本文選題：子癇前期 + 孕鼠��； 參考：《福建醫(yī)科大學》2014年碩士論文

【摘要】：第一部分子癇前期樣孕鼠模型的建立及輔酶Q10療效評價 目的 使用一氧化氮合成酶(nitric oxide synthase, NOS)抑制劑—左旋硝基精氨酸甲酯(L-nitro-arginine-methyl ester，L-NAME)建立子癇前期樣孕鼠模型，從血壓、24小時尿蛋白定量、肝功能、子代結(jié)局等方面評價輔酶Q10治療子癇前期樣孕鼠中的療效。 方法 1．子癇前期樣孕鼠模型建立：妊娠第10-14天給予L-NAME125mg/kg/d皮下注射，妊娠第2天開始隔日無創(chuàng)測量血壓，若較孕前血壓升高30mmHg或以上則認為建模成功。 2．實驗分組：選擇SD(Sprague-Dawley)孕鼠30只，隨機分為3組：正常妊娠組（n=10）及模型組（n=20，再分為輔酶Q10組與蒸餾水組，每組各10只孕鼠）。模型組：妊娠第10-14天予L-NAME125mg/kg/d皮下注射，正常妊娠組：妊娠第10-14天予同劑量生理鹽水皮下注射；輔酶Q10組：妊娠第15-20天予輔酶Q1060mg/kg/d灌胃；蒸餾水組：妊娠第15-20天予同劑量蒸餾水灌胃。 3．血壓、尿蛋白及肝功能測量：采用無創(chuàng)鼠尾血壓檢測方法，分別于孕前1周、妊娠第2天開始隔日測量血壓；收集妊娠前、妊娠第10天、妊娠第15天、妊娠第20天24小時尿液進行尿蛋白定量分析；妊娠第21天留取心臟血，進行丙氨酸氨基轉(zhuǎn)移酶（Alanine aminotransferase，ALT）及門冬氨酸氨基轉(zhuǎn)移酶（Aspartateaminotransferase，AST）檢測。 4．子代結(jié)局評價：妊娠第21天，剖宮術(shù)取出鼠胎、胎盤，計數(shù)正常鼠胎數(shù)，稱取鼠胎及胎盤重量。 結(jié)果 1．各組血壓變化：妊娠前、妊娠第10天正常妊娠組及模型組（輔酶Q10組與蒸餾水組）孕鼠血壓比較，差異均無統(tǒng)計學意義(P＞0.05)；妊娠第15天模型組血壓較孕前均升高，差異有統(tǒng)計學意義(P＜0.05)，亦高于正常妊娠組，差異有統(tǒng)計學意義(P＜0.05)；妊娠第21天蒸餾水組孕鼠血壓繼續(xù)維持在較高水平，與妊娠第15天比較，差異無統(tǒng)計學意義；而輔酶Q10組妊娠第21天血壓較妊娠第15天下降，且低于蒸餾水組，差異均有統(tǒng)計學意義(P＜0.05)，較正常妊娠組升高，差異有統(tǒng)計學意義(P＜0.05)。 2.各組24小時尿蛋白定量變化：比較三組孕鼠妊娠前、妊娠第10天24小時尿蛋白定量，差異無統(tǒng)計學意義；妊娠第15天，模型組24小時尿蛋白定量較妊娠前均升高，差異有統(tǒng)計學意義(P＜0.05)，亦高于正常妊娠組，差異有統(tǒng)計學意義(P＜0.05)；妊娠第20天，蒸餾水組尿蛋白高于正常妊娠組，輔酶Q10組尿蛋白低于蒸餾水組、高于正常妊娠組，分別比較，差異均有統(tǒng)計學意義(P＜0.05)。 3.各組肝功能變化：蒸餾水組ALT及AST均顯著高于正常妊娠組，輔酶Q10組ALT及AST則較蒸餾水組顯著降低，但仍較正常妊娠組升高，差異有統(tǒng)計學意義(P＜0.05)。 4.各組子代妊娠結(jié)局：蒸餾水組鼠胎數(shù)、鼠胎重量均低于正常妊娠組，差異均有統(tǒng)計學意義(P＜0.05)；輔酶Q10組鼠胎數(shù)、鼠胎重量均較蒸餾水組升高，差異均有統(tǒng)計學意義(P＜0.05)，低于正常妊娠組，差異均有統(tǒng)計學意義(P＜0.05)；模型組胎盤重量均低于正常妊娠組，差異均有統(tǒng)計學意義(P＜0.05)，輔酶Q10組胎盤重量高于蒸餾水組，差異均有統(tǒng)計學意義(P＜0.05)。 結(jié)論 妊娠第10-14天給予L-NAME125mg/kg/d皮下注射可以充分模擬子癇前期主要表現(xiàn)，成功建立子癇前期樣孕鼠模型；予輔酶Q10治療可以有效改善子癇前期樣孕鼠病理生理表現(xiàn)，改善子代結(jié)局。 第二部分輔酶Q10對子癇前期樣孕鼠的肝臟保護作用及其機制的研究 目的 通過分析子癇前期樣孕鼠肝臟氧化應激損傷水平及細胞凋亡水平的變化，探討輔酶Q10在治療子癇前期樣孕鼠肝臟中的抗氧化應激損傷及抗細胞凋亡的作用機制。 方法 1.實驗分組：選擇SD孕鼠50只，隨機分為正常妊娠組（n=10）與模型組（n=40，均為子癇前期樣孕鼠，再分為蒸餾水組、輔酶Q10組、硫酸鎂+輔酶Q10組及硫酸鎂組，每組各10只孕鼠）。妊娠第10-14天：模型組予左硝基精氨酸甲酯125mg/kg/d皮下注射，建立子癇前期樣孕鼠模型；正常妊娠組予同劑量生理鹽水皮下注射。妊娠第15-20天：蒸餾水組予蒸餾水1ml灌胃；輔酶Q10組予輔酶Q1060mg/kg/d灌胃；硫酸鎂+輔酶Q10組予硫酸鎂120mg/kg/d皮下注射+輔酶Q1060mg/kg/d灌胃；硫酸鎂組予硫酸鎂120mg/kg/d皮下注射+蒸餾水1ml灌胃。 2.氧化應激損傷水平及抗氧化酶檢測：檢測肝臟組織丙二醛（malondialdehyde，MDA）含量、過氧化物歧化酶（superoxide dismutase，SOD）及谷胱甘肽過氧化物酶（glutathione peroxidase, GSH-PX）活性。 3.細胞凋亡檢測：提取肝臟組織蛋白， Western-blot法檢測肝臟組織活化Caspase-3、Bcl-2及Bax蛋白表達水平。 結(jié)果 1.各組血壓變化：孕前正常妊娠組及模型組（蒸餾水組、輔酶Q10組、硫酸鎂+輔酶Q10組及硫酸鎂組）孕鼠血壓比較，差異無統(tǒng)計學意義（P＞0.05）；妊娠第15天模型組孕鼠血壓較孕前均升高，差異有統(tǒng)計學意義（P＜0.05），也高于正常妊娠組，差異有統(tǒng)計學意義（P＜0.05）；妊娠第21天蒸餾水組孕鼠血壓繼續(xù)維持在高水平，與妊娠第15天比較，差異無統(tǒng)計學意義（P＞0.05）；硫酸鎂+輔酶Q10組、硫酸鎂組、輔酶Q10組妊娠第21天血壓較妊娠第15天下降，且低于蒸餾水組，差異均有統(tǒng)計學意義（P＜0.05）；輔酶Q10組較正常妊娠組仍升高，差異有統(tǒng)計學意義（P＜0.05）。 2.各組24小時尿蛋白定量變化：比較各組孕鼠妊娠前、妊娠第10天24小時尿蛋白定量，差異無統(tǒng)計學意義；妊娠第15天，模型組（蒸餾水組、輔酶Q10組、硫酸鎂+輔酶Q10組及硫酸鎂組）24小時尿蛋白定量較孕前均升高，差異有統(tǒng)計學意義（P＜0.05），也高于正常妊娠組，差異有統(tǒng)計學意義（P＜0.05）。妊娠第20天，蒸餾水組尿蛋白高于正常妊娠組，差異有統(tǒng)計學意義（P＜0.05）；硫酸鎂+輔酶Q10組、硫酸鎂組、輔酶Q10組低于蒸餾水組，差異有統(tǒng)計學意義（P＜0.05），仍高于正常妊娠組，差異有統(tǒng)計學意義（P＜0.05）。 3.各組肝功能變化：蒸餾水組ALT及AST均高于正常妊娠組，差異有統(tǒng)計學意義（P＜0.05）；硫酸鎂+輔酶Q10組、硫酸鎂組、輔酶Q10組ALT及AST較蒸餾水組降低，較正常妊娠組升高，差異均有統(tǒng)計學意義（P＜0.05）。 4.各組子代妊娠結(jié)局：蒸餾水組鼠胎數(shù)、鼠胎重量均顯著低于正常妊娠組，硫酸鎂+輔酶Q10組、硫酸鎂組、輔酶Q10組鼠胎數(shù)、鼠胎重量均較蒸餾水組升高，差異有統(tǒng)計學意義（P＜0.05），三組之間比較，差異無統(tǒng)計學意義（P＞0.05）；仍低于正常妊娠組，差異有統(tǒng)計學意義（P＜0.05）。模型組胎盤重量均低于正常妊娠組，差異有統(tǒng)計學意義（P＜0.05），硫酸鎂+輔酶Q10組、硫酸鎂組及輔酶Q10組均高于蒸餾水組，差異有統(tǒng)計學意義（P＜0.05）；三組之間比較，差異無統(tǒng)計學意義（P＞0.05）。 5.各組肝臟GSH-PX及SOD活性水平：模型組肝臟GSH-PX及SOD活性水平均低于正常妊娠組，兩兩比較，差異均有統(tǒng)計學意義（P＜0.05）。輔酶Q10組與蒸餾水組、硫酸鎂組比較均升高，分別比較，差異均有統(tǒng)計學意義（P＜0.05）。硫酸鎂+輔酶Q10組與蒸餾水組比較升高，差異有統(tǒng)計學意義（P＜0.05）；與輔酶Q10組比較輕度升高，但差異無統(tǒng)計學意義（P＞0.05）；與硫酸鎂組比較亦升高，差異有統(tǒng)計學意義（P＜0.05）。硫酸鎂組與蒸餾水組比較升高，差異有統(tǒng)計學意義（P＜0.05）。 6.各組肝臟MDA水平：正常妊娠組肝臟MDA水平為（5.49±0.32）nmol/mg protein，模型組肝臟MDA水平均高于正常妊娠組，兩兩比較，差異均有統(tǒng)計學意義（P＜0.05）。蒸餾水組、輔酶Q10組、硫酸鎂+輔酶Q10組及硫酸鎂組分別為（9.38±0.51）nmol/mg protein、（6.80±0.32）nmol/mg protein、（6.86±0.45）nmol/mg protein及（7.73±0.28）nmol/mg protein，除輔酶Q10組與硫酸鎂+輔酶Q10組比較，差異無統(tǒng)計學意義（P＞0.05），其余各組間兩兩比較，差異均有統(tǒng)計學意義（P＜0.05）。 7.各組肝臟組織活化Caspase-3、Bcl-2及Bax蛋白水平：蒸餾水組孕鼠肝臟組織活化Caspase-3及Bax蛋白較正常妊娠組、硫酸鎂+輔酶Q10組、硫酸鎂組、輔酶Q10組增多，Bcl-2較正常妊娠組減少，差異均有統(tǒng)計學意義（P＜0.05），模型組經(jīng)過硫酸鎂、輔酶Q10治療后與蒸餾水組比較，活化Caspase-3及Bax蛋白表達減少（P＜0.05），Bcl-2蛋白表達增多，差異均有統(tǒng)計學意義（P＜0.05）。 結(jié)論 子癇前期樣孕鼠肝臟組織氧化應激水平、細胞凋亡水平均升高；輔酶Q10可以有效改善子癇前期樣孕鼠肝功能，減少肝臟細胞凋亡，具有保護肝臟的作用，其機制可能是通過抗氧化應激損傷和調(diào)節(jié)Bcl-2/Bax平衡從而抑制細胞凋亡。
[Abstract]:Establishment of Preeclampsia Model and Evaluation of Coenzyme Q10 in the First Part of Preeclampsia

Purpose

Using nitric oxide synthase ( NOS ) inhibitor , L - nitro - arginine methyl ester ( L - NAME ) to establish a rat model of pre - eclampsia , the effect of coenzyme Q10 in the treatment of pre - eclampsia was evaluated from the aspects of blood pressure , 24 - hour urinary protein , liver function and progeny outcome .

method

1 . The model of pre - eclampsia was established : L - NAME125mg / kg / day subcutaneous injection was administered on the 10th - 14th day of pregnancy , and the blood pressure was not measured on the 2nd day of gestation . If the blood pressure increased by 30 mmHg or above , the model was considered successful .

2 . Experimental group : Thirty - three Sprague - Dawley rats were randomly divided into three groups : normal pregnancy group ( n = 10 ) and model group ( n = 20 , subdivided into coenzyme Q10 group and distilled water group , 10 pregnant rats in each group ) . Model group : subcutaneous injection of L - NAME125mg / kg / day at 10 - 14 days of gestation , normal pregnancy group : subcutaneous injection with same dose of physiological saline at 10 - 14 days of pregnancy ;
Coenzyme Q10 group : coenzyme Q1060 mg / kg / day was administered to the stomach at the 15th to 20th days of pregnancy ;
Distilled water group : 15 - 20 days gestation , the same dose of distilled water was administered to the stomach .

3 . Measurement of blood pressure , urinary protein and liver function : The blood pressure was measured at 1 week before pregnancy and the blood pressure was measured on the 2nd day of gestation , respectively .
Urine protein was analyzed quantitatively in urine before pregnancy , gestation day 10 , gestation day 15 , gestation day 20 24 hours ;
Heart blood was collected on Day 21 of gestation and alanine aminotransferase ( ALT ) and aspartate aminotransferase ( AST ) were detected .

4 . Evaluation of the outcome of the progeny : 21 days of pregnancy , the uterus was taken out to remove the fetal placenta and the placenta , and the number of normal fetuses was counted , and the weight of the fetus and the placenta was weighed .

Results

1 . Blood pressure changes in each group : Before pregnancy , there was no significant difference in blood pressure between normal pregnant group and model group ( coenzyme Q10 group and distilled water group ) in the normal pregnancy group and the model group ( P > 0.05 ) .
The blood pressure of the 15th day of pregnancy was higher than that in normal pregnancy group ( P < 0.05 ) , and the difference was statistically significant ( P < 0.05 ) .
The blood pressure of pregnant rats in distilled water group was maintained at a high level in the 21st day of gestation , and the difference was not statistically significant compared with the 15th day of pregnancy .
Compared with the control group , the blood pressure of the coenzyme Q10 group was lower than that in the group of distilled water ( P < 0.05 ) , and the difference was statistically significant ( P < 0.05 ) .

2 . The quantitative changes of urinary protein 24 hours in each group : there was no significant difference in the amount of urinary protein 24 hours after pregnancy in three groups .
In the 15th day of gestation , the 24 - hour urinary protein of the model group was higher than that in the normal pregnancy group ( P < 0.05 ) , which was higher than that in the normal pregnancy group ( P < 0.05 ) .
The urinary protein in the distilled water group was higher than that in the normal pregnancy group on the 20th day of gestation , and the urinary protein in the coenzyme Q10 group was lower than that in the normal pregnancy group , which was higher than that in the normal pregnancy group , and the difference was statistically significant ( P < 0.05 ) .

3 . The changes of liver function in each group : ALT and AST in the distilled water group were significantly higher than those in the normal pregnancy group , but the ALT and AST in the coenzyme Q10 group were significantly lower than that in the distilled water group , but still higher in the normal pregnancy group ( P < 0.05 ) .

4 . The pregnant outcome of each group : the number of mice in the distilled water group and the weight of the fetus were lower than that of the normal pregnancy group ( P < 0.05 ) .
The number of Coenzyme Q10 group and the weight of fetus were higher than that in the distilled water group ( P < 0 . 05 ) , and the difference was statistically significant ( P < 0 . 05 ) .
The placental weight of the model group was lower than that in normal pregnancy group ( P < 0.05 ) , and the placental weight of coenzyme Q10 group was higher than that in distilled water group ( P < 0.05 ) .

Conclusion

Subcutaneous injection of L - NAME125mg / kg / day on the 10th to 14th days of pregnancy can fully simulate the main manifestations of the pre - eclampsia , and successfully establish a pre - eclampsia - like pregnant rat model ;
Coenzyme Q10 treatment can effectively improve the pathological physiology of pregnant mice in the early stage of eclampsia and improve the outcome of offspring .

Study on the Protective Effect of the Second Part of Coenzyme Q10 on the Liver of Preeclampsia - like Pregnant Rats and Its Mechanism

Purpose

The effects of coenzyme Q10 on oxidative stress injury and cell apoptosis in the liver of pregnant rats with pre - eclampsia were investigated by analyzing the changes of oxidative stress injury level and cell apoptosis level in pre - eclampsia - like pregnant rats .

method

1 . Experimental group : 50 SD pregnant rats were randomly divided into normal pregnancy group ( n = 10 ) and model group ( n = 40 , all were pre - eclampsia - like pregnant rats , then divided into distilled water group , coenzyme Q10 group , magnesium sulfate + coenzyme Q10 group and magnesium sulfate group , 10 pregnant rats in each group ) . 10 - 14 days of gestation : the model group was injected subcutaneously with left nitro - arginine methyl ester 125mg / kg / day to establish a pre - eclampsia - like pregnant rat model ;
The normal pregnancy group was administered subcutaneously with the same dose of physiological saline . Days 15 - 20 of gestation : 1 ml of distilled water was administered to the distilled water .
Coenzyme Q10 group was administered with coenzyme Q 101 mg / kg / d ;
Magnesium sulfate + coenzyme Q10 group was administered subcutaneously with 120 mg / kg / day magnesium sulfate + coenzyme Q1060 mg / kg / day ;
Magnesium sulfate group was given 120 mg / kg / day magnesium sulfate and 1 ml of distilled water .

2 . Oxidative stress injury level and anti - oxidation enzyme detection : detect the content of malondialdehyde ( MDA ) , superoxide dismutase ( SOD ) and glutathione peroxidase ( GSH - PX ) activity in liver .

3 . Apoptosis was detected by Western - blot . Caspase - 3 , Bcl - 2 and Bax protein expression levels were detected by Western - blot .

Results

1 . Blood pressure changes in each group : the blood pressure of pregnant rats in the normal pregnant group and the model group ( distilled water group , coenzyme Q10 group , magnesium sulfate + coenzyme Q10 group and magnesium sulfate group ) had no statistical significance ( P > 0.05 ) ;
The blood pressure of pregnant rats was higher than that in normal pregnancy group ( P < 0.05 ) , and the difference was statistically significant ( P < 0.05 ) .
The blood pressure of pregnant rats in distilled water group was maintained at a high level in the 21st day of gestation , and the difference was not statistically significant ( P > 0.05 ) compared with the 15th day of pregnancy .
The blood pressure of magnesium sulfate + coenzyme Q10 group , magnesium sulfate group and coenzyme Q10 group decreased on the 15th day of gestation and lower than that in distilled water group ( P < 0.05 ) .
There was significant difference between coenzyme Q10 group and normal pregnancy group ( P < 0.05 ) .

2 . Changes of urinary protein in 24 hours in each group : compared with that of pregnant rats in each group , there was no significant difference in the amount of urine protein in 24 hours of gestation at the 10th day of pregnancy ;
In the 15th day of gestation , the 24 - hour urinary protein of the model group ( distilled water group , coenzyme Q10 group , magnesium sulfate + coenzyme Q10 group and magnesium sulfate group ) was higher than that in normal pregnancy group ( P & lt ; 0.05 ) , and the difference was statistically significant ( P < 0.05 ) . The urinary protein of distilled water group was higher than that of normal pregnancy group ( P < 0.05 ) .
Magnesium sulfate + coenzyme Q10 group , magnesium sulfate group and coenzyme Q10 group were lower than that in distilled water group ( P < 0 . 05 ) , but the difference was statistically significant ( P < 0 . 05 ) .

3 . Changes of liver function in each group : ALT and AST in distilled water group were higher than those in normal pregnancy group ( P < 0.05 ) .
ALT and AST in the magnesium sulfate + coenzyme Q10 group , the magnesium sulfate group and the coenzyme Q10 group were lower than those in the distilled water group , and the difference was statistically significant ( P < 0.05 ) .

4 . The pregnant outcome of each group : the number of rats in the distilled water group and the weight of the fetus were significantly lower than that in the normal pregnancy group , the magnesium sulfate + coenzyme Q10 group , the magnesium sulfate group , the coenzyme Q10 group and the mouse embryo , the weight of the rats was higher than that in the distilled water group , the difference was statistically significant ( P < 0.05 ) , and the difference was not statistically significant ( P > 0.05 ) ;
The placental weight of the model group was lower than that in the normal pregnancy group ( P < 0 . 05 ) , the difference was statistically significant ( P < 0 . 05 ) , the magnesium sulfate + coenzyme Q10 group , the magnesium sulfate group and the coenzyme Q10 group were all higher than that of the distilled water group , the difference was statistically significant ( P < 0.05 ) ;
There was no significant difference between the three groups ( P > 0.05 ) .

5 . The levels of GSH - PX and SOD in the liver of each group were lower than those in normal pregnancy group ( P < 0 . 05 ) . Compared with the group of distilled water and magnesium sulfate ( P < 0.05 ) , there was significant difference between the coenzyme Q10 group and the distilled water group ( P < 0.05 ) .
Compared with coenzyme Q10 group , there was no significant difference ( P > 0.05 ) .
Compared with the magnesium sulfate group , the difference was statistically significant ( P < 0.05 ) . There was significant difference between the magnesium sulfate group and the distilled water group ( P < 0.05 ) .

6 . The level of MDA in liver of normal pregnancy group was ( 5.49 鹵 0.32 ) nmol / mg protein , and MDA level in the model group was higher than that in normal pregnancy group ( 9.38 鹵 0.51 ) nmol / mg protein , ( 6.80 鹵 0.32 ) nmol / mg protein , ( 6.86 鹵 0.45 ) nmol / mg protein and ( 7.73 鹵 0.28 ) nmol / mg protein , respectively .

7 . The levels of Caspase - 3 , Bcl - 2 and Bax protein in liver tissues of each group were higher than those in normal pregnancy group , magnesium sulfate + coenzyme Q10 group , magnesium sulfate group , coenzyme Q10 group , and Bcl - 2 group . The expression of Caspase - 3 and Bax protein decreased ( P < 0.05 ) .

Conclusion

The level of oxidative stress and the level of apoptosis in the liver tissues of pregnant rats with pre - eclampsia were increased .
Coenzyme Q10 can effectively improve the liver function of the pre - eclampsia - like pregnant mice , reduce the apoptosis of the liver cells , and have the function of protecting the liver , and the mechanism may be to inhibit apoptosis by resisting oxidative stress injury and regulating the Bcl - 2 / Bax balance .
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.244

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