【摘要】：目的： 1、明確參麥注射液改善心肌缺血再灌注損傷是否通過(guò)調(diào)節(jié)鈣調(diào)蛋白Phospholamban(PLB)的表達(dá)和磷酸化水平來(lái)改善細(xì)胞內(nèi)鈣濃度的機(jī)理；2、探討人參皂苷對(duì)缺血心臟的心功能的改善作用及可能的鈣相關(guān)的機(jī)理；3、比較參麥注射液復(fù)方制劑是否優(yōu)于人參皂苷單純制劑。 方法： 利用新生大鼠原代心肌細(xì)胞建立缺氧/復(fù)氧(I/R)模型,實(shí)驗(yàn)分為正常培養(yǎng)組(N)、正常培養(yǎng)+參麥組(N+SM)、正常培養(yǎng)+人參總皂苷組(N+TSPG)、正常培養(yǎng)+人參皂苷Rgl組(N+Rgl)、缺氧/復(fù)氧組(I/R)、缺氧/復(fù)氧組+參麥組(I/R+SM)、缺氧/復(fù)氧組+人參總皂苷組(I/R+TSPG)、缺氧/復(fù)氧+人參皂苷Rgl組(I/R+Rg1),采用流式細(xì)胞儀測(cè)定凋亡細(xì)胞百分率、細(xì)胞內(nèi)鈣的平均熒光值,實(shí)時(shí)熒光定量聚合酶式反應(yīng)(RT-PCR)法測(cè)定心肌細(xì)胞肌漿網(wǎng)鈣泵(SERCA)和PLB的mRNA表達(dá)量,Western Blot法測(cè)定心肌細(xì)胞SERCA和PLB、磷酸化PLB (p-PLB)的蛋白表達(dá)量。 結(jié)果： 心肌細(xì)胞缺氧／復(fù)氧后,PLBmRNA及蛋白表達(dá)無(wú)顯著變化,p-PLB蛋白表達(dá)和SERCA的mRNA及蛋白表達(dá)均有不同程度的下降,而I/R+Rgl組較I/R組上述變化無(wú)明顯差異(P0.05),I/R+SM組p-PLB蛋白表達(dá)、p-PLB/PLB、SERCA mRNA及蛋白表達(dá)均明顯升高(P0.01),PLB/SERCA明顯下降(P0.01),I/R+TSPG組SERCA mRNA及蛋白表達(dá)均明顯升高(P0.01),PLB/SERCA明顯下降(P0.05),p-PLB蛋白表達(dá)、p-PLB/PLB無(wú)明顯差異(P0.05)。I/R+SM組較I/R+TSPG組SERCA mRNA及蛋白表達(dá)明顯升高(P0.05)、PLB/SERCA明顯降低(P0.05)。I/R+SM組較I／R組凋亡細(xì)胞百分率均有不同程度下降(P0.05),I/R+SM組較I/R組細(xì)胞內(nèi)鈣的平均熒光值均有不同程度的降低(P0.01),I/R+TSPG組、I/R+Rgl組細(xì)胞凋亡率變化無(wú)明顯差異(P0.05),I/R+TSPG組細(xì)胞內(nèi)鈣離子濃度較I/R升高(P0.05),I/R+Rgl無(wú)明顯變化。 結(jié)論： 1、參麥注射液能通過(guò)調(diào)節(jié)鈣調(diào)蛋白PLB的磷酸化水平來(lái)改善細(xì)胞內(nèi)鈣濃度的機(jī)理來(lái)改善心肌缺血再灌注損傷；2、人參總皂苷可抑制缺血再灌注后心肌細(xì)胞內(nèi)鈣離子濃度,進(jìn)而改善心功能,此過(guò)程可能跟促進(jìn)SERCA表達(dá)有關(guān),但不通過(guò)鈣調(diào)蛋白PLB磷酸化途徑；3、參麥注射液復(fù)方制劑可能優(yōu)于人參皂苷單純制劑。
[Abstract]:Objective: 1. To determine whether Shenmai injection can improve the intracellular calcium concentration by regulating the expression and phosphorylation of calmodulin Phospholamban (PLB) in myocardial ischemia-reperfusion injury. (2) to investigate the effects of ginsenoside on cardiac function of ischemic heart and the possible calcium-related mechanism; (3) to compare whether the compound preparation of Shenmai injection is superior to the simple preparation of ginsenoside. Methods: neonatal rat cardiomyocytes were used to establish hypoxia / reoxygenation (I / R) model. The model was divided into normal culture group (N), normal culture group (N SM), normal cultured ginseng total saponin group (N TSPG),). Normal cultured ginsenoside Rgl group (N Rgl), hypoxia / reoxygenation group, hypoxia / reoxygenation group ginseng wheat group (I / R SM), hypoxia / reoxygenation group Ginseng total saponin group (I / R TSPG),) In hypoxia / reoxygenation ginsenoside Rgl group (I / R Rg1), the percentage of apoptotic cells and the average fluorescent value of intracellular calcium were measured by flow cytometry. The mRNA expression of sarcoplasmic reticulum calcium pump (SERCA) and PLB in cardiomyocytes was measured by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The protein expressions of SERCA and PLB, phosphorylated PLB (p-PLB) in cardiomyocytes were measured by, Western Blot method. Results: after hypoxia / reoxygenation, the expression of PLBmRNA and protein did not change significantly, but the expression of p-PLB protein and mRNA and protein of SERCA decreased to some extent. However, there was no significant difference between the two groups (P0.05). The expression of p-PLB protein, the expression of p-PLB, mRNA and protein in SM group were significantly increased (P0.01), and the expression of PLB/SERCA was significantly decreased (P0.01), but the expression of p-PLB protein in group I / R Rgl was significantly higher than that in group I / R (P < 0.01). The expression of SERCA mRNA and protein in TSPG group was significantly higher than that in control group (P0.01), while the expression of PLB/SERCA was significantly decreased (P0.05), and the expression of p-PLB protein was significantly decreased. There was no significant difference in p-PLB/PLB (P0.05). The expression of SERCA mRNA and protein in I / R SM group was significantly higher than that in I / R TSPG group (P 0.05). The percentage of apoptotic cells in PLB/SERCA SM group was significantly lower than that in I R group (P0.05), and the percentage of apoptotic cells was significantly decreased (P0.05). The average intracellular Ca ~ (2 +) fluorescence value in I / R SM group was significantly lower than that in I / R group (P0.01). There was no significant difference in apoptosis rate between I / R TSPG group and I / R Rgl group (P 0.05). The intracellular Ca ~ (2 +) concentration in I / R TSPG group was higher than that in I / R group (P 0.05), but there was no significant change in I / R Rgl. Conclusion: 1 Shenmai injection can improve the myocardial ischemia-reperfusion injury by regulating the phosphorylation level of calmodulin PLB to improve the intracellular calcium concentration. 2. Ginsenoside could inhibit intracellular calcium concentration and improve cardiac function after ischemia-reperfusion, which might be related to the promotion of SERCA expression, but not through calmodulin PLB phosphorylation pathway. 3. The compound preparation of Shenmai injection may be superior to the simple preparation of ginsenoside.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R542.2

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