本文選題：皮膚損傷 + 損傷時(shí)間判定��； 參考：《中國(guó)醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的 皮膚損傷愈合大致分為三個(gè)階段，即炎癥期、纖維增生期和組織重建期。多核粒細(xì)胞、單核細(xì)胞和成纖維細(xì)胞參與皮膚損傷愈合的全過(guò)程，清除損傷、壞死的皮膚組織和入侵的病原體并重建損傷組織，對(duì)于損傷愈合具有重要作用。在炎癥期和纖維增生期損傷區(qū)內(nèi)多核粒細(xì)胞、單核細(xì)胞和成纖維細(xì)胞的數(shù)量急劇增加；在組織重建期，這三種細(xì)胞的數(shù)量則顯著減少，直至傷口完全愈合。 粘著斑激酶(focal adhesion kinase，F(xiàn)AK，也稱pp~(125FAK))是人們最初在用v-src轉(zhuǎn)化細(xì)胞時(shí)發(fā)現(xiàn)的一個(gè)主要酪氨酸磷酸化蛋白，是整合素信號(hào)轉(zhuǎn)導(dǎo)中的關(guān)鍵分子之一，在細(xì)胞粘附過(guò)程中發(fā)揮著非常重要的作用。當(dāng)被整合素激活時(shí)FAK自身磷酸化，能夠直接或者通過(guò)相關(guān)激酶激活PI-3K(phosphatidylinositol 3-kinase，PI-3K)、MAPK(mitogen-activated protein kinase，MAPK)和STAT(signal transducer and activator of transcription，STAT)等信號(hào)轉(zhuǎn)導(dǎo)分子，進(jìn)而與細(xì)胞內(nèi)一些相關(guān)的信號(hào)轉(zhuǎn)導(dǎo)途徑相聯(lián)系，參與細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)過(guò)程。這些細(xì)胞內(nèi)分子的相互作用在信號(hào)轉(zhuǎn)導(dǎo)途徑中調(diào)節(jié)各種各樣的細(xì)胞功能，如擴(kuò)散、遷移、增殖、凋亡和細(xì)胞的存活。 本研究用免疫組織化學(xué)染色技術(shù)和Western blot方法檢測(cè)小鼠皮膚切創(chuàng)愈合過(guò)程中FAK和磷酸化FAK在多核粒細(xì)胞、單核細(xì)胞及成纖維細(xì)胞中的表達(dá)，進(jìn)而探討通過(guò)FAK的信號(hào)轉(zhuǎn)導(dǎo)途徑及其生物學(xué)作用，并討論通過(guò)檢測(cè)FAK和磷酸化FAK表達(dá)的規(guī)律推測(cè)皮膚損傷時(shí)間的可行性，進(jìn)一步揭示蛋白質(zhì)的磷酸化對(duì)皮膚損傷愈合的影響。 材料和方法 1、動(dòng)物模型的建立及分組 健康成年清潔級(jí)昆明系小鼠33只，雌雄不限，體重35-40g，隨機(jī)分為11組，每組3只，其中1組為正常對(duì)照組，其余各組做為實(shí)驗(yàn)組。乙醚氣體吸入麻醉，剪毛處理，常規(guī)手術(shù)消毒，在背部中央做一縱行長(zhǎng)1.5cm皮膚全層切口，深達(dá)筋膜。創(chuàng)面自然止血，不包扎，不施藥。切創(chuàng)后每只小鼠分籠飼養(yǎng)，給予消毒飼料及雙蒸水并保持傷口干燥、防止傷口感染。分別于傷后0h、3h、6h、12h、1d、3d、5d、7d、10d、14d將小鼠脫頸椎處死，取傷口處1.0cm×1.5cm皮膚組織。對(duì)照組小鼠取相同部位同等大小皮膚。每組1／2皮膚固定后石蠟包埋，制作5μm厚度連續(xù)切片；另1／2皮肽勻漿，離心，取上清。 2、FAK和磷酸化FAK的免疫組織化學(xué)染色(SP法) (1)切片脫蠟，水化； (2) 3％H_2O_2／PBS滅活內(nèi)源性過(guò)氧化物酶，室溫25min； (3) PBS洗滌切片后進(jìn)行抗原修復(fù)，置于10mM pH 6.0的檸檬酸緩沖液中，微波爐加熱至96℃，持續(xù)3min，，三次、計(jì)10min； (4)切片冷卻至室溫，用PBS(pH 7.2-7.4)洗滌切片后滴加正常山羊非免疫血清，室溫下保濕盒內(nèi)孵育25min； (5)滴加用抗體稀釋液1：200倍稀釋的兔抗小鼠FAK或磷酸化FAK多克隆抗體，保濕盒內(nèi)4℃孵育過(guò)夜； (6) PBS洗滌切片后滴加生物素標(biāo)記的山羊抗兔IgG抗體，保濕盒內(nèi)孵育25min； (7) PBS洗滌切片后滴加SP試劑，保濕盒內(nèi)孵育25min； (8) PBS洗滌切片后用新配制的DAB顯色3min； (9)蘇木素核復(fù)染1min，1％鹽酸-酒精中分化后在自來(lái)水中返藍(lán)10min； (10)梯度酒精脫水，二甲苯透明，中性樹(shù)膠封片。 實(shí)驗(yàn)中以PBS替代一抗作陰性對(duì)照，未見(jiàn)假陽(yáng)性。每組同時(shí)作HE染色。 3、陽(yáng)性細(xì)胞計(jì)數(shù)分析 顯微鏡×400倍下，在每張切片中隨機(jī)選擇10個(gè)視野計(jì)數(shù)包括多核粒細(xì)胞、單核細(xì)胞及成纖維細(xì)胞的陽(yáng)性細(xì)胞。計(jì)算每個(gè)視野中陽(yáng)性細(xì)胞與此三種細(xì)胞總數(shù)的比值。 (1)結(jié)果判斷 FAK和磷酸化FAK陽(yáng)性表達(dá)均位于細(xì)胞漿，用DAB顯色呈棕黃色。 (2)統(tǒng)計(jì)學(xué)分析 各損傷時(shí)間組數(shù)據(jù)應(yīng)用SPSS 11.0 for Windows統(tǒng)計(jì)軟件，采用單因素方差分析方法進(jìn)行統(tǒng)計(jì)學(xué)處理，數(shù)據(jù)以(?)±s表示。 4、FAK和磷酸化FAK的Western blot檢測(cè) (1)蛋白樣本制備； (2)蛋白定量； (3)電泳，100μg／lane，10％[w／v]SDS-PAGE，以標(biāo)準(zhǔn)分子量Marker作指示； (4)轉(zhuǎn)�。� (5)封閉抗原，5％脫脂牛奶和0.1％Tween-20封閉液； (6)兔抗小鼠FAK或磷酸化FAK多克隆抗體孵育； (7)羊抗兔抗體孵育，以GAPDH為內(nèi)參； (8) ECL顯色。 (9)結(jié)果判斷 檢測(cè)FAK和磷酸化FAK，在125kD顯示陽(yáng)性譜帶。 實(shí)驗(yàn)結(jié)果 1、皮膚損傷愈合過(guò)程的病理組織學(xué)變化 傷后3h組損傷區(qū)出現(xiàn)少量多核粒細(xì)胞。6h組見(jiàn)多核粒細(xì)胞數(shù)量明顯增加。12h組皮下組織內(nèi)有大量滲出的多核粒細(xì)胞。1d組損傷區(qū)有大量的多核粒細(xì)胞及單核細(xì)胞浸潤(rùn)。傷后3d～5d以單核細(xì)胞和成纖維細(xì)胞為主。傷后7d組表皮增生明顯，10d和14d組完全覆蓋傷口。 2、FAK和磷酸化FAK在皮膚切創(chuàng)愈合過(guò)程中損傷區(qū)及損傷周邊區(qū)的表達(dá) (1) FAK在對(duì)照組中小鼠的表皮、毛囊、皮脂腺、汗腺上皮細(xì)胞和血管內(nèi)皮細(xì)胞均呈陽(yáng)性染色。實(shí)驗(yàn)組傷后0h組織切片染色與對(duì)照組基本相同。3h開(kāi)始出現(xiàn)陽(yáng)性細(xì)胞，傷后6h～12h，大部分浸潤(rùn)的多核粒細(xì)胞和單核細(xì)胞為陽(yáng)性。1d～3d成纖維細(xì)胞大量增生，幾乎全部陽(yáng)性著色。5d～10d陽(yáng)性著色逐漸減弱，到14d呈最弱表達(dá)。 (2)磷酸化FAK在對(duì)照組中小鼠表皮、毛囊、皮脂腺、汗腺上皮細(xì)胞和血管內(nèi)皮細(xì)胞亦有陽(yáng)性染色，但其表達(dá)強(qiáng)度較FAK弱。實(shí)驗(yàn)組傷后3h開(kāi)始出現(xiàn)陽(yáng)性細(xì)胞，12h陽(yáng)性染色最為明顯，之后表達(dá)強(qiáng)度稍有減弱，5d～7d逐漸減弱，14d僅有微弱表達(dá)。 3、陽(yáng)性細(xì)胞計(jì)數(shù)及統(tǒng)計(jì)學(xué)分析 (1) FAK：傷后3h之內(nèi)，F(xiàn)AK的陽(yáng)性細(xì)胞率較低(9.34％±1.42％)，6h組陽(yáng)性細(xì)胞比率開(kāi)始升高(21.57％±0.91％)，12h陽(yáng)性細(xì)胞比率繼續(xù)升高(29.51％±0.76％)，1d組陽(yáng)性細(xì)胞比率升高明顯(36.22％±1.96％)，在傷后3d，陽(yáng)性細(xì)胞比率達(dá)到最高(53.48％±2.74％)。5d組陽(yáng)性細(xì)胞比率開(kāi)始下降(45.17％±3.21％)，7d組陽(yáng)性細(xì)胞比率繼續(xù)下降(39.96％±1.77％)，10d組該比率下降為(26.71％±2.33％)，傷后14d FAK陽(yáng)性細(xì)胞比率降到最低(12.15％±1.64％)。經(jīng)統(tǒng)計(jì)學(xué)方差分析，除0h組外FAK各時(shí)間段相鄰兩組之間陽(yáng)性細(xì)胞比率差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。 (2) phospho-FAK：傷后3h之內(nèi)，phospho-FAK的陽(yáng)性細(xì)胞率較低(5.48％±3.23％)，6h組陽(yáng)性細(xì)胞比率迅速升高(36.32％±3.76％)，12h組陽(yáng)性細(xì)胞比率最高，達(dá)(67.58％±2.87％)，1d組陽(yáng)性細(xì)胞比率稍有下降(55.65％±3.51％)，在傷后3d，陽(yáng)性細(xì)胞比率繼續(xù)下降(47.52％±3.89％)。5d組陽(yáng)性細(xì)胞比率下降為(39.71％±6.53％)，7d組陽(yáng)性細(xì)胞比率下降到(33.08％±3.19％)，10d組該比率繼續(xù)下降(20.14％±4.87％)，傷后14d陽(yáng)性細(xì)胞比率降到最低(8.29％+3.54％)。經(jīng)統(tǒng)計(jì)學(xué)方差分析，除0h組外phospho-FAK各時(shí)間段相鄰兩組之間陽(yáng)性細(xì)胞比率差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。 4、Western blot結(jié)果分析 FAK在對(duì)照組及實(shí)驗(yàn)組均有表達(dá)，位于125kD處。在實(shí)驗(yàn)組FAK表達(dá)強(qiáng)度變化不是十分明顯，但仍可見(jiàn)在3d時(shí)達(dá)到高峰。磷酸化FAK亦位于125kD處，在空白對(duì)照組及0h組表達(dá)較弱，在3h組表達(dá)略增強(qiáng)，6h開(kāi)始顯著增強(qiáng)，至12h組顯色最強(qiáng)，此后逐漸下降，至14d組呈弱陽(yáng)性表達(dá)。 結(jié)論 1、正常皮膚的表皮層、毛囊、皮脂腺和血管內(nèi)皮中表達(dá)FAK及磷酸化FAK，提示FAK在生理狀態(tài)下就發(fā)揮生物學(xué)作用。 2、小鼠皮膚切創(chuàng)愈合過(guò)程中，F(xiàn)AK和phospho-FAK在多核粒細(xì)胞、單核細(xì)胞及成纖維細(xì)胞中表達(dá)，并且在小鼠皮膚切創(chuàng)愈合過(guò)程中，F(xiàn)AK對(duì)于多核粒細(xì)胞和單核細(xì)胞的聚集、遷移及成纖維細(xì)胞的增殖起著重要作用。 3、在小鼠皮膚切創(chuàng)愈合過(guò)程中，F(xiàn)AK和phospho-FAK陽(yáng)性細(xì)胞率呈時(shí)間規(guī)律性變化，表明FAK和phospho-FAK可作為推斷皮膚切創(chuàng)損傷時(shí)間的指標(biāo)，且phospho-FAK要優(yōu)于FAK。
[Abstract]:objective
The healing of skin injury is divided into three stages, namely, inflammation, fibrous proliferation and tissue reconstruction. Multinuclear cells, monocytes and fibroblasts are involved in the whole process of skin injury healing. It is important to remove injury, necrotic skin tissue and invading pathogens and rebuild damaged tissues. The number of mononuclear cells and fibroblasts in the lesion area of the fibrous proliferation period increased dramatically, and the number of these three cells decreased significantly during the tissue reconstruction period until the wound was completely healed.
Focal adhesion kinase (FAK, also known as pp~ (125FAK)) is a major tyrosine phosphorylated protein found in v-src conversion cells. It is one of the key molecules in integrin signal transduction. It plays a very important role in cell adhesion process. When integrin is activated, FAK itself phosphorylation, can be used. Enough to activate PI-3K (phosphatidylinositol 3-kinase, PI-3K), MAPK (mitogen-activated protein kinase, MAPK) and STAT (signal transducer) and other signal transduction molecules, and then associated with some of the intracellular signal transduction pathways involved in cell signal transduction. These interactions in cells regulate various cellular functions in signal transduction pathways, such as proliferation, migration, proliferation, apoptosis and cell survival.
In this study, the expression of FAK and phosphorylated FAK in multinucleated granulocytes, monocytes and fibroblasts was detected by immunohistochemical staining and Western blot, and the signal transduction pathway and biological function of FAK were discussed, and the expression of FAK and phosphorylated FAK was discussed. The law predicts the feasibility of skin damage time, and further reveals the effect of protein phosphorylation on skin wound healing.
Materials and methods
1, establishment and grouping of animal models
33 healthy adult clean Kunming mice were randomly divided into 11 groups, with 3 rats and male and female, with 3 rats in each group, of which 1 were in the normal control group and the other groups were used as the experimental group. The ether gas inhalation anesthesia, the hair treatment, the routine operation disinfection, the long 1.5cm skin incision in the center of the back, the deep fascia, and the natural hemostasis of the wound. The mice were kept in cages, and each mouse was fed in cages. The mice were given disinfectant feed and double steamed water to keep the wound dry and prevent the wound infection. 0h, 3h, 6h, 12h, 1D, 3D, 5D, 7d, 10d, 14d, respectively, were sacrificed after the injury. The mice were removed from the cervical vertebra with the same size of the same size in the control group. Each group was 1 / 2 skin. After skin fixed, paraffin embedded, 5 m thickness slice was made, and 1 / 2 skin peptide homogenate was centrifugated to obtain the supernatant.
2, immunohistochemical staining of FAK and phosphorylated FAK (SP method).
(1) sliced dewaxing and hydrating;
(2) 3%H_2O_2 / PBS inactivated endogenous peroxidase and 25min at room temperature.
(3) after PBS washing, the antigen was repaired and placed in the citric acid buffer of 10mM pH 6. The microwave oven was heated to 96 degrees centigrade, lasting 3min, three times, counting 10min.
(4) slices were cooled to room temperature, washed with PBS (pH 7.2-7.4), and normal goat non immune serum was added. Incubated 25min was incubated at room temperature.
(5) Rabbit anti mouse FAK or phosphorylated FAK polyclonal antibody diluted with 1:200 diluted with antibody diluent was incubated at 4 C for overnight.
(6) after washing PBS sections, the Goat anti rabbit IgG antibody was labeled with biotin and incubated with 25min in the moisture box.
(7) after washing PBS sections, SP reagent was added and incubated with 25min in the moisture box.
(8) PBS was washed and sectioned with a newly developed DAB color 3min.
(9) hematoxylin was redyed 1min, and after 1% hydrochloric acid and alcohol, it was returned to blue 10min in tap water.
(10) gradient alcohol dehydration, xylene transparent, neutral gum seal.
In the experiment, PBS was used as a negative control and no false positive. No HE was detected in each group.
3, analysis of positive cell count
Under 400 times the microscope, 10 visual fields were randomly selected in each slice, including the multinucleated granulocyte, monocyte and fibroblast positive cells. The ratio of the positive cells to the total number of these three cells in each field of vision was calculated.
(1) result judgment
The positive expression of FAK and phosphorylated FAK was located in cytoplasm, and DAB showed a brownish yellow color.
(2) statistical analysis
The data of injury time group were processed by SPSS 11 for Windows statistical software, and the data were processed by one-way ANOVA. The data were expressed as (+) s.
Western blot detection of 4, FAK and phosphorylated FAK
(1) preparation of protein samples;
(2) protein quantification;
(3) electrophoresis, 100 g / lane, 10%[w / v]SDS-PAGE, with standard molecular weight Marker as an indicator.
(4) transfer printing;
(5) blocking antigen, 5% skim milk and 0.1%Tween-20 sealing solution;
(6) Rabbit anti mouse FAK or phosphorylated FAK polyclonal antibody was incubated.
(7) the sheep were incubated against rabbit antibodies and GAPDH was used as the internal reference.
(8) ECL color.
(9) result judgment
Detection of FAK and phosphorylated FAK showed positive bands in 125kD.
experimental result
1, histopathological changes in the healing process of skin injury
The number of multinucleated granulocytes in group 3h after injury was found in group.6h, and the number of multinucleated granulocytes increased obviously in group.12h. There was a large number of multinucleated granulocytes and mononuclear cells infiltrating in group.1d of group.12h. After injury, 3D to 5D was mainly monocyte and fibroblast. After injury, the epidermal hyperplasia of 7D group was obvious, 10d and 14 Group D completely covered the wound.
2, the expression of FAK and phosphorylated FAK in injured area and surrounding area during skin wound healing.
(1) FAK in the control group, the epidermis, the hair follicle, the sebaceous gland, the sweat gland epithelial cells and the vascular endothelial cells were positive staining. After the injury, the 0h tissue section of the experimental group was basically the same as the control group, and the positive cells began to appear in the same.3h. After the injury, 6h to 12h, most of the infiltrating multinucleated and mononuclear cells were positive for.1d to 3D fibroblasts. Almost all positive staining of.5d ~ 10d positive staining decreased gradually and the weakest expression was found in 14d.
(2) the positive staining of phosphorylated FAK in the mouse epidermis, hair follicle, sebaceous gland, sweat gland epithelial cells and vascular endothelial cells was also positive, but the expression intensity was weaker than that of FAK. The positive cells of 3H began to appear after the injury in the experimental group, the positive staining of 12h was the most obvious, the expression intensity was slightly weakened, the 5D to 7d gradually weakened, and the 14d only weak expression.
3, positive cell count and statistical analysis
(1) FAK: the positive cell rate of FAK was lower in 3h after injury (9.34% + 1.42%), the ratio of positive cells in 6h group began to rise (21.57% + 0.91%), the ratio of 12h positive cells continued to rise (29.51% + 0.76%), and the ratio of positive cells in group 1D increased significantly (36.22% + 1.96%). The ratio of positive cells to 3D after injury reached the highest (53.48% + 2.74%).5d The ratio of positive cells in the group began to decrease (45.17% + 3.21%), the ratio of positive cells in the 7d Group continued to decrease (39.96% + 1.77%), and the ratio of the 10d group decreased to (26.71% + 2.33%). The ratio of 14d FAK positive cells decreased to the lowest (12.15% + 1.64%) after injury. The positive cell ratio between the two groups adjacent to the 0h group except the FAK group was compared with the 0h group. The difference of rate was statistically significant (P < 0.01).
(2) phospho-FAK: the positive cell rate of phospho-FAK was lower (5.48% + 3.23%) after injury (5.48% + 3.23%). The ratio of positive cells in group 6h increased rapidly (36.32% + 3.76%). The ratio of positive cells in group 12h was the highest, reaching (67.58% + 2.87%). The ratio of positive cells in group 1D decreased slightly (55.65% + 3.51%). The percentage of positive cells in 3D after injury continued to decline (47.52 The ratio of positive cells in.5d group decreased to (39.71% + 6.53%), and the ratio of positive cells in group 7d decreased to (33.08% + 3.19%), and the ratio of 10d in group 10d continued to decrease (20.14% + 4.87%), and the ratio of 14d positive cells decreased to the lowest (8.29%+3.54%). By statistical analysis of variance, there were two adjacent groups of adjacent phospho-FAK except group 0h. The percentage of positive cells was statistically significant (P < 0.01).
4, Western blot results analysis
The expression of FAK in both the control group and the experimental group was in the 125kD. The expression intensity of FAK in the experimental group was not very obvious, but it still reached the peak at the time of 3D. The phosphorylated FAK was also located at 125kD, in the blank control group and the 0h group, the expression was slightly enhanced, the 6h began to increase, and the 12h group had the strongest color, and then gradually decreased to 1. Group 4D showed weak positive expression.
conclusion
1, the expression of FAK and phosphorylated FAK in the epidermis, hair follicle, sebaceous gland and vascular endothelium of normal skin indicates that FAK plays a biological role in physiological condition.
2, FAK and phospho-FAK are expressed in multinuclear cells, monocytes and fibroblasts during the wound healing process of mice skin, and FAK plays an important role in the aggregation, migration and proliferation of mononuclear cells and mononuclear cells in the healing process of mouse skin.
3, during the healing process of mouse skin, the rate of FAK and phospho-FAK positive cells changes regularly, indicating that FAK and phospho-FAK can be used as an index to deduce the time of skin incision injury, and phospho-FAK is better than FAK..
【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;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

2 許麗;楊應(yīng)周;譚衛(wèi)國(guó);吳清芳;張玉華;羅育希;;粘著斑激酶活性對(duì)氣道上皮細(xì)胞增殖的影響[J];實(shí)用臨床醫(yī)學(xué);2006年06期

3 梁光波,張國(guó)平,金惠銘,錢(qián)睿哲;粘著斑激酶在bFGF引起細(xì)胞遷移中的動(dòng)態(tài)變化及意義[J];生理學(xué)報(bào);2004年04期

4 尹航,汪麗蕙,彭旭,霍勇,夏春芳,唐朝樞;粘著斑激酶活化對(duì)平滑肌細(xì)胞粘附和遷移的影響[J];中國(guó)病理生理雜志;2002年06期

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