本文選題：SMAC + 肝衰竭　； 參考：《華中科技大學》2014年博士論文

【摘要】：[背景與目的] 肝衰竭是各種原因?qū)е碌母喂δ車乐厥Т鷥?出現(xiàn)以凝血功能障礙、腹水、肝性腦病、肝腎綜合征和出血等為主要表現(xiàn)的一種臨床癥候群,死亡率高達80%,目前尚缺乏特異的治療手段。研究發(fā)現(xiàn)肝細胞凋亡是肝衰竭發(fā)生發(fā)展的重要事件。肝衰竭時內(nèi)毒素血癥發(fā)生率高達80.0%,內(nèi)毒素(lipopolysaccharide, LPS)可以介導肝細胞的凋亡。我們課題組前期試驗發(fā)現(xiàn)第2個線粒體衍生的胱天蛋白酶激活劑(second mitochondria derived activator of caspase, SMAC)在體外可以單獨介導LPS誘導的肝細胞凋亡,體內(nèi)試驗也發(fā)現(xiàn)肝衰竭內(nèi)毒素血癥大鼠肝細胞的凋亡與SMAC密切相關。研究發(fā)現(xiàn)SMAC是凋亡的發(fā)生發(fā)展所必須的。RNA干擾(RNA interference, RNAi)是一種高效、特異性阻斷mRNA表達而導致轉(zhuǎn)錄后的基因沉默現(xiàn)象。應用RNAi抑制SMAC的表達,可能減輕肝衰竭內(nèi)毒素血癥小鼠肝細胞的凋亡,這將為肝衰竭內(nèi)毒素血癥的治療提供一個新的途徑,也有助于進一步明確SMAC在該凋亡中的作用。最后,研究HEV相關急性肝衰竭患者血清LPS水平,觀察該血清對人肝細胞生存和凋亡的影響,并初步探討內(nèi)毒素核心多糖抗體的保護作用。 [實驗方法] 一.細胞水平 針對人SMAC基因設計、合成并篩選出特異小干擾RNA (small interferingRNA, siRNA),脂質(zhì)體法將該特異干擾片段轉(zhuǎn)染肝細胞,再用內(nèi)毒素誘導細胞凋亡。轉(zhuǎn)染后不同的時間點(24h、36h、48h、60h)收集肝細胞,Real time PCR法檢測細胞SMAC mRNA。轉(zhuǎn)染后48h, western檢測肝細胞線粒體SMAC蛋白和胞漿caspase-9蛋白的表達,caspase-3活性檢測試劑盒檢測caspase-3活性,流式細胞儀檢測肝細胞的凋亡。 二.動物水平 構(gòu)建小鼠SMAC特異干擾慢病毒,測序鑒定構(gòu)建的正確性并測定其滴度。通過尾靜脈高壓注射技術將慢病毒導入小鼠肝臟。應用D-半乳糖氨(D-galactosanine, D-GalN)和LPS腹腔注射制作急性肝衰竭小鼠模型,觀察小鼠體征的變化并記錄小鼠死亡情況,留取小鼠血液和肝臟組織。全自動生化分析儀檢測小鼠血清谷丙轉(zhuǎn)氨酶(alanine aminotransferase, ALT)、谷草轉(zhuǎn)氨酶(aspartate aminotransferase,AST),鱟試劑檢測試劑盒檢測血清LPS水平,冰凍切片檢測慢病毒的感染效率,HE染色評定肝組織病理學改變,real time PCR法檢測肝組織SMAC mRNA, western檢測肝組織線粒體和胞漿SMAC蛋白、caspase-9蛋白的表達,Tunel染色觀察肝組織肝細胞的凋亡。 三.人體水平 收集13例HEV相關急性肝衰竭患者血清,采用鱟試劑基質(zhì)顯色法檢測血清LPS水平,用該血清孵育肝細胞,流式細胞儀檢測肝細胞凋亡率；再用內(nèi)毒素核心多糖抗體和該急性肝衰竭患者血清與肝細胞共培養(yǎng),流式細胞儀檢測肝細胞凋亡率。 四.統(tǒng)計學方法 計量資料用均數(shù)±標準差(x±S)表示,應用統(tǒng)計學軟件SPSS13.0進行多樣本均數(shù)兩兩比較的q檢驗方差分析,比較各樣本間的差異。P0.05為顯著性差異界限。 [結(jié)果] 1.3條siRNA (siRNA1、siRNA2、siRNA3)對人肝細胞SMAC mRNA均有不同程度的抑制作用,其中siRNA2的抑制效應最強,抑制效應達68.0%。 2.在轉(zhuǎn)染siRNA2的LPS誘導的肝細胞,線粒體SMAC蛋白、caspase-9蛋白、caspase-3酶活性均被抑制,肝細胞的凋亡率也被有效降低。 3.篩選并構(gòu)建了特異的小鼠SMAC干擾慢病毒載體,測序表明其構(gòu)建正確,慢病毒滴度為6E+8TU/ml。 4.熒光顯微鏡下,注射慢病毒的小鼠肝臟組織冰凍切片可見大量綠色熒光。 5.模型組小鼠血清ALT和AST水平較正常對照組明顯上升,特異序列組小鼠ALT和AST水平較模型組顯著降低。 6.模型組小鼠血清LPS水平較正常對照組明顯升高,特異序列組小鼠血清LPS水平較模型組顯著下降。 7.HE染色顯示,正常對照組小鼠肝組織無炎性細胞浸潤,無肝細胞壞死：模型組和無關序列組小鼠肝組織可見炎性細胞浸潤和肝細胞壞死；而特異序列組小鼠肝組織炎性細胞浸潤和肝細胞壞死較模型組明顯減少。 8.在肝衰竭模型小鼠,肝組織SMAC mRNA的表達水平較正常對照組小鼠明顯升高,線粒體SMAC蛋白表達降低,胞漿SMAC蛋白表達明顯增多,肝組織胞漿caspase-9蛋白表達量較正常組明顯增多,肝細胞凋亡增多,凋亡指數(shù)為33.0±±5.6%。而構(gòu)建的特異慢病毒,可以顯著降低肝組織SMAC mRNA的水平,下調(diào)線粒體和胞漿SMAC蛋白的表達,降低caspase-9的含量,減少肝組織肝細胞的凋亡。 9.HEV相關急性肝衰竭患者的血清LPS水平為0.26±0.02EU/ml,明顯高于健康者。 10.HEV相關急性肝衰竭患者血清孵育肝細胞后,細胞的凋亡率為5.83±0.42%,較對照組明顯增加,健康者的血清對細胞凋亡率沒有明顯影響。應用LPS抗體和急性肝衰竭患者血清共培養(yǎng),細胞凋亡率下降,但沒有統(tǒng)計學差異。 [結(jié)論] 1.通過抑制SMAC的表達,可以有效減少LPS誘導的肝細胞的凋亡及肝衰竭內(nèi)毒素血癥小鼠肝細胞的凋亡,為肝衰竭內(nèi)毒素血癥的治療提供了新的手段和理論基礎。同時也表明SMAC及其下游通路可能是LPS介導肝細胞凋亡的通路之一。 2.HEV相關急性肝衰竭患者血清含有較高濃度的LPS,該血清可以誘導肝細胞的凋亡。
[Abstract]:BACKGROUND & OBJECTIVE :

Liver failure is a clinical symptom caused by various causes , such as coagulation dysfunction , ascites , hepatic encephalopathy , hepatorenal syndrome , hemorrhage , etc .

EXPERIMENTAL METHOD

I . Cell level

Specific small interfering RNA ( siRNA ) was synthesized and screened for human SMAC gene . The specific interference fragment was transfected into hepatocytes by liposome method . After transfection , the cells were transfected with LPS . After transfection , the expression of SMAC protein and caspase - 9 protein were detected by real time PCR . The caspase - 3 activity was detected by caspase - 3 activity detection kit , and the apoptosis of hepatocytes was detected by flow cytometry .

II . Animal level

Mouse liver was induced by intraperitoneal injection of D - galactosanine ( D - GalN ) and lipopolysaccharide ( LPS ) by intraperitoneal injection of D - galactosanine ( D - GalN ) and LPS .

III . Human body level

The serum levels of LPS were measured by the method of chromogenic method in 13 patients with HEV - related acute liver failure , and the hepatocyte apoptosis rate was detected by incubating the hepatocytes with the serum .
then the serum of the endotoxin core polysaccharide antibody and the patient of the acute liver failure are co - cultured with the liver cells , and the apoptosis rate of the hepatocytes is detected by flow cytometry .

IV . Statistical method

The mean 鹵 standard deviation ( x 鹵 S ) of the measurement data showed that the statistical software SPSS 13.0 was used to carry out the q - test variance analysis of the two comparisons of the number of samples , and the difference between the samples was compared .

The result is not valid .

1.3 siRNA ( siRNA1 , siRNA2 , siRNA3 ) inhibited the expression of SMAC mRNA in human hepatocytes , in which siRNA2 had the strongest inhibitory effect and the inhibitory effect was 68.0 % .

2 . After transfection of siRNA2 - induced LPS - induced hepatocytes , mitochondrial SMAC protein , caspase - 9 protein and caspase - 3 activity were inhibited , and the apoptosis rate of hepatocytes was also effectively reduced .

3 . The specific mouse SMAC was screened and constructed to interfere with the slow virus vector . The sequencing showed that it was constructed correctly and the titer of slow virus was 6E + 8TU / ml .

4 . Under fluorescence microscope , a lot of green fluorescence was observed in frozen section of mouse liver tissue injected with slow virus .

5 . The serum ALT and AST levels in the model group were significantly higher than those in the normal control group , and the ALT and AST levels in the specific sequence group were significantly lower than those in the model group .

6 . The level of serum LPS in the model group was higher than that of the normal control group , and the level of LPS in the specific sequence group was significantly lower than that in the model group .

7 . HE staining showed that the liver tissues of normal control group had no inflammatory cell infiltration and no hepatocellular necrosis : inflammatory cell infiltration and hepatocellular necrosis were seen in liver tissues of model group and unrelated sequence group .
The inflammatory cell infiltration and necrosis of hepatocytes in the liver tissues of the specific sequence group were significantly reduced compared with the model group .

8 . In the model of liver failure , the expression level of SMAC mRNA in liver tissue was higher than that of normal control group , the expression of SMAC protein decreased , the expression of caspase - 9 protein in liver tissue was significantly increased , the apoptosis index was 33.0 鹵 5.6 % .

9 . The level of serum LPS in patients with HEV related acute liver failure was 0.26 鹵 0.02 EU / ml , which was significantly higher than that of healthy persons .

10 . After incubation of hepatocytes , the apoptosis rate of cells was 5.83 鹵 0.42 % , which was significantly higher than that in the control group . There was no significant difference in the apoptosis rate of the healthy subjects .

Conclusion

1 . By inhibiting the expression of SMAC , the apoptosis of liver cells induced by LPS can be effectively reduced and the apoptosis of liver cells in mice with hepatic failure can be effectively reduced . It provides a new means and theoretical basis for the treatment of hepatic failure endotoxaemia . It also shows that SMAC and its downstream pathway may be one of the pathways of LPS - mediated apoptosis .

2 . Patients with HEV - related acute hepatic failure had higher concentrations of LPS , which could induce apoptosis of hepatocytes .
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R575.3

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本文編號：1903670