發(fā)布時(shí)間：2018-02-02 11:59

  本文關(guān)鍵詞： 吡格列酮 燙傷 胰島素抵抗 核因子-κB 腫瘤壞死因子α 小鼠　出處：《天津醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 探討吡格列酮對(duì)嚴(yán)重燙傷小鼠創(chuàng)面組織愈合過程中TNF-α和NF-κB的表達(dá)影響。 方法 1.建立30%全身體表面積(TBSA)Ⅲ°燙傷小鼠模型 選擇健康SPF級(jí)ICR小鼠130只(天津醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供),雌雄各半,體質(zhì)量29～32g,鼠齡6～8周。小鼠背部按30%TBSA用4%Na2S試劑退毛,腹腔注射4%水合氯醛(40mg·kg-1)麻醉,麻醉成功后將小鼠背部置于100℃熱水中燙12s,制成燙傷面積為30%的Ⅲ°燙傷模型(病理切片證實(shí),正常組不燙傷),燙傷后立即腹腔注射乳酸鈉林格液(40mg·kg-1)復(fù)蘇。 2.實(shí)驗(yàn)動(dòng)物的分組 隨機(jī)分成4組,正常組10只,對(duì)照組(Ⅰ)、實(shí)驗(yàn)Ⅱ組、實(shí)驗(yàn)Ⅲ組各40只。建模成功后于每日晨8時(shí)Ⅰ組予以0.5mL·d-1生理鹽水灌胃,Ⅱ、Ⅲ組分別予以10mg·kg-1·d-1、40mg·kg-1-d-1比格列酮灌胃(根據(jù)預(yù)實(shí)驗(yàn)和參考文獻(xiàn)確定2個(gè)劑量)。吡格列酮每日晨8時(shí)灌胃一次,連續(xù)2周。 3.觀察指標(biāo) (1)空腹血糖(FBG) 實(shí)驗(yàn)動(dòng)物禁食過夜,于晨8時(shí)斷頭迅速取血。正常組10只一次性處死,Ⅰ、Ⅱ、Ⅲ組分別于1、3、7、14d各處死10只。各組處死小鼠時(shí)用一次性血糖試紙和血糖儀即時(shí)測(cè)量FBG。 (2)空腹胰島素(FIns) 各組分別于1、3、7、14d各處死10只小鼠時(shí)(正常組10只一次性處死),收集血液標(biāo)本2mL于3000r/min、30min、4℃離心。收集血清0.5mL于-80℃保存,用于空腹胰島素測(cè)定。實(shí)驗(yàn)結(jié)束時(shí),將制備好的0.5mL血清置于樣品杯中,放置于全自動(dòng)化學(xué)發(fā)光免疫分析儀上(運(yùn)用化學(xué)發(fā)光免疫法測(cè)量FIns的含量),調(diào)整好檢測(cè)參數(shù)后開始檢測(cè),讀取光量子數(shù),根據(jù)標(biāo)準(zhǔn)曲線計(jì)算出FIns含量。 (3)計(jì)算IR值 利用穩(wěn)態(tài)模式法(HOMA)計(jì)算IR值：胰島素抵抗指數(shù)(HOMA-IR)=FBG(mmol/L)×FIns(mIU/L)/22.5。 (4)創(chuàng)面組織中TNF-α(?)(?)NF-κB的表達(dá) 將創(chuàng)面組織標(biāo)本常規(guī)石蠟包埋,厚度5μm連續(xù)切片。常規(guī)脫蠟至水后,免疫組織化學(xué)染色(SABC法)檢測(cè)TNF-α和NF-κB的表達(dá)。免疫組織化學(xué)染色(DAB顯色)結(jié)果：陽(yáng)性呈棕黃色顆粒(主要在細(xì)胞漿和/或細(xì)胞核中表達(dá))。顯微鏡下觀察并截圖：運(yùn)用計(jì)算機(jī)圖像處理軟件進(jìn)行分析,分別隨機(jī)計(jì)數(shù)每張切片5個(gè)無重復(fù)高倍視野(×400),測(cè)定平均灰度值,以灰度值表示TNF-α和NF-κB的表達(dá)情況。 4.統(tǒng)計(jì)方法 采用SPSS18.0進(jìn)行數(shù)據(jù)的統(tǒng)計(jì)和處理。數(shù)據(jù)均以x+s表示,資料采用單因素方差分析(ANOVA),若有統(tǒng)計(jì)學(xué)差異采用LSD-t檢驗(yàn),以P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.各組小鼠空腹血糖(FBG)的測(cè)定 各組處死小鼠時(shí)用一次性血糖試紙和血糖儀即時(shí)測(cè)量FBG,檢測(cè)結(jié)果顯示Ⅰ、Ⅱ、Ⅲ組FBG與正常組比較具有明顯差異(P0.05)。 2.各組小鼠空腹血清胰島素(FIns)的測(cè)定 運(yùn)用化學(xué)發(fā)光免疫法測(cè)量FIns的含量,Ⅰ、Ⅱ、Ⅲ組FIns與正常組比較具有明顯差異(P0.05)。 3.各組小鼠胰島素抵抗(IR) 同一時(shí)相點(diǎn)Ⅰ、Ⅱ、Ⅲ組HOMA-IR值兩兩比較均存在顯著性差異(P0.01)；同一組內(nèi)不同時(shí)相點(diǎn)HOMA-IR值兩兩比較,均存在差異(P0.05或P0.01)；且分別與正常組HOMA-IR值比較,亦均存在差異(P0.05)。 4.各組小鼠創(chuàng)面組織中TNF-α的表達(dá) Ⅰ、Ⅱ、Ⅲ組同一時(shí)相點(diǎn)和同一組內(nèi)不同時(shí)相點(diǎn),TNF-α灰度值兩兩之間分別進(jìn)行比較,均有顯著性差異(P0.01)。 5.各組小鼠創(chuàng)面組織中NF-κB的表達(dá) Ⅰ、Ⅱ、Ⅲ組同一時(shí)相點(diǎn)和同一組內(nèi)不同時(shí)相點(diǎn),NF-κB灰度值兩兩之間分別進(jìn)行比較,均有顯著性差異(P0.01)。 結(jié)論 1.傷后Ⅰ、Ⅱ、Ⅲ組的小鼠空腹血糖(FBG)、空腹血清胰島素(FIns)較正常組明顯升高,證明30%全身體表面積(TBSA)Ⅲ°燙傷小鼠模型建模成功(病理切片證實(shí)),產(chǎn)生明顯胰島素抵抗(IR)。 2.Ⅰ組與Ⅱ、Ⅲ組空腹血糖(FBG)、空腹血清胰島素值(FIns)比較,具有顯著性差異(P0.05),證明吡格列酮能有效控制胰島素抵抗(IR)。 3. Ⅰ、Ⅱ、Ⅲ組之間不同時(shí)相點(diǎn)TNF-α灰度值兩兩比較和同一組內(nèi)不同時(shí)相點(diǎn)兩兩比較,均有顯著性差異(P0.01)。Ⅰ、Ⅱ、Ⅲ組之間不同時(shí)相點(diǎn)NF-κB灰度值兩兩比較和同一組內(nèi)不同時(shí)相點(diǎn)兩兩比較,均有顯著性差異(P0.01)。吡格列酮能有效抑制燒(燙)傷后胰島素抵抗(IR),進(jìn)而抑制創(chuàng)面組織中TNF-a和NF-κB的表達(dá),改善和促進(jìn)創(chuàng)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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R644

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