本文選題：小鼠胚胎成纖維細(xì)胞 + 胚胎干細(xì)胞�。� 參考：《濱州醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：通過過表達(dá)Wnt和FGF1在擬胚體上的表達(dá),檢測(cè)擬胚體中Flk-1、CD133、CD34陽性細(xì)胞的表達(dá)情況,探討過表達(dá)Wnt對(duì)Shh表達(dá)的影響,研究Wnt和FGF1對(duì)小鼠胚胎干細(xì)胞造血分化的作用及信號(hào)通路的調(diào)控。 方法：1.小鼠胚胎干細(xì)胞的培養(yǎng)：購買ICR小鼠,取孕13.5天的小鼠用于分離小鼠胚胎成纖維細(xì)胞；取第3-5代對(duì)數(shù)生長(zhǎng)期細(xì)胞制備飼養(yǎng)層細(xì)胞。將ES細(xì)胞D3細(xì)胞接種于飼養(yǎng)層細(xì)胞上,待細(xì)胞呈明顯集落狀態(tài)時(shí),利用差速貼壁法去除飼養(yǎng)層細(xì)胞,懸滴法制備擬胚體,48h后收集。 2.過表達(dá)Wnt對(duì)小鼠胚胎干細(xì)胞造血分化的作用：懸滴法收集擬胚體后分別按氯化鋰濃度不同分成3組：對(duì)照組,5mmol/L LiCl組和10mmoI/L LiCl組,再用懸浮法將其各培養(yǎng)3、5、7、9天,利用免疫熒光法及IMAGE-PRO PLUS圖像分析系統(tǒng)對(duì)各組擬胚體中Flk-1+、CD133+、CD34+細(xì)胞進(jìn)行測(cè)量分析；利用流式細(xì)胞儀檢測(cè)擬胚體中Flk-1+/CD133+的共表達(dá)情況；通過RT-PCR法檢測(cè)最適濃度5mmol/L組的Flk-1基因的表達(dá)。 3.過表達(dá)Wnt對(duì)Shh表達(dá)的影響：利用免疫熒光法檢測(cè)Shh在EB中的表達(dá)情況,IMAGE-PRO PLUS圖像分析系統(tǒng)對(duì)各組擬胚體中Shh表達(dá)陽性細(xì)胞進(jìn)行檢測(cè),探討Wnt信號(hào)對(duì)胚胎干細(xì)胞造血分化的調(diào)控相關(guān)機(jī)制。 4.FGF1在小鼠胚胎干細(xì)胞造血分化中的作用：利用免疫熒光法檢測(cè)FGF1在ES細(xì)胞與EB中的表達(dá),懸滴法收集擬胚體后,在分化培養(yǎng)液中分別添加1ug/L、2ug/L和5ug/L的FGF1,分別培養(yǎng)3、5、7、9天后收集所得擬胚體,用流式細(xì)胞術(shù)檢測(cè)其中Flk-1+、CD133+細(xì)胞率。 結(jié)果：1.成功培養(yǎng)擴(kuò)增大量小鼠胚胎成纖維細(xì)胞、飼養(yǎng)層細(xì)胞、胚胎干細(xì)胞； 2.(1)過表達(dá)Wnt后不同時(shí)間與不同濃度的Flk-1+、CD133+細(xì)胞量不同。不同時(shí)間比較Flk-1+細(xì)胞的平均吸光度值,結(jié)果顯示培養(yǎng)5天組顯著高于其他組(P0.05)；不同濃度間比較Flk-1+細(xì)胞的平均吸光度值,結(jié)果顯示在5mmol/L組最高(P0.05)；而CD133+細(xì)胞量在不同時(shí)間組別內(nèi)平均吸光度值5天組顯著高于其他組(P0.05)；不同濃度間平均吸光度值比較,對(duì)照組最高(P0.05)；實(shí)驗(yàn)組與對(duì)照組相比CD34+細(xì)胞有顯著增加(P0.05)；在相同時(shí)間內(nèi)平均吸光度值比較,5mmol/L組最高(P0.05),相同濃度下比較結(jié)果顯示CD34+細(xì)胞呈先增長(zhǎng)后下降的趨勢(shì)；(2)流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示Flk-1+/CD133+細(xì)胞率在5天組的5mmol/L組最高(P0.05);(3)RT-PCR檢測(cè)5mmol/L組不同時(shí)間的Flk-1的表達(dá)情況,5天最明顯。 3.在相同時(shí)間內(nèi)比較測(cè)量所得Shh表達(dá)陽性的細(xì)胞數(shù)值,結(jié)果顯示5mmol/L組高于其他組(P0.05),相同濃度下比較結(jié)果顯示5天組整體高于其他組。 4.不同濃度的FGF1處理后,5天內(nèi)Flk-1+與CD133+細(xì)胞相對(duì)于正常對(duì)照組其總體呈負(fù)增長(zhǎng)趨勢(shì),而7天后Flk-1+與CD133+細(xì)胞呈快速增長(zhǎng),與對(duì)照組相比呈增長(zhǎng)趨勢(shì)(P0.05)。 結(jié)論：1.凍存大量小鼠胚胎成纖維細(xì)胞、飼養(yǎng)層細(xì)胞和胚胎干細(xì)胞； 2.Wnt可以促進(jìn)擬胚體中Flk-1+、CD133+、CD34+細(xì)胞的形成,但是這一作用受時(shí)間和濃度的雙重影響,其最適濃度為5mmol/L,最佳時(shí)間為5天； 3.Wnt可以調(diào)控Shh的表達(dá),但存在時(shí)間濃度的依賴性,可以推測(cè)Wnt在胚胎干細(xì)胞造血分化中發(fā)揮作用與Shh信號(hào)通路有關(guān)。 4.初步證明Wnt和FGF1在小鼠胚胎干細(xì)胞造血分化過程中具有重要作用；
[Abstract]:Objective: to detect the expression of Wnt and FGF1 in the embryoid body, to detect the expression of Flk-1, CD133, CD34 positive cells in the embryoid body, to explore the effect of Wnt on the expression of Shh, and to study the effect of Wnt and FGF1 on the hematopoietic differentiation of mouse embryonic stem cells and the modulation of signal transduction pathway.
Methods: 1. mouse embryonic stem cells were cultured: ICR mice were purchased, the mice of 13.5 days of pregnancy were used to separate the mouse embryonic fibroblasts, and the feeder layer cells were prepared by the 3-5 generation of logarithmic growth phase cells. The ES cell D3 cells were inoculated on the feeder cells, and the feeder layer was removed by differential adherence. The embryoid body was prepared by suspension drop method and collected after 48h.
2. the effect of overexpression of Wnt on the hematopoietic differentiation of mouse embryonic stem cells: the suspension method was divided into 3 groups according to the concentration of lithium chloride, respectively: the control group, the 5mmol/L LiCl group and the 10mmoI/L LiCl group. The suspension method was used to train them for 3,5,7,9 days with the suspension method. The immune fluorescence method and the IMAGE-PRO PLUS image analysis system were used to determine Flk- in each embryo. 1+, CD133+, CD34+ cells were measured and analyzed. The co expression of Flk-1+/CD133+ in the embryoid body was detected by flow cytometry, and the expression of the Flk-1 gene in the most suitable concentration 5mmol/L group was detected by RT-PCR.
3. the effect of overexpression of Wnt on the expression of Shh: the expression of Shh in EB was detected by immunofluorescence. The IMAGE-PRO PLUS image analysis system was used to detect the positive cells of Shh expression in the embryoid bodies, and the mechanism of regulating the hematopoietic differentiation of embryonic stem cells by Wnt signal was discussed.
The role of 4.FGF1 in the hematopoietic differentiation of mouse embryonic stem cells: the expression of FGF1 in ES cells and EB was detected by immunofluorescence. After the suspension method was used to collect the embryoid body, 1ug/L, 2ug/L and 5ug/L FGF1 were added to the differentiation culture medium respectively, and the embryos were collected from the 3,5,7,9 days, respectively, and Flk-1+ and CD133+ were detected by flow cytometry. Cytosolic rate.
Results: 1. a large number of mouse embryonic fibroblasts, feeder layer cells and embryonic stem cells were successfully cultured.
2. (1) after overexpression of Wnt and different concentrations of Flk-1+, CD133+ cells were different. The average absorbance of Flk-1+ cells was compared at different times. The results showed that the 5 day group was significantly higher than the other groups (P0.05). The average absorbance of Flk-1+ cells was compared between different concentrations, and the results showed the highest (P0.05) in the 5mmol/L group, and CD133+ cells in the 5mmol/L group. The average absorbance value in 5 days group in different time groups was significantly higher than that in other groups (P0.05); the average absorbance between different concentrations was higher than the control group (P0.05); the experimental group was significantly increased (P0.05) compared with the control group (P0.05); the average absorbance value of the 5mmol/L group at the same time (P0.05) was the highest (P0.05), and the same concentration was compared. The results showed that the CD34+ cells showed a tendency to increase first and then decrease; (2) the results of flow cytometry showed that the Flk-1+/CD133+ cell rate was the highest in the group 5mmol/L of the 5 day group (P0.05), and (3) RT-PCR was used to detect the expression of Flk-1 in the 5mmol/L group at different times, the most obvious in the 5 day.
3. the number of positive cells expressed in Shh was measured at the same time. The results showed that the 5mmol/L group was higher than the other groups (P0.05). The comparison of the same concentration showed that the group of 5 days was higher than that of the other groups.
4. after 5 days of FGF1 treatment with different concentrations, the total Flk-1+ and CD133+ cells showed a negative growth trend compared with the normal control group, while the Flk-1+ and CD133+ cells increased rapidly after 7 days, and showed a growing trend compared with the control group (P0.05).
Conclusion: 1., a large number of mouse embryonic fibroblasts, feeder layer cells and embryonic stem cells were cryopreserved.
2.Wnt can promote the formation of Flk-1+, CD133+, and CD34+ cells in the embryoid body, but this effect is influenced by time and concentration, and the optimum concentration is 5mmol/L, and the optimum time is 5 days.
3.Wnt can regulate the expression of Shh, but there is a time concentration dependence. It can be speculated that Wnt plays a role in hematopoietic differentiation of embryonic stem cells and is related to Shh signaling pathway.
4. it is preliminarily proved that Wnt and FGF1 play an important role in hematopoietic differentiation of mouse embryonic stem cells.

【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 陳春華;秦瑩;;胚胎干細(xì)胞定向分化為胰島素分泌細(xì)胞的研究進(jìn)展[J];國(guó)際生物醫(yī)學(xué)工程雜志;2006年02期

2 熊吉信;劉小春;楊春江;劉兆軒;;轉(zhuǎn)化生長(zhǎng)因子β_1聯(lián)合血管內(nèi)皮細(xì)胞生長(zhǎng)因子體外誘導(dǎo)胚胎干細(xì)胞向血管內(nèi)皮細(xì)胞分化[J];中國(guó)組織工程研究與臨床康復(fù);2007年20期

3 何志旭,黃紹良,周其峰,李樹濃;體外定向誘導(dǎo)小鼠胚胎干細(xì)胞發(fā)育為造血干/祖細(xì)胞方法的初步研究[J];中國(guó)病理生理雜志;2003年04期

4 徐長(zhǎng)憲,于靈芝,郭蘭敏,趙躍然,劉愛蓮,郭雨霽;小鼠胚胎干細(xì)胞體外定向分化為心肌細(xì)胞的實(shí)驗(yàn)研究[J];中華醫(yī)學(xué)雜志;2005年26期

，

本文編號(hào)：1817738