甘氨酸在出血性休克大白鼠中的作用
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摘要
实验目的：观察甘氨酸是否对出血性休克所致的脏器损伤具有保护作用以及能否降低死亡率，同时阐明其机制。
实验方法：1．失血性休克模型的建立：Wister大鼠(体重250-300g)雌雄不限，用4.5％异戊巴比妥(80mg／kg)在25℃室温下腹腔内给药麻醉。右股动脉插入套管针，连接低压分析仪，监测血压；左颈静脉插管给予甘氨酸、生理盐水和自体血(复苏通路)；右颈动脉插管，另一端连接肝素化玻璃注射器中，进行放血诱导休克，在5分钟内使血压下降并维持平均动脉压30-35mmHg。通过输、抽少量血维持低血压状态55分钟。低血压60分钟后用60％自体血回输5分钟和2倍回输血量的生理盐水回输55分钟进行复苏。复苏前大白鼠随机分为三组，每组20只：休克组(Shock)、甘氨酸治疗组(Glycine+Shock)和假休克组(sham)(只进行手术操作不放血诱导休克)。甘氨酸60mg／kg溶于0.5ml生理盐水中在复苏开始时输入左颈静脉，相同体积的生理盐水给予休克组。2．Cr，CK AST，ALT和ALKP检测：分别于休克前，休克终末，复苏终末及复苏后18小时采集血样，血清离心，检测肌酐(Cr)，肌酸磷酸激酶(CK)，谷草转氨酶(AST)，谷丙转氨酶(ALT)和碱性磷酸酶(ALKP)。3．肝脏Kupffer细胞的分离和培养：复苏后2小时，肝脏在活体时经门静脉插管，用无Ca~(2+)无Mg~(2+) Hanks'平衡盐溶液(HBSS)在37℃下进行灌流(以20ml／分钟，灌流10分钟)，随后用含有0.025％Ⅳ型胶原酶的Hanks'溶液在37℃下以23ml／分的速度灌流7分钟，当肝脏被充分消化后，取下放入Ⅳ型胶原酶缓冲液中并切成小碎块，37℃水浴振荡5分钟，悬浮液用150目网过滤，然后在4℃以50*g离心10分钟，除去下层细胞团块，上层液加入25％+50％Percoll梯度介质中在4℃800*g离心10分钟，抽取25％与50％界面之间细胞平板片状团块，用Hanks'液冲洗再离心，细胞移入培养液中，放入37℃5％CO_2孵育箱中孵育1小时，非贴壁的上皮细胞通过换液除去而贴壁细胞为kupffer细胞。细胞培养24小时后做[Ca~(2+)]i和
TNF一Q检测。4.细胞内钙(【Caz+〕i)测定:使用荧光钙指示剂Fura一2
和荧光显微镜进行检测。含有kupffer细胞的培养皿在37℃下用
1一IO00ng/ml内毒素(LPS)刺激，在340和380nm激发波长和510nm
的发射波长检测显微镜视野下每个kupffer细胞Fura一2荧光密度的
变化。细胞内钙浓度由以下公式来确定:〔Caz+]，=Kd{(R一Rmin)/
(Rmax一R)}/FO/Fs)。5.在特定介质中从培养的kupffer细胞测定
TNF一Q:游离的Kupffer细胞种植在含有培养液的24孔板中培养。
24小时后Kupffer细胞在含有l一1000ng/mL的LPS、5%鼠血清、10mM
HEPES和抗生素的R即1 1 640液体中刺激4小时。上层清液用酶联免
疫吸附测定法检测TNF一Q(ELISA)。6.病理组织学观察:复苏后18
小时取大白鼠肺、心、肝和肾脏，用l%多聚甲醛固定，石蜡包埋，切
片，H一E染色，光镜观察组织病理变化。7.统计学分析:使用SPSS n .0
版本分析软件，应用方差、卡方和t检验。
实验结果:1.失血性休克后大白鼠的生存率:甘氨酸治疗组生存
呈现出甘氨酸剂量依赖性。本实验对甘氨酸剂量对生存率影响实验观
察72小时，结果得出最大生存率为77.8%，剂量大约为6Omg/kg。休克
组10只大鼠接受生理盐水，18小时生存率为70%，24小时生存率为
50%，48小时生存率为30%，72小时为20%。甘氨酸治疗组(9只)，18
小时生存率100%，24小时88.9%，48小时77.8%，72小时77.8%。
72小时两组生存率经卡方(Chi一square)检验差异显著(P<0 .05)。
甘氨酸能明显降低出血性休克大白鼠的死亡率。2.甘氨酸对肌酸磷
酸激酶，转氨酶及肌配的影响:肌酸磷酸激酶(CK)反映心肌和骨骼肌
损伤的指标，在休克期没有明显升高，但复苏后18小时明显增高，高
于正常值3倍以上。CK的升高可被甘氨酸有效阻止，经t检验，两
组差异非常显著(P<0 .01);谷草转氨酶(AST)，谷丙转氨酶(ALT)以及
碱性磷酸酶(ALKP)反映肝脏损害的指标，18小时明显升高，甘氨酸同
样可明显减少其升高，经t检验，两组差异非常显著(P<0 .01);Cr
反映肾损害的指标，18小时明显增高，甘氨酸明显降低其血中浓度，
经t检验，差异非常显著(P<0 .01)。这提示甘氨酸具有脏器保护作用。
3.出血性休克脏器损害病理组织形态学改变以及甘氨酸的作用:复苏
后18小时分别取休克组，假休克组和甘氨酸组大白鼠的肺，心，肝，肾
脏，中性福尔马林固定，石蜡包埋，常规H一E染色。在假休克组，没有明
显病理性变化;在休克组，肺组织中可见明显的肺泡壁增厚，上皮增生，
毛细血管扩张充血，肺泡壁间质细胞增生，巨噬细胞增多，毛细血管周
围淋巴细胞，浆细胞侵润，形成血管套样改变;在心肌组织中，休克组
呈现心肌细胞水肿，有中度、重度变性等;肝组织病理，休克组肝细胞
肿大变性，水肿，肝窦变窄;肾组织中，休克组，近曲小管广泛水肿变性，
管腔变窄。甘氨酸治疗组显示上述组织的病变程度明显减轻。这也提
示甘氨酸具有脏器保护作用。4.甘氨酸对Kupffer细胞内钙水平变
化的影响:三组大白鼠，复苏后二小时，肝Kupffer细胞进行游离单细
胞荧光检查。加入LPS(l，10，100，10O0ng/ml
Objective: This study investigates whether glycine prevents the organs of rat from injury and decreases mortality after hemorrhagic shock. Methods: 1. Hemorrhagic shock model: Wister rats(250-300g) were anesthetized with 4.5% amobarbital sodium(80mg/Kg i.p.) in room temperature(25℃). Blood pressure was monitored via polyethylene tubing(Intima 24GA) inserted into the right femoral artery, using a low pressure analyzer(Polygraph RM6000). The left jugular vein was cannulated(Intima 22GA) to administer glycine, normal saline and shed blood. The right carotid artery was cannulated with polyethylene tubing(Intima 22GA), and shock was induced over 5min by withdrawing blood into a heparinized glass syringe until mean arterial pressure was reduced to 30-35mmHg. Constant pressure was maintained by further withdrawal of small amount of blood as necessary for 55min. After 60 min of hypotension, rats were resuscitated by transfusion of 60%shed blood over 5 min, followed by normal saline(twice the shed blood volume) for
55 min. Prior to resuscitation, animals were randomly assigned to three groups(20 rats each group): shock group; glycine treatment group and sham group(operation was performed not to induce shock). Glycine(60mg/Kg) dissolved in 0.5mL normal saline was injected into the left jugular vein, and an equal volume of normal saline was given to shock group. 2. Determination of creatine phosphokinase(CK), aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(ALKP) and creatinine(Cr): Blood samples were collected before and at the end of shock, at the end of resuscitation and 18 h later. Plasma was centrifuged, stored at -80℃ for determining Cr,CK,AST,ALT and ALKP. 3. Kupffer cell preparation and culture: Two hours after resuscitation, the liver was perfused through the portal vein with Ca2+- and Mg2+-free Hanks' balanced salt solution(HBSS) at 37C
for 10 min at a flow rate of 20mL/min. Subsequently, perfusion was with HBSS containing 0.025% collagenase IV at37C for 7min at a flow rate of 23mL/min. After the liver was digested, it was excised and cut into small pieces in collagenase buffer. The suspension was filtered through nylon gauze mesh and the filtrate was centrifuged at 50*g for 10 min at 4 C. The suspension was centrifuged on a density cushion of 25%+50% Percoll at 800*g for 10 min, and cells fraction between 25% and 50% Percoll was collected and washed with buffer again, cells were seeded into 2mL RPMI1640 at 37C and incubated with 5% CO2. Non-αdherent cells were removed after 1 h by replacing buffer, and adherent cells(Kupffer cells) were cultured for 24 h prior to [Ca2+]i and TNF- a measurement. 4. Measurement of intracellular Ca2+([Ca2+]i): Intracellular calcium was measured flurometrically using the fluorescent calcium indicator dye fura-2 and a microspectroflurometer interfaced with an inverted microscope. Kupffer cells were placed in chambers with buffer at 37 C and stimulated with 1-1000ng/mL endotoxin. Changes in fluorescence intensity of fura-2 at excitation wavelengths of 340nm and 380nm and emission at 510nm were monitored in individual Kupffer cells. Intracellular calcium was determined from the equation: [Ca2+]i = Kd{(R-Rmin)/(Rmax-R)}/(Fo/Fs). 5. Measurement of TNF- a in conditioned media from cultured Kupffer cells: Isolated Kupffer cells were seeded onto 24-well plates and cultured in RPMI 1640. After 24 h in primary culture Kupffer cells were stimulated with RPMI 1640 containing 1-1000ng/mL LPS, 5% rat serum, 10mM HEPES and antibiotics for 4 h. Superaatants were collected and analyzed for TNF- a using an enzyme-linked immunosorbent assay(ELISA) kit. 6. Histological observation: Eighteen hours after resuscitation, lungs, heart, liver and kidneys of rat were excised and fixed with 1% paraformaldehyde. Fixed tissues were embedded in paraffin and processed for light microscope. Sections were stained with hematoxylin and eosin. 7. Statistical analysis: SPSS 11.0 software was used and
Chi-square test, ANOVA and t-test. Results: 1. Survival rate after hemor
实验方法：1．失血性休克模型的建立：Wister大鼠(体重250-300g)雌雄不限，用4.5％异戊巴比妥(80mg／kg)在25℃室温下腹腔内给药麻醉。右股动脉插入套管针，连接低压分析仪，监测血压；左颈静脉插管给予甘氨酸、生理盐水和自体血(复苏通路)；右颈动脉插管，另一端连接肝素化玻璃注射器中，进行放血诱导休克，在5分钟内使血压下降并维持平均动脉压30-35mmHg。通过输、抽少量血维持低血压状态55分钟。低血压60分钟后用60％自体血回输5分钟和2倍回输血量的生理盐水回输55分钟进行复苏。复苏前大白鼠随机分为三组，每组20只：休克组(Shock)、甘氨酸治疗组(Glycine+Shock)和假休克组(sham)(只进行手术操作不放血诱导休克)。甘氨酸60mg／kg溶于0.5ml生理盐水中在复苏开始时输入左颈静脉，相同体积的生理盐水给予休克组。2．Cr，CK AST，ALT和ALKP检测：分别于休克前，休克终末，复苏终末及复苏后18小时采集血样，血清离心，检测肌酐(Cr)，肌酸磷酸激酶(CK)，谷草转氨酶(AST)，谷丙转氨酶(ALT)和碱性磷酸酶(ALKP)。3．肝脏Kupffer细胞的分离和培养：复苏后2小时，肝脏在活体时经门静脉插管，用无Ca~(2+)无Mg~(2+) Hanks'平衡盐溶液(HBSS)在37℃下进行灌流(以20ml／分钟，灌流10分钟)，随后用含有0.025％Ⅳ型胶原酶的Hanks'溶液在37℃下以23ml／分的速度灌流7分钟，当肝脏被充分消化后，取下放入Ⅳ型胶原酶缓冲液中并切成小碎块，37℃水浴振荡5分钟，悬浮液用150目网过滤，然后在4℃以50*g离心10分钟，除去下层细胞团块，上层液加入25％+50％Percoll梯度介质中在4℃800*g离心10分钟，抽取25％与50％界面之间细胞平板片状团块，用Hanks'液冲洗再离心，细胞移入培养液中，放入37℃5％CO_2孵育箱中孵育1小时，非贴壁的上皮细胞通过换液除去而贴壁细胞为kupffer细胞。细胞培养24小时后做[Ca~(2+)]i和
TNF一Q检测。4.细胞内钙(【Caz+〕i)测定:使用荧光钙指示剂Fura一2
和荧光显微镜进行检测。含有kupffer细胞的培养皿在37℃下用
1一IO00ng/ml内毒素(LPS)刺激，在340和380nm激发波长和510nm
的发射波长检测显微镜视野下每个kupffer细胞Fura一2荧光密度的
变化。细胞内钙浓度由以下公式来确定:〔Caz+]，=Kd{(R一Rmin)/
(Rmax一R)}/FO/Fs)。5.在特定介质中从培养的kupffer细胞测定
TNF一Q:游离的Kupffer细胞种植在含有培养液的24孔板中培养。
24小时后Kupffer细胞在含有l一1000ng/mL的LPS、5%鼠血清、10mM
HEPES和抗生素的R即1 1 640液体中刺激4小时。上层清液用酶联免
疫吸附测定法检测TNF一Q(ELISA)。6.病理组织学观察:复苏后18
小时取大白鼠肺、心、肝和肾脏，用l%多聚甲醛固定，石蜡包埋，切
片，H一E染色，光镜观察组织病理变化。7.统计学分析:使用SPSS n .0
版本分析软件，应用方差、卡方和t检验。
实验结果:1.失血性休克后大白鼠的生存率:甘氨酸治疗组生存
呈现出甘氨酸剂量依赖性。本实验对甘氨酸剂量对生存率影响实验观
察72小时，结果得出最大生存率为77.8%，剂量大约为6Omg/kg。休克
组10只大鼠接受生理盐水，18小时生存率为70%，24小时生存率为
50%，48小时生存率为30%，72小时为20%。甘氨酸治疗组(9只)，18
小时生存率100%，24小时88.9%，48小时77.8%，72小时77.8%。
72小时两组生存率经卡方(Chi一square)检验差异显著(P<0 .05)。
甘氨酸能明显降低出血性休克大白鼠的死亡率。2.甘氨酸对肌酸磷
酸激酶，转氨酶及肌配的影响:肌酸磷酸激酶(CK)反映心肌和骨骼肌
损伤的指标，在休克期没有明显升高，但复苏后18小时明显增高，高
于正常值3倍以上。CK的升高可被甘氨酸有效阻止，经t检验，两
组差异非常显著(P<0 .01);谷草转氨酶(AST)，谷丙转氨酶(ALT)以及
碱性磷酸酶(ALKP)反映肝脏损害的指标，18小时明显升高，甘氨酸同
样可明显减少其升高，经t检验，两组差异非常显著(P<0 .01);Cr
反映肾损害的指标，18小时明显增高，甘氨酸明显降低其血中浓度，
经t检验，差异非常显著(P<0 .01)。这提示甘氨酸具有脏器保护作用。
3.出血性休克脏器损害病理组织形态学改变以及甘氨酸的作用:复苏
后18小时分别取休克组，假休克组和甘氨酸组大白鼠的肺，心，肝，肾
脏，中性福尔马林固定，石蜡包埋，常规H一E染色。在假休克组，没有明
显病理性变化;在休克组，肺组织中可见明显的肺泡壁增厚，上皮增生，
毛细血管扩张充血，肺泡壁间质细胞增生，巨噬细胞增多，毛细血管周
围淋巴细胞，浆细胞侵润，形成血管套样改变;在心肌组织中，休克组
呈现心肌细胞水肿，有中度、重度变性等;肝组织病理，休克组肝细胞
肿大变性，水肿，肝窦变窄;肾组织中，休克组，近曲小管广泛水肿变性，
管腔变窄。甘氨酸治疗组显示上述组织的病变程度明显减轻。这也提
示甘氨酸具有脏器保护作用。4.甘氨酸对Kupffer细胞内钙水平变
化的影响:三组大白鼠，复苏后二小时，肝Kupffer细胞进行游离单细
胞荧光检查。加入LPS(l，10，100，10O0ng/ml
Objective: This study investigates whether glycine prevents the organs of rat from injury and decreases mortality after hemorrhagic shock. Methods: 1. Hemorrhagic shock model: Wister rats(250-300g) were anesthetized with 4.5% amobarbital sodium(80mg/Kg i.p.) in room temperature(25℃). Blood pressure was monitored via polyethylene tubing(Intima 24GA) inserted into the right femoral artery, using a low pressure analyzer(Polygraph RM6000). The left jugular vein was cannulated(Intima 22GA) to administer glycine, normal saline and shed blood. The right carotid artery was cannulated with polyethylene tubing(Intima 22GA), and shock was induced over 5min by withdrawing blood into a heparinized glass syringe until mean arterial pressure was reduced to 30-35mmHg. Constant pressure was maintained by further withdrawal of small amount of blood as necessary for 55min. After 60 min of hypotension, rats were resuscitated by transfusion of 60%shed blood over 5 min, followed by normal saline(twice the shed blood volume) for
55 min. Prior to resuscitation, animals were randomly assigned to three groups(20 rats each group): shock group; glycine treatment group and sham group(operation was performed not to induce shock). Glycine(60mg/Kg) dissolved in 0.5mL normal saline was injected into the left jugular vein, and an equal volume of normal saline was given to shock group. 2. Determination of creatine phosphokinase(CK), aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(ALKP) and creatinine(Cr): Blood samples were collected before and at the end of shock, at the end of resuscitation and 18 h later. Plasma was centrifuged, stored at -80℃ for determining Cr,CK,AST,ALT and ALKP. 3. Kupffer cell preparation and culture: Two hours after resuscitation, the liver was perfused through the portal vein with Ca2+- and Mg2+-free Hanks' balanced salt solution(HBSS) at 37C
for 10 min at a flow rate of 20mL/min. Subsequently, perfusion was with HBSS containing 0.025% collagenase IV at37C for 7min at a flow rate of 23mL/min. After the liver was digested, it was excised and cut into small pieces in collagenase buffer. The suspension was filtered through nylon gauze mesh and the filtrate was centrifuged at 50*g for 10 min at 4 C. The suspension was centrifuged on a density cushion of 25%+50% Percoll at 800*g for 10 min, and cells fraction between 25% and 50% Percoll was collected and washed with buffer again, cells were seeded into 2mL RPMI1640 at 37C and incubated with 5% CO2. Non-αdherent cells were removed after 1 h by replacing buffer, and adherent cells(Kupffer cells) were cultured for 24 h prior to [Ca2+]i and TNF- a measurement. 4. Measurement of intracellular Ca2+([Ca2+]i): Intracellular calcium was measured flurometrically using the fluorescent calcium indicator dye fura-2 and a microspectroflurometer interfaced with an inverted microscope. Kupffer cells were placed in chambers with buffer at 37 C and stimulated with 1-1000ng/mL endotoxin. Changes in fluorescence intensity of fura-2 at excitation wavelengths of 340nm and 380nm and emission at 510nm were monitored in individual Kupffer cells. Intracellular calcium was determined from the equation: [Ca2+]i = Kd{(R-Rmin)/(Rmax-R)}/(Fo/Fs). 5. Measurement of TNF- a in conditioned media from cultured Kupffer cells: Isolated Kupffer cells were seeded onto 24-well plates and cultured in RPMI 1640. After 24 h in primary culture Kupffer cells were stimulated with RPMI 1640 containing 1-1000ng/mL LPS, 5% rat serum, 10mM HEPES and antibiotics for 4 h. Superaatants were collected and analyzed for TNF- a using an enzyme-linked immunosorbent assay(ELISA) kit. 6. Histological observation: Eighteen hours after resuscitation, lungs, heart, liver and kidneys of rat were excised and fixed with 1% paraformaldehyde. Fixed tissues were embedded in paraffin and processed for light microscope. Sections were stained with hematoxylin and eosin. 7. Statistical analysis: SPSS 11.0 software was used and
Chi-square test, ANOVA and t-test. Results: 1. Survival rate after hemor
引文
1．盛志勇，胡森主编．多器官功能障碍综合征．第1版，北京：科学出版社1999：18-61
2. Csaba Szabo and Timothy R.Billiar: Novel Roles of Nitric Oxide in Hemorrhage Shock. Shock, Vol. 12,No. 1,p1-9,1999
3. Abello PA, Buchman TG, Bulkley GB: Shock and multiple organ failure. Adv Exp Med Biol 366:253-268, 1994.
4. Camco CJ, Meakins JL, Marshall JC, Fry D, Maier RV: Multiple-organ failure syndrome. Arch Surg 121:196-208, 1986.
5. John Menezes, Christian Hierholzer, Simon C.Watkins etc: A novel nitric oxide scavenger decreases liver injury and improves survival after hemorrhagic shock. Am.J.Physiol. 277(Gastrointest.liver physiol.40): G144-G151,1999.
6. Jeffrey A. Kline, Lisa R. Thornton, etc.: Heart function after severe hemorrhagic shock. Shock. Vol. 12 No.6 p454-461,1999.
7. Marzi I, Maier M, Herzog C, Bauer M: Influence of pentoxifylline and albifylline on liver microcirculation and leukocyte adhesion after hemorrhagic shock in the rat. J Trauma 40:90-96, 1996.
8．胡森，盛志勇，周宝桐．多器官功能障碍综合征早期诊断依据和实验预警指标的探讨．解放军医学杂志，1996，21：5
9．胡森，盛志勇，周宝桐等．双相迟发型多器官功能障碍综合征(MODS)动物模型的研究．中华创伤杂志，1996，12：3
2. Csaba Szabo and Timothy R.Billiar: Novel Roles of Nitric Oxide in Hemorrhage Shock. Shock, Vol. 12,No. 1,p1-9,1999
3. Abello PA, Buchman TG, Bulkley GB: Shock and multiple organ failure. Adv Exp Med Biol 366:253-268, 1994.
4. Camco CJ, Meakins JL, Marshall JC, Fry D, Maier RV: Multiple-organ failure syndrome. Arch Surg 121:196-208, 1986.
5. John Menezes, Christian Hierholzer, Simon C.Watkins etc: A novel nitric oxide scavenger decreases liver injury and improves survival after hemorrhagic shock. Am.J.Physiol. 277(Gastrointest.liver physiol.40): G144-G151,1999.
6. Jeffrey A. Kline, Lisa R. Thornton, etc.: Heart function after severe hemorrhagic shock. Shock. Vol. 12 No.6 p454-461,1999.
7. Marzi I, Maier M, Herzog C, Bauer M: Influence of pentoxifylline and albifylline on liver microcirculation and leukocyte adhesion after hemorrhagic shock in the rat. J Trauma 40:90-96, 1996.
8．胡森，盛志勇，周宝桐．多器官功能障碍综合征早期诊断依据和实验预警指标的探讨．解放军医学杂志，1996，21：5
9．胡森，盛志勇，周宝桐等．双相迟发型多器官功能障碍综合征(MODS)动物模型的研究．中华创伤杂志，1996，12：3