng)面均出現(xiàn)瘢痕愈合,顏色呈淡紅或蒼白色,組間比較瘢痕面積大小關(guān)系提示:c組d組b組a組。d組瘢痕組織較硬;a、b組兩組瘢痕質(zhì)韌,組間無(wú)明顯差異;而c組位于兩者之間。4、he染色后鏡下觀察,術(shù)后第3天:a、b兩組創(chuàng)面情況類似,可見(jiàn)炎性細(xì)胞、成纖維細(xì)胞散在分布,但新生毛細(xì)血管數(shù)量不多;d組鏡下見(jiàn)細(xì)胞結(jié)構(gòu)混亂,炎性細(xì)胞及成纖維細(xì)胞數(shù)量多,分布凌亂,且見(jiàn)豐富毛細(xì)血管。c組炎性細(xì)胞及成纖維細(xì)胞不及d組豐富,但遠(yuǎn)多于a、b兩組。術(shù)后第42天:d組瘢痕組織內(nèi)大量成纖維細(xì)胞、細(xì)胞外基質(zhì)和毛細(xì)血管分布,部分呈環(huán)形或螺旋樣分布,炎性細(xì)胞數(shù)量不多,纖維組織散在分布,無(wú)明顯條理性;c組瘢痕組織顏色較d組淺,鏡下可見(jiàn)成纖維細(xì)胞明顯少于d組,排列尚規(guī)則,膠原纖維排列較d組整齊,細(xì)胞外基質(zhì)不多,散在分布少量毛細(xì)血管;而a組、b組瘢痕組織增生不明顯,鏡下見(jiàn)成纖維細(xì)胞排列較緊密、有序,纖維組織排列整齊有序。5、肥大細(xì)胞計(jì)數(shù):切片行甲苯胺藍(lán)染色法后,在顯微鏡下進(jìn)行計(jì)數(shù),發(fā)現(xiàn)各組間肥大細(xì)胞總數(shù)差異無(wú)統(tǒng)計(jì)學(xué)意義,但a、b兩組脫顆粒細(xì)胞數(shù)明顯少于D組,C組位于兩者之間,相應(yīng)脫顆粒肥大細(xì)胞數(shù)/肥大細(xì)胞總數(shù)比例:D組C組A組≈B組。6、類胰蛋白酶計(jì)數(shù):術(shù)后第3天,接受到刺激信號(hào)后的肥大細(xì)胞開(kāi)始表達(dá)類胰蛋白酶,D組類胰蛋白酶陽(yáng)性肥大細(xì)胞數(shù)量明顯少于A組、B組,C組數(shù)量位于兩者之間。類胰蛋白酶陽(yáng)性肥大細(xì)胞數(shù):B組A組C組D組。結(jié)論:1、色甘酸鈉可以顯著抑制肥大細(xì)胞脫顆粒,減輕早期炎癥反應(yīng);但不影響局部組織中肥大細(xì)胞數(shù)量,且不影響創(chuàng)面愈合時(shí)間;2、色甘酸鈉可以有效減少創(chuàng)面愈合后瘢痕的形成;3、通過(guò)對(duì)比使用不同劑量色甘酸鈉,發(fā)現(xiàn)抑制大鼠肥大細(xì)胞脫顆粒的最佳有效劑量為20mg/kg;4、色甘酸鈉影響肥大細(xì)胞類胰蛋白酶釋放,可能是其影響成纖維細(xì)胞活化,并最終減少瘢痕形成的機(jī)制之一。
[Abstract]:Objective: This study intends to establish a rat model of deep wounds, mast cells using mast cell membrane stabilizer cromolyn sodium intervention in the process of wound healing effect, through the observation of the healing scar hyperplasia and detected the number of mast cells, tryptase and other related indicators, to explore the feasibility of mast cells as targets for prevention and treatment of scar. Methods: 40 male SD rats were randomly divided into the A, B, C, D four groups, 10 rats in each group, A, B, C group, A group for a large dose of sodium cromoglycate (40mg/kg) group, B group middle dose of cromolyn sodium group (20mg/kg), C group of small dose of cromolyn sodium group (10mg/kg), group D was negative. The use of hair removal agent back hair removal to all subjects after anesthesia conditions on the back on both sides of the spine with a scalpel to form two symmetrical area of about 15mm*10mm*2mm the cutting depth to wound wound. 2% dilute Release the iodophor solution after disinfection to be exposed, the daily observation of back wounds in rats (whether there is abnormal blood seepage and seepage). Respectively at 30min before operation; after twenty-fourth, forty-eighth, seventy-second hours of A, B, C group all subjects corresponding dose of intraperitoneal injection of sodium cromoglycate solution, group D injection equal volume of saline. The initial recording wound area, regular observation of wound size, and after the 3D take the left side of the back wound tissue were fixed, dehydration and conventional HE staining on tissue, toluidine blue staining (mast cell count), immunohistochemical staining (tryptase) treatment. The recording time of all experimental objects the wound healing, the number of groups were compared. Postoperative 42d were all subjects, and collect the scar right back, routine HE stain on it. Mast cell count: tissue sections using toluidine blue staining method, use Image-pro6.0 diagram Image analysis software system in double blind high magnification (* 400) continuous count per unit area (/mm2), each of 5 randomly selected fields, the counting of mast cells in total, the average value of the experimental data and the final object. Degranulation and degranulation were not defined off. The particles after mast cell membrane is not complete, fuzzy boundaries, and could be seen around the scattered particles. And the complete form of mast cells relatively complete rules, and the surrounding tissue of the border clear. Tryptase analysis method: immunohistochemical staining and image analysis software system with image-pro6.0, double blind high magnification (* 400) count per unit area (/mm2), the same in each section was randomly selected 5 horizons, which records of tryptase positive mast cell number, and take the average value as the final result. Results: 1, the initial wound area: through the analysis of variance were compared between the two groups after Then 22 can get P0.05, suggesting that all initial experiments showed no significant difference.2 the wound area, wound healing time: by the analysis of variance were compared between the two groups after 22 can get P0.05, suggesting that all subjects had no significant difference.3 wound healing time, scar area and hardness: forty-second days after operation, see all subjects back scar wounds were healed, the color was light red or pale, prompting groups scar area size relationship: C group D group B group a group.D group scar is hard; a, B two groups of scar quality and tough, no significant differences between two groups; group C in between.4 and he staining microscopically observed after third days after operation: A, B two groups were similar, inflammatory cells, fibroblasts scattered, but the number of new capillaries; D group microscopically, cell structure disorder, inflammatory cells and fibroblast cell number, distribution Messy, and abundant capillary.C group of inflammatory cells and fibroblasts in D group than the rich, but far more than a, B two groups. After forty-second days, the scar tissue in the D group, a large number of fibroblasts and extracellular matrix and capillary distribution, part of a circular or spiral like distribution, the number of inflammatory cells not much, fibrous tissue scattered, no obvious rational; scar tissue in group C than in group D color light microscope fibroblasts significantly less than group D, are still rules, collagen fibers were compared with the D group neatly, extracellular matrix is not much, scattered in small capillaries; and group A, B group of scar tissue hyperplasia was not obvious, microscopically fibroblasts arranged more closely and orderly, neat and orderly.5 fiber tissue, mast cell count: sections were stained with toluidine blue staining, were counted under the microscope, found no statistically significant difference between the groups, the total number of mast cells, but a, B two Group degranulation was less than the D group, C group is located in between the corresponding number of mast cells degranulation of mast cells / total ratio: D group C group A group is B group.6, tryptase count: third days after operation, to accept the signal after the start of stimulation of mast cell tryptase expression quantity. D group of tryptase positive mast cells was significantly less than that of A group, B group, C group is located between the two. The number of tryptase positive mast cell number: B group A group C group D group. Conclusion: 1, cromolyn sodium can significantly inhibit the degranulation of mast cells, reducing inflammatory reaction; but does not affect the the number of mast cells in the local tissues, and does not affect the wound healing time; 2, cromolyn sodium can effectively reduce the wound healing after scar formation; 3, cromolyn sodium with different doses by comparison, found that the best effective dose inhibits rat mast cell degranulation of 20mg/kg; 4, cromolyn sodium. The release of the ringing mast cell tryptase may be one of the mechanisms that affect the activation of fibroblasts and ultimately reduce the formation of cicatricial cells.

【學(xué)位授予單位】：西南醫(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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