创伤性脑损伤大鼠RSTN基因表达特征及RSTN对创伤后胰岛素敏感性的影响作用
|
摘要
目的
建立自由落体致颅脑损伤SD大鼠模型,探讨创伤后脑损伤TBI抵抗素基因表达的特征及其对创伤后胰岛素敏感性的影响作用。方法
清洁级SD(Spruge-Dawley)雄性大鼠216只,体重300～350g。计算机编号随机抽取方法均分为6组：正常对照组,假手术组,轻度损伤组,重度损伤组,外源性磷酸脂多糖(LPS)注射组,RSTN抗体注射组。每组36只。大鼠用10%水合氯醛(0.35g/kg体重)腹腔内注射麻醉后,固定于立体定向仪消毒并切开头皮(沿中线偏左侧)分离骨膜,于紧挨冠状缝后中线旁2mm用牙科电钻开一直径5mm的圆形窗,硬膜保持完整。采用自制改进型Feeney自由落体创伤性脑损伤装置造成大鼠左顶叶局限性脑损伤：用20g砝码于10cm处通过一金属导杆坠落,撞击置于硬膜上的圆锥上致轻型损伤,另用40g砝码于25cm高处坠落致重型损伤。外源性磷酸脂多糖(LPS)注射组(以下简称LPS组)用10%水合氯醛(0.35g/kg体重)腹腔内注射麻醉大鼠后,将其头部固定于立体定向仪上,以前囟为零点值,于前囟后5mm,中线左侧4mm处,用牙科电钻钻开颅骨,切开硬脑膜,用微量注射器自大脑表面进针(垂直)1.8mm,缓慢注射5ul LPS溶液(25ug),留针2min,缓慢拔针,缝合皮肤,松解大鼠,待其然苏醒。RSTN抗体注射组(以下简称RSTN组)取重度损伤组大鼠,按1mg/只剂量腹腔注射。正常对照组(以下简称正组)不做任何处理。假手术组(以下简称假组)仅切开头皮和颅骨开窗,不致脑损伤。对重度损伤组和RSTN组于术后24小时,1周,4周应用Longa评分法进行神经行为学评分。各组均于术后12小时,24小时,72小时,1周,2周,4周尾静脉采血,供ELISA法检测血糖,血清胰岛素,血清抵抗素并以-ln(FPG(血糖)×FZNS(胰岛素))计算定量胰岛素敏感性检测指数(QUICKI),同时处死大鼠,取同侧和对侧海马、下丘脑、皮层组织,供逆转录-聚合酶链式反应(RT-PCR)法检测抵抗素mRNA及Western blot法检测抵抗素蛋白的表达。
结果
(1)血清抵抗素浓度在正常组和假手术组各时间点之间均没有显著差异(各P均>0.05)。以假手术组为基准,轻度损伤组、重度损伤组、LPS组和RSTN组在建模成功后的各个检测时点(12h、24h、72h、1W、2w和4w)均出现血清抵抗素水平的显著升高(各P均<0.05)；各组平均血清抵抗素浓度：重度损伤组>RSTN组>LPS组>轻度损伤组>假手术组(各P均<0.05)。组内血清抵抗素随时间的变化趋势：轻度损伤组与重度损伤组均于12h出现升高,持续升高至4w达到峰值(各P均<0.05)。LPS注射组在12h开始升高,72h达到峰值,到1w时出现下降,持续下降到4w(各P均<0.05);RSTN组12h升高至重度损伤组水平(P>0.05),24h时出现下降,72h时又出现升高,持续升高至4w达到峰值(各P均<0.05)。比较重度损伤组和RSTN组血清抵抗素浓度发现两者差异最大的时点出现在2w：RSTN组均值较重度损伤组下降了53.4%(P<0.05)。
(2)Resistin的表达(1mRNA和蛋白)在正常组和假手术组之间没有显著差异(P>0.05)。抵抗素在脑内不同组织(海马、下丘脑、皮层)中呈现表达随时间变化趋势一致的有：以假手术组为基准,轻度损伤组到4W才开始出现表达量显著提高(P<0.05)。重度损伤组在12小时表达量就升高,一直到4w持续升高表达(各P均<0.05)。LPS组在12小时表达量就升高,一直到1w持续升高表达,到2w开始表达量下降并持续下降到4w(各P均<0.05)。RSTN注射组在12小时表达量就升高,一直到4w持续升高表达(各P均<0.05)。抵抗素在脑内不同组织中呈现表达随时间的变化趋势不一致的为：重度损伤组和RSTN组的皮层部位mRNA的表达在4w时均呈现下降的趋势(各P均<0.05)。与基准值相比,各部位能检测到的Resistin有统计学意义的表达升高在损伤同侧组织中均显著高于对侧(各P均<0.05)。在本研究涉及的检测部位海马、下丘脑和皮层中,抵抗素表达量的升高以海马部位最明显,其次依次是皮层和下丘脑(P<0.05)。比较重度损伤组和RSTN组之间各时点抵抗素mRNA的表达量均为重度损伤组>RSTN组,但两者差异最大的时点出现在4w：RSTN组较重度损伤组下降了77.5‰,不过与1w时点的下降74.3%无统计学差异(P>0.05),两者差异最小的时点出现在24h：RSTN组较重度损伤组下降了60.3%,与上述时点间差异有统计学意义(P<0.05)。
(3)血糖和血清胰岛素浓度在正常组和假手术组各时间点之间均没有显著差异(各P均>0.05)。以假手术组为基准,轻度损伤组在12h、24h、72h显著升高(各P均<0.05)；重度损伤组在12h、1w、2w、4w显著升高(各P均<0.05);LPS组在12h、24h显著升高(各P均<0.05)；RSTN组在1w、2w、4w显著升高(各P均<0.05)。各组平均血糖和血清胰岛素浓度：重度损伤组>RSTN组>LPS组>轻度损伤组>假手术组(各P均<0.05)。组内血糖和血清胰岛素随时间的变化趋势：轻度损伤组12h开始升高,24h达到峰值,72h出现下降(各P均<0.05),到1w时降至假手术组水平(P>0.05)。重度损伤组12h开：始升高,持续升高至1w达到峰值,2w时下降一直到4w时仍未恢复到假手术组水水平(各P均<0.05)；LPS注射组在12h开始升高,24h达到峰值(各P均<0.05),72h已降至假手术组水平(P>0.05)；RSTN组12h升高至重度损伤组水平(P>0.05),24h时出现下降,72h时又出现升高,到1w时达到峰值,随后有所下降,持续降到4w时仍未恢复到假手术组水平(P均<0.05)。
(4)根据血糖和血清胰岛素浓度以公式法计算获得的QUICKI来表示的胰岛素敏感指数：同样,QUICKI在正常组和假手术组各时间点之间均没有显著差异(各P均>0.05)。以假手术组为基准,轻度损伤组在12h、24h、72h显著降低(各P均<0.05)；重度损伤组在12h、24h、72h、1w、2w、4w显著降低(各P均<0.05)；LPS组在12h、24h显著降低(各P均<0.05);RSTTN组在12h、72h、1w、2w、4w显著降低(各P均<0.05)。平均组间QUICKI在数值上比较：重度损伤组0.05)。重度损伤组12h开始降低,持续升高至1w达到谷值,2w时已有升高但仍低于假手术组,到4w时仍未恢复到假手术组水平(各P均<0.05)；LPS注射组在12h开始降低,24h达到谷值(各P均<0.05),72h已升至假手术组水平(P>0.05)；RSTN组12h降低至重度损伤组水平(P>0.05),24h时已升高,但低于假手术组水平(P<0.05),72h又降低,到1w时达到谷值,随后有所升高,持续到4w时仍未恢复到假手术组水平(各P均<0.05)。
(5)所有组别(除LPS组之外)样本的血清抵抗素指标与根据血糖和血清胰岛素指标计算获得的胰岛素敏感指数指标两者进一步做相关分析,在排除时间点的协变量因素后显示,血清抵抗素与胰岛素敏感指数呈显著负相关(r=-0.764,P=0.000<0.05)。
(6)将重度损伤组和RSTN注射组之间进行24h、1w和4w的大鼠神经功能评分,发现：组间比较,重度损伤组与RSTN组在24h,1w和4w的神经功能评分均有统计学意义,重度损伤组高于RSTN组(P<0.05);组内分析,以假手术组的评分0为基准,重度损伤组在致伤后24h评分升高,1w达到峰值,随后4w时出现下降(各P均<0.05)。RSTN组趋势相同(各P均<0.05)。
结论
(1)创伤性脑损伤发生后,血清抵抗素浓度在伤后12小时即出现升高,且呈现持续升高的趋势直至伤后4周以上,同时升高的程度与损伤的程度有关,损伤越重,升高越明显。抵抗素在mRNA和蛋白层面的表达也呈现类似于血清抵抗素的升高和变化趋势,但存在大脑内不同组织／器官的差异化表达,这种差异化不仅体现在表达量上(海马>皮层>下丘脑,伤侧>对侧),还反映到表达量随时间的变化趋势上(皮层部位抵抗素mRNA的表达在伤后四周时呈现下降的趋势)。甚至反映为抵抗素抗体发挥拮抗抵抗素效应作用的位点和时点上(注射抵抗素抗体后,能够在伤后2周对血清抵抗素浓度产生下降53.4%的拈抗效应,以及在伤后1周和4周对海马组织内抵抗素的表达分别产生下降77.5%和下降74.3%的影响)。
(2)创伤性脑损伤发生后,血糖和血清胰岛素呈现完全一致的变化趋势：在伤后12小时即出现显著升高,升高的峰值和持续时程因损伤程度轻重而异,损伤越重,升高的峰值越高,持续升高的时程也越长,然后在1-4周内逐渐下降,恢复或不完全恢复到致伤前水平。胰岛素敏感性指数则呈现相反的变化趋势：即胰岛素敏感性下降(胰岛素抵抗)随着TBI的发生而出现,最早见于伤后12h,根据伤情轻重不同,逐渐于24小时—1周内达到谷值,然后逐渐恢复,在1—4周内恢复或不完全恢复到致伤前水平。
(3)经过统计分析,血清抵抗素与胰岛素敏感指数呈显著负相关,即在创伤性脑损伤状态下,血清抵抗素越高,机体的胰岛素敏感性越低,即机体的胰岛素抵抗越强。提示抵抗素可能在创伤性脑损伤大鼠发生胰岛素抵抗的过程中发挥重要的影响作用。推测抵抗素与机体发生应激性胰岛素抵抗关系密切,TBI条件下,抵抗素发生致脑损害的机理有可能是通过影响胰岛素敏感性发挥作用,两者之间可能以影响代谢为共同手段和桥梁来引起脑损害尤其是继发性脑损害。控制抵抗素可能是减轻脑损害的有效途径。
(4)通过设立外源性脂多糖局部注射产生的单纯炎性脑损伤作对比分析,发现创伤性脑损伤引起的血清抵抗素水平的变化并不能认为仅仅是炎性反应的结果,推测创伤性脑损伤的致伤机理区别于单纯炎性反应的复杂性,以及单纯用抵抗素作为炎症介质参与炎性反应来解释创伤性脑损伤发生发展的过程存在局限性。
(5)通过经腹腔注射抵抗素抗体来干预抵抗素的生理效应后观察糖代谢及胰岛素敏感性指标的变化以及对相应时段的神经功能评分的影响,可以认为血清抵抗索与神经功能之间存在一定关联。控制血清抵抗素水平有可能“接或间接发挥一定程度的神经功能保护效应。血清抵抗素还有望作为侧面反映神经功能的生化指标甚至作为将来临床促进神经功能康复的干预位点。
Objective
The model established in SD rat were produced by the method of freely falling body, to investigate the features of gene expression of the resistin following traumatic brain injury and its effect of insulin sensitivity after the trauma.
Methods
A total of 216 clean level SD (Spruge-Dawley) male rats were involved in the study, weight 300-350 g. Divided them into six groups using Computer Numbers randomly methods:normal control group, sham operation group, mild injury group, severe injury group, exogenous phosphoric acid lipopolysaccharide (LPS) injection group and RSTN antibody injection group.(N=36). With 10% hydration chlorine aldehyde (0.35g/kg) intra-abdominal inject into rats,fix the head to the stereotactic instrument, cut off the top fur, disinfect the skin, cut the scalp along the midline left, separate the periosteum, with dental drill in the midline 2 mm, close to coronary seam open a 5 mm in diameter of circular window, hard film intact. The left parietal lobe brain damage limitation of rats caused by the self-made improved Feeney traumatic brain injury free fall device:with 20 g farmar in 10 cm high through a metal guide bar to fall, hits on the cone of hard film bring about light damage, the other with 40 g farmar in 25 cm, falling to heavy damage. Exogenous phosphoric acid lipopolysaccharide (LPS) injection group (hereinafter referred to as LPS group) with 10% hydration chlorine aldehyde (0.35 g/kg weight) intra-abdominal inject into rats,then its head is fixed to the stereotactic instrument, anterior fontanelle before for zero value, before after anterior fontanelle in 5 mm, the midline left 4 mm points, using electric drill drill a skull, cut dural, before after anterior fontanelle in 5 mm, the midline left 4 mm in the brain surface with trace syringe needle into the vertical 1.8 mm, slow inject 5 ul LPS solution (25 ug), reserve 2 min, slow pull needle, skin suture, loose solution rats, and back to animal room make its natural waking up. RSTN antibody injection group (hereinafter referred to as RSTN group) take severe damage rats, according to 1 mg/only intraperitoneal injection dose. Normal control group (hereinafter referred to as are group) don't do anything.JiaShouShu group (hereinafter referred to as the false group) only cut scalp and skull open window, from brain damage. In the most severely damage and RSTN group after postoperative at 24 hours,1 week,4 weeks application of neural behavior Longa rating score. Each byte is in postoperative 12 hours,24 hours,72 hours, one week, two weeks,4 weeks tail venous blood sampling for ELISA test blood sugar, serum insulin, serum resistin and based on the calculation of quantitative insulin sensitivity test index (QUICKI), and put to death the rat,take out with side the hippocampus, hypothalamus,cortex organization, for the reverse transcription-the polymerase chain reaction (RT-PCR) method to detect resistin mRNA element and Western blot method to detect resistin protein expression.
Results
(1) The concentration of serum resistin in normal group and sham operation group group have no significant difference at each time point (all P>0.05).Take sham operation group as a benchmark, light damage group, severe damage group, LPS group and RSTN group in each test date after the success of the modeling(12 h,24 h,72 h,1 w, 2 w and 4 w) all appear significantly increased in serum resistin level (all P< 0.05); The average concentration of serum resistin:severe damage group>RSTN group>LPS group>mild damage> sham operation group (all P< 0.05).Serum resistin in the group with time changing trends:The mild injury group and the severe injury group were elevated after 12 hours,and continue to rise 4w after peak (all P< 0.05).LPS injection group in the 12 h beginning to rise, and 72 h reached the peak, then decreased in 1w and last 4w (all P< 0.05); RSTN group rise to the level of severe damage group in 12 h (P> 0.05), while falling in 24 h,72 h appear again when the rise, continue to rise to 4 w peak (all P< 0.05). Compared with each point between the severe group and RSTN group, the biggest difference point of the concentration of serum resistin is appear in 2 w: RSTN group was dropped 53.4% compared with the severe damage group.
(2) Resistin expression (mRNA and protein) has no significant differences between normal group and shame operation group.Resistin expression in the different organization of brain (the hippocampus, hypothalamus, cortex) presentation changing with time trend consistent:to shame operation group as a benchmark, mild damage group to 4 w began to appear expression is significantly improve (P< 0.05). Severe damage in 12 hours of expression is increased, has been to 4 w rising each expression (P< 0.05). LPS group in 12 hours expression is increased, has been to 1 w rising in the expression, to 2w expression begin to decrease and continue to decline to 4w (all P< 0.05). RSTN injection group in 12 hours expression is increased, has been to 4 w rising each expression (P< 0.05). RSTN in the brain in different organization presentation changing with time trend is not the same as:severe damage group and RSTN group of cortex parts of mRNA expression in 4 w are presented down trend (all P< 0.05). Compared with the benchmark, who can detect all the RSTN is statistically significant increase in the expression of damage on the same side in the organization were significantly higher than for each side (P< 0.05).In this research involves the detection place seahorses, head issuing grave and cortex, the increase of the expression of resistin in the hippocampus is the most obvious, followed is the cortex and the hypothalamus (P< 0.05). Compared with each point between the severe group and RSTN group,both of the expression of mRNA of resistin are severe damage group> RSTN groups, but the biggest difference at appeared in 4 w:RSTN group was dropped 77.5% compared with the severe damage group, but with 1 w drop 74.3% is no statistical difference (P> 0.05), minimum points of the difference in appear in 24 h:RSTN group was dropped 60.3%, and the difference between the point was statistically significant (P< 0.05).
(3) Blood glucose and insulin concentrations in serum normal group and shame operation group between different time points they have no significant difference (P> 0.05 each). To shame operation group as a benchmark, mild damage group in the 12 h,24 h,72 h the significantly increased (P< 0.05); Severe damage group in the 12 h,24 h,72 h,1 w,2 w,4 w significantly increased (all P< 0.05); LPS group in the 12 h,24 h the significantly increased (P< 0.05); RSTN group in the 12 h,72 h,1 w,2 w,4 w significantly increased (all P< 0.05). The average blood sugar and serum insulin concentrations:severe damage group> RSTN group> LPS group> mild damage> shame operation group (the group> P< 0.05). In the group blood sugar and serum insulin changing with time trend:the mild damage group 12 h beginning to rise,24 h peak,72 h the fall (P< 0.05), to 1 w drop to shame operation group when level (P> 0.05). Severe damage group 12 h beginning to rise, rising to 1 w peak,2 w falls has been to 4 w still has not returned to shame operation group level (all P< 0.05); LPS injection group in the 12 h beginning to rise,24 h peak (all P< 0.05),72 h has been reduced to a level shame operation group (P> 0.05); RSTN group 12 h rise to severe damage level group (P > 0.05), when 24 h fall,72 h appear again when the rise, to 1 w peak when, then declined, continued down to four w still has not returned to the level shame operation group (P< 0.05).
(4) According to blood sugar and serum insulin concentrations to formula calculation method for the presentation of the QUICKI insulin sensitivity index:also, QUICKI in normal group and shame operation group between different time points they have no significant difference (P> 0.05 each). To shame operation group as a benchmark, mild damage group in the 12h,24 h,72h significantly reduced (all P< 0.05); Severe damage group in the 12h,24h,72h, 1w,2w,4w significantly reduced (all P< 0.05); LPS group in the 12h,24h significantly reduced (all P< 0.05); RSTN group in the 12 h,72 h, 1 w,2 w,4 w significantly reduced (all P< 0.05). Average QUICKI between groups in numerical comparison:severe damage group< RSTN group< LPS group< slightly damaged< shame operation group (the group P< 0.05). In the group QUICKI changing with time trend analysis:mild damage group 12 h began to drop,24 h reach valley value,72 h rise but still lower than the existing shame operation group (all P< 0.05), to 1 w, has increased the level shame operation to group (P> 0.05). Severe damage group in the 12 h began to drop, continue to rise to 1 w reach valley value,2 w had been rising but still lower than the shame operation group, to 4 w still has not returned to shame operation group level (all P< 0.05); LPS injection group in the 12 h began to drop, 24 h reach valley value (all P< 0.05),72 h has risen to the level shame operation group (P> 0.05); RSTN group 12 h reduce to severe damage level group (P> 0.05),24 h, has increased, but lower than the level shame operation group (P< 0.05),72 h and reduce to 1 w, at the valley, then have increased, values continue to 4 w still has not returned to shame operation group level (all P< 0.05).
(5) All categories (except LPS outside group) sample of serum resistin index and according to the blood glucose and insulin index calculation of serum for insulin sensitivity index index both further related analysis, eliminating time points of production that variables, serum resistin and insulin sensitivity index is the significant negative correlation (r=-0.764, P=0.000<0.05).
(6) heavy damage group and RSTN injection group between 24 h,1 w,4 w rats and neurobehavioral scores, found that compared between groups, severe damage group and RSTN group in the 24 h,1 w and 4 w neurobehavioral scores are statistically significant, severe damage group was higher than RSTN group (P< 0.05); In the group analysis, to the score 0 shame operation group as the benchmark, severe damage in group of injury within 24 h score is increased,1 w peak, then 4 w begin to fall (P< 0.05). RSTN group's trend was the same(P< 0.05).
Conclusions
(1) After the occurrence of the traumatic brain injury, the concentration of serum resistin in 12 hours after injury that appear rise, and present the trend of rising until more than 4 weeks after injury, and the extent of increase is in relation to the extent of the damage, damage is heavier, the more obvious increase. Expression of resistin in the mRNA and protein level also presents that similar to the rise and change trend of the serum resistin, but which exist in different organization/organs of brain have differently, the differentiation is not only reflected in the quantity of expression (the hippocampus> the cortex> the hypothalamus, injury> to side), but also reflected in the expression of changing with time trend (the expression of the mRNA resistin in cortex has declined after four weeks of injury). Even for anti-resistin to reflect to play antagonistic action toward to the loci and point of effect of resistin (After the anti-resistin was injected, the concentration of serum resistin had dropped 53.4% in two weeks after injury, and the expression of resistin in the hippocampus generate decreased by 77.5% and 74.3% respectively in 1 w and 4 w after injury).
(2) After the occurrence of the traumatic brain injury, blood sugar and serum insulin present the coherent variation trend completely:in 12 hours after injury is significant increases, the peak and duration increased because of the damage degree of process and different, damage is heavier, the higher the peak of the rising, rising schedule is also much longer, then down gradually in 1-4 weeks, recovery or not fully restored to the level before the injury. Insulin sensitivity index revealed a opposite trend:the decrease of insulin sensitivity (insulin resistance) appears with the occurrence of TBI,and in the earliest injury after 12 h, according to different weight injury, gradually reach the valley value in 24 hours-1 weeks, and then returned gradually, in 1-4 weeks restored or not fully restored to the level before the injury.
(3) Through the statistical analysis, serum resistin and insulin sensitivity index is the significant negative correlation, namely in traumatic brain injury state, serum resistin is higher,the lower insulin sensitivity of the body, in which the body's resistance to insulin is stronger. Tip resistin may play an important role in the process of insulin resistance that happened in traumatic brain injury rat.
(4) Through the establishment of exogenous lipopolysaccharide local injections of produce of pure inflammatory brain damage as contrast analysis, traumatic brain injury that caused the level of serum resistin changes and can't think is just the result of the inflammatory response, traumatic brain injury that the cause of injury difference from the complexity of simple inflammatory reaction and pure with resistin as inflammatory mediators reaction to explain traumatic brain injuries development process and occurrence limitations.
(5) Through the intraperitoneal injection of Anti-resistin to intervene the physiological effect of resistin and then observed sugar metabolism and insulin sensitivity index changes, and to the influence of neurobehavioral scores in the corresponding period, we can think that exist certain relevance between serum resistin and nerve function. Control serum resistin levels may be directly or indirectly play a certain degree of neural function protection effect. Serum resistin also is expected to as a reflection of neurological function biochemical indicators even as the future clinical stimulative nerve function recovery intervention sites.
建立自由落体致颅脑损伤SD大鼠模型,探讨创伤后脑损伤TBI抵抗素基因表达的特征及其对创伤后胰岛素敏感性的影响作用。方法
清洁级SD(Spruge-Dawley)雄性大鼠216只,体重300～350g。计算机编号随机抽取方法均分为6组：正常对照组,假手术组,轻度损伤组,重度损伤组,外源性磷酸脂多糖(LPS)注射组,RSTN抗体注射组。每组36只。大鼠用10%水合氯醛(0.35g/kg体重)腹腔内注射麻醉后,固定于立体定向仪消毒并切开头皮(沿中线偏左侧)分离骨膜,于紧挨冠状缝后中线旁2mm用牙科电钻开一直径5mm的圆形窗,硬膜保持完整。采用自制改进型Feeney自由落体创伤性脑损伤装置造成大鼠左顶叶局限性脑损伤：用20g砝码于10cm处通过一金属导杆坠落,撞击置于硬膜上的圆锥上致轻型损伤,另用40g砝码于25cm高处坠落致重型损伤。外源性磷酸脂多糖(LPS)注射组(以下简称LPS组)用10%水合氯醛(0.35g/kg体重)腹腔内注射麻醉大鼠后,将其头部固定于立体定向仪上,以前囟为零点值,于前囟后5mm,中线左侧4mm处,用牙科电钻钻开颅骨,切开硬脑膜,用微量注射器自大脑表面进针(垂直)1.8mm,缓慢注射5ul LPS溶液(25ug),留针2min,缓慢拔针,缝合皮肤,松解大鼠,待其然苏醒。RSTN抗体注射组(以下简称RSTN组)取重度损伤组大鼠,按1mg/只剂量腹腔注射。正常对照组(以下简称正组)不做任何处理。假手术组(以下简称假组)仅切开头皮和颅骨开窗,不致脑损伤。对重度损伤组和RSTN组于术后24小时,1周,4周应用Longa评分法进行神经行为学评分。各组均于术后12小时,24小时,72小时,1周,2周,4周尾静脉采血,供ELISA法检测血糖,血清胰岛素,血清抵抗素并以-ln(FPG(血糖)×FZNS(胰岛素))计算定量胰岛素敏感性检测指数(QUICKI),同时处死大鼠,取同侧和对侧海马、下丘脑、皮层组织,供逆转录-聚合酶链式反应(RT-PCR)法检测抵抗素mRNA及Western blot法检测抵抗素蛋白的表达。
结果
(1)血清抵抗素浓度在正常组和假手术组各时间点之间均没有显著差异(各P均>0.05)。以假手术组为基准,轻度损伤组、重度损伤组、LPS组和RSTN组在建模成功后的各个检测时点(12h、24h、72h、1W、2w和4w)均出现血清抵抗素水平的显著升高(各P均<0.05)；各组平均血清抵抗素浓度：重度损伤组>RSTN组>LPS组>轻度损伤组>假手术组(各P均<0.05)。组内血清抵抗素随时间的变化趋势：轻度损伤组与重度损伤组均于12h出现升高,持续升高至4w达到峰值(各P均<0.05)。LPS注射组在12h开始升高,72h达到峰值,到1w时出现下降,持续下降到4w(各P均<0.05);RSTN组12h升高至重度损伤组水平(P>0.05),24h时出现下降,72h时又出现升高,持续升高至4w达到峰值(各P均<0.05)。比较重度损伤组和RSTN组血清抵抗素浓度发现两者差异最大的时点出现在2w：RSTN组均值较重度损伤组下降了53.4%(P<0.05)。
(2)Resistin的表达(1mRNA和蛋白)在正常组和假手术组之间没有显著差异(P>0.05)。抵抗素在脑内不同组织(海马、下丘脑、皮层)中呈现表达随时间变化趋势一致的有：以假手术组为基准,轻度损伤组到4W才开始出现表达量显著提高(P<0.05)。重度损伤组在12小时表达量就升高,一直到4w持续升高表达(各P均<0.05)。LPS组在12小时表达量就升高,一直到1w持续升高表达,到2w开始表达量下降并持续下降到4w(各P均<0.05)。RSTN注射组在12小时表达量就升高,一直到4w持续升高表达(各P均<0.05)。抵抗素在脑内不同组织中呈现表达随时间的变化趋势不一致的为：重度损伤组和RSTN组的皮层部位mRNA的表达在4w时均呈现下降的趋势(各P均<0.05)。与基准值相比,各部位能检测到的Resistin有统计学意义的表达升高在损伤同侧组织中均显著高于对侧(各P均<0.05)。在本研究涉及的检测部位海马、下丘脑和皮层中,抵抗素表达量的升高以海马部位最明显,其次依次是皮层和下丘脑(P<0.05)。比较重度损伤组和RSTN组之间各时点抵抗素mRNA的表达量均为重度损伤组>RSTN组,但两者差异最大的时点出现在4w：RSTN组较重度损伤组下降了77.5‰,不过与1w时点的下降74.3%无统计学差异(P>0.05),两者差异最小的时点出现在24h：RSTN组较重度损伤组下降了60.3%,与上述时点间差异有统计学意义(P<0.05)。
(3)血糖和血清胰岛素浓度在正常组和假手术组各时间点之间均没有显著差异(各P均>0.05)。以假手术组为基准,轻度损伤组在12h、24h、72h显著升高(各P均<0.05)；重度损伤组在12h、1w、2w、4w显著升高(各P均<0.05);LPS组在12h、24h显著升高(各P均<0.05)；RSTN组在1w、2w、4w显著升高(各P均<0.05)。各组平均血糖和血清胰岛素浓度：重度损伤组>RSTN组>LPS组>轻度损伤组>假手术组(各P均<0.05)。组内血糖和血清胰岛素随时间的变化趋势：轻度损伤组12h开始升高,24h达到峰值,72h出现下降(各P均<0.05),到1w时降至假手术组水平(P>0.05)。重度损伤组12h开：始升高,持续升高至1w达到峰值,2w时下降一直到4w时仍未恢复到假手术组水水平(各P均<0.05)；LPS注射组在12h开始升高,24h达到峰值(各P均<0.05),72h已降至假手术组水平(P>0.05)；RSTN组12h升高至重度损伤组水平(P>0.05),24h时出现下降,72h时又出现升高,到1w时达到峰值,随后有所下降,持续降到4w时仍未恢复到假手术组水平(P均<0.05)。
(4)根据血糖和血清胰岛素浓度以公式法计算获得的QUICKI来表示的胰岛素敏感指数：同样,QUICKI在正常组和假手术组各时间点之间均没有显著差异(各P均>0.05)。以假手术组为基准,轻度损伤组在12h、24h、72h显著降低(各P均<0.05)；重度损伤组在12h、24h、72h、1w、2w、4w显著降低(各P均<0.05)；LPS组在12h、24h显著降低(各P均<0.05);RSTTN组在12h、72h、1w、2w、4w显著降低(各P均<0.05)。平均组间QUICKI在数值上比较：重度损伤组
(5)所有组别(除LPS组之外)样本的血清抵抗素指标与根据血糖和血清胰岛素指标计算获得的胰岛素敏感指数指标两者进一步做相关分析,在排除时间点的协变量因素后显示,血清抵抗素与胰岛素敏感指数呈显著负相关(r=-0.764,P=0.000<0.05)。
(6)将重度损伤组和RSTN注射组之间进行24h、1w和4w的大鼠神经功能评分,发现：组间比较,重度损伤组与RSTN组在24h,1w和4w的神经功能评分均有统计学意义,重度损伤组高于RSTN组(P<0.05);组内分析,以假手术组的评分0为基准,重度损伤组在致伤后24h评分升高,1w达到峰值,随后4w时出现下降(各P均<0.05)。RSTN组趋势相同(各P均<0.05)。
结论
(1)创伤性脑损伤发生后,血清抵抗素浓度在伤后12小时即出现升高,且呈现持续升高的趋势直至伤后4周以上,同时升高的程度与损伤的程度有关,损伤越重,升高越明显。抵抗素在mRNA和蛋白层面的表达也呈现类似于血清抵抗素的升高和变化趋势,但存在大脑内不同组织／器官的差异化表达,这种差异化不仅体现在表达量上(海马>皮层>下丘脑,伤侧>对侧),还反映到表达量随时间的变化趋势上(皮层部位抵抗素mRNA的表达在伤后四周时呈现下降的趋势)。甚至反映为抵抗素抗体发挥拮抗抵抗素效应作用的位点和时点上(注射抵抗素抗体后,能够在伤后2周对血清抵抗素浓度产生下降53.4%的拈抗效应,以及在伤后1周和4周对海马组织内抵抗素的表达分别产生下降77.5%和下降74.3%的影响)。
(2)创伤性脑损伤发生后,血糖和血清胰岛素呈现完全一致的变化趋势：在伤后12小时即出现显著升高,升高的峰值和持续时程因损伤程度轻重而异,损伤越重,升高的峰值越高,持续升高的时程也越长,然后在1-4周内逐渐下降,恢复或不完全恢复到致伤前水平。胰岛素敏感性指数则呈现相反的变化趋势：即胰岛素敏感性下降(胰岛素抵抗)随着TBI的发生而出现,最早见于伤后12h,根据伤情轻重不同,逐渐于24小时—1周内达到谷值,然后逐渐恢复,在1—4周内恢复或不完全恢复到致伤前水平。
(3)经过统计分析,血清抵抗素与胰岛素敏感指数呈显著负相关,即在创伤性脑损伤状态下,血清抵抗素越高,机体的胰岛素敏感性越低,即机体的胰岛素抵抗越强。提示抵抗素可能在创伤性脑损伤大鼠发生胰岛素抵抗的过程中发挥重要的影响作用。推测抵抗素与机体发生应激性胰岛素抵抗关系密切,TBI条件下,抵抗素发生致脑损害的机理有可能是通过影响胰岛素敏感性发挥作用,两者之间可能以影响代谢为共同手段和桥梁来引起脑损害尤其是继发性脑损害。控制抵抗素可能是减轻脑损害的有效途径。
(4)通过设立外源性脂多糖局部注射产生的单纯炎性脑损伤作对比分析,发现创伤性脑损伤引起的血清抵抗素水平的变化并不能认为仅仅是炎性反应的结果,推测创伤性脑损伤的致伤机理区别于单纯炎性反应的复杂性,以及单纯用抵抗素作为炎症介质参与炎性反应来解释创伤性脑损伤发生发展的过程存在局限性。
(5)通过经腹腔注射抵抗素抗体来干预抵抗素的生理效应后观察糖代谢及胰岛素敏感性指标的变化以及对相应时段的神经功能评分的影响,可以认为血清抵抗索与神经功能之间存在一定关联。控制血清抵抗素水平有可能“接或间接发挥一定程度的神经功能保护效应。血清抵抗素还有望作为侧面反映神经功能的生化指标甚至作为将来临床促进神经功能康复的干预位点。
Objective
The model established in SD rat were produced by the method of freely falling body, to investigate the features of gene expression of the resistin following traumatic brain injury and its effect of insulin sensitivity after the trauma.
Methods
A total of 216 clean level SD (Spruge-Dawley) male rats were involved in the study, weight 300-350 g. Divided them into six groups using Computer Numbers randomly methods:normal control group, sham operation group, mild injury group, severe injury group, exogenous phosphoric acid lipopolysaccharide (LPS) injection group and RSTN antibody injection group.(N=36). With 10% hydration chlorine aldehyde (0.35g/kg) intra-abdominal inject into rats,fix the head to the stereotactic instrument, cut off the top fur, disinfect the skin, cut the scalp along the midline left, separate the periosteum, with dental drill in the midline 2 mm, close to coronary seam open a 5 mm in diameter of circular window, hard film intact. The left parietal lobe brain damage limitation of rats caused by the self-made improved Feeney traumatic brain injury free fall device:with 20 g farmar in 10 cm high through a metal guide bar to fall, hits on the cone of hard film bring about light damage, the other with 40 g farmar in 25 cm, falling to heavy damage. Exogenous phosphoric acid lipopolysaccharide (LPS) injection group (hereinafter referred to as LPS group) with 10% hydration chlorine aldehyde (0.35 g/kg weight) intra-abdominal inject into rats,then its head is fixed to the stereotactic instrument, anterior fontanelle before for zero value, before after anterior fontanelle in 5 mm, the midline left 4 mm points, using electric drill drill a skull, cut dural, before after anterior fontanelle in 5 mm, the midline left 4 mm in the brain surface with trace syringe needle into the vertical 1.8 mm, slow inject 5 ul LPS solution (25 ug), reserve 2 min, slow pull needle, skin suture, loose solution rats, and back to animal room make its natural waking up. RSTN antibody injection group (hereinafter referred to as RSTN group) take severe damage rats, according to 1 mg/only intraperitoneal injection dose. Normal control group (hereinafter referred to as are group) don't do anything.JiaShouShu group (hereinafter referred to as the false group) only cut scalp and skull open window, from brain damage. In the most severely damage and RSTN group after postoperative at 24 hours,1 week,4 weeks application of neural behavior Longa rating score. Each byte is in postoperative 12 hours,24 hours,72 hours, one week, two weeks,4 weeks tail venous blood sampling for ELISA test blood sugar, serum insulin, serum resistin and based on the calculation of quantitative insulin sensitivity test index (QUICKI), and put to death the rat,take out with side the hippocampus, hypothalamus,cortex organization, for the reverse transcription-the polymerase chain reaction (RT-PCR) method to detect resistin mRNA element and Western blot method to detect resistin protein expression.
Results
(1) The concentration of serum resistin in normal group and sham operation group group have no significant difference at each time point (all P>0.05).Take sham operation group as a benchmark, light damage group, severe damage group, LPS group and RSTN group in each test date after the success of the modeling(12 h,24 h,72 h,1 w, 2 w and 4 w) all appear significantly increased in serum resistin level (all P< 0.05); The average concentration of serum resistin:severe damage group>RSTN group>LPS group>mild damage> sham operation group (all P< 0.05).Serum resistin in the group with time changing trends:The mild injury group and the severe injury group were elevated after 12 hours,and continue to rise 4w after peak (all P< 0.05).LPS injection group in the 12 h beginning to rise, and 72 h reached the peak, then decreased in 1w and last 4w (all P< 0.05); RSTN group rise to the level of severe damage group in 12 h (P> 0.05), while falling in 24 h,72 h appear again when the rise, continue to rise to 4 w peak (all P< 0.05). Compared with each point between the severe group and RSTN group, the biggest difference point of the concentration of serum resistin is appear in 2 w: RSTN group was dropped 53.4% compared with the severe damage group.
(2) Resistin expression (mRNA and protein) has no significant differences between normal group and shame operation group.Resistin expression in the different organization of brain (the hippocampus, hypothalamus, cortex) presentation changing with time trend consistent:to shame operation group as a benchmark, mild damage group to 4 w began to appear expression is significantly improve (P< 0.05). Severe damage in 12 hours of expression is increased, has been to 4 w rising each expression (P< 0.05). LPS group in 12 hours expression is increased, has been to 1 w rising in the expression, to 2w expression begin to decrease and continue to decline to 4w (all P< 0.05). RSTN injection group in 12 hours expression is increased, has been to 4 w rising each expression (P< 0.05). RSTN in the brain in different organization presentation changing with time trend is not the same as:severe damage group and RSTN group of cortex parts of mRNA expression in 4 w are presented down trend (all P< 0.05). Compared with the benchmark, who can detect all the RSTN is statistically significant increase in the expression of damage on the same side in the organization were significantly higher than for each side (P< 0.05).In this research involves the detection place seahorses, head issuing grave and cortex, the increase of the expression of resistin in the hippocampus is the most obvious, followed is the cortex and the hypothalamus (P< 0.05). Compared with each point between the severe group and RSTN group,both of the expression of mRNA of resistin are severe damage group> RSTN groups, but the biggest difference at appeared in 4 w:RSTN group was dropped 77.5% compared with the severe damage group, but with 1 w drop 74.3% is no statistical difference (P> 0.05), minimum points of the difference in appear in 24 h:RSTN group was dropped 60.3%, and the difference between the point was statistically significant (P< 0.05).
(3) Blood glucose and insulin concentrations in serum normal group and shame operation group between different time points they have no significant difference (P> 0.05 each). To shame operation group as a benchmark, mild damage group in the 12 h,24 h,72 h the significantly increased (P< 0.05); Severe damage group in the 12 h,24 h,72 h,1 w,2 w,4 w significantly increased (all P< 0.05); LPS group in the 12 h,24 h the significantly increased (P< 0.05); RSTN group in the 12 h,72 h,1 w,2 w,4 w significantly increased (all P< 0.05). The average blood sugar and serum insulin concentrations:severe damage group> RSTN group> LPS group> mild damage> shame operation group (the group> P< 0.05). In the group blood sugar and serum insulin changing with time trend:the mild damage group 12 h beginning to rise,24 h peak,72 h the fall (P< 0.05), to 1 w drop to shame operation group when level (P> 0.05). Severe damage group 12 h beginning to rise, rising to 1 w peak,2 w falls has been to 4 w still has not returned to shame operation group level (all P< 0.05); LPS injection group in the 12 h beginning to rise,24 h peak (all P< 0.05),72 h has been reduced to a level shame operation group (P> 0.05); RSTN group 12 h rise to severe damage level group (P > 0.05), when 24 h fall,72 h appear again when the rise, to 1 w peak when, then declined, continued down to four w still has not returned to the level shame operation group (P< 0.05).
(4) According to blood sugar and serum insulin concentrations to formula calculation method for the presentation of the QUICKI insulin sensitivity index:also, QUICKI in normal group and shame operation group between different time points they have no significant difference (P> 0.05 each). To shame operation group as a benchmark, mild damage group in the 12h,24 h,72h significantly reduced (all P< 0.05); Severe damage group in the 12h,24h,72h, 1w,2w,4w significantly reduced (all P< 0.05); LPS group in the 12h,24h significantly reduced (all P< 0.05); RSTN group in the 12 h,72 h, 1 w,2 w,4 w significantly reduced (all P< 0.05). Average QUICKI between groups in numerical comparison:severe damage group< RSTN group< LPS group< slightly damaged< shame operation group (the group P< 0.05). In the group QUICKI changing with time trend analysis:mild damage group 12 h began to drop,24 h reach valley value,72 h rise but still lower than the existing shame operation group (all P< 0.05), to 1 w, has increased the level shame operation to group (P> 0.05). Severe damage group in the 12 h began to drop, continue to rise to 1 w reach valley value,2 w had been rising but still lower than the shame operation group, to 4 w still has not returned to shame operation group level (all P< 0.05); LPS injection group in the 12 h began to drop, 24 h reach valley value (all P< 0.05),72 h has risen to the level shame operation group (P> 0.05); RSTN group 12 h reduce to severe damage level group (P> 0.05),24 h, has increased, but lower than the level shame operation group (P< 0.05),72 h and reduce to 1 w, at the valley, then have increased, values continue to 4 w still has not returned to shame operation group level (all P< 0.05).
(5) All categories (except LPS outside group) sample of serum resistin index and according to the blood glucose and insulin index calculation of serum for insulin sensitivity index index both further related analysis, eliminating time points of production that variables, serum resistin and insulin sensitivity index is the significant negative correlation (r=-0.764, P=0.000<0.05).
(6) heavy damage group and RSTN injection group between 24 h,1 w,4 w rats and neurobehavioral scores, found that compared between groups, severe damage group and RSTN group in the 24 h,1 w and 4 w neurobehavioral scores are statistically significant, severe damage group was higher than RSTN group (P< 0.05); In the group analysis, to the score 0 shame operation group as the benchmark, severe damage in group of injury within 24 h score is increased,1 w peak, then 4 w begin to fall (P< 0.05). RSTN group's trend was the same(P< 0.05).
Conclusions
(1) After the occurrence of the traumatic brain injury, the concentration of serum resistin in 12 hours after injury that appear rise, and present the trend of rising until more than 4 weeks after injury, and the extent of increase is in relation to the extent of the damage, damage is heavier, the more obvious increase. Expression of resistin in the mRNA and protein level also presents that similar to the rise and change trend of the serum resistin, but which exist in different organization/organs of brain have differently, the differentiation is not only reflected in the quantity of expression (the hippocampus> the cortex> the hypothalamus, injury> to side), but also reflected in the expression of changing with time trend (the expression of the mRNA resistin in cortex has declined after four weeks of injury). Even for anti-resistin to reflect to play antagonistic action toward to the loci and point of effect of resistin (After the anti-resistin was injected, the concentration of serum resistin had dropped 53.4% in two weeks after injury, and the expression of resistin in the hippocampus generate decreased by 77.5% and 74.3% respectively in 1 w and 4 w after injury).
(2) After the occurrence of the traumatic brain injury, blood sugar and serum insulin present the coherent variation trend completely:in 12 hours after injury is significant increases, the peak and duration increased because of the damage degree of process and different, damage is heavier, the higher the peak of the rising, rising schedule is also much longer, then down gradually in 1-4 weeks, recovery or not fully restored to the level before the injury. Insulin sensitivity index revealed a opposite trend:the decrease of insulin sensitivity (insulin resistance) appears with the occurrence of TBI,and in the earliest injury after 12 h, according to different weight injury, gradually reach the valley value in 24 hours-1 weeks, and then returned gradually, in 1-4 weeks restored or not fully restored to the level before the injury.
(3) Through the statistical analysis, serum resistin and insulin sensitivity index is the significant negative correlation, namely in traumatic brain injury state, serum resistin is higher,the lower insulin sensitivity of the body, in which the body's resistance to insulin is stronger. Tip resistin may play an important role in the process of insulin resistance that happened in traumatic brain injury rat.
(4) Through the establishment of exogenous lipopolysaccharide local injections of produce of pure inflammatory brain damage as contrast analysis, traumatic brain injury that caused the level of serum resistin changes and can't think is just the result of the inflammatory response, traumatic brain injury that the cause of injury difference from the complexity of simple inflammatory reaction and pure with resistin as inflammatory mediators reaction to explain traumatic brain injuries development process and occurrence limitations.
(5) Through the intraperitoneal injection of Anti-resistin to intervene the physiological effect of resistin and then observed sugar metabolism and insulin sensitivity index changes, and to the influence of neurobehavioral scores in the corresponding period, we can think that exist certain relevance between serum resistin and nerve function. Control serum resistin levels may be directly or indirectly play a certain degree of neural function protection effect. Serum resistin also is expected to as a reflection of neurological function biochemical indicators even as the future clinical stimulative nerve function recovery intervention sites.
引文
1.白祥军,李占飞.完善创伤急救医学体系建设的必要性.中华急诊医学杂志,2010，19(5)：456-458.
2. Langlois JA, Rutland-Brown W, Thomas KE. Traumatic brain injury in the United States:emergency department visits, hospitalizations, and deaths. Atlanta(GA): Centers for Disease Control and Prevention, National Center for Injury Prevention ant Control,2006.
3. Gerhart KA,Mellick DC,Weintraub AH. Vionlence-related traumatic brain injury population-based study[J].J Trauma,2003,55(6):1045-1053.
4. McNair ND. Traumatic brain injm'y, Nurs Clin North Am,1999,34(3):637-659.
5. Andersson EH, Bjorkland R, Emanuelson I, et al. Epidemiology of traumatic brain injury:a population based study in western Sweden. Acta Neurol Scand.2003, 107(4):256-259.
6. Anonymous.NIH Consensus Development Panel on Rehabilitation of Persons With Traumatic Brain Injury. JAMA,1999,282(10):974-983.
7. Servadei F, Antonelli V, Betti L, et al. Regional brain injury epidemiology as the basis for planning brain injury treatment. The Romagna(Italy)experience. J Neurosurg Sci,2002,46(3-4):111-119.
8. Gururaj G. Epidemiology of traumatic brain injuries:Indian seenario. Neurol Res, 2002,24(1):24-28.
9.赵雅度.神经系统外伤[M].北京：人民军医出版社,2001,3-5.
10.王忠诚,赵元立.加强颅脑外伤临床基础研究,提倡规范化治疗，中华神经外科杂志.2001,17(3)：133-134.
11. Marshall LF. Head injury:recent past, present, and future. Neurosurgery,2000, 47(3):546-561.
12. Moinard C, Neveux N, Royo N, Genthon C, Marchand-Verrecchia C, Plotkine M, Cynober L.Characterization of the alteration of nutritional state in brain injury induced by fluid percussion in rats. Intens. Care Med 2005,31:281-288.
13. Thompson HJ, Tkacs NC, Saatman KE, Raghupathi R, McIntosh TK. Hyperthermia following traumatic brain injury:a critical evaluation. Neurobiol Dis,2003,12:163-173.
14. Ahima RS, Qi Y, Singhal NS. Adipokines that link obesity and diabetes to the hypothalamus. Prog Brain Res,2006,153:155-174.
15. Trayhurn P, Wood IS. Adipokines:inflammation and the pleiotropic role of white adipose tissue. Br J. Nutr,2004,92:347-355.
16. Wilkinson M, Brown R, Imran SA, Ur E. Adipokine gene expression in brain and pituitary gland.Neuroendocrinology,2007,86:191-209.
17. Dash PK, Kobori N, Moore AN. A molecular description of brain trauma pathophysiology using microarray technology:an overview. Neurochem. Res,2004,29:1275-1286.
18. Matzilevich DA, Rall JM, Moore AN, Grill RJ, Dash PK. High-density microarray analysis of hippocampal gene expression following experimental brain injury. J. Neurosci Res,2002;67:646-663.
19. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes[J]. Nature,2001,409 (3):307-312.
20. Morash B, Willkinson D, Ur E, et al. Resistin expression and regulation in mouse pituitary. FEBS Lett,2002,526:26-30.
21.乔智慧,唐兰芬.抵抗素与炎症相关性疾病研究进展.医学综述,2010,16(11)：1632-1634.
22. Reilly MP, Lehrke M,Wolfe ML, et al. Resistin is an inflammatory marker of atherosclerosis in humans[J]. Circulation,2005,111(7):932-939.
23. Kaser S, KaserA, SandhoferA, et al. Resistin messenger-RNA expression is increased by p roinflammatory cytokines in vitro [J].Biochem Biophys Res Commun,2003, 309 (2):286-290.
24. Kawanami D, Maemura K, Takeda N, et al. Direct recip rocal effects of resistin and adiponectin on vascular endothelial cells:a new insight into adipocytokine2endothelial cell interactions[J]. Biochem Biophys Res Commun,2004, 314 (2):415-419.
25. Hartman HB, Hu X, Tyler KX, et al. Mechanisms regulating adipocyte expression of resistin. J Biol Chem,2002,277:19754-19761.
26. Nagaev I, Smith U. Insulin resistance and type diabetes are not related to resistin expression in human fat cells or skeletal muscle. Biochem Biophys Res Commun, 2001,285:561-564.
27. Levy JR, Davenport B, Clore JN, et al. Lipid metabolism and resistin gene expression in insulin-resistant Fischer 344 rats. Am J Physiol Endocrinol Metab, 2002,282:E626-E633.
28. Brown R, Thompson HJ, Imran SA, et al. Traumatic brain injury induces adipokine gene expression in rat brain. Neurosci Lett,2008,432:73-78.
29. Wiesner G, Brown RE, Robertson GS, et al. Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ischaemic mouse brain. Neuroreport,2006,17:1195-1198.
30. Dong X-Q, Hu Y-Y, Yu W-H, et al. High concentrations of resistin in the peripheral blood of patients with acute basal ganglia hemorrhage are associated with poor outcome. J Crit Care,2009, doi:10.1016/j.jcrc.2009.09.008.
31. Oktay A,Aliefendioglu D, Caglayan O, et al. Plasma resistin levels of term newborn infants with hypoxic-ischemic encephalopathy[J]. Arch Dis Child,2008,6 (11):92-93.
32.杨松斌,王克义,竹方龙,等.重型脑外伤患者血浆抵抗索水平动态变化与预后的关系.中国临床神经科学,2011,19(3)：265-269.
33. Andrade CF, Waddell TK, Keshavjee S, et al. Innate immunity and organ transplaniation:the potential role of toll-like receptors. Am J Trans,2005,5:969-975.
34.李秀钧主编.胰岛素抵抗综合征[M].第一版.北京：人民卫生出版社,2001.22-25
35. Ljungqvist O, Nygren J, Thorell A. Insulin resistance and elective surgery[J].Surgery,2000,128(5):757-760.
36.王清华,徐如祥,李良平,等.大鼠不同程度脑损伤模型的建立[J].创伤外科杂志,2000；2(1)：42-44.
37. Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke,1989,20(1):84-91.
38.吴旭,王保捷,张国华,等.大鼠脑损伤分级自由落体打击模型的建立.中国法医学杂志,2005,20(1)：1-3.
39. Feeney DM, Boyeson MG, Lima RT, et al. Responses to cortical injury:Methodology and local effects of contusions in the rat[J]. Brain Res,1981,211:67-77.
40. Dixon CE, Lyeth BG, Povlishock JK, et al. A fluid percussion model of experimental brain injury in the rat[J]. J Neurosurg,1987,67:110-119.
41.李力仙,王天佑,钟震宇.自由落体和液压致大鼠脑损伤的对比研究.中华实验外科杂志.1999,16：447-8.
42. Luigi Brandi, Donatella Santoro, Andrea Natali, et al.Insulin resistance of stress:site and mechanisms.Clinical Science.1993;85:525-535.
43. Shaw Jhf,Wolfe RR.An integrated analysis of glucose,fat and protein metabolism in severely traumatized patients.Studies in the basal state and the response to total parenteral nutrition.Ann Surg.1989;209:63-71.
44.李光伟,潘孝仁,Lillioja S,等.检测人群胰岛素敏感性的一项新指数.中华内科杂志,1993,12(10)：656.
45. Soop M, Nygren J, Myrenfors P, et al. Preoperative oral carbohydrate treatment attenuates immediate postoperative insulin resistance. Am J Physiol Endocrinol Metab.2001;280:E576-583.
46. Nygren J, Soop M, Thorell A, et al. Preoperative oral carbohydrates and postoperative insulin resistance. Clinical Nutrition.1999; 18(2):117-120.
47. Nygren J, Soop M, Thorell A, et al. Preoperative oral carbohydrate administration reduces postoperative insulin resistance. Clinical Nutrition.1998; 17:65-71.
48.管玉龙,董培青,潘玉春等.冠状动脉搭桥围术期糖代谢的变化研究.中华外科杂志.1999,37(11)703.
49.杜智勇,史忠,杨天德.体外循环心内直视手术中胰岛素受体TPK活性的变化及意义.体外循环杂志,2002；4(3)：81-87.
50.李军.手术创伤后胰岛素抵抗的防治.[J]国外医学外科学2001；28(6)：341-3
1.李秀钧主编.胰岛素抵抗综合征[M].第一版.北京：人民卫生出版社,2001.22-25.
2. Ljungqvist O, Nygren J, Thorell A. Insulin resistance and elective surgery[J].Surgery,2000,128[5]:757-760.
3.林兴建,张颖冬.抵抗素与胰岛素抵抗[J].国外医学脑血管疾病分册,2004,12[7]：548-551.
4. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes[J]. Nature,2001,409[3]:307-312.
5. Zeyda M, Stulnig TM. Obesity, inflammation, and insulin resistance--a mini-review.Gerontology,2009,55[4]:379-386.
6. Bastard JP, Maachi M, Lagathu C, et al.Recent advances in the relationship between obesity, inflammation, and insulin resistance.Eur Cytokine Netw.2006,17[1]:4-12.
7. Gerrits AJ, Gitz E, Koekman CA,et al. Induction of insulin resistance by the adipokines resistin, leptin, PAI-1 and RBP4 in human megakaryocytes.Haematologica,2012 Apr 4.DOI:10.3324/haematol.2011.054916.
8. Kim KH, Lee K, Moon YS, et al. A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J Biol Chem,2001,276:11252-11256.
9. 乔智慧,唐兰芬.抵抗素与炎症相关性疾病研究进展.医学综述,2010,16[11]：1632-1634.
10. Schulze PC, Biolo A, Gopal D,et al. Dynamics in insulin resistance and plasma levels of adipokines in patients with acute decompensated and chronic stable heart failure.J Card Fail.2011,17[12]:1004-1011.
11. Fraulob JC, Souza-Mello V, Aguila MB,et al.Beneficial effects of rosuvastatin on insulin resistance, adiposity, inflammatory markers and non-alcoholic fatty liver disease in mice fed on a high-fat diet.Clin Sci (Lond).2012,123[4]:259-270.
12. De Vinuesa SG, Goicoechea M, Kanter J,et al. Insulin resistance, inflammatory biomarkers, and adipokines in patients with chronic kidney disease:effects of angiotensin Ⅱ blockade.J Am Soc Nephrol.2006,17[12 Suppl 3]:S206-212.
13.章建梁,秦永文,郑兴,等.血清抵抗素浓度与血压的相关性研究.解放军医学杂志,2003,28：241-242.
14. Melone M, Wilsie L, Palyha O, et al.Discovery of a new role of human resistin in hepatocyte low-density lipoprotein receptor suppression mediated in part by proprotein convertase subtilisin/kexin type 9.J Am Coll Cardiol,2012,59[19]:1697-1705.
15. De Luis DA, Gonzalez Sagrado M, et al.Resistin levels and inflammatory markers in patients with morbid obesity.Nutr Hosp.2010,25[4]:630-634.
16. Kozlowska L, Rydzewski A, Fiderkiewicz B, Adiponectin, resistin and leptin response to dietary intervention in diabetic nephropathy.J Ren Nutr. 2010,20[4]:255-262.
17. Hartman HB, Hu X, Tyler KX, et al. Mechanisms regulating adipocyte expression of resistin. J Biol Chem,2002,277:19754-19761.
18. Olszanecka-Glinianowicz M, Kuglin D, Dabkowska-Huc A, et al.Serum adiponectin and resistin in relation to insulin resistance and markers of hyperandrogenism in lean and obese women with polycystic ovary syndrome.Eur J Obstet Gynecol Reprod Biol,2011,154[1]:51-56.
19. Gharibeh MY, Al Tawallbeh GM, Abboud MM,et al. Correlation of plasma resistin with obesity and insulin resistance in type 2 diabetic patients.Diabetes Metab, 2010,36[6 Pt 1]:443-449.
20. Wilkinson M, Brown R, Imran SA, et al. Adipokine gene expression in brain and pituitary gland. Neuroendocrinology,2007,86:191-209.
21. Wiesner G, Brown RE, Robertson GS, et al. Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ischaemic mouse brain. Neuroreport,2006,17:1195-1198.
22. Brown R, Thompson HJ, Imran SA, et al. Traumatic brain injury induces adipokine gene expression in rat brain. Neurosci Lett,2008,432:73-78.
23. Dong X-Q, Hu Y-Y, Yu W-H, et al. High concentrations of resistin in the peripheral blood of patients with acute basal ganglia hemorrhage are associated with poor outcome. J Crit Care,2009, doi:10.1016/j.jcrc.2009.09.008.
24. Oktay A,Aliefendioglu D, Caglayan O, et al. Plasma resistin levels of term newborn infants with hypoxic-ischemic encephalopathy[J]. Arch Dis Child,2008,6 [11]:92-93.
25.杨松斌,王克义,竹方龙,等.重型脑外伤患者血浆抵抗索水平动态变化与预后的关系.中国临床神经科学,2011,19[3]：265-269.
26. Dash PK, Kobori N, Moore AN. A molecular description of brain trauma pathophysiology using microarray technology:an overview. Neurochem,Res 2004,29:1275-1286.
27. Matzilevich DA, Rall JM, Moore AN, Grill RJ, Dash PK. High-density microarray analysis of hippocampal gene expression following experimental brain injury. J. Neurosci. Res 2002,67:646-663.
28. Moinard C, Neveux N, Royo N,et al. Marchand-Verrecchia C, Plotkine M, Cynober L.Characterization of the alteration of nutritional state in brain injury induced by fluid percussion in rats. Intens. Care Med 2005,31:281-288.
2. Langlois JA, Rutland-Brown W, Thomas KE. Traumatic brain injury in the United States:emergency department visits, hospitalizations, and deaths. Atlanta(GA): Centers for Disease Control and Prevention, National Center for Injury Prevention ant Control,2006.
3. Gerhart KA,Mellick DC,Weintraub AH. Vionlence-related traumatic brain injury population-based study[J].J Trauma,2003,55(6):1045-1053.
4. McNair ND. Traumatic brain injm'y, Nurs Clin North Am,1999,34(3):637-659.
5. Andersson EH, Bjorkland R, Emanuelson I, et al. Epidemiology of traumatic brain injury:a population based study in western Sweden. Acta Neurol Scand.2003, 107(4):256-259.
6. Anonymous.NIH Consensus Development Panel on Rehabilitation of Persons With Traumatic Brain Injury. JAMA,1999,282(10):974-983.
7. Servadei F, Antonelli V, Betti L, et al. Regional brain injury epidemiology as the basis for planning brain injury treatment. The Romagna(Italy)experience. J Neurosurg Sci,2002,46(3-4):111-119.
8. Gururaj G. Epidemiology of traumatic brain injuries:Indian seenario. Neurol Res, 2002,24(1):24-28.
9.赵雅度.神经系统外伤[M].北京：人民军医出版社,2001,3-5.
10.王忠诚,赵元立.加强颅脑外伤临床基础研究,提倡规范化治疗，中华神经外科杂志.2001,17(3)：133-134.
11. Marshall LF. Head injury:recent past, present, and future. Neurosurgery,2000, 47(3):546-561.
12. Moinard C, Neveux N, Royo N, Genthon C, Marchand-Verrecchia C, Plotkine M, Cynober L.Characterization of the alteration of nutritional state in brain injury induced by fluid percussion in rats. Intens. Care Med 2005,31:281-288.
13. Thompson HJ, Tkacs NC, Saatman KE, Raghupathi R, McIntosh TK. Hyperthermia following traumatic brain injury:a critical evaluation. Neurobiol Dis,2003,12:163-173.
14. Ahima RS, Qi Y, Singhal NS. Adipokines that link obesity and diabetes to the hypothalamus. Prog Brain Res,2006,153:155-174.
15. Trayhurn P, Wood IS. Adipokines:inflammation and the pleiotropic role of white adipose tissue. Br J. Nutr,2004,92:347-355.
16. Wilkinson M, Brown R, Imran SA, Ur E. Adipokine gene expression in brain and pituitary gland.Neuroendocrinology,2007,86:191-209.
17. Dash PK, Kobori N, Moore AN. A molecular description of brain trauma pathophysiology using microarray technology:an overview. Neurochem. Res,2004,29:1275-1286.
18. Matzilevich DA, Rall JM, Moore AN, Grill RJ, Dash PK. High-density microarray analysis of hippocampal gene expression following experimental brain injury. J. Neurosci Res,2002;67:646-663.
19. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes[J]. Nature,2001,409 (3):307-312.
20. Morash B, Willkinson D, Ur E, et al. Resistin expression and regulation in mouse pituitary. FEBS Lett,2002,526:26-30.
21.乔智慧,唐兰芬.抵抗素与炎症相关性疾病研究进展.医学综述,2010,16(11)：1632-1634.
22. Reilly MP, Lehrke M,Wolfe ML, et al. Resistin is an inflammatory marker of atherosclerosis in humans[J]. Circulation,2005,111(7):932-939.
23. Kaser S, KaserA, SandhoferA, et al. Resistin messenger-RNA expression is increased by p roinflammatory cytokines in vitro [J].Biochem Biophys Res Commun,2003, 309 (2):286-290.
24. Kawanami D, Maemura K, Takeda N, et al. Direct recip rocal effects of resistin and adiponectin on vascular endothelial cells:a new insight into adipocytokine2endothelial cell interactions[J]. Biochem Biophys Res Commun,2004, 314 (2):415-419.
25. Hartman HB, Hu X, Tyler KX, et al. Mechanisms regulating adipocyte expression of resistin. J Biol Chem,2002,277:19754-19761.
26. Nagaev I, Smith U. Insulin resistance and type diabetes are not related to resistin expression in human fat cells or skeletal muscle. Biochem Biophys Res Commun, 2001,285:561-564.
27. Levy JR, Davenport B, Clore JN, et al. Lipid metabolism and resistin gene expression in insulin-resistant Fischer 344 rats. Am J Physiol Endocrinol Metab, 2002,282:E626-E633.
28. Brown R, Thompson HJ, Imran SA, et al. Traumatic brain injury induces adipokine gene expression in rat brain. Neurosci Lett,2008,432:73-78.
29. Wiesner G, Brown RE, Robertson GS, et al. Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ischaemic mouse brain. Neuroreport,2006,17:1195-1198.
30. Dong X-Q, Hu Y-Y, Yu W-H, et al. High concentrations of resistin in the peripheral blood of patients with acute basal ganglia hemorrhage are associated with poor outcome. J Crit Care,2009, doi:10.1016/j.jcrc.2009.09.008.
31. Oktay A,Aliefendioglu D, Caglayan O, et al. Plasma resistin levels of term newborn infants with hypoxic-ischemic encephalopathy[J]. Arch Dis Child,2008,6 (11):92-93.
32.杨松斌,王克义,竹方龙,等.重型脑外伤患者血浆抵抗索水平动态变化与预后的关系.中国临床神经科学,2011,19(3)：265-269.
33. Andrade CF, Waddell TK, Keshavjee S, et al. Innate immunity and organ transplaniation:the potential role of toll-like receptors. Am J Trans,2005,5:969-975.
34.李秀钧主编.胰岛素抵抗综合征[M].第一版.北京：人民卫生出版社,2001.22-25
35. Ljungqvist O, Nygren J, Thorell A. Insulin resistance and elective surgery[J].Surgery,2000,128(5):757-760.
36.王清华,徐如祥,李良平,等.大鼠不同程度脑损伤模型的建立[J].创伤外科杂志,2000；2(1)：42-44.
37. Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats [J]. Stroke,1989,20(1):84-91.
38.吴旭,王保捷,张国华,等.大鼠脑损伤分级自由落体打击模型的建立.中国法医学杂志,2005,20(1)：1-3.
39. Feeney DM, Boyeson MG, Lima RT, et al. Responses to cortical injury:Methodology and local effects of contusions in the rat[J]. Brain Res,1981,211:67-77.
40. Dixon CE, Lyeth BG, Povlishock JK, et al. A fluid percussion model of experimental brain injury in the rat[J]. J Neurosurg,1987,67:110-119.
41.李力仙,王天佑,钟震宇.自由落体和液压致大鼠脑损伤的对比研究.中华实验外科杂志.1999,16：447-8.
42. Luigi Brandi, Donatella Santoro, Andrea Natali, et al.Insulin resistance of stress:site and mechanisms.Clinical Science.1993;85:525-535.
43. Shaw Jhf,Wolfe RR.An integrated analysis of glucose,fat and protein metabolism in severely traumatized patients.Studies in the basal state and the response to total parenteral nutrition.Ann Surg.1989;209:63-71.
44.李光伟,潘孝仁,Lillioja S,等.检测人群胰岛素敏感性的一项新指数.中华内科杂志,1993,12(10)：656.
45. Soop M, Nygren J, Myrenfors P, et al. Preoperative oral carbohydrate treatment attenuates immediate postoperative insulin resistance. Am J Physiol Endocrinol Metab.2001;280:E576-583.
46. Nygren J, Soop M, Thorell A, et al. Preoperative oral carbohydrates and postoperative insulin resistance. Clinical Nutrition.1999; 18(2):117-120.
47. Nygren J, Soop M, Thorell A, et al. Preoperative oral carbohydrate administration reduces postoperative insulin resistance. Clinical Nutrition.1998; 17:65-71.
48.管玉龙,董培青,潘玉春等.冠状动脉搭桥围术期糖代谢的变化研究.中华外科杂志.1999,37(11)703.
49.杜智勇,史忠,杨天德.体外循环心内直视手术中胰岛素受体TPK活性的变化及意义.体外循环杂志,2002；4(3)：81-87.
50.李军.手术创伤后胰岛素抵抗的防治.[J]国外医学外科学2001；28(6)：341-3
1.李秀钧主编.胰岛素抵抗综合征[M].第一版.北京：人民卫生出版社,2001.22-25.
2. Ljungqvist O, Nygren J, Thorell A. Insulin resistance and elective surgery[J].Surgery,2000,128[5]:757-760.
3.林兴建,张颖冬.抵抗素与胰岛素抵抗[J].国外医学脑血管疾病分册,2004,12[7]：548-551.
4. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes[J]. Nature,2001,409[3]:307-312.
5. Zeyda M, Stulnig TM. Obesity, inflammation, and insulin resistance--a mini-review.Gerontology,2009,55[4]:379-386.
6. Bastard JP, Maachi M, Lagathu C, et al.Recent advances in the relationship between obesity, inflammation, and insulin resistance.Eur Cytokine Netw.2006,17[1]:4-12.
7. Gerrits AJ, Gitz E, Koekman CA,et al. Induction of insulin resistance by the adipokines resistin, leptin, PAI-1 and RBP4 in human megakaryocytes.Haematologica,2012 Apr 4.DOI:10.3324/haematol.2011.054916.
8. Kim KH, Lee K, Moon YS, et al. A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J Biol Chem,2001,276:11252-11256.
9. 乔智慧,唐兰芬.抵抗素与炎症相关性疾病研究进展.医学综述,2010,16[11]：1632-1634.
10. Schulze PC, Biolo A, Gopal D,et al. Dynamics in insulin resistance and plasma levels of adipokines in patients with acute decompensated and chronic stable heart failure.J Card Fail.2011,17[12]:1004-1011.
11. Fraulob JC, Souza-Mello V, Aguila MB,et al.Beneficial effects of rosuvastatin on insulin resistance, adiposity, inflammatory markers and non-alcoholic fatty liver disease in mice fed on a high-fat diet.Clin Sci (Lond).2012,123[4]:259-270.
12. De Vinuesa SG, Goicoechea M, Kanter J,et al. Insulin resistance, inflammatory biomarkers, and adipokines in patients with chronic kidney disease:effects of angiotensin Ⅱ blockade.J Am Soc Nephrol.2006,17[12 Suppl 3]:S206-212.
13.章建梁,秦永文,郑兴,等.血清抵抗素浓度与血压的相关性研究.解放军医学杂志,2003,28：241-242.
14. Melone M, Wilsie L, Palyha O, et al.Discovery of a new role of human resistin in hepatocyte low-density lipoprotein receptor suppression mediated in part by proprotein convertase subtilisin/kexin type 9.J Am Coll Cardiol,2012,59[19]:1697-1705.
15. De Luis DA, Gonzalez Sagrado M, et al.Resistin levels and inflammatory markers in patients with morbid obesity.Nutr Hosp.2010,25[4]:630-634.
16. Kozlowska L, Rydzewski A, Fiderkiewicz B, Adiponectin, resistin and leptin response to dietary intervention in diabetic nephropathy.J Ren Nutr. 2010,20[4]:255-262.
17. Hartman HB, Hu X, Tyler KX, et al. Mechanisms regulating adipocyte expression of resistin. J Biol Chem,2002,277:19754-19761.
18. Olszanecka-Glinianowicz M, Kuglin D, Dabkowska-Huc A, et al.Serum adiponectin and resistin in relation to insulin resistance and markers of hyperandrogenism in lean and obese women with polycystic ovary syndrome.Eur J Obstet Gynecol Reprod Biol,2011,154[1]:51-56.
19. Gharibeh MY, Al Tawallbeh GM, Abboud MM,et al. Correlation of plasma resistin with obesity and insulin resistance in type 2 diabetic patients.Diabetes Metab, 2010,36[6 Pt 1]:443-449.
20. Wilkinson M, Brown R, Imran SA, et al. Adipokine gene expression in brain and pituitary gland. Neuroendocrinology,2007,86:191-209.
21. Wiesner G, Brown RE, Robertson GS, et al. Increased expression of the adipokine genes resistin and fasting-induced adipose factor in hypoxic/ischaemic mouse brain. Neuroreport,2006,17:1195-1198.
22. Brown R, Thompson HJ, Imran SA, et al. Traumatic brain injury induces adipokine gene expression in rat brain. Neurosci Lett,2008,432:73-78.
23. Dong X-Q, Hu Y-Y, Yu W-H, et al. High concentrations of resistin in the peripheral blood of patients with acute basal ganglia hemorrhage are associated with poor outcome. J Crit Care,2009, doi:10.1016/j.jcrc.2009.09.008.
24. Oktay A,Aliefendioglu D, Caglayan O, et al. Plasma resistin levels of term newborn infants with hypoxic-ischemic encephalopathy[J]. Arch Dis Child,2008,6 [11]:92-93.
25.杨松斌,王克义,竹方龙,等.重型脑外伤患者血浆抵抗索水平动态变化与预后的关系.中国临床神经科学,2011,19[3]：265-269.
26. Dash PK, Kobori N, Moore AN. A molecular description of brain trauma pathophysiology using microarray technology:an overview. Neurochem,Res 2004,29:1275-1286.
27. Matzilevich DA, Rall JM, Moore AN, Grill RJ, Dash PK. High-density microarray analysis of hippocampal gene expression following experimental brain injury. J. Neurosci. Res 2002,67:646-663.
28. Moinard C, Neveux N, Royo N,et al. Marchand-Verrecchia C, Plotkine M, Cynober L.Characterization of the alteration of nutritional state in brain injury induced by fluid percussion in rats. Intens. Care Med 2005,31:281-288.