本文選題：失血性休克　切入點(diǎn)：血管低反應(yīng)性　出處：《第三軍醫(yī)大學(xué)》2013年碩士論文

【摘要】：失血休克、膿毒癥等臨床重癥患者在經(jīng)歷缺血缺氧、內(nèi)毒素釋放、腸道細(xì)菌移位等多重打擊后，在失代償期容易出現(xiàn)嚴(yán)重的組織損傷，出現(xiàn)全身炎癥反應(yīng)綜合癥(SIRS)、多器官功能障礙(MODS)甚至發(fā)生多器官功能衰竭(MOF)。探究其原因，除了全身炎癥失控和能量代謝障礙以外，休克后血管反應(yīng)性降低也是另一重要原因。血管低反應(yīng)性即血管對血管舒縮藥物的失敏。血管低反應(yīng)性不僅影響著休克的發(fā)生發(fā)展，還制約著休克的復(fù)蘇和預(yù)后。目前認(rèn)為休克后血管低反應(yīng)性的發(fā)生機(jī)制與鈣失敏(calcium desensitization)、腎上腺素能受體失敏、血管平滑肌細(xì)胞(VSMC)膜超極化有關(guān)。線粒體通透性轉(zhuǎn)換孔(MPTP)是線粒體內(nèi)膜上的一個重要的非選擇性通道，研究表明MPTP在細(xì)胞死亡中發(fā)揮了重要作用，并參與了多種疾病的發(fā)生，例如MPTP開放參與了缺血再灌注損傷；MPTP通過線粒體凋亡途徑參與了休克后血管高滲透性的發(fā)生。失血性休克后血管組織MPTP是否異常開放？MPTP是否參與了失血性休克后血管低反應(yīng)性的發(fā)生？MPTP開放通過Caspase依賴的凋亡途徑參與了細(xì)胞凋亡和疾病發(fā)生，研究表明Caspase能激活Rho激酶并參與心臟肥大和心衰的發(fā)生，而本實(shí)驗(yàn)室證實(shí)Rho激酶參與調(diào)控休克后血管鈣敏感性及血管反應(yīng)性，MPTP是否通過Caspase和Rho激酶從而調(diào)節(jié)血管鈣敏感性及血管反應(yīng)性？目前還未見相關(guān)報(bào)道。 據(jù)此，本研究利用大鼠失血性休克模型，以腸系膜上動脈(SMA)為研究對象，利用MPTP特異性關(guān)閉劑環(huán)孢素A(CsA)和開放劑蒼術(shù)苷(ATR)為工具藥，探討了MPTP在失血性休克后血管低反應(yīng)性中的作用。具體內(nèi)容包括兩大部分：(1)MPTP是否參與失血性休克后血管低反應(yīng)性的發(fā)生；(2)失血性休克后MPTP調(diào)節(jié)血管反應(yīng)性的機(jī)制。 主要實(shí)驗(yàn)方法： 取正常及失血性休克大鼠腸系膜上動脈(SMA)，采用離體血管環(huán)張力測定技術(shù)，用血管環(huán)對梯度濃度去甲腎上腺素(NA)的收縮反應(yīng)測定血管反應(yīng)性，在去極化狀態(tài)下(120mmol/L K+)，用血管環(huán)對梯度濃度Ca2+的收縮反應(yīng)測定血管的鈣敏感性，用分光光度法測定線粒體在540nm處的吸光度變化反映MPTP的開放和關(guān)閉狀態(tài)，用分光光度法測定SMA405nm處的吸光度反映Caspase-3的活性，用western blotting測定SMA肌球蛋白磷酸酶調(diào)節(jié)亞單位(MYPT)磷酸化水平表示Rho激酶活性。實(shí)驗(yàn)分兩部分：第一部分實(shí)驗(yàn)觀察失血性休克大鼠血管反應(yīng)性變化和鈣敏感性變化及MPTP開放的變化，以及通過觀察MPTP特異性開放劑ATR和關(guān)閉劑CsA是否可以通過MPTP的開放關(guān)閉來調(diào)節(jié)血管反應(yīng)性及鈣敏感性，以證實(shí)MPTP在失血性休克血管低反應(yīng)性形成中的作用。第二部分實(shí)驗(yàn)先在體觀察休克后Caspase-3活性和Rho激酶活性的變化，以及ATR和CsA對其的影響，以證實(shí)MPTP與Caspase-3和Rho激酶的關(guān)系。離體實(shí)驗(yàn)，缺氧處理血管環(huán)測定血管反應(yīng)性及鈣敏感性及MPTP開放狀態(tài)，以模擬失血性休克模型；在ATR孵育處理下觀察Rho激酶特異性抑制劑Y27632、Caspase-3特異性抑制劑Z-DEVD-FMK對Rho激酶、血管鈣敏感性及血管反應(yīng)性的影響，以明確MPTP是否通過Caspase-3、Rho激酶調(diào)節(jié)血管反應(yīng)性及鈣敏感性。 主要研究結(jié)果： 1．失血性休克后腸系膜上動脈血管(SMA)反應(yīng)性和鈣敏感性存在雙相變化，表現(xiàn)為休克早期(10min-0.5h)SMA血管環(huán)對NE和鈣的反應(yīng)性升高，量-效曲線左移，最大收縮力(Emax)升高(p0.05或p0.01)；隨著時間的延長，血管環(huán)對NE和鈣的反應(yīng)性逐漸下降，表現(xiàn)為休克中晚期(1h-4h)，其量-效曲線明顯右移，，最大收縮力(Emax)明顯降低(p0.01)。休克早期(10min-0.5h)腸系膜上動脈MPTP的吸光度比值出現(xiàn)下降趨勢(表示MPTP開放)，時-效曲線下移，吸光度變化值增大，但無顯著性差異(p0.05)；休克中晚期(1h-4h)腸系膜上動脈MPTP的吸光度比值明顯下降，時-效曲線下移，吸光度變化值顯著增大(p0.05或p0.01)。說明失血性休克中晚期血管MPTP存在異常開放。 2．MPTP的開放劑ATR(5mg/kg)可使休克0.5h后腸系膜上動脈MPTP的吸光度時-效曲線下移，吸光度變化值顯著增加(p0.05)，并使SMA血管環(huán)對NE和鈣的量-效曲線右移，使NE和Ca2+的Emax降低(p0.05或p0.01)；而MPTP的關(guān)閉劑CsA(10mg/kg)則可使休克2.0h后腸系膜上動脈MPTP的吸光度時-效曲線上移，吸光度變化值顯著降低(p0.05)，并使SMA血管環(huán)對NE和鈣的量-效曲線左移，使NE和Ca2+的Emax升高(p0.05或p0.01)，提示MPTP在失血性休克血管低反應(yīng)性和鈣失敏的發(fā)生中起重要作用。 3.失血性休克后Rho激酶活性出現(xiàn)雙相變化，表現(xiàn)為休克早期(10min-0.5h)MYPT磷酸化水平升高(代表Rho激酶活性升高)(p0.05或p0.01)，而休克晚期(4h)MYPT磷酸化水平降低(p0.05)；休克早期(10min)Caspase-3活性無顯著變化，休克中晚期(1h-4h)Caspase-3活性增加(p0.01)；MPTP開放劑ATR可使休克0.5h后SMA的caspase-3活性增加(p0.05)和MYPT磷酸化水平降低，而MPTP的關(guān)閉劑CsA則可使休克2.0h后caspase-3活性降低(p0.01)和MYPT磷酸化水平升高，說明MPTP調(diào)節(jié)失血性休克后血管組織caspase-3活性和Rho激酶活性。 4．缺氧處理SMA10min、0.5h后，SMA血管環(huán)對NE和鈣的反應(yīng)性升高(P 0.05)，缺氧處理1.0h、2.0h，SMA血管環(huán)對NE和鈣的反應(yīng)性降低(P 0.05)，MPTP的吸光度變化值顯著增加(P 0.05)，說明缺氧處理可模擬失血性休克。在ATR(20μmol/L)孵育下,給予Rho激酶抑制劑Y27632(10-5mol/L)顯著降低SMA血管環(huán)對NE和鈣的反應(yīng)性(P0.05)，給予Caspase-3抑制劑Z-DEVD-FMK(100μmol/L)顯著升高SMA血管環(huán)對NE和鈣的反應(yīng)性(P0.05)，同時MYPT磷酸化水平升高(P0.05)，提示MPTP通過Caspase-3、Rho激酶調(diào)節(jié)血管反應(yīng)性和鈣敏感性。 結(jié)論： 1.失血性休克血管MPTP存在異常開放；MPTP在失血性休克血管低反應(yīng)性和鈣失敏的發(fā)生中起重要作用。 2. MPTP對失血性休克Caspase-3活性和Rho激酶活性有重要的調(diào)節(jié)作用，MPTP對Caspase-3活性有正向調(diào)節(jié)作用，對Rho激酶活性有負(fù)向調(diào)節(jié)作用。 3. MPTP可通過Caspase-3、Rho激酶調(diào)節(jié)血管反應(yīng)性及鈣敏感性。
[Abstract]:In addition to systemic inflammatory reaction and energy metabolism disorder , the mechanism of hyporeactivity is not only the development of shock , but also the resuscitation and prognosis of shock . The study shows that MPTP plays an important role in cell death and participates in the occurrence of various diseases , such as MPTP is open to ischemia - reperfusion injury .
MPTP is involved in the occurrence of vascular hyporeactivity following hemorrhagic shock . Does the MPTP participate in the occurrence of hyporesponsiveness after hemorrhagic shock ? MPTP is involved in the occurrence of apoptosis and heart failure through caspase - dependent apoptotic pathway . The study shows that Caspase can activate rho kinase and participate in cardiac hypertrophy and heart failure .

In this study , the effects of MPTP on the hyporesponsiveness of hemorrhagic shock were investigated by using the rat hemorrhagic shock model with MPTP - specific closing agent cyclosporin A ( CsA ) and open agent ( ATR ) . The specific contents included two parts : ( 1 ) whether MPTP was involved in the occurrence of hyporeactivity after hemorrhagic shock ;
( 2 ) MPTP after hemorrhagic shock regulates the mechanism of vascular reactivity .

Main experimental methods :

The effects of MPTP on the activity of Caspase - 3 and the activity of Caspase - 3 were measured by means of spectrophotometry . The effects of MPTP on the activity of Caspase - 3 and the activity of Caspase - 3 were measured .
The effects of the specific inhibitor Y27632 , Caspase - 3 specific inhibitor Z - DEVD - FMK on rho kinase , calcium sensitivity and vascular reactivity were observed under ATR incubation .

Key findings :

1 . There was a biphasic change in the reactivity and calcium sensitivity of SMA in the mesenteric artery after hemorrhagic shock . The response to NE and Ca increased at the early stage of shock ( 10 min - 0.5 h ) , and the maximal contraction force ( Emax ) increased ( p < 0.05 or p0.01 ) .
In the early stage of shock ( 1h - 4h ) , the maximal contractile force ( Emax ) decreased significantly ( P 0.01 ) , while the maximum contraction force ( Emax ) decreased significantly ( P 0.01 ) .
In the middle and late stages of shock ( 1h - 4h ) , the absorbance ratio of MPTP in the mesenteric artery decreased significantly , the time - effect curve moved down , and the absorbance changed significantly ( p < 0.05 or p0.01 ) . The abnormal opening of MPTP in the middle and late stage of hemorrhagic shock was described .

2 . MPTP ( 5 mg / kg ) could decrease the absorbance of MPTP in mesenteric artery after 0.5 h shock , and the change in absorbance was significantly increased ( p < 0.05 ) , and the amount of NE and Ca 2 + in SMA blood vessel was decreased ( p < 0.05 or p0.01 ) .
However , CsA ( 10 mg / kg ) of MPTP decreased the absorbance of MPTP on mesenteric artery after shock 2.0h . The change in absorbance was significantly decreased ( p < 0.05 ) , and the Emax of NE and Ca 2 + increased ( p < 0.05 or p0.01 ) . It was suggested that MPTP played an important role in hyporeactivity of hemorrhagic shock and loss of calcium .

3 . After hemorrhagic shock , there were two phase changes after hemorrhagic shock , which showed that the phosphorylation level of MYPT increased in the early stage of shock ( 10 min - 0.5 h ) ( p0.01 or p0.01 ) , while the phosphorylation level of MYPT decreased ( p . 05 ) at the late stage of shock ( 4h ) ;
Caspase - 3 activity was not significantly changed in the early stage of shock ( 10min ) , and the activity of Caspase - 3 in the middle and late stage of shock was increased ( p0.01 ) .
The activity of caspase - 3 and the phosphorylation of MYPT decreased after shock 0.5h , and the activity of caspase - 3 decreased ( p0.01 ) and the level of MYPT phosphorylation increased , indicating that MPTP regulated the activity of caspase - 3 and the activity of caspase - 3 after hemorrhagic shock .

4 . The response of SMA to NE and Ca was significantly increased ( P0.05 ) . The response of SMA to NE and Ca was significantly increased ( P0.05 ) . At the same time , the expression of Caspase - 3 inhibitor Z - DEVD - FMK ( 100 渭mol / L ) increased significantly ( P0.05 ) .

Conclusion :

1 . There was an abnormal opening of MPTP in hemorrhagic shock .
MPTP plays an important role in the hyporeactivity of hemorrhagic shock and the occurrence of calcium loss .

2 . MPTP plays an important role in regulating the activity of Caspase - 3 in hemorrhagic shock . MPTP has a positive regulatory effect on Caspase - 3 activity .

3 . MPTP can regulate vascular reactivity and calcium sensitivity through Caspase - 3 and rho kinase .

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R631.4

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