本文選題：皮膚挫傷 + NFкB　； 參考：《中國醫(yī)科大學(xué)》2004年碩士論文

【摘要】： 前言 皮膚挫傷是法醫(yī)學(xué)鑒定中常見的損傷，因此，皮膚挫傷時(shí)間的推斷成為多年來國內(nèi)外法醫(yī)學(xué)界研究的熱點(diǎn)內(nèi)容。 最初根據(jù)挫傷皮膚顏色的變化推斷挫傷時(shí)間，但由于人種膚色、年齡等個(gè)體差異，無法精確判斷皮膚挫傷時(shí)間，只能粗略估計(jì)。后來通過光學(xué)顯微鏡觀察皮膚挫傷后炎癥反應(yīng)中炎性細(xì)胞的數(shù)量來推斷挫傷時(shí)間，但由于各種變化的時(shí)相互相重疊，給確切推斷挫傷時(shí)間帶來了困難，所以，人們一直在尋找可以相對(duì)準(zhǔn)確地推斷皮膚挫傷時(shí)間的方法。隨著醫(yī)學(xué)界對(duì)創(chuàng)傷后分子病理學(xué)認(rèn)識(shí)的提高，創(chuàng)傷后生物物質(zhì)的變化越來越受到人們的重視，也為法醫(yī)工作者推斷皮膚挫傷時(shí)間提供了新的方法。 核轉(zhuǎn)錄因子NFκB(nuclear factor kappa B，NFκB)是一種重要的生物因子，調(diào)控多種細(xì)胞因子如生長因子、細(xì)胞因子、粘附分子等編碼基因的表達(dá)，近來研究表明，NFκB與創(chuàng)傷關(guān)系密切。IκBα因其抑制NFκB活性，也越來越受到人們的重視，故本實(shí)驗(yàn)應(yīng)用免疫組織化學(xué)方法觀察NFκB家族的重要成員NFκB P65與其抑制因子IκBα在大鼠皮膚挫傷后不同時(shí)間在多核粒細(xì)胞、單核巨噬細(xì)胞及成纖維細(xì)胞中的表達(dá)變化，揭示其與皮膚挫傷時(shí)間的關(guān)系，為皮膚挫傷時(shí)間的判定提供新的理論依據(jù)。 材料和方法 1．動(dòng)物模型制作及分組 健康成年Sprague-Dawley大鼠33只，雌雄不限，體重180±20g，隨機(jī)分為11組，每組3只，其中10組作為實(shí)驗(yàn)組，1組作為對(duì)照組。用250g重錘在150cm高度自由落下，造成右后肢股四頭肌處皮膚挫傷，分別于挫傷后0h，3h，6h，9h，12h，1d，3d，，5d，7d，10d將大鼠脫頸椎處死，于挫傷處取1.5cm×1.5cm皮膚組織。對(duì)照組大鼠取與挫傷大鼠相同部位同等大小皮膚組織作為對(duì)照。4％多聚甲醛／PBS固定后石蠟包埋，連續(xù)5μm厚度切片。 2．實(shí)驗(yàn)方法 應(yīng)用免疫組織化學(xué)SABC法，步驟如下: (1)切片脫蠟水化; (2)3%過氧化氫孵育20而n，微波抗原修復(fù)(PH6 .0的檸檬酸緩沖液 中，微波爐加熱至沸騰，間隔smin，連續(xù)三次); (3)正常山羊血清孵育，室溫下20面n; (4)加人1:200倍稀釋的兔抗鼠多克隆NFKB巧5抗體，IKBa抗體，4℃ 過夜; (5)滴加生物素標(biāo)記的山羊抗兔I嬌抗體，保濕盒內(nèi)25℃溫箱中孵育 20min; (6)滴加SABC試劑，保濕盒內(nèi)25℃溫箱中孵育20而n; (7)DAB顯色5一10而n，水洗、蘇木素復(fù)染、透明、中性樹膠封片。以上 抗體及SABC試劑盒均購自武漢博士德生物有限公司。 3.統(tǒng)計(jì)學(xué)分析 采用SPSS for而ndows n .0軟件包，應(yīng)用方差分析進(jìn)行統(tǒng)計(jì)學(xué)處理。 實(shí)驗(yàn)結(jié)果 1.NFKB P65與IKBa在挫傷修復(fù)過程中的表達(dá)變化 正常對(duì)照組皮膚中，NFKB P65與IKBa在表皮基底細(xì)胞層、棘細(xì)胞層、 顆粒細(xì)胞層和真皮層皮脂腺、汗腺上皮細(xì)胞呈陽性表達(dá)。實(shí)驗(yàn)組標(biāo)本中，傷 后Oh皮膚染色與正常皮膚基本相同;傷后3h組，在浸潤的少量多核粒細(xì)胞 的胞質(zhì)中NFKB P65與IKBa呈陽性表達(dá);傷后6h一12h，表達(dá)增加，傷后ld， 在浸潤的幾乎所有的單核細(xì)胞和多核粒細(xì)胞的胞質(zhì)和胞核中NFKB巧5呈 強(qiáng)陽性表達(dá)，IKBa也在大多數(shù)細(xì)胞中表達(dá);傷后3d組，成纖維細(xì)胞大量增 生，陽性細(xì)胞主要以成纖維細(xì)胞為主;5d一10d組陽性細(xì)胞數(shù)逐漸減少。 2.陽性細(xì)胞計(jì)數(shù)及統(tǒng)計(jì)學(xué)分析 2.1 NFKB P65陽性細(xì)胞計(jì)數(shù):傷后3h，NFKB P65陽性細(xì)胞率為(3. 20%士0.58%);傷后6h，NFKB P65陽性細(xì)胞率開始升高(11.33%士1. 53%);gh一12h繼續(xù)升高，ld達(dá)高峰(97.33%土1.53%);傷后3d，陽性細(xì) 胞率開始下降(79.33%土3.05%);sd一7d繼續(xù)下降，傷后10d，陽性細(xì)胞 率下降至(17.67%士2.52%)。 2.2 IKBa陽性細(xì)胞計(jì)數(shù):傷后3h，IKBa陽性細(xì)胞率為(3.33%*0. 58%)，傷后6h，IKBa陽性細(xì)胞率開始升高(1 1 .00%士1 .00%)，gh一12h繼 續(xù)升高，ld達(dá)高峰(84.67%土2.52%);傷后3d，陽性細(xì)胞率開始下降(73. 00%土2.00%)，sd一7d繼續(xù)下降，傷后10d，陽性細(xì)胞率下降至(13.33% 土1 .53%)。 2 .3統(tǒng)計(jì)學(xué)方差分析:經(jīng)統(tǒng)計(jì)學(xué)方差分析，相鄰兩組陽性細(xì)胞率均存在 顯著差異(P0.01)，且各組與1d組相比陽性細(xì)胞率均存在顯著差異(P 0 .01)。 討論 核轉(zhuǎn)錄因子NFKB(nuelear factor kappaB，NFKB)是1986年由Sen和 Baltimere首先從B淋巴細(xì)胞抽提物中檢測到的一種能與免疫球蛋白k鏈 基因增強(qiáng)子B位點(diǎn)結(jié)合，調(diào)控免疫球蛋白k鏈基因轉(zhuǎn)錄的核蛋白因子。細(xì) 胞未受到刺激時(shí)，細(xì)胞中的NFKB處于未活化狀態(tài)，不具有調(diào)節(jié)基因轉(zhuǎn)錄的 功能，此時(shí)，NFKB成員通常以同源二聚體或異源二聚體形式與其抑制蛋白 IKB結(jié)合形成三聚體復(fù)合物，以非活性形式存在于細(xì)胞質(zhì)中。當(dāng)細(xì)胞受到 病毒、放射線、氧自由基、細(xì)胞因子如TNFa、IL一1等細(xì)胞外因素刺激時(shí)， NFKB從NFKB一IKB復(fù)合物中解離出來，并迅速移位至細(xì)胞核，從而發(fā)揮轉(zhuǎn) 錄調(diào)節(jié)功能。越來越多的研究表明，NFKB在炎癥反應(yīng)、免疫應(yīng)答中起重要 作用。 NFKB P65是NFKB家族中最為重要的成員，而IKBa是NFKB的主要抑 制蛋白之一。本實(shí)驗(yàn)應(yīng)用免疫組織化學(xué)技術(shù)觀察到在皮膚挫傷修復(fù)過程中 NFKB P65與 IKBa在皮膚挫傷區(qū)及周邊區(qū)?
[Abstract]:Preface
Skin contusion is a common injury in forensic identification. Therefore, the inference of the time of skin contusion has become a hot topic in the field of forensic medicine at home and abroad for many years.
The initial contusion time was deduced based on the changes in the skin color of the contusion, but the time of Skin Contusion could not be accurately judged by the individual difference of skin color and age. The amount of inflammatory cells in the inflammatory reaction after the skin contusion was observed by the optical microscope. It is difficult to accurately infer the time of contusion, so people have been looking for a relatively accurate method to infer the time of skin contusion. With the improvement of the posttraumatic molecular pathology in the medical field, the changes in the biological substances after the trauma are being paid more and more attention to, and for the forensic workers to infer the skin contusion. A new way is provided.
The nuclear transcription factor NF kappa B (nuclear factor kappa B, NF kappa B) is an important biological factor that regulates the expression of a variety of cytokines, such as growth factors, cytokines and adhesion molecules. Recent studies have shown that NF kappa B is closely related to the trauma of.I kappa B alpha because it inhibits the activity of nuclear kappa. Therefore, it has also been paid more and more attention. Therefore, this experiment is applied to this experiment. The expression of NF kappa B P65, an important member of the NF kappa B family, and its inhibitory factor I kappa B alpha in the rat skin contusion, and the changes in the expression of the mononuclear macrophages and fibroblasts at different time after the skin contusion in rats were observed by immunohistochemical method. The relationship between the time of Skin Contusion and the time of skin contusion was revealed, which provided a new theoretical basis for the determination of the time of skin contusion.
Materials and methods
1. animal model making and grouping
33 healthy adult Sprague-Dawley rats, male and female, 180 + 20g, were randomly divided into 11 groups, 3 in each group, of which 10 groups were used as experimental group and 1 group as control group. The 250g weight was used as the control group. The skin contusion of the four head of the right hind leg of the right hind limb was caused by 0h, 3h, 6h, 9h, 12h, 1D, 3D, 5D. 1.5cm x 1.5cm skin tissue was taken at the contusion site. The same size skin tissue of the control group was taken as the control.4% paraformaldehyde / PBS fixed paraffin embedded in the control group, and the thickness of the skin was 5 mu m continuously.
2. experimental method
The immunohistochemical SABC method was used as follows:
(1) the slices were dewaxing and hydrated;
(2) 3% H2O2 incubated with 20 and N, microwave antigen repair (PH6.0 citric acid buffer)
The microwave oven was heated to boiling, separated by smin for three consecutive times.
(3) the normal goat serum was incubated and 20 N at room temperature.
(4) adding 1:200 times diluted Rabbit anti mouse polyclonal NFKB Qiao 5 antibody, IKBa antibody, 4 degrees centigrade
Pass the night;
(5) the Goat anti rabbit I antibody labeled with biotin was incubated in the incubator at 25 degrees centigrade incubator.
20min;
(6) SABC reagent was added to the incubator and incubated with 20 and N in the 25 degree incubator.
(7) DAB chromogenic 5 1 10 and N, washed, hematoxylin dyed, transparent, neutral gum wrap.
Antibodies and SABC kit were purchased from Wuhan doctorate biological Co., Ltd.
3. statistical analysis
Using SPSS for and ndows n.0 software package, ANOVA was used for statistical analysis.
experimental result
Expression changes of 1.NFKB P65 and IKBa during contusion repair
In the skin of normal control group, NFKB P65 and IKBa were found in the basal cell layer and spinous cell layer of epidermis.
The granular cell layer and the sebaceous glands of the dermis were positive for sweat gland epithelial cells.
After Oh, the skin staining was basically the same as that of the normal skin. In the 3H group, a small number of multinucleated granulocytes were infiltrated.
The expression of NFKB P65 and IKBa in the cytoplasm was positive, and the expression of 6h 12h increased after injury, LD after injury.
In the cytoplasm and nucleus of almost all mononuclear cells and multinucleated granulocytes, NFKB was 5.
IKBa was also expressed in most of the cells. After 3D, there was a large increase in fibroblasts.
The positive cells were mainly fibroblasts, while the number of positive cells in 5D 10d group gradually decreased.
2. positive cells count and statistical analysis
2.1 NFKB P65 positive cell count: 3h after injury, NFKB P65 positive cell rate was (3.
20%, 0.58%). The rate of 6h, NFKB P65 positive cells began to increase after injury (11.33% 1.).
53%); GH 12h continued to rise, LD reached the peak (97.33% soil 1.53%); 3D after injury, positive.
The cell rate began to decrease (79.33% soil 3.05%); SD 1 7d continued to decline, and 10d positive cells after injury.
The rate dropped to (17.67%. 2.52%).
2.2 IKBa positive cell count: 3h after injury, IKBa positive cell rate was (3.33%*0.
58%) after injury, the positive rate of 6h and IKBa cells began to increase (11% 1%), and GH one 12h continued.
The LD reached the peak (84.67% soil 2.52%) and the percentage of positive cells began to decrease after 3D (73.
00% soil 2%), SD 7d continued to decline, 10d after injury, the percentage of positive cells decreased to (13.33%
Soil 1.53%).
2.3 statistical variance analysis: by statistical variance analysis, the positive rates of two adjacent groups all exist.
Significant difference (P0.01), and the rates of positive cells in each group were significantly different from those in group 1D (P
0.01).
discuss
The nuclear transcription factor NFKB (Nuelear factor kappaB, NFKB) was formed in 1986 by Sen and
Baltimere first detected a K chain from immunoglobulin in B lymphocyte extracts.
B enhancer locates the nuclear protein factor that regulates the transcription of immunoglobulin K chain gene.
When the cell is not stimulated, the NFKB in the cell is not activated and does not regulate gene transcription.
At this time, NFKB members usually form homologous two dimers or heterologous two dimers and their inhibitory proteins.
IKB forms a trimer complex and forms in the cytoplasm in inactive form.
When virus, radiation, oxygen free radicals, cytokines such as TNFa, IL 1 and other extracellular factors are stimulated,
NFKB dissociated from the NFKB IKB complex and rapidly shifted to the nucleus, thus making the switch.
More and more studies have shown that NFKB plays an important role in inflammatory response and immune response.
Effect.
NFKB P65 is the most important member of NFKB family, and IKBa is the main inhibition of NFKB.
In this experiment, immunohistochemical staining was used to observe the process of Skin Contusion repair.
NFKB P65 and IKBa in the skin contusion area and surrounding area?

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2004
【分類號(hào)】：D919

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