Wnt、FGF1对胚胎干细胞造血分化的作用研究

发布时间：2018-04-29 01:17

本文选题：小鼠胚胎成纤维细胞 + 胚胎干细胞　；参考：《滨州医学院》2012年硕士论文

【摘要】：目的：通过过表达Wnt和FGF1在拟胚体上的表达,检测拟胚体中Flk-1、CD133、CD34阳性细胞的表达情况,探讨过表达Wnt对Shh表达的影响,研究Wnt和FGF1对小鼠胚胎干细胞造血分化的作用及信号通路的调控。方法：1.小鼠胚胎干细胞的培养：购买ICR小鼠,取孕13.5天的小鼠用于分离小鼠胚胎成纤维细胞；取第3-5代对数生长期细胞制备饲养层细胞。将ES细胞D3细胞接种于饲养层细胞上,待细胞呈明显集落状态时,利用差速贴壁法去除饲养层细胞,悬滴法制备拟胚体,48h后收集。 2.过表达Wnt对小鼠胚胎干细胞造血分化的作用：悬滴法收集拟胚体后分别按氯化锂浓度不同分成3组：对照组,5mmol/L LiCl组和10mmoI/L LiCl组,再用悬浮法将其各培养3、5、7、9天,利用免疫荧光法及IMAGE-PRO PLUS图像分析系统对各组拟胚体中Flk-1+、CD133+、CD34+细胞进行测量分析；利用流式细胞仪检测拟胚体中Flk-1+/CD133+的共表达情况；通过RT-PCR法检测最适浓度5mmol/L组的Flk-1基因的表达。 3.过表达Wnt对Shh表达的影响：利用免疫荧光法检测Shh在EB中的表达情况,IMAGE-PRO PLUS图像分析系统对各组拟胚体中Shh表达阳性细胞进行检测,探讨Wnt信号对胚胎干细胞造血分化的调控相关机制。 4.FGF1在小鼠胚胎干细胞造血分化中的作用：利用免疫荧光法检测FGF1在ES细胞与EB中的表达,悬滴法收集拟胚体后,在分化培养液中分别添加1ug/L、2ug/L和5ug/L的FGF1,分别培养3、5、7、9天后收集所得拟胚体,用流式细胞术检测其中Flk-1+、CD133+细胞率。结果：1.成功培养扩增大量小鼠胚胎成纤维细胞、饲养层细胞、胚胎干细胞； 2.(1)过表达Wnt后不同时间与不同浓度的Flk-1+、CD133+细胞量不同。不同时间比较Flk-1+细胞的平均吸光度值,结果显示培养5天组显著高于其他组(P0.05)；不同浓度间比较Flk-1+细胞的平均吸光度值,结果显示在5mmol/L组最高(P0.05)；而CD133+细胞量在不同时间组别内平均吸光度值5天组显著高于其他组(P0.05)；不同浓度间平均吸光度值比较,对照组最高(P0.05)；实验组与对照组相比CD34+细胞有显著增加(P0.05)；在相同时间内平均吸光度值比较,5mmol/L组最高(P0.05),相同浓度下比较结果显示CD34+细胞呈先增长后下降的趋势；(2)流式细胞术检测结果显示Flk-1+/CD133+细胞率在5天组的5mmol/L组最高(P0.05);(3)RT-PCR检测5mmol/L组不同时间的Flk-1的表达情况,5天最明显。 3.在相同时间内比较测量所得Shh表达阳性的细胞数值,结果显示5mmol/L组高于其他组(P0.05),相同浓度下比较结果显示5天组整体高于其他组。 4.不同浓度的FGF1处理后,5天内Flk-1+与CD133+细胞相对于正常对照组其总体呈负增长趋势,而7天后Flk-1+与CD133+细胞呈快速增长,与对照组相比呈增长趋势(P0.05)。结论：1.冻存大量小鼠胚胎成纤维细胞、饲养层细胞和胚胎干细胞； 2.Wnt可以促进拟胚体中Flk-1+、CD133+、CD34+细胞的形成,但是这一作用受时间和浓度的双重影响,其最适浓度为5mmol/L,最佳时间为5天； 3.Wnt可以调控Shh的表达,但存在时间浓度的依赖性,可以推测Wnt在胚胎干细胞造血分化中发挥作用与Shh信号通路有关。 4.初步证明Wnt和FGF1在小鼠胚胎干细胞造血分化过程中具有重要作用；
[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.

【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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