【摘要】：目的探討在大鼠心臟移植缺血再灌注損傷(Ischemia-Reperfusion injury,IRI)過程中血清和糖皮質激素調節(jié)激酶1(Serum-and glucocorticoid regulated kinase 1,SGK-1)的表達變化;研究SGK-1是否參與心臟移植IRI所引起的心肌細胞凋亡;分析地塞米松能否改變SGK-1的表達,能否起到保護心臟移植IRI心肌細胞的作用。方法1、通過大鼠頸部異位心臟移植方法,建立大鼠同異基因心臟移植組(Syngeneic heart transplantation SHT/Allogeneic heart transplantation AHT,SHT組Lewis(Lew:RT11)移植到Lewis(Lew:RT11)大鼠);AHT組:Wistar(WF:RT1u)移植到Lewis(Lew:RT11)大鼠)IRI模型,部分供體在心臟移植前預先用不同濃度的地塞米松(0.05,0.5,and 2mg/BWkg)預先處理。2、通過RT-PCR方法,分析SGK-1 mRNA在AHT/SHT組IRI過程中表達變化。3、利用免疫組化學方法(Immunohistochemistry)、蛋白印記方法(Western Blot)、免疫熒光共定位染色法方法(Double Immunofluorescent Staining),分析在AHT/SHT組IRI過程中SGK-1表達變化、組織分布、細胞定位情況。4、利用免疫熒光共定位染色法方法,研究分析SGK-1表達變化與免疫細胞、細胞凋亡相關Marker之間的關系。5、通過HE染色方法,觀察大鼠心臟移植術后急性排斥反應及心臟移植IRI心肌細胞的病理變化過程。6、通過統(tǒng)計學法方,綜合分析實驗數據并得出統(tǒng)計學結論。結果1、大鼠AHT/SHT組IRI模型中,通過RT-PCR、蛋白印記等實驗方法,發(fā)現(xiàn)SGK-1在mRNA及蛋白質水平都發(fā)生了表達改變。在術后1 h,mRNA及蛋白質表達開始升高,6 h升高明顯,12 h達到表達高峰,24 h后恢復到接近6 h表達水平。2、通過免疫組化結果,我們分別在AHT/SHT組移植物的心肌細胞上觀察到SGK-1陽性細胞的表達,通過免疫熒光共定位染色結果,進一步明確了SGK-1在心肌細胞中表達,且表達趨勢與蛋白印記結果具有一致性。3、通過免疫熒光共定位染色結果,我們發(fā)現(xiàn)SGK-1與急性排斥相關Marker之間沒有明顯的共定位現(xiàn)象。而與凋亡Marker之間存在共定位現(xiàn)象。4、通過蛋白印記、免疫組化、免疫熒光等實驗分析發(fā)現(xiàn):地塞米松預處理后SGK-1的表達明顯比未用地塞米松處理組高。5、HE染色顯示:地塞米松預處理后心臟移植IRI所引起的心肌細胞損傷明顯減少。結論1、SGK-1在心臟移植后表達上調,且主要定位于心肌細胞中。2、SGK-1參與了心臟移植IRI引起的心肌細胞凋亡過程,并起到抗心肌細胞凋亡的作用,其可能機制與NF-kB凋亡途徑相關。對SGK-1的深入研究可為今后抑制心臟移植IRI心肌細胞凋亡提供有效的作用靶點。3、地塞米松能夠增加SGK-1的表達并起到減少心肌細胞損傷的作用。這將可能為減少IRI后心肌細胞的凋亡提供一個新的理論依據并將成為新的治療靶點。
[Abstract]:Objective to investigate the changes of serum and glucocorticoid regulated kinase 1 (Serum-and glucocorticoid regulated kinase 1) SGK-1 expression during ischemia-reperfusion injury (Ischemia-Reperfusion injury,IRI) in rat heart transplantation. To study whether SGK-1 is involved in cardiomyocyte apoptosis induced by cardiac transplantation IRI and whether dexamethasone can change the expression of SGK-1 and protect the cardiac myocytes from heart transplantation IRI. Methods 1. By using the method of cervical heterotopic heart transplantation in rats, Lewis (Lew:RT11) in (Syngeneic heart transplantation SHT/Allogeneic heart transplantation AHT,SHT group was transplanted to Lewis (Lew:RT11) group. AHT group (: Wistar (WF:RT1u) was transplanted into Lewis (Lew:RT11) rat) IRI model. Some donors were pretreated with different concentrations of dexamethasone (0.05U 0.5 and 2mg/BWkg) before heart transplantation. 2. RT-PCR method was used. The expression of SGK-1 mRNA in IRI of AHT/SHT group was analyzed. 3. The (Immunohistochemistry), protein imprinting method, (Western Blot), immunofluorescence co-localization staining method and (Double Immunofluorescent Staining), method were used to analyze the expression of IRI in AHT/SHT group. The changes of SGK-1 expression, tissue distribution and cellular localization during IRI in AHT/SHT group were analyzed. 4. Immunofluorescence co-localization staining method was used to study the changes of SGK-1 expression and immune cells. The relationship between apoptosis-related Marker. 5. By means of HE staining, the pathological process of acute rejection and cardiac allograft IRI cardiomyocytes after cardiac transplantation in rats was observed. Comprehensive analysis of experimental data and draw statistical conclusions. Results 1. In the IRI model of rat AHT/SHT group, the expression of SGK-1 in mRNA and protein levels was changed by RT-PCR, protein imprinting and other experimental methods. The expression of mRNA and protein began to increase at 1 h after operation, increased significantly at 6 h, reached the peak at 12 h, and recovered to nearly 6 h after 24 h. We observed the expression of SGK-1 positive cells in cardiac myocytes of AHT/SHT group, and further clarified the expression of SGK-1 in cardiac myocytes by immunofluorescence co-localization staining. The expression trend was consistent with the result of protein imprinting. 3. By immunofluorescence co-localization staining, we found that there was no obvious co-localization between SGK-1 and Marker associated with acute rejection. The expression of SGK-1 was significantly higher after dexamethasone pretreatment than that without dexamethasone treatment. HE staining showed that cardiac injury induced by IRI was significantly reduced after dexamethasone preconditioning. Conclusion 1 the expression of SGK-1 was up-regulated after cardiac transplantation and was mainly located in cardiac myocytes. 2SGK-1 participated in the process of cardiomyocyte apoptosis induced by cardiac transplantation IRI and played an anti-apoptosis role in cardiac myocytes. The possible mechanism is related to the apoptosis pathway of NF-kB. The further study of SGK-1 can provide an effective target for inhibiting cardiomyocyte apoptosis in cardiac transplantation IRI in the future. 3. Dexamethasone can increase the expression of SGK-1 and reduce myocardial cell injury. This may provide a new theoretical basis for the reduction of cardiomyocyte apoptosis after IRI and become a new therapeutic target.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R654.2

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