小鼠皮肤切创愈合过程中FAK和磷酸化FAK表达及其时间规律性变化的研究

发布时间：2018-06-11 15:42

本文选题：皮肤损伤 + 损伤时间判定　；参考：《中国医科大学》2007年硕士论文

【摘要】： 目的皮肤损伤愈合大致分为三个阶段，即炎症期、纤维增生期和组织重建期。多核粒细胞、单核细胞和成纤维细胞参与皮肤损伤愈合的全过程，清除损伤、坏死的皮肤组织和入侵的病原体并重建损伤组织，对于损伤愈合具有重要作用。在炎症期和纤维增生期损伤区内多核粒细胞、单核细胞和成纤维细胞的数量急剧增加；在组织重建期，这三种细胞的数量则显著减少，直至伤口完全愈合。粘着斑激酶(focal adhesion kinase，FAK，也称pp~(125FAK))是人们最初在用v-src转化细胞时发现的一个主要酪氨酸磷酸化蛋白，是整合素信号转导中的关键分子之一，在细胞粘附过程中发挥着非常重要的作用。当被整合素激活时FAK自身磷酸化，能够直接或者通过相关激酶激活PI-3K(phosphatidylinositol 3-kinase，PI-3K)、MAPK(mitogen-activated protein kinase，MAPK)和STAT(signal transducer and activator of transcription，STAT)等信号转导分子，进而与细胞内一些相关的信号转导途径相联系，参与细胞信号转导过程。这些细胞内分子的相互作用在信号转导途径中调节各种各样的细胞功能，如扩散、迁移、增殖、凋亡和细胞的存活。本研究用免疫组织化学染色技术和Western blot方法检测小鼠皮肤切创愈合过程中FAK和磷酸化FAK在多核粒细胞、单核细胞及成纤维细胞中的表达，进而探讨通过FAK的信号转导途径及其生物学作用，并讨论通过检测FAK和磷酸化FAK表达的规律推测皮肤损伤时间的可行性，进一步揭示蛋白质的磷酸化对皮肤损伤愈合的影响。材料和方法 1、动物模型的建立及分组健康成年清洁级昆明系小鼠33只，雌雄不限，体重35-40g，随机分为11组，每组3只，其中1组为正常对照组，其余各组做为实验组。乙醚气体吸入麻醉，剪毛处理，常规手术消毒，在背部中央做一纵行长1.5cm皮肤全层切口，深达筋膜。创面自然止血，不包扎，不施药。切创后每只小鼠分笼饲养，给予消毒饲料及双蒸水并保持伤口干燥、防止伤口感染。分别于伤后0h、3h、6h、12h、1d、3d、5d、7d、10d、14d将小鼠脱颈椎处死，取伤口处1.0cm×1.5cm皮肤组织。对照组小鼠取相同部位同等大小皮肤。每组1／2皮肤固定后石蜡包埋，制作5μm厚度连续切片；另1／2皮肽匀浆，离心，取上清。 2、FAK和磷酸化FAK的免疫组织化学染色(SP法) (1)切片脱蜡，水化； (2) 3％H_2O_2／PBS灭活内源性过氧化物酶，室温25min； (3) PBS洗涤切片后进行抗原修复，置于10mM pH 6.0的柠檬酸缓冲液中，微波炉加热至96℃，持续3min，，三次、计10min； (4)切片冷却至室温，用PBS(pH 7.2-7.4)洗涤切片后滴加正常山羊非免疫血清，室温下保湿盒内孵育25min； (5)滴加用抗体稀释液1：200倍稀释的兔抗小鼠FAK或磷酸化FAK多克隆抗体，保湿盒内4℃孵育过夜； (6) PBS洗涤切片后滴加生物素标记的山羊抗兔IgG抗体，保湿盒内孵育25min； (7) PBS洗涤切片后滴加SP试剂，保湿盒内孵育25min； (8) PBS洗涤切片后用新配制的DAB显色3min； (9)苏木素核复染1min，1％盐酸-酒精中分化后在自来水中返蓝10min； (10)梯度酒精脱水，二甲苯透明，中性树胶封片。实验中以PBS替代一抗作阴性对照，未见假阳性。每组同时作HE染色。 3、阳性细胞计数分析显微镜×400倍下，在每张切片中随机选择10个视野计数包括多核粒细胞、单核细胞及成纤维细胞的阳性细胞。计算每个视野中阳性细胞与此三种细胞总数的比值。 (1)结果判断 FAK和磷酸化FAK阳性表达均位于细胞浆，用DAB显色呈棕黄色。 (2)统计学分析各损伤时间组数据应用SPSS 11.0 for Windows统计软件，采用单因素方差分析方法进行统计学处理，数据以(?)±s表示。 4、FAK和磷酸化FAK的Western blot检测 (1)蛋白样本制备； (2)蛋白定量； (3)电泳，100μg／lane，10％[w／v]SDS-PAGE，以标准分子量Marker作指示； (4)转印； (5)封闭抗原，5％脱脂牛奶和0.1％Tween-20封闭液； (6)兔抗小鼠FAK或磷酸化FAK多克隆抗体孵育； (7)羊抗兔抗体孵育，以GAPDH为内参； (8) ECL显色。 (9)结果判断检测FAK和磷酸化FAK，在125kD显示阳性谱带。实验结果 1、皮肤损伤愈合过程的病理组织学变化伤后3h组损伤区出现少量多核粒细胞。6h组见多核粒细胞数量明显增加。12h组皮下组织内有大量渗出的多核粒细胞。1d组损伤区有大量的多核粒细胞及单核细胞浸润。伤后3d～5d以单核细胞和成纤维细胞为主。伤后7d组表皮增生明显，10d和14d组完全覆盖伤口。 2、FAK和磷酸化FAK在皮肤切创愈合过程中损伤区及损伤周边区的表达 (1) FAK在对照组中小鼠的表皮、毛囊、皮脂腺、汗腺上皮细胞和血管内皮细胞均呈阳性染色。实验组伤后0h组织切片染色与对照组基本相同。3h开始出现阳性细胞，伤后6h～12h，大部分浸润的多核粒细胞和单核细胞为阳性。1d～3d成纤维细胞大量增生，几乎全部阳性着色。5d～10d阳性着色逐渐减弱，到14d呈最弱表达。 (2)磷酸化FAK在对照组中小鼠表皮、毛囊、皮脂腺、汗腺上皮细胞和血管内皮细胞亦有阳性染色，但其表达强度较FAK弱。实验组伤后3h开始出现阳性细胞，12h阳性染色最为明显，之后表达强度稍有减弱，5d～7d逐渐减弱，14d仅有微弱表达。 3、阳性细胞计数及统计学分析 (1) FAK：伤后3h之内，FAK的阳性细胞率较低(9.34％±1.42％)，6h组阳性细胞比率开始升高(21.57％±0.91％)，12h阳性细胞比率继续升高(29.51％±0.76％)，1d组阳性细胞比率升高明显(36.22％±1.96％)，在伤后3d，阳性细胞比率达到最高(53.48％±2.74％)。5d组阳性细胞比率开始下降(45.17％±3.21％)，7d组阳性细胞比率继续下降(39.96％±1.77％)，10d组该比率下降为(26.71％±2.33％)，伤后14d FAK阳性细胞比率降到最低(12.15％±1.64％)。经统计学方差分析，除0h组外FAK各时间段相邻两组之间阳性细胞比率差异有统计学意义(P＜0.01)。 (2) phospho-FAK：伤后3h之内，phospho-FAK的阳性细胞率较低(5.48％±3.23％)，6h组阳性细胞比率迅速升高(36.32％±3.76％)，12h组阳性细胞比率最高，达(67.58％±2.87％)，1d组阳性细胞比率稍有下降(55.65％±3.51％)，在伤后3d，阳性细胞比率继续下降(47.52％±3.89％)。5d组阳性细胞比率下降为(39.71％±6.53％)，7d组阳性细胞比率下降到(33.08％±3.19％)，10d组该比率继续下降(20.14％±4.87％)，伤后14d阳性细胞比率降到最低(8.29％+3.54％)。经统计学方差分析，除0h组外phospho-FAK各时间段相邻两组之间阳性细胞比率差异有统计学意义(P＜0.01)。 4、Western blot结果分析 FAK在对照组及实验组均有表达，位于125kD处。在实验组FAK表达强度变化不是十分明显，但仍可见在3d时达到高峰。磷酸化FAK亦位于125kD处，在空白对照组及0h组表达较弱，在3h组表达略增强，6h开始显著增强，至12h组显色最强，此后逐渐下降，至14d组呈弱阳性表达。结论 1、正常皮肤的表皮层、毛囊、皮脂腺和血管内皮中表达FAK及磷酸化FAK，提示FAK在生理状态下就发挥生物学作用。 2、小鼠皮肤切创愈合过程中，FAK和phospho-FAK在多核粒细胞、单核细胞及成纤维细胞中表达，并且在小鼠皮肤切创愈合过程中，FAK对于多核粒细胞和单核细胞的聚集、迁移及成纤维细胞的增殖起着重要作用。 3、在小鼠皮肤切创愈合过程中，FAK和phospho-FAK阳性细胞率呈时间规律性变化，表明FAK和phospho-FAK可作为推断皮肤切创损伤时间的指标，且phospho-FAK要优于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..
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919

【参考文献】