重组人促红细胞生成素诱导人源羊水干细胞向心肌前体细胞的分化及其Wnt信号通路机制的研究

发布时间：2018-01-11 16:12

本文关键词：重组人促红细胞生成素诱导人源羊水干细胞向心肌前体细胞的分化及其Wnt信号通路机制的研究　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：观察不同浓度重组人促红细胞生成素（recombinant humanerythropoietin，rhEPO）对人源羊水干细胞（amniotic fluid stem cells，AFSC)向心肌前体细胞分化的作用及观察该分化过程中可能参与的Wnt信号机制。方法: ⑴扩增、纯化AFSC。 ⑵倒置显微镜下观察体外培养的羊水干细胞的一般生物学特性。采用流式细胞术检测AFSC的CD29及CD34的表达情况。 ⑶本实验分为对照组，rhEPO组，rhEPO+LiCl组。 ⑷对照组一直使用常规培养基培养；rhEPO组用对照组的培养基中加入rhEPO组成的诱导培养基培养，且rhEPO的终浓度分别为1.O、5．0、10.0、20.0U／ml，干预24h后换成与对照组相同的培养基培养；rhEPO+LiCl组用上述浓度的rhEPO组成的诱导培养基培养并在诱导分化液中各加上5mmol/L的Wnt信号激动剂LiCl干预24h，然后换成与对照组相同的培养基培养。 ⑸将鉴定后的细胞种于10*10的小玻片上，按上述要求进行干预，48h后，用免疫荧光法检测羊水干细胞在诱导前及诱导早期的促红细胞生成素受体（EPOR）的表达情况。用免疫组化检测β-catenin及p-GSK-3β (Ser~9)的表达情况。 ⑹2周后在倒置显微镜下观察诱导分化后的细胞，计算有形态改变的心肌前体细胞数占总细胞数的比率为分化率，并比较rhEPO组与对照组的分化率的差异。 ⑺14天后收集各组细胞，，提取总的RNA，应用RT-PCR检测各组心肌早期转录因子GATA-4、Nkx2.5mRNA的表达情况。 ⑻28天后应用Western blot检测各组细胞心肌特异蛋白(β-MHC、cTnT)的表达情况。结果: ⑴羊水内的细胞接种后原代静养7天，第8天在倒置显微镜下可见集落细胞团，集落分布不均，大小不一，集落周边仅有很少量细胞，集落中心细胞易可见老化凋亡的细胞。羊水干细胞生长快，传代后细胞平铺时体积大，核仁大，胞浆丰富，细胞生长较密时，细胞似成纤维样细胞，排列成漩涡状。 ⑵AFSC经流式细胞仪检测显示AFSC表型为CD29~+为97.04%，CD34~+为0.61%。 ⑶免疫荧光观察到AFSC在诱导前和诱导早期均有EPOR的表达。 ⑷诱导分化第14天，倒置显微镜下可见细胞由长梭形逐渐变短增宽，相邻的细胞间有融合现象，似类肌管样结构。不同浓度的rhEPO诱导的分化率有差异，但都显著高于对照组（P0.05），且以5.0U/mLrhEPO的诱导分化率最高（P0.05）。 ⑸在诱导分化14天后，与未干预组比较，各浓度rhEPO组及各浓度rhEPO+LiCl组干预后细胞的GATA-4、Nkx2.5mRNA的表达明显上调（P0.05），以5．0U／m1的作用最显著；与rhEPO组比较，rhEPO+Licl组能显著干预上调诱导细胞的GATA-4、Nkx2.5mRNA的表达（P0.05），以5.0U／m1rhEPO+5mmol/L的LiCl干预作用最强。 ⑹经诱导28天后，各干预组的细胞的心肌特异相关蛋白β-MHC、cTnT的表达明显上调（P0.05），以5．0U／m1的作用最显著，而rhEPO与Wnt信号激动剂共同作用强于单用rhEPO。 ⑺诱导过程中各组均有如前所述的Wnt信号通路中的蛋白表达，与对照组比较，rhEPO及rhEPO+LiCl组β-catenin及p-GSK-3β的表达的增多（P0.05），与rhEPO比较，rhEPO+LiCl组β-catenin p-GSK-3β的表达强于rhEPO组。结论: ⑴人源羊水中存在AFSC，其性质类似于间充质干细胞及胚胎干细胞。 ⑵外源性rhEPO成素能作为一种化学诱导剂应用于干细胞的研究。 ⑶外源性rhEPO早期干预能剂量依赖性促进人源羊水干细胞向心肌前体细胞分化。 ⑷外源性rhEPO可能通过启动Wnt信号诱导人源羊水干细胞分化为心肌前体细胞。
[Abstract]:Objective:
Objective To observe the effect of different concentrations of recombinant HumanErythropoietin (rhEPO) on the differentiation of human amniotic fluid stem cells (AFSC) into cardiac precursor cells and observe the Wnt signaling mechanism that may participate in the differentiation process.
Method:
The amplification and purification of AFSC.
It was observed under inverted microscope in vitro biological characteristics of amniotic fluid stem cells. The general expression by CD29 and CD34 AFSC by flow cytometry.
The present experiment was divided into control group, rhEPO group, rhEPO+LiCl group.
The control group has been using conventional culture medium; adding rhEPO group with control group culture medium composed of rhEPO inducing culture medium, and the final concentration of rhEPO was 1.O / ml, 5.0,10.0,20.0U, 24h after the intervention and control group in the same medium culture; group rhEPO+LiCl with the concentration of rhEPO. Induction medium and in the differentiation medium induced Wnt signal 5mmol/L agonist LiCl intervention 24h, then replaced with the control group culture medium for the same.
The identification of cells after a 10*10 of the small slides, intervention, according to the requirements of 48h, detection of amniotic fluid stem cells before induction and induction of erythropoietin early hormone receptor by immunofluorescence (EPOR). The expression of -catenin and p-GSK-3 for detecting beta beta (Ser~9) of the immune group the expression.
It observed after 2 weeks after induction of differentiation of cells in the inverted microscope, calculate the ratio of morphological changes of the cardiac precursor cells accounted for the total number of cells for differentiation rate, and compared with the rhEPO group and the control group differentiation rate differences.
14 days later, cells were collected to extract total RNA, using RT-PCR to detect early cardiac transcription factor GATA-4, Nkx2.5mRNA expression.
28 days later, the application of Western blot to detect the expression of cardiac specific protein (beta -MHC, cTnT) expression.
Result:
The amniotic fluid cells after inoculation of primary rest 7 days, eighth days under the inverted microscope in the colony cells, colony distribution is uneven, the size of a colony around only a small quantity of cells, colony center cells visible to the aging cell apoptosis. Amniotic fluid stem cells grew faster after passage, cells tile when large volume, large nucleoli, abundant cytoplasm, dense cell growth, cell like fibroblast like cells, arranged in a spiral shape.
The AFSC by flow cytometry showed that AFSC phenotype was CD29~+ 97.04%, CD34~+ 0.61%.
The immunofluorescence was observed in AFSC before induction and early had EPOR expression.
The fourteenth days of differentiation, under the inverted microscope, cells from fusiform became short gradually widened, adjacent cell fusion phenomenon, like myotube like structure. There are differences in different concentrations of rhEPO induced differentiation rate, but significantly higher than that of the control group (P0.05), and to induce the highest differentiation rate of 5.0U/ mLrhEPO the (P0.05).
In the differentiation of 14 days, compared with the untreated group, the concentration of rhEPO group and the concentration of rhEPO+LiCl group after the intervention of GATA-4 cells, up-regulated expression of Nkx2.5mRNA (P0.05), 5.0U / M1 the most significant; compared with rhEPO group, rhEPO+Licl group significantly increased intervention induced cell GATA-4, Nkx2.5mRNA the expression of LiCl (P0.05), the intervention effect of the strongest 5.0U / m1rhEPO+5mmol/L.
It was after 28 days of induction, the intervention group of cells of cardiac specific protein beta -MHC, up-regulated expression of cTnT (P0.05), 5.0U / M1 had significant effect, while the rhEPO and Wnt signal agonist interaction is stronger than a single rhEPO.
The expression of Wnt signaling pathway, such as the proteins in the induction process in each group, compared with control group, rhEPO group and rhEPO+LiCl beta -catenin and p-GSK-3 beta expression increased (P0.05), compared with rhEPO group the expression of rhEPO+LiCl beta -catenin p-GSK-3 beta is stronger than the rhEPO group.
Conclusion:
The presence of AFSC derived from human amniotic fluid, similar to the nature of mesenchymal stem cells and embryonic stem cells.
The exogenous rhEPO can be used as a chemical inducing agent used in stem cell research.
The early intervention of exogenous rhEPO can dose dependently promote human amniotic fluid stem cells differentiate into myocardial precursor cells.
The exogenous rhEPO may start by Wnt signal induced by human amniotic fluid stem cells to differentiate into cardiac precursor cells.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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