本文選題：熱休克蛋白25 + 心肌梗死��； 參考：《南京師范大學(xué)》2014年碩士論文

【摘要】：背景和目的 小分子熱休克蛋白作為一類在進(jìn)化上高度保守的蛋白,其在哺乳動物中生物學(xué)作用的重要性早已引起廣泛關(guān)注。該家族中的Hsp25(鼠源)分子量為25kDa；而其人源的同源蛋白為Hsp27,分子量為27kDa。研究表明,Hsp25/Hsp27與心血管疾病發(fā)生發(fā)展之間存在重要關(guān)系。本課題組以往研究表明,過表達(dá)Hsp27能減輕急性心肌缺血/再灌注損傷,縮小心肌梗死面積,提示Hsp25/Hsp27具有潛在的心臟保護(hù)功能。但是Hsp25/27對心肌梗塞后的心肌重塑、中長期存活率的影響,都不清楚。因此,本課題組前期在構(gòu)建Hsp25心肌特異性敲除小鼠基礎(chǔ)上,發(fā)現(xiàn)Hsp25基因缺失顯著增加心肌梗塞小鼠的死亡率,其死亡原因主要是心臟破裂。本實驗將從細(xì)胞外基質(zhì)重塑的角度,探討Hsp25基因?qū)π墓：笮募￠g質(zhì)重塑的影響,以期為揭示Hsp25對心梗后疤痕修復(fù)的調(diào)節(jié)作用提供理論依據(jù)。 實驗方法 1動物模型：本實驗室通過與南京大學(xué)模式動物研究所合作構(gòu)建了Hsp25基因心肌特異性敲除小鼠(Hsp25knockout mice,Hsp25KO),飼養(yǎng)在南京大學(xué)模式所的無特定病原體(Specific Pathogen Free,SPF)動物房中。對照組為具有同樣背景的野生型小鼠(Wild Type,WT) 2小鼠基因型的鑒定： 1)用聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase Chain Reaction, PCR)確定小鼠的基因型； 2)用蛋白質(zhì)免疫印跡法(Western Blot)檢測小鼠心肌中Hsp25的蛋白表達(dá)水平。 3小鼠心肌梗死(Myocardial Infarction,MI)模型的構(gòu)建：戊巴比妥納麻醉小鼠后,取仰臥位,呼吸機(jī)機(jī)械通氣。備皮、開胸、暴露出心臟,撕開心包膜,在冠狀動脈左前降支三分之一處進(jìn)行永久性結(jié)扎,關(guān)胸,縫皮,30min之后撤下呼吸機(jī),讓小鼠自然蘇醒。 4心功能評價：分別對假手術(shù)(sham)、MI后1天、3天的KO小鼠和WT小鼠進(jìn)行異氟烷氣體麻醉,取仰臥位,使用超聲心動圖測定心臟多項心功能指標(biāo),包括左心室射血分?jǐn)?shù)(EF%)、心室短軸縮短率(FS%)、舒張期左室內(nèi)徑(LVIDd)、收縮期左室內(nèi)徑(LVIDs)等。 5小鼠心臟組織分區(qū)分離：斷頸處死小鼠,迅速開胸,將心臟取出置于4℃的PBS中,首先截取結(jié)扎線以上的組織為梗死遠(yuǎn)端區(qū)(Infarct remote left ventricle;Infarct remote LV)；剩下的組織按照梗死界限進(jìn)行分離,梗死的區(qū)域稱為(Infar leftventricle;Infarct LV);另一部分組織稱為梗死周圍區(qū)(Infarct border left ventricle; Infarct border LV)。 6細(xì)胞外基質(zhì)的膠原檢測：將梗死區(qū)的心肌置于戊二醛固定,送至南京醫(yī)科大學(xué)電鏡室進(jìn)行制片,透射電子顯微鏡(Transmission electron microscope,TEM)觀察心臟組織超微結(jié)構(gòu)變化。7統(tǒng)計學(xué)方法：使用SPSS17.0進(jìn)行數(shù)據(jù)處理。數(shù)據(jù)以均數(shù)士標(biāo)準(zhǔn)差(x士s)表示,兩組數(shù)據(jù)之間的比較用兩樣本均數(shù)檢驗(t檢驗),多組數(shù)據(jù)之間的比較用單因素方差分析(ANOVA),P0.05表示有顯著性差異,P0.01表示有極顯著性差異。 實驗結(jié)果 1WT鼠心肌梗死后,其心肌中的Hsp25蛋白表達(dá)量會隨著時間的延長而逐漸上調(diào)。 2構(gòu)建Hsp25心肌特異敲除的小鼠模型,其野生型(WT)小鼠的心肌中的Hsp25表達(dá)水平正常,而基因敲除型(KO)小鼠心肌中的Hsp25表達(dá)水平與之相比明顯下降。 3Hsp25基因敲除后,不影響小鼠的正常發(fā)育以及生殖功能。 4小鼠MI后,在統(tǒng)計的兩周時間內(nèi)WT小鼠的存活率在75%,而KO小鼠與之相比,其存活率明顯下降,并且在造模后的五天時間內(nèi)全部死亡,尸體解剖發(fā)現(xiàn)90%的小鼠是因為心臟破裂而死。 5MI后1天和3天的心功能追蹤顯示,KO小鼠心功能較WT小鼠顯著降低。 6通過對MI后1天后3天的心臟梗死區(qū)的的細(xì)胞外基質(zhì)研究發(fā)現(xiàn),KO小鼠的Ⅰ型和Ⅲ型膠原較WT小鼠在蛋白水平有明顯下降。 7通過對MI后1天后的心臟梗死區(qū)的的細(xì)胞外基質(zhì)研究發(fā)現(xiàn),KO小鼠的基質(zhì)金屬酶(matrix metalloproteinases,MMP)2和9較WT小鼠在蛋白水平有明顯升高。 結(jié)論 Hsp25基因?qū)τ谛募」Ｋ赖膭?chuàng)傷修復(fù)存在保護(hù)作用,該作用與調(diào)節(jié)細(xì)胞外機(jī)制重塑有關(guān)。
[Abstract]:Background and purpose
As a class of highly conserved proteins, small molecular heat shock proteins have been widely concerned in the importance of biological action in mammals. The molecular weight of Hsp25 (Shu Yuan) in this family is 25kDa, and the homologous protein of the human source is Hsp27, and the molecular weight of 27kDa. shows that Hsp25/Hsp27 and cardiovascular disease occur and develop. Previous studies have shown that overexpression of Hsp27 can reduce acute myocardial ischemia / reperfusion injury and narrow the area of myocardial infarction, suggesting that Hsp25/Hsp27 has potential cardiac protective function. However, the effect of Hsp25/27 on myocardial remodeling after myocardial infarction and the effect of medium and long term survival is not clear. Therefore, this research group On the basis of the early construction of Hsp25 specific knockout mice, we found that Hsp25 gene deletion significantly increased the mortality of myocardial infarction mice. The main cause of death was cardiac rupture. The effect of Hsp25 gene on myocardial interstitial remodeling after myocardial infarction was discussed from the angle of extracellular matrix remodeling, in order to reveal the scar of Hsp25 after myocardial infarction. The regulation of scar repair provides a theoretical basis.
Experimental method
1 animal model: in this laboratory, the Hsp25 gene Hsp25knockout mice (Hsp25KO) was constructed in collaboration with the Nanjing University model animal research institute, which was raised in the Specific Pathogen Free, SPF animal room of the Nanjing University model. The control group was a wild type mouse with the same background (Wild T). Ype, WT)
2 Identification of the genotypes of mice:
1) polymerase chain reaction (Polymerase Chain Reaction) (PCR) was used to identify the genotype of mice.
2) protein expression level of Hsp25 in murine myocardium was detected by Western Blot.
The construction of 3 Myocardial Infarction (MI) model in mice: after pentobarbital was anesthetized in mice, the supine position, ventilator mechanical ventilation, skin preparation, open chest, exposure of heart, tear film, 1/3 permanent ligation of the left anterior descending coronary artery, closed chest, suture, and 30min after 30min were removed to make the mice natural Suzhou. Wake up.
4 cardiac function evaluation: Isoflurane gas anesthesia was performed on false operation (sham), 1 days after MI, 3 days of KO mice and WT mice. Cardiac multinomial cardiac function indexes were measured by echocardiography, including left ventricular ejection fraction (EF%), short axis shortening rate (FS%), diastolic left ventricular diameter (LVIDd), systolic left ventricular diameter (LVIDs), etc.
5 division of the mouse heart tissue Division: cut the neck to death in mice, quickly open the chest, put the heart out of the PBS at 4 degrees C, first intercepted the tissue above the ligation line for the distal infarct region (Infarct remote left ventricle; Infarct remote LV); the remaining tissues were separated according to the boundary of the infarct, the infarct area called (Infar leftventricle; Infa) RCT LV); another part is called Infarct border left ventricle (Infarct border LV).
6 collagen detection of extracellular matrix: the myocardium in the infarct area was fixed by glutaraldehyde and sent to the electron microscope room of Nanjing Medical University. Transmission electron microscope (TEM) was used to observe the ultrastructural changes of the heart tissue,.7 statistical method: data processing with SPSS17.0. X s) said that the comparison between the two groups of data was tested by two samples (t test), and the comparison between the multiple groups of data was made by the single factor variance analysis (ANOVA), and the P0.05 showed significant differences, and the P0.01 indicated a very significant difference.
experimental result
After 1WT myocardial infarction, the expression of Hsp25 protein in myocardium gradually increases with time.
2 a mouse model of Hsp25 specific knockout was constructed, and the expression level of Hsp25 in the myocardium of its wild type (WT) mice was normal, while the Hsp25 expression level in the myocardium of the gene knockout (KO) mice was significantly lower than that in the myocardium.
Knockout of 3Hsp25 gene did not affect the normal development and reproductive function of mice.
After 4 mice MI, the survival rate of WT mice was 75% during the two week period of statistics, and the survival rate of KO mice decreased significantly compared with that of the mice, and all died within five days after the model. The autopsy found that 90% of the mice died of the heart rupture.
Cardiac function tracing on the 1 and 3 days after 5MI showed that the heart function of KO mice was significantly lower than that of WT mice.
6 it was found that type I and type III collagen in KO mice were significantly lower than that of WT mice in the WT mice through the study of the extracellular matrix in the 3 days after 1 days of the heart infarction.
7 it was found that the matrix metalloproteinases (matrix metalloproteinases, MMP) 2 and 9 in KO mice were significantly higher in the protein level than in the WT mice through the study of the extracellular matrix of the heart infarction area after MI 1 days later.
conclusion
Hsp25 gene plays a protective role in wound healing of myocardial infarction, which is related to remodeling of extracellular mechanism.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R542.22

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Leukolysin/MMP25/MT6-MMP: a novel matrix metalloproteinase specifically expressed in the leukocyte lineage[J];Cell Research;1999年04期

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本文編號：2068863