【摘要】：研究背景 近年來，急性高血糖引起人們越來越多的關注。此種高血糖往往發(fā)生在重度燒傷、感染、創(chuàng)傷、心梗和大手術后等危重癥狀態(tài)下，表現(xiàn)為血糖迅速升高和患者既往無糖尿病病史的特點，而由于在多項臨床研究中，血糖升高幅度與機體損傷程度、患者死亡率等不良預后指標高度相關,因而又常被稱為“危重癥糖尿病”。 在急性高血糖的同時，患者往往出現(xiàn)高胰島素血癥和糖耐量受損，提示其存在胰島素抵抗現(xiàn)象。而在嚴重燒傷、體外循環(huán)術后、心梗急性期等患者均有用胰島素控制血糖效果不佳的報道，進一步反映危重癥情況下機體對胰島素敏感性降低。作為慢性病“共同土壤”的胰島素抵抗往往經年累月才能出現(xiàn)，而危重癥患者所出現(xiàn)的急性胰島素抵抗（AIR）則具有快速發(fā)生的特點，僅需要幾天，幾小時甚至數分鐘就可出現(xiàn)，這種在出現(xiàn)時間上的巨大差異是否意味著二者在產生機制和其它表現(xiàn)形式上有所不同，其臨床意義何在，目前均不清楚。 另外，從Greet Van den Berghe等人進行的Leuven研究開始，人們就開始關注用胰島素嚴格控制血糖水平對ICU病人預后的影響。但隨后名為“NICE-SUGAR”的多中心大樣本隨機對照研究得出了強化胰島素治療對患者死亡率沒有影響，且會增加低血糖風險的陰性結論。由于褒貶不一，強化胰島素治療目前仍難以在臨床廣泛應用。但是否AIR的出現(xiàn)是導致強化胰島素治療效果欠佳的重要原因，成為當前亟待回答的重要問題。 在大面積重度燒傷病人和相應燒、燙傷動物模型中，高血糖的出現(xiàn)均較為典型。因此，本研究擬以重度燙傷大鼠作為實驗動物模型，深入研究高血糖和急性胰島素抵抗出現(xiàn)的特點，以期有助于指導臨床危重癥患者的診療工作。研究目的 1.觀察重度燙傷大鼠早期高血糖特點及其與大鼠死亡率之間的關系。 2.探究重度燙傷后大鼠是否存在急性胰島素抵抗和其臨床意義。 實驗方法 將80只雄性SD大鼠（200-220g）腹、背部固定區(qū)域剃毛后隨機分為（1）假傷組(37℃溫水處理)；（2）燙傷組（95℃熱水處理，背部燙傷15s，腹部8s）；（3）早期胰島素治療組（燙傷后立即皮下注射胰島素2.5IU/kg，目的在于控制燙傷后第1個高血糖峰）；（4）晚期胰島素治療組（于燙傷后2.5h皮下注射胰島素2.5IU/kg，目的是控制燙傷后出現(xiàn)的第2個高血糖峰），每組20只，乙醚麻醉后用自制模具造成40%總體表面積（TBSA）重度燙傷，腹腔注射生理鹽水抗休克（40ml/kg）后分組給予不同處理。 1.于大鼠燙傷前和燙傷后30min、1h、3h、6h、12h分別剪尾采血觀察急性期各組大鼠血糖變化； 2.于大鼠燙傷前和燙傷后30min、1h、3h、6h、12h頸動脈取血，靜置離心后取血清，用放免法測定各時間點胰島素、胰高血糖素和糖皮質激素的變化； 3.計算燙傷組大鼠HOMA-IR指數［血清胰島素(μU/ml)×血糖(mM)/22.5］作為胰島素抵抗程度參考，采用腹腔注射葡萄糖耐量實驗（IPGTT）和胰島素敏感性實驗(IST)分別測定燙傷后3h和10h組大鼠的胰島素敏感性； 4.在燙傷后3h測定各組大鼠血流動力學指標：平均動脈壓（MABP）、左室收縮壓（LVSP）、心率（HR）、左室收縮/舒張壓最大變化速率（±LVdP/dtmax）和皮膚微循環(huán)灌注情況； 5.用Western-blot測定假傷組和燙傷組大鼠心肌、骨骼肌組織pAkt/Akt，pGSK3β/GSK3β和peNOS/eNOS在燙傷后30min、1h、3h、6h、12h的表達量與活性變化； 6.分別記錄各組大鼠燙傷后12h內生存曲線與14d內燙傷大鼠的體重、糖耐量和存活率變化情況。 實驗結果 1.重度燙傷大鼠在傷后12h內出現(xiàn)兩個血糖峰值，分別是在燙傷后30min（7.4±0.3mM,較基礎血糖升高13%, n=32, P0.05）和燙傷后3h（10.0±1.0mM,較基礎血糖升高35%, n=31, P0.01）。進行相關分析發(fā)現(xiàn)兩血糖峰值之間呈線性相關（r2=0.787, P0.01, n=31）。以燙傷后3h大鼠血糖值中位數（8mM）作為分組依據，可發(fā)現(xiàn)燙傷后較低血糖組大鼠生存率（BG8mM,92%）明顯高于高血糖組（BG8mM,44%）（n=12-16, P0.05）。 2.與假傷組相比，燙傷組大鼠血清胰高血糖素在傷后30min顯著升高（n=6, P0.05），之后在6h逐漸回落（n=6, P0.05），，而燙傷組糖皮質激素和胰島素在燙傷后12h內較假傷組始終處于顯著升高水平(n=6,P0.05)。 3. HOMA-IR值提示重度燙傷后大鼠出現(xiàn)胰島素抵抗。在燙傷后3h和10h行腹腔糖耐量實驗和胰島素敏感性實驗，結果表明燙傷后存在急性胰島素抵抗（AIR）現(xiàn)象，且在燙傷后3h機體胰島素敏感性最低(n=6,P0.05)。 4. Western-blot結果顯示，燙傷組大鼠心肌和骨骼肌中pAkt/Akt,pGSK3β/GSK3β和peNOS/eNOS比值均隨時間發(fā)生變化，表現(xiàn)為燙傷后30min起開始升高，至1h達到峰值(n=4, P0.05)，3h后逐漸下降，至12h趨于穩(wěn)定。表明在重度燙傷所致的應激條件下，盡管機體胰島素水平升高，但其信號通路下游分子在傷后3h出現(xiàn)激活障礙。 5.與假傷組相比，大鼠在燙傷后3h MABP（97.6±5.7vs.126.7±6.7mmHg, n=6, P0.05），LVSP（97.6±6.7vs.146.7±7.7mmHg, n=6,P0.01），HR（348.8±17.4vs.450±17.1bpm, n=6, P0.01），±LVdP/dtmax（2746.8±432.4vs.5581±384.7mmHg/s, n=6, P0.01；1955±244.7vs.4532±439.0mmHg/s, n=6, P0.01）和體表微循環(huán)灌注均顯著降低（n=6, P0.05），表明重度燙傷后心血管功能明顯受損。 6.與燙傷組相比，在燙傷后立即給予胰島素治療可以抑制隨后出現(xiàn)的30min與3h血糖峰值，顯著提高燙傷后3h與10h的胰島素敏感性（n=6,P0.05），增強3h心臟血流動力學和體表微循環(huán)灌注（n=6, P0.05），最終使早期胰島素治療組大鼠燙傷后12h內存活率較燙傷組顯著提高（93%vs.71%, n=34-43, P0.05），14d內存活狀態(tài)（體重，糖耐量和生存率）也較燙傷組明顯改善（n=6-43, P0.05）。 7.在燙傷后2.5h予以同等劑量胰島素治療后，與燙傷組相比，雖然能暫時降低燙傷后3h血糖峰值，但隨后另一血糖峰會在傷后8h出現(xiàn)，12h內大鼠存活率較燙傷組有下降趨勢但未見顯著差異(55.6%vs.71%, n=18-34, P=0.17)，且14d內存活狀態(tài)（體重，糖耐量和生存率）與燙傷組相比無改善（n=6-34, P=0.21）。 結論 1．重度燙傷大鼠在急性期會出現(xiàn)兩個高血糖峰，其中第2個血糖峰的出現(xiàn)往往與預后不良密切相關。 2．從整體胰島素敏感性和分子信號兩方面證明重度燙傷大鼠出現(xiàn)急性胰島素抵抗現(xiàn)象（AIR）,且在燙傷后3h最為嚴重。 3．分別干預兩個血糖峰，發(fā)現(xiàn)針對第一個血糖峰（H1）的胰島素治療不僅能有效降低血糖，還可減輕AIR，改善心功能和體表微循環(huán)灌注，最終提高大鼠存活率;而用同等劑量胰島素控制第2個血糖峰（H2）則難以發(fā)揮相應保護作用，推測其機制可能與AIR的出現(xiàn)有關。
[Abstract]:Research background
In recent years, acute hyperglycemia has attracted more and more attention. This hyperglycemia often occurs in severe burns, infections, trauma, myocardial infarction, and major postoperative critical conditions, characterized by rapid rise in blood sugar and no history of diabetes in the patient, but the increase in blood sugar and the body's injury process in many clinical studies It is highly correlated with adverse prognostic indicators such as mortality, and is often referred to as "critical diabetes".
At the same time of acute hyperglycemia, patients often have hyperinsulinemia and impaired glucose tolerance, suggesting the presence of insulin resistance. In severe burns, patients with acute myocardial infarction, after cardiopulmonary bypass, are used to report the poor effect of insulin on blood glucose control, further reflecting the sensitivity of the body to insulin sensitivity in critical conditions. Low. Insulin resistance, as a chronic disease "common soil", often occurs over the years, and the acute insulin resistance (AIR) of critically ill patients has a rapid occurrence, only a few days, a few hours or even a few minutes. Whether the huge difference in time means that the two are producing the machine. It is unclear what the clinical significance of the system differs from other forms.
In addition, from the Leuven study conducted by Greet Van den Berghe and others, people began to pay attention to the effect of insulin on the prognosis of ICU patients strictly controlled by insulin. However, the subsequent multicenter, randomized controlled study called "NICE-SUGAR" has concluded that intensive insulin therapy does not affect the mortality of patients and increases the low blood pressure. The negative conclusion of sugar risk is still difficult to be widely used in clinical practice because of mixed treatment. But whether the emergence of AIR is an important cause of the poor effect of intensive insulin therapy and is an important question to be answered urgently.
In the large area of severe burn patients and the corresponding burn and scald animal models, the appearance of hyperglycemia is more typical. Therefore, this study intends to use severe scald rats as an experimental animal model to further study the characteristics of hyperglycemia and acute insulin resistance in order to help guide the diagnosis and treatment of clinical critical patients.
1. to observe the relationship between hyperglycemia and mortality in severely scalded rats.
2. to investigate whether acute insulin resistance and its clinical significance exist after severe scald in rats.
Experimental method
After shaving 80 male SD rats (200-220g), the back fixed area was randomly divided into (1) false injury group (37 C warm water treatment); (2) scald group (95 C hot water treatment, back scald 15s, abdominal 8s); (3) early insulin treatment group (immediately after scald, subcutaneous injection of insulin 2.5IU/kg, aimed at controlling first hyperglycemia peaks after scald); (4) late late. The insulin treatment group (2.5h subcutaneous injection of insulin 2.5IU/kg after scald was designed to control second hyperglycemia peaks in each group), 20 in each group. After eether anesthesia, the total surface area (TBSA) of the total surface area (TBSA) was severely scalded, and the intraperitoneal injection of saline against Hugh (40ml/kg) was given to different treatments.
1. blood samples were taken from rats before scalding and scalding for 30min, 1H, 3h, 6h and 12h respectively to observe the blood glucose changes in each group during acute phase.
2. the blood of 30min, 1H, 3h, 6h, 12h was taken before and after scald in rats. The serum was taken after static centrifugation. The changes of insulin, glucagon and glucocorticoid were measured by radioimmunoassay at all time points.
3. the HOMA-IR index [serum insulin (micron) (mM) /22.5] of rats in the scald group was calculated as a reference for insulin resistance. The insulin sensitivity was measured by intraperitoneal injection of glucose tolerance test (IPGTT) and insulin sensitivity test (IST), respectively, in group 3H and 10h group after scald.
4. after the scald, the hemodynamic indexes of the rats were measured by 3H: mean arterial pressure (MABP), left ventricular systolic pressure (LVSP), heart rate (HR), the maximum change rate of left ventricular systolic / diastolic pressure (+ LVdP/dtmax) and skin microcirculation perfusion.
5. the myocardium of rats in the injured group and the scald group was measured by Western-blot. The expression and activity of 30min, 1H, 3h, 6h, 12h in the skeletal muscle tissue pAkt/Akt, pGSK3 beta /GSK3 beta and peNOS/eNOS were changed after the scald.
6. the survival curves of 12h after scald and the changes of body weight, glucose tolerance and survival rate of scalded rats in each 14d group were recorded respectively.
experimental result
In 1. severe scald rats, there were two peak blood glucose peaks in 12h after injury, which were 30min (7.4 + 0.3mM, 13%, n=32, P0.05) after scald and 3H (10 + 1.0mM, 35%, n=31, P0.01) after scald. The correlation analysis showed that the peak of two blood sugar showed linear correlation (r2=0.787, P0.01, n=31). 3 after scald. The median blood glucose value (8mM) of H rats was used as a basis for grouping, and the survival rate of rats in the lower blood glucose group after scald (BG8mM, 92%) was significantly higher than that in the hyperglycemia group (BG8mM, 44%) (n=12-16, P0.05).
2. compared with the false injury group, the serum glucagon in the scald group increased significantly after the injury (n=6, P0.05), and then decreased gradually in the 6h (n=6, P0.05), while the glucocorticoid and insulin in the scald group remained significantly higher than the false group in the scald group (n=6, P0.05) in the scald group after the burn.
3. HOMA-IR values showed insulin resistance in rats after severe scald. In 3H and 10h after scald, intraperitoneal glucose tolerance test and insulin sensitivity test showed that there was acute insulin resistance (AIR) after scald, and the insulin sensitivity was the lowest in 3h after scald (n=6, P0.05).
4. Western-blot results showed that the ratio of pAkt/Akt, pGSK3 beta, /GSK3 beta and peNOS/eNOS in the myocardium and skeletal muscle of the scalded rats varied with time, which showed that 30min began to rise after scald, to the peak of 1H (n=4, P0.05), and the 3H decreased gradually to 12h, indicating that the pancreas in severe scald caused by the stress conditions, despite the body pancreas. The level of islet is increased, but the downstream molecules of signaling pathway are activated after 3H.
5. 3H MABP (97.6 + 5.7vs.126.7 + 6.7mmHg, n=6, P0.05), LVSP (97.6 + 6.7vs.146.7 + 7.7mmHg, n=6, P0.01) after scald, LVSP (97.6 + 6.7vs.146.7 + 7.7mmHg, n=6, P0.01). Perfusion significantly decreased (n=6, P0.05), indicating severe cardiovascular injury after severe scald.
6. compared with the scald group, insulin treatment immediately after scald could inhibit the subsequent peak of 30min and 3H blood glucose, significantly increase the insulin sensitivity of 3H and 10h after scald (n=6, P0.05), enhance 3H cardiac hemodynamics and body surface microcirculation perfusion (n=6, P0.05), and eventually lead to the survival of the early insulin treatment group after the scald in the rat 12h. Compared with the scald group, the survival rate was significantly increased (93% vs. 71%, n = 34-43, P 0.05). The living state (body weight, glucose tolerance and survival rate) in 14 days was also significantly improved (n = 6-43, P 0.05).
7. after the same dose of insulin after the scald, 2.5h was compared with the scald group, although it could temporarily reduce the peak of 3H blood sugar after scald, but then the other blood sugar summit appeared at 8h after injury. The survival rate in 12h rats was lower than that in the scald group, but there was no significant difference (55.6%vs.71%, n=18-34, P=0.17), and the memory of 14d (weight, glucose tolerance) There was no improvement in the volume and survival rate compared with the scald group (n=6-34, P=0.21).
conclusion
1. Two hyperglycemic peaks appear in the acute phase of Severe Scalded rats, and the second one is often closely related to poor prognosis.
2. Acute insulin resistance (AIR) was found in severely scalded rats in terms of overall insulin sensitivity and molecular signaling, and it was most severe at 3 hours after scald.
3. intervention of two blood glucose peaks, and it is found that insulin therapy against the first glucose peak (H1) can not only effectively reduce blood sugar, but also reduce AIR, improve cardiac function and body surface microcirculation perfusion, and ultimately improve the survival rate of rats, while the same dose of insulin control second blood glucose peaks (H2) is difficult to play the corresponding protective effect, speculate the mechanism It may be related to the emergence of AIR.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363

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