本文選題：丹參注射液 + 心臟干細胞　； 參考：《南京中醫(yī)藥大學(xué)》2013年博士論文

【摘要】：目的：通過分別對體外、體內(nèi)小鼠心肌細胞和心臟干細胞的研究,了解丹參注射液聯(lián)合心臟干細胞移植治療對小鼠急性心肌梗死的影響。方法：(1)給予小鼠心肌細胞缺氧培養(yǎng)的同時,給予不同劑量丹參注射液干預(yù),并以空白對照,檢測Annex in V-PI、Caspase-3、CCK-8,通過Real-Time PCR檢測Akt、BAD、FGF的基因表達。(2)體外小鼠心臟干細胞培養(yǎng),并行Sca-1+鑒定,給予缺氧培養(yǎng),同時給予不同劑量丹參注射液干預(yù),并以空白對照,檢測Caspase-3、CCK-8, Edu檢測心臟干細胞DNA的合成。(3)以不同劑量丹參注射液喂養(yǎng)小鼠1周,并以空白對照,檢測小鼠血常規(guī)和肝腎功能指標(biāo)；將小鼠心臟組織切片檢查,熒光檢測Sca-1+細胞數(shù)量；Real-Time PCR檢測VEGF、IL-6、BAD的基因表達。(4)結(jié)扎小鼠冠狀動脈前降支制作小鼠急性心肌梗死模型,并在心肌梗死周圍區(qū)域心肌內(nèi)注射體外培養(yǎng)的小鼠心臟干細胞,以不同劑量的丹參注射液喂養(yǎng)1周,并以空白對照。取小鼠心臟組織,予CD31檢測、Ki-67染色、Masson檢測、TUNEL檢測,采用Western blot方法檢測小鼠心臟內(nèi)的Akt表達。結(jié)果：(1)小鼠心肌細胞缺氧培養(yǎng)后,丹參注射液高劑量組的Annexin Ⅴ-PI與其它兩組相比顯著性降低；丹參注射液低劑量和高劑量組的Caspase-3與對照組相比顯著降低；CCK-8OD值丹參注射液高劑量組最高,對照組最低,各組間有顯著性差異；FGF基因表達在丹參注射液高劑量組最高,對照組最低,各組之間均有顯著性差異；BAD基因表達在丹參注射液高劑量組最低,對照組最高,各組之間均有顯著性差異；丹參注射液高劑量和低劑量組的Akt基因表達與對照組相比顯著增高,高、低劑量組之間沒有顯著性差異。(2)體外小鼠心臟干細胞缺氧培養(yǎng)后,丹參注射液高劑量組Caspase-3的0D值與低劑量組和對照組相比顯著下降,低劑量組、對照組之間沒有顯著性差異；丹參注射液高劑量組的CCK-8OD值與低劑量組和對照組相比顯著增高,低劑量組和對照組之間沒有顯著性差異；丹參注射液高劑量和低劑量組的EdU陽性細胞數(shù)與對照組相比顯著增高,高、低劑量組之間沒有顯著性差異。(3)丹參注射液喂養(yǎng)一周的小鼠,各組間的血細胞、肝腎功能無顯著性差別；心臟組織中的Sca-1+心臟成體干細胞的數(shù)量,以丹參注射液高劑量最多,對照組最少,各組間有顯著性差異；VEGF和IL-6的基因表達,均以丹參注射液高劑量組最高,對照組最低,各組間有顯著性差異；丹參注射液高劑量組的Bad基因表達與低劑量組和對照組相比顯著降低,低劑量組和對照組之間沒有顯著性差異。(4)對小鼠急性心肌梗死給予心臟干細胞移植,并喂養(yǎng)丹參注射液一周后,反映梗死周圍區(qū)域新生毛細血管密度的CD31,以丹參注射液高劑量最多,對照組最少,各組間有顯著性差異；檢測細胞核增殖的Ki67染色丹參注射液高劑量組與對照組和低劑量組相比顯著升高,對照組、低劑量組之間沒有顯著性差異:Masson檢測反映的纖維化程度,丹參注射液高劑量組與對照組和低劑量組相比顯著減輕,對照組、低劑量組之間沒有顯著性差異；TUNEL檢測顯示細胞凋亡,丹參注射液高劑量最少,對照組最多,各組間有顯著性差異；磷酸化Akt蛋白表達水平上,以丹參注射液高劑量最多,對照組最少,各組間有顯著性差異；總Akt水平上,丹參注射液高劑量和低劑量組與對照組相比顯著升高,高、低劑量組之間沒有顯著性差異。結(jié)論：本研究通過體內(nèi)、體外多層次實驗證明丹參注射液可以通過Akt通路,有效提高心肌干細胞移植治療心肌梗死的效率,促進組織血管新生,抑制細胞凋亡及組織纖維化,最終起到抑制心梗后心室重構(gòu),保護心功能的作用。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R542.22

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