本文關(guān)鍵詞： 神經(jīng)干細胞 轉(zhuǎn)鐵蛋白受體 磁共振成像 超小順磁性氧化鐵　出處：《安徽醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究背景 神經(jīng)干細胞（neural stem cell, NSC）是一種能夠自我增殖、自我更新、并能夠分化成神經(jīng)元和膠質(zhì)細胞的干細胞，可以用作修復神經(jīng)功能損傷的種子細胞，但神經(jīng)功能改善與干細胞移植之間的相關(guān)性還未完全明確。動物實驗采用無創(chuàng)性活體動態(tài)示蹤技術(shù)有助于活體評價干細胞移植的療效，，是干細胞治療走向臨床前必須解決的問題。磁共振分子影像學因其高組織空間分辨率、無硬性偽信號、無輻射等優(yōu)點而得到越來越多的應用。目前，超小順磁性氧化鐵（USPIO）是最常用的體內(nèi)外細胞磁共振標記物，其存在著明顯的缺點就是隨著細胞的傳代而出現(xiàn)信號遞減，這限制了其不能長期動態(tài)的觀察，但利用磁共振報告基因轉(zhuǎn)染細胞方法可以達到無創(chuàng)動態(tài)觀察移植的細胞變化。 目的 1.合成Tf-USPIO納米微粒以及建立穩(wěn)定表達TfR的神經(jīng)干細胞。 2.檢測TfR神經(jīng)干細胞吸收Tf-USPIO能否在磁共振上成像，為下一步動物活體實驗提供基礎(chǔ)。 方法 1.利用慢病毒轉(zhuǎn)染神經(jīng)干細胞，通過流式細胞儀篩選出穩(wěn)定表達TfR的神經(jīng)干細胞，運用WB檢測其表達水平。將Tf與USPIO進行偶聯(lián)，合成Tf-USPIO納米微粒，通過DLS方法測出USPIO和Tf-USPIO的直徑分別為36±0.7nm，43.5±0.08nm。 2.將Tf-USPIO與USPIO分別于TfR神經(jīng)神經(jīng)干細胞共培養(yǎng)，通過普魯士藍染色、電鏡以及ICP方法檢測細胞中的鐵離子含量，并用CCK-8試劑檢測其對干細胞的毒性，利用7.0T磁共振檢測其成像結(jié)果。 結(jié)果 1.慢病毒成功的轉(zhuǎn)染神經(jīng)干細胞，通過流式細胞儀器篩選出穩(wěn)定表達的神經(jīng)干細胞，并用WB證實了TfR的過表達；通過偶聯(lián)的方法合成了穩(wěn)定的Tf-USPIO微粒。 2. Tf-USPIO與USPIO分別與TfR神經(jīng)干細胞孵育后，普魯士藍染色、電鏡及ICP檢測鐵離子濃度證實Tf-USPIO比USPIO在TfR神經(jīng)干細胞中吸收的明顯增多（P0.05）,CCK-8結(jié)果顯示二者對神經(jīng)干細胞的毒性較小（P0.05）,磁共振成像結(jié)果亦證實Tf-USPIO吸收量更多。 結(jié)論 1.成功的構(gòu)建了TfR神經(jīng)干細胞系，合成出穩(wěn)定的Tf-USPIO納米微粒。 2.Tf-USPIO特異性的與TfR神經(jīng)干細胞結(jié)合，并且可在磁共振上成像，可以作為一種磁共振對比劑。 3.Tf-USPIO在體外可以作為TfR神經(jīng)干細胞的特異性示蹤劑，為下一步活體成像實驗研究奠定基礎(chǔ)。
[Abstract]:Research background Neural stem cells (NSCs) are stem cells capable of self-proliferation, self-renewal, and differentiation into neurons and glial cells. It can be used as seed cells to repair neurological damage. However, the correlation between neural function improvement and stem cell transplantation has not been completely clear. In animal experiments, the use of non-invasive dynamic tracer technique is helpful to evaluate the efficacy of stem cell transplantation in vivo. Mr molecular imaging has been applied more and more because of its high tissue spatial resolution, no hard pseudo-signal, no radiation and so on. Ultrasmall paramagnetic iron oxide (USPIO) is the most commonly used magnetic resonance marker in vitro and in vivo. This limited its long-term dynamic observation, but the method of MRI report gene transfection could be used to observe the changes of transplanted cells in a non-invasive manner. Purpose 1. Synthesis of Tf-USPIO nanoparticles and establishment of neural stem cells stably expressing TfR. 2. Detecting whether TfR neural stem cells can absorb Tf-USPIO on magnetic resonance imaging can provide the basis for animal experiment in vivo. Method 1. Neural stem cells were transfected with lentivirus. Neural stem cells expressing TfR stably were screened by flow cytometry, and the expression level was detected by WB. TF was coupled with USPIO. Tf-USPIO nanoparticles were synthesized and the diameters of USPIO and Tf-USPIO were 36 鹵0.7 nm and 43.5 鹵0.08 nm, respectively, measured by DLS. 2. Tf-USPIO and USPIO were co-cultured in TfR neural stem cells. The iron ion content was detected by Prussian blue staining, electron microscopy and ICP. The toxicity of stem cells was detected by CCK-8 reagent and the imaging results were detected by 7.0T magnetic resonance imaging. Results 1. Lentivirus was successfully transfected into neural stem cells. The stable expressed neural stem cells were screened by flow cytometry, and the overexpression of TfR was confirmed by WB. Stable Tf-USPIO particles were synthesized by coupling method. 2. After Tf-USPIO and USPIO were incubated with TfR neural stem cells, Prussian blue staining was used. Electron microscopy and ICP showed that the absorption of Tf-USPIO in TfR neural stem cells was significantly higher than that of USPIO (P0.05). The results of CCK-8 showed that the toxicity of the two to neural stem cells was less than that of P0.05, and magnetic resonance imaging also confirmed that the absorption of Tf-USPIO was more. Conclusion 1. TfR neural stem cell lines were successfully constructed and stable Tf-USPIO nanoparticles were synthesized. 2. Tf-USPIO specifically binds to TfR neural stem cells and can be used as a magnetic resonance contrast agent. 3. Tf-USPIO can be used as a specific tracer for TfR neural stem cells in vitro.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R743.3

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