血清和糖皮质激素调节激酶1在心脏移植缺血再灌注损伤中的表达及意义
发布时间：2018-11-22 14:49
【摘要】：目的探讨在大鼠心脏移植缺血再灌注损伤(Ischemia-Reperfusion injury,IRI)过程中血清和糖皮质激素调节激酶1(Serum-and glucocorticoid regulated kinase 1,SGK-1)的表达变化;研究SGK-1是否参与心脏移植IRI所引起的心肌细胞凋亡;分析地塞米松能否改变SGK-1的表达,能否起到保护心脏移植IRI心肌细胞的作用。方法1、通过大鼠颈部异位心脏移植方法,建立大鼠同异基因心脏移植组(Syngeneic heart transplantation SHT/Allogeneic heart transplantation AHT,SHT组Lewis(Lew:RT11)移植到Lewis(Lew:RT11)大鼠);AHT组:Wistar(WF:RT1u)移植到Lewis(Lew:RT11)大鼠)IRI模型,部分供体在心脏移植前预先用不同浓度的地塞米松(0.05,0.5,and 2mg/BWkg)预先处理。2、通过RT-PCR方法,分析SGK-1 mRNA在AHT/SHT组IRI过程中表达变化。3、利用免疫组化学方法(Immunohistochemistry)、蛋白印记方法(Western Blot)、免疫荧光共定位染色法方法(Double Immunofluorescent Staining),分析在AHT/SHT组IRI过程中SGK-1表达变化、组织分布、细胞定位情况。4、利用免疫荧光共定位染色法方法,研究分析SGK-1表达变化与免疫细胞、细胞凋亡相关Marker之间的关系。5、通过HE染色方法,观察大鼠心脏移植术后急性排斥反应及心脏移植IRI心肌细胞的病理变化过程。6、通过统计学法方,综合分析实验数据并得出统计学结论。结果1、大鼠AHT/SHT组IRI模型中,通过RT-PCR、蛋白印记等实验方法,发现SGK-1在mRNA及蛋白质水平都发生了表达改变。在术后1 h,mRNA及蛋白质表达开始升高,6 h升高明显,12 h达到表达高峰,24 h后恢复到接近6 h表达水平。2、通过免疫组化结果,我们分别在AHT/SHT组移植物的心肌细胞上观察到SGK-1阳性细胞的表达,通过免疫荧光共定位染色结果,进一步明确了SGK-1在心肌细胞中表达,且表达趋势与蛋白印记结果具有一致性。3、通过免疫荧光共定位染色结果,我们发现SGK-1与急性排斥相关Marker之间没有明显的共定位现象。而与凋亡Marker之间存在共定位现象。4、通过蛋白印记、免疫组化、免疫荧光等实验分析发现:地塞米松预处理后SGK-1的表达明显比未用地塞米松处理组高。5、HE染色显示:地塞米松预处理后心脏移植IRI所引起的心肌细胞损伤明显减少。结论1、SGK-1在心脏移植后表达上调,且主要定位于心肌细胞中。2、SGK-1参与了心脏移植IRI引起的心肌细胞凋亡过程,并起到抗心肌细胞凋亡的作用,其可能机制与NF-kB凋亡途径相关。对SGK-1的深入研究可为今后抑制心脏移植IRI心肌细胞凋亡提供有效的作用靶点。3、地塞米松能够增加SGK-1的表达并起到减少心肌细胞损伤的作用。这将可能为减少IRI后心肌细胞的凋亡提供一个新的理论依据并将成为新的治疗靶点。
[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.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2
[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.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2
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