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

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

【摘要】：目的： 觀察不同濃度重組人促紅細胞生成素（recombinant humanerythropoietin，rhEPO）對人源羊水干細胞（amniotic fluid stem cells，AFSC)向心肌前體細胞分化的作用及觀察該分化過程中可能參與的Wnt信號機制。 方法: ⑴擴增、純化AFSC。 ⑵倒置顯微鏡下觀察體外培養(yǎng)的羊水干細胞的一般生物學特性。采用流式細胞術(shù)檢測AFSC的CD29及CD34的表達情況。 ⑶本實驗分為對照組，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組成的誘導培養(yǎng)基培養(yǎng)并在誘導分化液中各加上5mmol/L的Wnt信號激動劑LiCl干預24h，然后換成與對照組相同的培養(yǎng)基培養(yǎng)。 ⑸將鑒定后的細胞種于10*10的小玻片上，按上述要求進行干預，48h后，用免疫熒光法檢測羊水干細胞在誘導前及誘導早期的促紅細胞生成素受體（EPOR）的表達情況。用免疫組化檢測β-catenin及p-GSK-3β (Ser~9)的表達情況。 ⑹2周后在倒置顯微鏡下觀察誘導分化后的細胞，計算有形態(tài)改變的心肌前體細胞數(shù)占總細胞數(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的表達情況。 ⑻28天后應(yīng)用Western blot檢測各組細胞心肌特異蛋白(β-MHC、cTnT)的表達情況。 結(jié)果: ⑴羊水內(nèi)的細胞接種后原代靜養(yǎng)7天，第8天在倒置顯微鏡下可見集落細胞團，集落分布不均，大小不一，集落周邊僅有很少量細胞，集落中心細胞易可見老化凋亡的細胞。羊水干細胞生長快，傳代后細胞平鋪時體積大，核仁大，胞漿豐富，細胞生長較密時，細胞似成纖維樣細胞，排列成漩渦狀。 ⑵AFSC經(jīng)流式細胞儀檢測顯示AFSC表型為CD29~+為97.04%，CD34~+為0.61%。 ⑶免疫熒光觀察到AFSC在誘導前和誘導早期均有EPOR的表達。 ⑷誘導分化第14天，倒置顯微鏡下可見細胞由長梭形逐漸變短增寬，相鄰的細胞間有融合現(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的誘導分化率最高（P0.05）。 ⑸在誘導分化14天后，與未干預組比較，各濃度rhEPO組及各濃度rhEPO+LiCl組干預后細胞的GATA-4、Nkx2.5mRNA的表達明顯上調(diào)（P0.05），以5．0U／m1的作用最顯著；與rhEPO組比較，rhEPO+Licl組能顯著干預上調(diào)誘導細胞的GATA-4、Nkx2.5mRNA的表達（P0.05），以5.0U／m1rhEPO+5mmol/L的LiCl干預作用最強。 ⑹經(jīng)誘導28天后，各干預組的細胞的心肌特異相關(guān)蛋白β-MHC、cTnT的表達明顯上調(diào)（P0.05），以5．0U／m1的作用最顯著，而rhEPO與Wnt信號激動劑共同作用強于單用rhEPO。 ⑺誘導過程中各組均有如前所述的Wnt信號通路中的蛋白表達，與對照組比較，rhEPO及rhEPO+LiCl組β-catenin及p-GSK-3β的表達的增多（P0.05），與rhEPO比較，rhEPO+LiCl組β-catenin p-GSK-3β的表達強于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)干細胞及胚胎干細胞。 ⑵外源性rhEPO成素能作為一種化學誘導劑應(yīng)用于干細胞的研究。 ⑶外源性rhEPO早期干預能劑量依賴性促進人源羊水干細胞向心肌前體細胞分化。 ⑷外源性rhEPO可能通過啟動Wnt信號誘導人源羊水干細胞分化為心肌前體細胞。
[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.

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

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