本文關(guān)鍵詞： 法醫(yī)病理學(xué) 皮膚挫傷 caspase-6 挫傷時(shí)間推斷　出處：《中國(guó)醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 探討皮膚挫傷修復(fù)過(guò)程中，caspase-6在挫傷區(qū)及挫傷周邊區(qū)不同時(shí)間表達(dá)的變化規(guī)律。 方法 1、動(dòng)物模型的建立和分組 健康成年雄性SD大鼠30只，體質(zhì)量220-250g，隨機(jī)分為10組，每組3只，其中9組為實(shí)驗(yàn)組，1組為正常對(duì)照組。用2％戊巴比妥鈉(0.45mg／kg體重)腹腔注射麻醉后，參照Kami等的大鼠骨骼肌急性鈍挫傷模型制作方法，用重500g的自制打擊器，從54cm高處自由垂直落下打擊動(dòng)物右小腿皮膚。分別于傷后3h、6h、12h、1d、3d、5d、7d、10d、14d將大鼠脫頸椎處死，取做好標(biāo)記的皮膚組織，對(duì)照組大鼠同樣方法處理后不作打擊，處死后取與損傷組大鼠相同部位，大小相等的皮膚組織作為對(duì)照，OCT包埋后，液氮速凍，5μm厚度切片。 2、免疫組織化學(xué)染色(SP法) (1)室溫晾干；(2)3％H_2O_2／PBS，室溫孵育20min；(3)0.3％Triton X-100中20min；(4)非免疫血清封閉，室溫孵育20min；(5)一抗1∶100倍稀釋，4℃孵育過(guò)夜；(6)生物素化兔抗山羊二抗，室溫孵育20min；(7)SP試劑，室溫孵育20min；(8)DAB顯色，3min，蘇木素核復(fù)染、透明、中性樹(shù)膠封片。同時(shí)進(jìn)行常規(guī)H．E染色。caspase-6抗體購(gòu)自美國(guó)Santa Cruz公司、SP試劑盒購(gòu)自北京中杉金橋生物試劑公司。 3、Caspase-6的Western blot檢測(cè) (1)蛋白定量；(2)電泳；(3)轉(zhuǎn)��；(4)封閉抗原，采用含有0.1％Tween-20的5％脫脂奶粉封閉液；(5)山羊抗大鼠caspase-6抗體孵育；(6)兔抗山羊二抗孵育；(7)ECL顯色。 結(jié)果 1、皮膚挫傷的宏觀所見(jiàn) 傷后3h組出血、水腫明顯，呈鮮紅色；6h組出血、水腫程度減輕；1d組挫傷區(qū)仍有水腫，出血明顯減輕；3d組水腫幾乎全部吸收，挫傷處皮膚呈棕黃色；5d組挫傷區(qū)皮膚僅可見(jiàn)呈淺黃色改變；7d～14d組挫傷區(qū)皮膚與正常對(duì)照組皮膚相同。 2、組織學(xué)所見(jiàn) 傷后3h組挫傷處表皮層與真皮層分離，真皮內(nèi)毛細(xì)血管擴(kuò)張充血，少量多型核細(xì)胞浸潤(rùn)；6h組挫傷周邊區(qū)中性粒細(xì)胞浸潤(rùn)增多；12h組單個(gè)核細(xì)胞開(kāi)始大量浸潤(rùn)，1d達(dá)高峰；3d組多型核細(xì)胞明顯減少，成纖維細(xì)胞和單核細(xì)胞大量浸潤(rùn)；5d組膠原成分增多，細(xì)胞數(shù)目明顯減少；7d～14d大量膠原纖維形成。 3、Caspase-6在皮膚損傷修復(fù)過(guò)程中損傷區(qū)及損傷周邊區(qū)的表達(dá) 對(duì)照組標(biāo)本中，caspase-6只在表皮層、毛囊、皮脂腺處表達(dá)。挫傷組標(biāo)本中，傷后3h組挫傷區(qū)真皮層內(nèi)有成纖維細(xì)胞浸潤(rùn)，6h組成纖維細(xì)胞陽(yáng)性染色進(jìn)一步增多，12h、1d、3d組達(dá)到高峰，5d組成纖維細(xì)胞陽(yáng)性染色減少，7d組、10d組成纖維細(xì)胞陽(yáng)性染色繼續(xù)下降，14d組降至最低。傷后3h組挫傷區(qū)皮下組織處開(kāi)始有多型核細(xì)胞浸潤(rùn)，6h組、12h組挫傷區(qū)浸潤(rùn)的細(xì)胞數(shù)量逐漸增多，caspase-6陽(yáng)性細(xì)胞主要為多型核細(xì)胞和單個(gè)核細(xì)胞，1d組多型核細(xì)胞和單個(gè)核細(xì)胞數(shù)目最多，3d組出現(xiàn)大量成纖維細(xì)胞陽(yáng)性染色，5d～14d組caspase-6陽(yáng)性細(xì)胞以成纖維細(xì)胞為主。免疫組織化學(xué)染色陰性對(duì)照組切片caspase-6無(wú)陽(yáng)性染色。 陽(yáng)性細(xì)胞計(jì)數(shù)及統(tǒng)計(jì)學(xué)分析：傷后3h組，陽(yáng)性細(xì)胞比率為(25.78％±1.38％)，6h組陽(yáng)性細(xì)胞比率繼續(xù)增高(36.21％±4.25％)，12h組陽(yáng)性細(xì)胞比率增高明顯(47.70％±5.14％)，1d組陽(yáng)性細(xì)胞比率進(jìn)一步增高(50.62％±5.42％)，3d組caspase-6陽(yáng)性細(xì)胞率達(dá)到最高(54.58％±5.64％)。傷后5d組開(kāi)始下降(28.39％±4.50％)，7d、10d組進(jìn)一步下降，且陽(yáng)性細(xì)胞率基本維持在一個(gè)相對(duì)穩(wěn)定的水平(23.75％±3.12％，，20.43％±2.36％)，14d組陽(yáng)性細(xì)胞率下降至(11.31％±2.19％)。 經(jīng)統(tǒng)計(jì)學(xué)方差分析，各時(shí)間段相鄰兩組之間caspase-6陽(yáng)性細(xì)胞率相比均存在顯著差異(P＜0.01)。 4、Western blot分析 實(shí)驗(yàn)組中，1d、3d、5d組可見(jiàn)明顯的10kD caspase-6活化片段。 結(jié)論 大鼠皮膚挫傷修復(fù)過(guò)程中，caspase-6在挫傷后的炎癥反應(yīng)中發(fā)揮重要作用；同時(shí)，caspase-6在挫傷區(qū)內(nèi)多型核細(xì)胞、單個(gè)核細(xì)胞及成纖維細(xì)胞中的表達(dá)呈規(guī)律性變化，可對(duì)挫傷時(shí)間的推斷提供參考。
[Abstract]:objective
To investigate the changes of caspase-6 expression in the contusion area and the peripheral zone of contusion during the repair of skin contusion.
Method
1, establishment and grouping of animal models
30 adult male SD rats, weighing 220-250g, were randomly divided into 10 groups, 3 rats in each group, the 9 groups as the experimental group, the 1 group was the normal control group. With 2% sodium pentobarbital (0.45mg / kg body weight) intraperitoneal injection of anesthesia, method of making acute blunt skeletal muscle contusion in rats model reference Kami, homemade blow 500g, free fall from a height of 54cm vertical strike animal right calf skin. After injury, 3h, 6h, 12h, 1D, 3D, 5D, 7d, 10d, 14d rats were sacrificed by cervical dislocation, and marked the skin tissue of rats in the control group the same method does not blow after sacrificed and injury rats in the same parts of equal size skin tissue as control, OCT embedding, quick-frozen in liquid nitrogen, 5 mu m thick slices.
2, immunohistochemical staining (SP)
Dry at room temperature (1); (2) 3%H_2O_2 / PBS, incubated at room temperature for 20min; (3) 20min 0.3%Triton X-100; (4) non immune serum blocking, and incubated at room temperature for 20min; (5) - 1: 100 times dilution, 4 C incubated overnight; (6) the biotinylated anti rabbit two anti goat, incubated at room temperature for 20min; (7) SP reagent, incubated at room temperature for 20min; (8) DAB color, 3min, hematoxylin nuclear staining, transparent, neutral balata. Routine H.E staining of.Caspase-6 antibody was purchased from American Santa Cruz company at the same time, SP Kit was purchased from Beijing golden bridge the biological reagent company.
3, Western blot detection of Caspase-6
(1) protein quantification; (2) electrophoresis; (3) transfer; (4) blocking antigen, using 5% skim milk powder containing 0.1%Tween-20, and (5) Goat anti rat caspase-6 antibody incubation; (6) Rabbit anti goat two anti incubation; (7) ECL color.
Result
1, the macroscopic view of the skin contusion
The group of 3H hemorrhage, edema, bright red; group 6h hemorrhage, edema decreased in 1D group; the contusion area still have edema, hemorrhage significantly reduced; 3D group edema is almost completely absorbed, the skin contusion was brown; 5D group of Skin Contusion area only visible pale yellow; 7d ~ 14d contusion group the skin and the normal control group the same skin.
2, histology
The group of 3H injured epidermis and dermis, dermis angiotelectasis hyperemia, a few polymorphonuclear cell infiltration; 6h group of surrounding area increased neutrophil infiltration; 12h group began a large number of mononuclear cells infiltration, reached the peak at 1D; 3D group of polymorphonuclear cells significantly decreased, into a large number of fibroblasts and infiltration mononuclear cells; group 5D collagen increased, the number of cells was significantly reduced; 7d ~ 14d a large number of collagen fibers.
3, Caspase-6 expression in the damaged area and the surrounding area during the repair of skin injury
Samples in the control group, caspase-6 only in the epidermis, hair follicles and sebaceous glands. The expression of contusion group specimens after injury, contusion group 3H in dermal fibroblast infiltration, 6h positive staining cells further increased, 12h, 1D, 3D group reached the peak, 5D fibroblasts positive staining decreased 7d group, 10d positive staining cells continued to decline, fell to the lowest in group 14d after injury in 3H group. The subcutaneous tissue contusion area starting with polymorphonuclear cell infiltration, 6h group, 12h group the number of cells infiltrating the contusion area gradually increased, caspase-6 positive cells were polymorphonuclear cells and mononuclear cells, 1D group polymorphonuclear cells and mononuclear cell number, a large number of fibroblasts positive staining of 3D group, 5D group 14d ~ caspase-6 positive cells with fibroblast. Immunohistochemical staining in negative control group were caspase-6 positive staining.
Positive cell counting and statistical analysis: after injury in 3H group, positive cells ratio was (25.78% + 1.38%), the ratio of positive cells in the 6h group continue to increase (36.21% + 4.25%) 12h group, the rate of positive cells increased significantly (47.70% + 5.14%), 1D group further increased the rate of positive cells (50.62% + 5.42%), 3D group caspase-6 the rate of positive cells reached the highest (54.58% + 5.64%). The group of 5D began to decline (28.39% + 4.50%), 7d group, 10d decreased further, and the positive cell rate remained at a relatively stable level (23.75% + 3.12%, 20.43% + 2.36%), 14d group (positive cell rate dropped to 11.31% + 2.19%).
The statistical analysis of variance showed that there were significant differences in the rate of caspase-6 positive cells between the two adjacent groups (P < 0.01).
4, Western blot analysis
In the experimental group, 1D, 3D, and 5D groups showed significant 10kD caspase-6 activation fragments.
conclusion
In the process of Skin Contusion repair, caspase-6 plays an important role in the inflammatory response after contusion. Meanwhile, the expression of caspase-6 changes regularly in various types of nuclear cells, mononuclear cells and fibroblasts in the contusion area, which can provide a reference for the inference of contusion time.

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：D919

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 杜宇,官大威,趙銳;小鼠皮膚切創(chuàng)愈合過(guò)程中caspase-6、-7表達(dá)的免疫組織化學(xué)研究[J];中國(guó)法醫(yī)學(xué)雜志;2003年05期

2 杜宇,官大威,趙銳;皮膚切創(chuàng)愈合中caspase-3表達(dá)的免疫組化研究[J];法醫(yī)學(xué)雜志;2004年01期

3 彭湃,郭樹(shù)忠,韓巖,靳安民;VEGF對(duì)創(chuàng)傷組織中KDR,bFGF和PDGF mRNA表達(dá)的影響[J];中國(guó)美容醫(yī)學(xué);2004年03期

4 黃暉,賴西南,王正國(guó),王麗麗;創(chuàng)傷愈合中P物質(zhì)對(duì)表皮干細(xì)胞遷移及受體表達(dá)的作用[J];中華創(chuàng)傷雜志;2004年03期

本文編號(hào)：1496682