【摘要】： 目的:1.探討維甲酸(RA)、參麥、抗壞血酸作為誘導(dǎo)劑誘導(dǎo)人胚胎生殖細(xì)胞(EG細(xì)胞)體外分化為心肌細(xì)胞的可行性;2.觀察人胚胎生殖細(xì)胞經(jīng)三種誘導(dǎo)劑誘導(dǎo)不同時(shí)間后的分化狀態(tài);3.尋求能使人胚胎生殖細(xì)胞體外分化為心肌細(xì)胞的更安全可靠且高效的誘導(dǎo)劑。 方法:取5-10周流產(chǎn)人胚胎生殖腺嵴和背側(cè)腸系膜,進(jìn)行組織塊培養(yǎng)。以源于自身胚胎組織的成纖維細(xì)胞作為飼養(yǎng)層,在培養(yǎng)液中添加白血病抑制因子(LIF),觀察人EG細(xì)胞生長(zhǎng)及集落形成情況。將培養(yǎng)得到的細(xì)胞消化成單細(xì)胞懸液,差速貼壁1h去除成纖維細(xì)胞(MEF),細(xì)胞計(jì)數(shù)后以1×105/ml的初始密度接種于100mm的培養(yǎng)皿進(jìn)行懸浮培養(yǎng),培養(yǎng)液中去除了LIF。培養(yǎng)5天后,小心挑出類(lèi)胚體(EBs)接種于24孔板中,然后分別添加含不同濃度RA、參麥、抗壞血酸的誘導(dǎo)液進(jìn)行誘導(dǎo)分化培養(yǎng),觀察細(xì)胞的分化狀態(tài)。取誘導(dǎo)不同時(shí)間(1周、2周、3周、4周)后的細(xì)胞做免疫細(xì)胞化學(xué)染色,鑒定誘導(dǎo)后細(xì)胞心肌特異轉(zhuǎn)錄因子GATA-4和心肌肌鈣蛋白T(cTnT)的表達(dá)情況。用統(tǒng)計(jì)學(xué)方法計(jì)算人EG細(xì)胞經(jīng)三種誘導(dǎo)劑誘導(dǎo)后的心肌樣細(xì)胞轉(zhuǎn)化率,從而找出各種誘導(dǎo)劑的最佳誘導(dǎo)濃度,并比較三種誘導(dǎo)劑的誘導(dǎo)效果。 結(jié)果:經(jīng)RA、參麥、抗壞血酸三種誘導(dǎo)劑誘導(dǎo)1周后,EBs周邊長(zhǎng)出的細(xì)胞呈梭形或不規(guī)則形;誘導(dǎo)2周,細(xì)胞之間的突起相互連接;誘導(dǎo)3周,細(xì)胞之間的連接更加緊密,可見(jiàn)幾個(gè)細(xì)胞連接形成閏盤(pán)樣結(jié)構(gòu);誘導(dǎo)4周,細(xì)胞呈條索狀排列,走向趨于一致。誘導(dǎo)1周后細(xì)胞即出現(xiàn)GATA-4弱表達(dá),核呈棕黃色,數(shù)量較少;誘導(dǎo)2周,陽(yáng)性細(xì)胞數(shù)量增多且顏色加深,核橢圓形,染成棕色;誘導(dǎo)3周GATA-4的表達(dá)達(dá)高峰,核長(zhǎng)橢圓形染色為棕褐色;誘導(dǎo)4周,陽(yáng)性細(xì)胞數(shù)量較3周時(shí)減少且顏色變淡。誘導(dǎo)2周cTnT開(kāi)始有弱表達(dá),胞漿染成淺棕色;誘導(dǎo)3周,cTnT的表達(dá)增強(qiáng),胞漿內(nèi)出現(xiàn)橫紋樣陽(yáng)性結(jié)構(gòu),但主要分布于核周區(qū),染色為棕色;誘導(dǎo)4周,胞漿陽(yáng)性反應(yīng)產(chǎn)物明顯增強(qiáng)且分布廣泛,染色為棕褐色。對(duì)照組GATA-4和cTnT陰性表達(dá)。RA誘導(dǎo)人EG細(xì)胞向心肌細(xì)胞分化的最佳誘導(dǎo)濃度為10-8mol/l或10-9mol/l,誘導(dǎo)效率最高達(dá)(81.7±0.41)%;參麥的最佳誘導(dǎo)濃度為1g/l,誘導(dǎo)效率最高達(dá)(57.0±3.25)%;抗壞血酸的最佳誘導(dǎo)濃度為0.1mg/ml,誘導(dǎo)分化率最高達(dá)(85.2±1.79)%。 結(jié)論:RA、參麥和抗壞血酸三種誘導(dǎo)劑均能誘導(dǎo)人EG細(xì)胞向心肌細(xì)胞分化,誘導(dǎo)后的細(xì)胞GATA-4和cTnT陽(yáng)性表達(dá)。抗壞血酸和傳統(tǒng)的誘導(dǎo)劑RA一樣,對(duì)人EG細(xì)胞向心肌細(xì)胞分化具有很強(qiáng)的誘導(dǎo)作用,而且是一種更安全高效的誘導(dǎo)劑。參麥雖然誘導(dǎo)效率低一點(diǎn),但與RA相比較,更安全可靠,所以也是一種比較好的誘導(dǎo)劑。
[Abstract]:Objective: 1. To explore the feasibility of inducing human embryonic germ cells (EG cells) to differentiate into cardiomyocytes by (RA), and ascorbic acid as inducers in vitro. 2. To observe the differentiation state of human embryonic germ cells induced by three inducers for different time. To seek a safer and more effective inducer to differentiate human embryonic germ cells into cardiomyocytes in vitro. Methods: gonadal ridge and dorsal mesentery were collected from 5-10 weeks aborted human embryo and cultured with tissue mass. The growth and colony formation of human EG cells were observed by adding leukemia inhibitory factor (LIF),) into the culture medium with fibroblasts derived from their own embryonic tissues as the feeder layer. The cultured cells were digested into single cell suspensions, the fibroblast (MEF), cells were removed from the fibroblasts for 1 h, and then the cells were seeded into the culture dish of 100mm at the initial density of 1 脳 105/ml. The LIF. was removed from the culture medium. After 5 days of culture, the embryoid (EBs) was carefully selected and inoculated into the 24-well plate, and then the differentiation of the cells was observed by adding different concentrations of RA, and ascorbic acid respectively. The expression of cardiomyocyte specific transcription factor (GATA-4) and cardiac troponin (T (cTnT) were detected by immunocytochemical staining at different time (1 week, 2 weeks, 3 weeks and 4 weeks) after induction. The myocardial cell transformation rate of human EG cells induced by three inducers was calculated by statistical method, and the optimal concentration of the inducers was found out, and the induction effects of the three inducers were compared. Results: after 1 week of induction by RA, and ascorbic acid, the cells around EBs were fusiform or irregular, and the processes were connected with each other after 2 weeks of induction. After 3 weeks of induction, the connection between the cells became more close, and several cells formed intercalated disk-like structure, and at 4 weeks, the cells were arranged in stripe shape and tended to be in the same direction. After 1 week of induction, the cells showed weak expression of GATA-4, the nucleus was brownish yellow, and the number of the positive cells increased at 2 weeks, and the nucleus was oval and stained brown. After 3 weeks of induction, the expression of GATA-4 reached its peak and the nucleus was stained brown with long ellipse, and at 4 weeks, the number of positive cells decreased and the color became lighter than that at week 3. After 2 weeks of induction, the expression of cTnT began to be weakly expressed, and the cytoplasm was stained with light brown color. After 3 weeks of induction, the expression of cTnT increased, and the striated positive structure appeared in the cytoplasm, but mainly distributed in the perinuclear area and stained brown. After 4 weeks of induction, the cytoplasmic positive reaction products were significantly increased and widely distributed and stained brown. The negative expression of GATA-4 and cTnT in the control group showed that the best concentration of RA induced human EG cells to differentiate into cardiomyocytes was 10-8mol/l or 10-9 mol / L, and the highest induction efficiency was (81.7 鹵0.41)%. The best inductive concentration of Shenmai was 1 g / l, the highest induction efficiency was (57.0 鹵3.25)%, and the optimum concentration of ascorbic acid was 0.1 mg / ml, and the highest induced differentiation rate was (85.2 鹵1.79)%. Conclusion: both RA, and ascorbic acid can induce human EG cells to differentiate into cardiomyocytes and induce the positive expression of GATA-4 and cTnT. Ascorbic acid, like the traditional inducer RA, can induce the differentiation of human EG cells into cardiomyocytes, and it is a safer and more efficient inducer. Although the induction efficiency of Shenmai is lower, it is more safe and reliable than RA, so it is also a good inducer.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

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相關(guān)期刊論文 前10條

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