吡格列酮对严重烫伤小鼠创面组织中NF-κB和TNF-α表达的影响

发布时间：2018-02-02 11:59

本文关键词： 吡格列酮烫伤胰岛素抵抗核因子-κB 肿瘤坏死因子α 小鼠　出处：《天津医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的探讨吡格列酮对严重烫伤小鼠创面组织愈合过程中TNF-α和NF-κB的表达影响。方法 1.建立30%全身体表面积(TBSA)Ⅲ°烫伤小鼠模型选择健康SPF级ICR小鼠130只(天津医科大学实验动物中心提供),雌雄各半,体质量29～32g,鼠龄6～8周。小鼠背部按30%TBSA用4%Na2S试剂退毛,腹腔注射4%水合氯醛(40mg·kg-1)麻醉,麻醉成功后将小鼠背部置于100℃热水中烫12s,制成烫伤面积为30%的Ⅲ°烫伤模型(病理切片证实,正常组不烫伤),烫伤后立即腹腔注射乳酸钠林格液(40mg·kg-1)复苏。 2.实验动物的分组随机分成4组,正常组10只,对照组(Ⅰ)、实验Ⅱ组、实验Ⅲ组各40只。建模成功后于每日晨8时Ⅰ组予以0.5mL·d-1生理盐水灌胃,Ⅱ、Ⅲ组分别予以10mg·kg-1·d-1、40mg·kg-1-d-1比格列酮灌胃(根据预实验和参考文献确定2个剂量)。吡格列酮每日晨8时灌胃一次,连续2周。 3.观察指标 (1)空腹血糖(FBG) 实验动物禁食过夜,于晨8时断头迅速取血。正常组10只一次性处死,Ⅰ、Ⅱ、Ⅲ组分别于1、3、7、14d各处死10只。各组处死小鼠时用一次性血糖试纸和血糖仪即时测量FBG。 (2)空腹胰岛素(FIns) 各组分别于1、3、7、14d各处死10只小鼠时(正常组10只一次性处死),收集血液标本2mL于3000r/min、30min、4℃离心。收集血清0.5mL于-80℃保存,用于空腹胰岛素测定。实验结束时,将制备好的0.5mL血清置于样品杯中,放置于全自动化学发光免疫分析仪上(运用化学发光免疫法测量FIns的含量),调整好检测参数后开始检测,读取光量子数,根据标准曲线计算出FIns含量。 (3)计算IR值利用稳态模式法(HOMA)计算IR值：胰岛素抵抗指数(HOMA-IR)=FBG(mmol/L)×FIns(mIU/L)/22.5。 (4)创面组织中TNF-α(?)(?)NF-κB的表达将创面组织标本常规石蜡包埋,厚度5μm连续切片。常规脱蜡至水后,免疫组织化学染色(SABC法)检测TNF-α和NF-κB的表达。免疫组织化学染色(DAB显色)结果：阳性呈棕黄色颗粒(主要在细胞浆和/或细胞核中表达)。显微镜下观察并截图：运用计算机图像处理软件进行分析,分别随机计数每张切片5个无重复高倍视野(×400),测定平均灰度值,以灰度值表示TNF-α和NF-κB的表达情况。 4.统计方法采用SPSS18.0进行数据的统计和处理。数据均以x+s表示,资料采用单因素方差分析(ANOVA),若有统计学差异采用LSD-t检验,以P0.05为差异有统计学意义。结果 1.各组小鼠空腹血糖(FBG)的测定各组处死小鼠时用一次性血糖试纸和血糖仪即时测量FBG,检测结果显示Ⅰ、Ⅱ、Ⅲ组FBG与正常组比较具有明显差异(P0.05)。 2.各组小鼠空腹血清胰岛素(FIns)的测定运用化学发光免疫法测量FIns的含量,Ⅰ、Ⅱ、Ⅲ组FIns与正常组比较具有明显差异(P0.05)。 3.各组小鼠胰岛素抵抗(IR) 同一时相点Ⅰ、Ⅱ、Ⅲ组HOMA-IR值两两比较均存在显著性差异(P0.01)；同一组内不同时相点HOMA-IR值两两比较,均存在差异(P0.05或P0.01)；且分别与正常组HOMA-IR值比较,亦均存在差异(P0.05)。 4.各组小鼠创面组织中TNF-α的表达 Ⅰ、Ⅱ、Ⅲ组同一时相点和同一组内不同时相点,TNF-α灰度值两两之间分别进行比较,均有显著性差异(P0.01)。 5.各组小鼠创面组织中NF-κB的表达 Ⅰ、Ⅱ、Ⅲ组同一时相点和同一组内不同时相点,NF-κB灰度值两两之间分别进行比较,均有显著性差异(P0.01)。结论 1.伤后Ⅰ、Ⅱ、Ⅲ组的小鼠空腹血糖(FBG)、空腹血清胰岛素(FIns)较正常组明显升高,证明30%全身体表面积(TBSA)Ⅲ°烫伤小鼠模型建模成功(病理切片证实),产生明显胰岛素抵抗(IR)。 2.Ⅰ组与Ⅱ、Ⅲ组空腹血糖(FBG)、空腹血清胰岛素值(FIns)比较,具有显著性差异(P0.05),证明吡格列酮能有效控制胰岛素抵抗(IR)。 3. Ⅰ、Ⅱ、Ⅲ组之间不同时相点TNF-α灰度值两两比较和同一组内不同时相点两两比较,均有显著性差异(P0.01)。Ⅰ、Ⅱ、Ⅲ组之间不同时相点NF-κB灰度值两两比较和同一组内不同时相点两两比较,均有显著性差异(P0.01)。吡格列酮能有效抑制烧(烫)伤后胰岛素抵抗(IR),进而抑制创面组织中TNF-a和NF-κB的表达,改善和促进创面的愈合。
[Abstract]:objective
To investigate the effect of pioglitazone on the expression of TNF- - alpha and NF- - kappa B in the healing process of wound tissue in severely scalded mice.
Method
1. establishment of a 30% total body surface area (TBSA) model of scald in mice
Choose healthy SPF 130 ICR mice (experimental animal center of Medical University Of Tianjin), male and female, body mass of 29 to 32g rats aged 6~8 weeks. Mice back by 30%TBSA using 4%Na2S reagent back hair, intraperitoneal injection of 4% chloral hydrate (40mg - kg-1) anesthesia, after the success of anesthesia will be placed in the back of mice in hot water of 100 DEG C hot 12s, burn area was made scald model 30% (pathologically, the normal group did not burn, scald) immediately after intraperitoneal injection of sodium lactate Ringer's solution (40mg - kg-1) recovery.
2. groups of experimental animals
Were randomly divided into 4 groups, 10 rats in the normal group, the control group (I), experimental group II, III experimental group with 40 rats in each group. After the success of modeling in the daily morning 8 when I 0.5mL group received D-1 normal saline, group II, III respectively to 10mg - kg-1 - d-1,40mg - kg-1-d-1 pioglitazone orally (according to the pre experiment and references to determine the 2 dose). Pioglitazone daily morning 8 gavage once, for 2 consecutive weeks.
3. observation index
(1) fasting blood glucose (FBG)
Animals fasted overnight, and blood was taken off at 8 in the morning. 10 rats in the normal group were sacrificed at once. Group I, II and III were killed at 1,3,7,14d. 10 rats were sacrificed in each group. The FBG. was immediately measured by single blood glucose test paper and blood glucose meter.
(2) fasting insulin (FIns)
Rats in each group were 10 rats of each group were sacrificed 1,3,7,14d mice (10 rats in normal group were sacrificed), blood samples were collected from 2mL to 3000r/min, 30min, 4 DEG C centrifugal. Serum was collected at -80 deg.c for 0.5mL preservation, fasting insulin were measured. At the end of the experiment, the prepared 0.5mL serum sample cup, placed in the automatic chemiluminescence immunoassay analyzer (content using chemiluminescence immunoassay measurement FIns), adjust the detection parameters after detection, read quantum number, FIns content was calculated according to the standard curve.
(3) calculating the IR value
The IR value of insulin resistance index (HOMA-IR) =FBG (mmol/L) * FIns (mIU/L) /22.5. is calculated by the steady-state model method (HOMA)
(4) expression of TNF- alpha (?) (?) NF- kappa B in the wound tissue
The wound tissues were embedded in paraffin, the thickness of 5 m continuous sections. Conventional dewaxing to water, immunohistochemical staining (SABC method) to detect the expression of TNF- alpha and NF- kappa B. Immunohistochemical staining (DAB staining) results: positive brownish yellow granules (mainly expressed in the cytoplasm and / or in the nucleus). Observe and screenshots under the microscope: carries on the analysis using the computer image processing software, respectively, counting each slice 5 non repeat HPF (* 400), the determination of the average gray value, the gray value of the expression of TNF- alpha and NF- kappa B.
4. statistical methods
Data and statistics were processed by SPSS18.0. Data were all expressed in x+s. Data were analyzed by one-way ANOVA (ANOVA). If there was statistical difference, LSD-t test was used. The difference was statistically significant.
Result
1. the determination of fasting blood glucose (FBG) in mice of each group
In each group, FBG was immediately measured by disposable blood glucose test paper and blood glucose meter. The results showed that FBG in group I, II and III was significantly different from that in normal group (P0.05).
2. the determination of serum insulin (FIns) in the fasting serum of mice in each group
The content of FIns was measured by chemiluminescent immunoassay. The FIns in group I, II, and group III was significantly different from that of the normal group (P0.05).
3. insulin resistance (IR) in mice of each group
There were significant differences in the HOMA-IR value 22 at the same time point I, II and III groups (P0.01). There were differences (HOMA-IR or P0.05) in the same time points in the same group, and there were differences (P0.05 or P0.01) in the same time points, and there was also a difference between the HOMA-IR values of the same group and the normal group (P0.05).
4. expression of TNF- alpha in the wound tissue of mice in each group
In group I, II, and group III, the same phase point and the same group are not at the same time, and the TNF- alpha gray value 22 is compared, and there are significant differences (P0.01).
5. expression of NF- kappa B in the wound tissue of mice
In group I, II, and group III, the same phase point and the same group were different in the same group, and the NF- kappa B gray value 22 were compared, and there were significant differences (P0.01).
conclusion
1., after injury, the fasting blood glucose (FBG) and fasting serum insulin (FIns) in group I, II and III were significantly higher than those in the normal group. It was proved that 30% of the whole body surface area (TBSA) and third degree scald mice were successfully modeled (confirmed by pathological sections), resulting in significant insulin resistance (IR).
2. there was a significant difference between group I and group II and group 3 in fasting blood glucose (FBG) and fasting serum insulin (FIns) (P0.05), indicating that pioglitazone can effectively control insulin resistance (IR).
3. I, II, III group between different time points TNF- a gray value 22 compared with the same group in different time points of 22, there were significant differences (P0.01). I, II, III group between different time points of NF- kappa B 22 gray value compared with the same group at different time 22, there were significant differences (P0.01). Pioglitazone can inhibit burn (scald) insulin resistance after injury (IR), and inhibit the expression of TNF-a and NF- K B in the wound tissue, improve and promote wound healing.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R644

【参考文献】