丹参注射液联合心脏干细胞移植治疗对急性心肌梗死小鼠的影响

发布时间：2018-06-01 21:23

本文选题：丹参注射液 + 心脏干细胞　；参考：《南京中医药大学》2013年博士论文

【摘要】：目的：通过分别对体外、体内小鼠心肌细胞和心脏干细胞的研究,了解丹参注射液联合心脏干细胞移植治疗对小鼠急性心肌梗死的影响。方法：(1)给予小鼠心肌细胞缺氧培养的同时,给予不同剂量丹参注射液干预,并以空白对照,检测Annex in V-PI、Caspase-3、CCK-8,通过Real-Time PCR检测Akt、BAD、FGF的基因表达。(2)体外小鼠心脏干细胞培养,并行Sca-1+鉴定,给予缺氧培养,同时给予不同剂量丹参注射液干预,并以空白对照,检测Caspase-3、CCK-8, Edu检测心脏干细胞DNA的合成。(3)以不同剂量丹参注射液喂养小鼠1周,并以空白对照,检测小鼠血常规和肝肾功能指标；将小鼠心脏组织切片检查,荧光检测Sca-1+细胞数量；Real-Time PCR检测VEGF、IL-6、BAD的基因表达。(4)结扎小鼠冠状动脉前降支制作小鼠急性心肌梗死模型,并在心肌梗死周围区域心肌内注射体外培养的小鼠心脏干细胞,以不同剂量的丹参注射液喂养1周,并以空白对照。取小鼠心脏组织,予CD31检测、Ki-67染色、Masson检测、TUNEL检测,采用Western blot方法检测小鼠心脏内的Akt表达。结果：(1)小鼠心肌细胞缺氧培养后,丹参注射液高剂量组的Annexin Ⅴ-PI与其它两组相比显著性降低；丹参注射液低剂量和高剂量组的Caspase-3与对照组相比显著降低；CCK-8OD值丹参注射液高剂量组最高,对照组最低,各组间有显著性差异；FGF基因表达在丹参注射液高剂量组最高,对照组最低,各组之间均有显著性差异；BAD基因表达在丹参注射液高剂量组最低,对照组最高,各组之间均有显著性差异；丹参注射液高剂量和低剂量组的Akt基因表达与对照组相比显著增高,高、低剂量组之间没有显著性差异。(2)体外小鼠心脏干细胞缺氧培养后,丹参注射液高剂量组Caspase-3的0D值与低剂量组和对照组相比显著下降,低剂量组、对照组之间没有显著性差异；丹参注射液高剂量组的CCK-8OD值与低剂量组和对照组相比显著增高,低剂量组和对照组之间没有显著性差异；丹参注射液高剂量和低剂量组的EdU阳性细胞数与对照组相比显著增高,高、低剂量组之间没有显著性差异。(3)丹参注射液喂养一周的小鼠,各组间的血细胞、肝肾功能无显著性差别；心脏组织中的Sca-1+心脏成体干细胞的数量,以丹参注射液高剂量最多,对照组最少,各组间有显著性差异；VEGF和IL-6的基因表达,均以丹参注射液高剂量组最高,对照组最低,各组间有显著性差异；丹参注射液高剂量组的Bad基因表达与低剂量组和对照组相比显著降低,低剂量组和对照组之间没有显著性差异。(4)对小鼠急性心肌梗死给予心脏干细胞移植,并喂养丹参注射液一周后,反映梗死周围区域新生毛细血管密度的CD31,以丹参注射液高剂量最多,对照组最少,各组间有显著性差异；检测细胞核增殖的Ki67染色丹参注射液高剂量组与对照组和低剂量组相比显著升高,对照组、低剂量组之间没有显著性差异:Masson检测反映的纤维化程度,丹参注射液高剂量组与对照组和低剂量组相比显著减轻,对照组、低剂量组之间没有显著性差异；TUNEL检测显示细胞凋亡,丹参注射液高剂量最少,对照组最多,各组间有显著性差异；磷酸化Akt蛋白表达水平上,以丹参注射液高剂量最多,对照组最少,各组间有显著性差异；总Akt水平上,丹参注射液高剂量和低剂量组与对照组相比显著升高,高、低剂量组之间没有显著性差异。结论：本研究通过体内、体外多层次实验证明丹参注射液可以通过Akt通路,有效提高心肌干细胞移植治疗心肌梗死的效率,促进组织血管新生,抑制细胞凋亡及组织纤维化,最终起到抑制心梗后心室重构,保护心功能的作用。
[Abstract]:Objective: To investigate the effect of Salvia miltiorrhiza injection combined with cardiac stem cell transplantation on acute myocardial infarction in mice. Methods: (1) at the same time, different doses of Salvia miltiorrhiza injection were given at the same time, and An was given to the mice. NEX in V-PI, Caspase-3, CCK-8, the gene expression of Akt, BAD, FGF was detected by Real-Time PCR. (2) in vitro culture of mouse heart stem cells, parallel Sca-1+ identification, hypoxic culture, and different doses of Salvia miltiorrhiza injection intervention. The mice were fed with Salvia miltiorrhiza for 1 weeks, and the blood routine and liver and kidney function indexes were measured in blank control. The mice heart tissue sections were examined and the number of Sca-1+ cells was detected by fluorescence; Real-Time PCR was used to detect the gene expression of VEGF, IL-6, BAD. (4) the acute myocardial infarction model of mice was made by ligating the anterior descending branch of the coronary artery and the myocardial infarction was made in the myocardial infarction. The murine cardiac stem cells were injected into the myocardium in the surrounding region of the dead, and the mice were fed with different doses of Salvia miltiorrhiza for 1 weeks. The mice heart tissue was taken with CD31 detection, Ki-67 staining, Masson detection and TUNEL detection. The Western blot method was used to detect the expression of Akt in the heart of mice. Results: (1) the hypoxia of murine cardiomyocytes After culture, the Annexin V -PI in the high dose group of Salvia miltiorrhiza injection was significantly lower than the other two groups, and the Caspase-3 of the low dose and high dose group of Salvia miltiorrhiza injection was significantly lower than that of the control group; the high dose group with CCK-8OD value of Salvia miltiorrhiza injection was the highest, the control group was the lowest, and the FGF gene expression was injected into Salvia miltiorrhiza. The high dose group was the highest and the control group was the lowest. The BAD gene expression was the lowest in the high dose group of Salvia miltiorrhiza injection and the highest in the control group. The Akt gene expression in the high and low dose groups of Salvia miltiorrhiza injection was significantly higher than that in the control group, and there was no significant difference between the low dose group and the low dose group. (2) the 0D value of Caspase-3 in the high dose group of Salvia miltiorrhiza injection was significantly lower than that of the low dose group and the control group. There was no significant difference between the low dose group and the control group. The CCK-8OD value of the high dose group of Salvia miltiorrhiza injection was significantly higher than the low dose group and the control group, and the low dose group was significantly higher than the control group. There was no significant difference between the control group and the control group. The number of EdU positive cells in the high and low dose groups of Salvia miltiorrhiza injection was significantly higher than that of the control group. There was no significant difference between the high and low dose groups. (3) there was no significant difference between the blood cells and the liver and kidney function of the mice fed by Salvia miltiorrhiza injection for one week, and the Sca-1+ heart in the heart tissue. The number of somatic stem cells was the most high dose of Salvia miltiorrhiza injection, and the control group was the least. The expression of VEGF and IL-6 was the highest in the high dose group of Salvia miltiorrhiza injection and the lowest in the control group. The expression of Bad gene in the high dose group of Salvia miltiorrhiza injection was compared with the low dose group and the control group. There was no significant difference between the low dose group and the control group. (4) the heart stem cell transplantation was given to the acute myocardial infarction in mice. After one week feeding Salvia miltiorrhiza injection, the CD31 of the newborn capillaries density around the infarct area was reflected, with the most high dose of Salvia miltiorrhiza injection and the least in the control group. There was a significant difference between the groups. The high dose group of Salvia miltiorrhiza injection with Ki67 staining was significantly higher than the control group and the low dose group. There was no significant difference between the control group and the low dose group: the degree of fibrosis reflected by the Masson test, the ratio of the high dose group of Salvia miltiorrhiza injection to the control group and the low dose group was significantly reduced, and there was no significant difference between the control group and the low dose group. TUNEL detection showed cell apoptosis, the high dose of Salvia miltiorrhiza injection was the least, the control group was the most, and there were significant differences among the groups. The high dose of Salvia miltiorrhiza was the most high dose of Salvia miltiorrhiza injection and the control group was the least. The high dose and low dose group of Salvia miltiorrhiza injection and the low dose group were in the high and low dose groups on the total Akt level. There is no significant difference between the group and the group. Conclusion: through the body, the multilevel experiment in vitro proves that the injection of Salvia miltiorrhiza can effectively improve the efficiency of myocardial stem cell transplantation in the treatment of myocardial infarction, promote the angiogenesis, inhibit the apoptosis and the fibrosis of the tissue. It can inhibit ventricular remodeling and protect cardiac function after myocardial infarction.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R542.22

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