本文關(guān)鍵詞：重組人促紅細(xì)胞生成素誘導(dǎo)人源羊水干細(xì)胞向心肌前體細(xì)胞的分化及其Wnt信號通路機(jī)制的研究　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 重組人促紅細(xì)胞生成素 人源羊水干細(xì)胞 心肌前體細(xì)胞 分化 Wnt 干細(xì)胞

【摘要】：目的： 觀察不同濃度重組人促紅細(xì)胞生成素（recombinant humanerythropoietin，rhEPO）對人源羊水干細(xì)胞（amniotic fluid stem cells，AFSC)向心肌前體細(xì)胞分化的作用及觀察該分化過程中可能參與的Wnt信號機(jī)制。 方法: ⑴擴(kuò)增、純化AFSC。 ⑵倒置顯微鏡下觀察體外培養(yǎng)的羊水干細(xì)胞的一般生物學(xué)特性。采用流式細(xì)胞術(shù)檢測AFSC的CD29及CD34的表達(dá)情況。 ⑶本實(shí)驗(yàn)分為對照組，rhEPO組，rhEPO+LiCl組。 ⑷對照組一直使用常規(guī)培養(yǎng)基培養(yǎng)；rhEPO組用對照組的培養(yǎng)基中加入rhEPO組成的誘導(dǎo)培養(yǎng)基培養(yǎng)，且rhEPO的終濃度分別為1.O、5．0、10.0、20.0U／ml，干預(yù)24h后換成與對照組相同的培養(yǎng)基培養(yǎng)；rhEPO+LiCl組用上述濃度的rhEPO組成的誘導(dǎo)培養(yǎng)基培養(yǎng)并在誘導(dǎo)分化液中各加上5mmol/L的Wnt信號激動(dòng)劑LiCl干預(yù)24h，然后換成與對照組相同的培養(yǎng)基培養(yǎng)。 ⑸將鑒定后的細(xì)胞種于10*10的小玻片上，按上述要求進(jìn)行干預(yù)，48h后，用免疫熒光法檢測羊水干細(xì)胞在誘導(dǎo)前及誘導(dǎo)早期的促紅細(xì)胞生成素受體（EPOR）的表達(dá)情況。用免疫組化檢測β-catenin及p-GSK-3β (Ser~9)的表達(dá)情況。 ⑹2周后在倒置顯微鏡下觀察誘導(dǎo)分化后的細(xì)胞，計(jì)算有形態(tài)改變的心肌前體細(xì)胞數(shù)占總細(xì)胞數(shù)的比率為分化率，并比較rhEPO組與對照組的分化率的差異。 ⑺14天后收集各組細(xì)胞，，提取總的RNA，應(yīng)用RT-PCR檢測各組心肌早期轉(zhuǎn)錄因子GATA-4、Nkx2.5mRNA的表達(dá)情況。 ⑻28天后應(yīng)用Western blot檢測各組細(xì)胞心肌特異蛋白(β-MHC、cTnT)的表達(dá)情況。 結(jié)果: ⑴羊水內(nèi)的細(xì)胞接種后原代靜養(yǎng)7天，第8天在倒置顯微鏡下可見集落細(xì)胞團(tuán)，集落分布不均，大小不一，集落周邊僅有很少量細(xì)胞，集落中心細(xì)胞易可見老化凋亡的細(xì)胞。羊水干細(xì)胞生長快，傳代后細(xì)胞平鋪時(shí)體積大，核仁大，胞漿豐富，細(xì)胞生長較密時(shí)，細(xì)胞似成纖維樣細(xì)胞，排列成漩渦狀。 ⑵AFSC經(jīng)流式細(xì)胞儀檢測顯示AFSC表型為CD29~+為97.04%，CD34~+為0.61%。 ⑶免疫熒光觀察到AFSC在誘導(dǎo)前和誘導(dǎo)早期均有EPOR的表達(dá)。 ⑷誘導(dǎo)分化第14天，倒置顯微鏡下可見細(xì)胞由長梭形逐漸變短增寬，相鄰的細(xì)胞間有融合現(xiàn)象，似類肌管樣結(jié)構(gòu)。不同濃度的rhEPO誘導(dǎo)的分化率有差異，但都顯著高于對照組（P0.05），且以5.0U/mLrhEPO的誘導(dǎo)分化率最高（P0.05）。 ⑸在誘導(dǎo)分化14天后，與未干預(yù)組比較，各濃度rhEPO組及各濃度rhEPO+LiCl組干預(yù)后細(xì)胞的GATA-4、Nkx2.5mRNA的表達(dá)明顯上調(diào)（P0.05），以5．0U／m1的作用最顯著；與rhEPO組比較，rhEPO+Licl組能顯著干預(yù)上調(diào)誘導(dǎo)細(xì)胞的GATA-4、Nkx2.5mRNA的表達(dá)（P0.05），以5.0U／m1rhEPO+5mmol/L的LiCl干預(yù)作用最強(qiáng)。 ⑹經(jīng)誘導(dǎo)28天后，各干預(yù)組的細(xì)胞的心肌特異相關(guān)蛋白β-MHC、cTnT的表達(dá)明顯上調(diào)（P0.05），以5．0U／m1的作用最顯著，而rhEPO與Wnt信號激動(dòng)劑共同作用強(qiáng)于單用rhEPO。 ⑺誘導(dǎo)過程中各組均有如前所述的Wnt信號通路中的蛋白表達(dá)，與對照組比較，rhEPO及rhEPO+LiCl組β-catenin及p-GSK-3β的表達(dá)的增多（P0.05），與rhEPO比較，rhEPO+LiCl組β-catenin p-GSK-3β的表達(dá)強(qiáng)于rhEPO組。 結(jié)論: ⑴人源羊水中存在AFSC，其性質(zhì)類似于間充質(zhì)干細(xì)胞及胚胎干細(xì)胞。 ⑵外源性rhEPO成素能作為一種化學(xué)誘導(dǎo)劑應(yīng)用于干細(xì)胞的研究。 ⑶外源性rhEPO早期干預(yù)能劑量依賴性促進(jìn)人源羊水干細(xì)胞向心肌前體細(xì)胞分化。 ⑷外源性rhEPO可能通過啟動(dòng)Wnt信號誘導(dǎo)人源羊水干細(xì)胞分化為心肌前體細(xì)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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