本文選題：骨髓基質(zhì)細(xì)胞 + 神經(jīng)干細(xì)胞　； 參考：《第一軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 目前，對(duì)中樞神經(jīng)系統(tǒng)(central nervous system，CNS)病的治療目的多為緩解癥狀和控制疾病的發(fā)展。近年來(lái)研究發(fā)現(xiàn)，在成年哺乳動(dòng)物腦組織中存在神經(jīng)干細(xì)(neural stem cells，NSCs)，這些NSCs可以分化成神經(jīng)組織，表明CNS也存在再生修復(fù)能力，為研究CNS損傷的修復(fù)提出了希望。干細(xì)胞(stem cells，SCs)是指有多種分化潛能和自我更新能力的細(xì)胞，這種多潛能細(xì)胞存在于成年個(gè)體的許多組織和胚胎中。近年來(lái)，隨著SCs研究的深入，骨髓基質(zhì)細(xì)胞(bone marrow derived stroma cells，BMSCs)被證明具有向NSCs分化的潛能，同時(shí)BMSCs還具有取材方便、來(lái)源廣泛、免疫原性低、遺傳性狀穩(wěn)定等優(yōu)勢(shì)，因此目前已成為SCs移植替代治療最理想的種子細(xì)胞來(lái)源之一，應(yīng)用NSCs特別是BMSCs源性NSCs分化調(diào)控和移植修復(fù)CNS功能亦成為研究重點(diǎn)。 到目前為止，不論是CNS還是周圍神經(jīng)系統(tǒng)(peripheral nervous system，PNS)疾病，絕大多數(shù)經(jīng)NSCs移植治療后，判斷其臨床癥狀的改善是否由移植細(xì)胞所致，以及移植后神經(jīng)再生的鑒定主要采用免疫組織化學(xué)的方法檢測(cè)。該方法必須獲取組織而不可能進(jìn)行活體評(píng)價(jià)，臨床及科研工作中渴望能無(wú)創(chuàng)性的對(duì)NSCs移植后的存活、遷徙及功能性分化進(jìn)行活體研究。 隨著超順磁性磁共振成像(magnetic resonance imaging，MRI)對(duì)比劑的研制成功及MRI設(shè)備及方法的快速發(fā)展，為無(wú)創(chuàng)傷性地在活體內(nèi)動(dòng)態(tài)監(jiān)測(cè)移植后骨髓基質(zhì)細(xì)胞的存活、遷移、生存狀態(tài)等情況提供了可能。目前，MRI可以提供25～50μm的分辨率，接近單一細(xì)胞的水平，使其在理論上可以用來(lái)對(duì)移植的細(xì)胞進(jìn)行活體示蹤。氧化鐵類中的超順磁性氧化鐵(superparamagnetic iron oxide，SPIO)和超小順磁性氧化鐵(ultrasmall superparamagnetic iron oxide，USPIO)是較為理想的MRI示蹤劑，已經(jīng)成功在體外用于多種細(xì)胞的標(biāo)記。菲立磁(feridex，F(xiàn)E)是經(jīng)過(guò)美國(guó)食品及藥物管理局(food and drug administration，F(xiàn)DA)認(rèn)可的臨床上應(yīng)用的SPIO類MRI造影劑之一，借助轉(zhuǎn)染試劑多聚賴氨酸(poly-l-lysine，PLL)，F(xiàn)E已成功標(biāo)記多種哺乳動(dòng)物SCs。目前國(guó)內(nèi)專家學(xué)者已經(jīng)開(kāi)始了關(guān)于FE的試驗(yàn)研究，系統(tǒng)研究了大鼠、家兔及恒河猴的FE標(biāo)記BMSCs的培養(yǎng)及其向NSCs的轉(zhuǎn)化，向外傷動(dòng)物模型的移植，利用MRI影像學(xué)定位檢查標(biāo)記NSCs在腦內(nèi)的成活和遷移情況，并運(yùn)用免疫織組化學(xué)、透射電子顯微鏡等技術(shù)觀察NSCs生長(zhǎng)、分化和突觸的形成及與腦組織整合情況，得到了肯定的實(shí)驗(yàn)結(jié)果。但目前針對(duì)FE標(biāo)記前后BMSCs生物學(xué)活性的檢測(cè)，所做的研究還不夠完善，尚未見(jiàn)相關(guān)研究的系列報(bào)道。本課題擬在細(xì)胞水平，用不同濃度的FE特異性標(biāo)記BMSCs，并于體外對(duì)標(biāo)記前后的BMSCs活性、增殖、分化能力、凋亡等情況進(jìn)行研究，探索不同濃度FE對(duì)標(biāo)記BMSCs的影響及標(biāo)記BMSCs的最佳濃度，為今后的進(jìn)一步研究工作提供技術(shù)參考。 第一章新西蘭兔BMSCs體外分離培養(yǎng)和誘導(dǎo)分化的實(shí)驗(yàn)研究 目的：觀察BMSCs體外培養(yǎng)、擴(kuò)增和誘導(dǎo)分化為NSCs的情況，熟悉并掌握細(xì)胞培養(yǎng)技術(shù)，為下一步實(shí)驗(yàn)奠定基礎(chǔ)。 方法： 1．以新西蘭大白兔為實(shí)驗(yàn)對(duì)象，無(wú)菌條件下行髂骨穿刺取骨髓，梯度密度離心法分離獲取新西蘭兔BMSCs。 2．骨髓源性NSCs誘導(dǎo)培養(yǎng)及純化：原代培養(yǎng)第3天后，應(yīng)用NSCs培養(yǎng)基，加入胎牛血清(FBS 1％終濃度)、白血病抑制因子(LIF 10ng／ml)、堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF 10ng／ml)進(jìn)行體外培養(yǎng)、誘導(dǎo)、增殖。培養(yǎng)1～2周后得到較純化的BMSCs，可進(jìn)行細(xì)胞傳代培養(yǎng)，在培養(yǎng)過(guò)程中加入FBS以及維甲酸(RA 0.5μg／ml)以誘導(dǎo)BMSCs向NSCs分化。 3．CK-2型倒置相差光學(xué)顯微鏡追蹤觀察細(xì)胞生長(zhǎng)情況并拍照。 4．免疫細(xì)胞化學(xué)鑒定：采用SABC法，利用抗神經(jīng)巢蛋白(Nestin)、神經(jīng)元特異性烯醇化酶(NSE)、膠質(zhì)原纖維酸性蛋白(GFAP)等免疫細(xì)胞化學(xué)方法對(duì)BMSCs分化成的NSCs、神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞分別進(jìn)行特異性鑒定。 結(jié)果： 1．倒置相差顯微鏡觀察：接種后的10小時(shí)內(nèi)，BMSCs開(kāi)始貼壁，貼壁細(xì)胞培養(yǎng)48小時(shí)后有分裂增殖，然后逐漸形成島嶼狀細(xì)胞克隆團(tuán)，給予適當(dāng)?shù)姆只瘲l件，10～20天后，這些細(xì)胞能分化成具有細(xì)長(zhǎng)突起、多種形態(tài)的細(xì)胞。 2．經(jīng)免疫細(xì)胞化學(xué)鑒定，早期的培養(yǎng)細(xì)胞在沒(méi)有給予誘導(dǎo)分化時(shí)可陽(yáng)性表達(dá)Nestin，表明具有SCs特性，證實(shí)已經(jīng)形成BMSCs源性NSCs(BMSCs-D-NSCs)；培養(yǎng)細(xì)胞經(jīng)誘導(dǎo)分化后能分化出神經(jīng)元樣細(xì)胞和膠質(zhì)細(xì)胞樣細(xì)胞，免疫細(xì)胞化學(xué)檢測(cè)可見(jiàn)有NSE、GFAP的表達(dá)。 結(jié)論： 1．新西蘭大白兔的BMSCs能在體外培養(yǎng)和擴(kuò)增，具有增殖能力，能表達(dá)Nestin抗原，具有NSCs特征。 2．本實(shí)驗(yàn)所采用的培養(yǎng)方案(NSCs培養(yǎng)基)適合于兔BMSCs向NSCs轉(zhuǎn)分化，源自BMSCs的NSCs具有分化潛能，在合適的誘導(dǎo)分化條件下分化出的細(xì)胞能表達(dá)神經(jīng)系細(xì)胞(神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞)的特征性抗原。 3．BMSCs來(lái)源豐富，體外分離、擴(kuò)增容易，可作為自體移植的種子細(xì)胞。 第二章FE標(biāo)記新西蘭兔BMSCs的細(xì)胞生物學(xué)研究 目的：初步探索利用不同濃度FE和轉(zhuǎn)染試劑特異性標(biāo)記新西蘭兔BMSCs，于體外對(duì)標(biāo)記前后的BMSCs活性、增殖、分化能力、凋亡等情況進(jìn)行研究，探索FE標(biāo)記細(xì)胞的最佳濃度，為今后的進(jìn)一步研究工作提供技術(shù)參考。 方法： 1．實(shí)驗(yàn)動(dòng)物和骨髓采集、BMSCs的分離擴(kuò)增同第一部分。 2．FE標(biāo)記細(xì)胞的濃度及分組：首先將FE分別稀釋成10μg／ml、25μg／ml、50μg／ml和75μg／ml四個(gè)不同濃度，并分別和PLL(1.5μg／ml)等體積混合，室溫下振蕩搖勻，，30分鐘后將FE-PLL復(fù)合物(FE-PLL)加入細(xì)胞培養(yǎng)基中，F(xiàn)E(Fe)和PLL的終濃度分別為5μg／ml、12.5μg／ml、25μg／ml、37.5μg／ml和0.75μg／ml。試驗(yàn)最后分組：a．純BMSC-D-NSC組；b．0.75PLL組：c．5FE-0.75PLL組；d．12.5FE-0.75PLL組；e．25FE-0.75PLL組；f．37.5FE-0.75PLL組(n=10)。 3．倒置顯微鏡和透射電鏡觀察：觀察各組標(biāo)記細(xì)胞的形態(tài)學(xué)變化。 4．細(xì)胞內(nèi)鐵的鑒定：采用普魯士蘭染色和透射電鏡對(duì)細(xì)胞內(nèi)鐵和標(biāo)記效率進(jìn)行檢測(cè)。 5．FE-PLL標(biāo)記BMSCs的滲漏性檢測(cè)：FE標(biāo)記標(biāo)記NSCs與神經(jīng)元共培養(yǎng)以檢測(cè)標(biāo)記細(xì)胞是否會(huì)發(fā)生FE的滲漏。 6．生長(zhǎng)曲線的測(cè)定：采用CCK-8法鑒定各組細(xì)胞的活力。 7．流式細(xì)胞儀(flow cytometry，F(xiàn)CM)檢測(cè)凋亡：對(duì)細(xì)胞凋亡情況進(jìn)行分析。 8．BMSCs標(biāo)記后細(xì)胞MRI：MRI掃描對(duì)象分為5組，4.7T MRI掃描序列包括軸面T_2快速自旋回波(Fast spin echo，F(xiàn)SE)序列，T_2~*梯度回波(Gradient echo，GRE)序列掃描。 9．統(tǒng)計(jì)分析：應(yīng)用SPSS13.0統(tǒng)計(jì)軟件，細(xì)胞吸光度及凋亡率數(shù)據(jù)均采用重復(fù)測(cè)量的方差分析進(jìn)行處理，組間兩兩比較采用SNK法，檢驗(yàn)水準(zhǔn)α=0.05。 結(jié)果： 1．倒置顯微鏡和透射電鏡觀察：倒置相差顯微鏡下在不同培養(yǎng)階段的標(biāo)記組和未標(biāo)記組BMSCs相比較，各組之間細(xì)胞形態(tài)無(wú)明顯差異。組間主要差別表現(xiàn)為FE-PLL標(biāo)記的BMSCs顏色呈淡黃色～深黃色。透射電鏡下大部分標(biāo)記的BMSCs其大小形態(tài)細(xì)胞器無(wú)明顯變化，胞質(zhì)內(nèi)散在分布含膜的囊泡樣包涵體結(jié)構(gòu)，少部分標(biāo)記細(xì)胞可見(jiàn)凋亡表現(xiàn)，f組凋亡細(xì)胞較多。 2．細(xì)胞內(nèi)鐵的鑒定：電鏡結(jié)果顯示FE-PLL復(fù)合物標(biāo)記的BMSCs胞質(zhì)內(nèi)含有許多包裹鐵顆粒的囊泡。普魯士藍(lán)染色顯示FE-PLL復(fù)合物標(biāo)記BMSCs胞質(zhì)內(nèi)出現(xiàn)細(xì)小的藍(lán)色鐵顆粒，c～f組藍(lán)色依次加深。 3．FE標(biāo)記效率的組間比較：各FE組細(xì)胞均被不同程度標(biāo)記，光鏡下計(jì)數(shù)后統(tǒng)計(jì)表明隨著FE濃度的增加其標(biāo)記效率亦增加。電鏡下觀察結(jié)果表明隨各組濃度增高，胞質(zhì)內(nèi)涵FE的含膜囊泡結(jié)構(gòu)也隨之增高，說(shuō)明其含鐵量亦隨之增加。 4．酶聯(lián)免疫檢測(cè)儀檢測(cè)OD值及生長(zhǎng)曲線測(cè)定結(jié)果：各組細(xì)胞相比，OD值有顯著性差異，a、b、c、d、e組無(wú)顯著性差異，f組同其他a～e組比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.001)，說(shuō)明FE-PLL復(fù)合物在25μg／ml以下時(shí)，對(duì)細(xì)胞活力無(wú)顯著影響。 5．FE-PLL標(biāo)記NSCs的滲漏性檢測(cè)：FE-PLL復(fù)合物標(biāo)記NSCs與大腦皮層神經(jīng)元共同培養(yǎng)后，普魯士蘭染色顯示標(biāo)記NSCs呈陽(yáng)性，未標(biāo)記的大腦皮層神經(jīng)元內(nèi)呈陰性。 6．FCM檢測(cè)細(xì)胞凋亡：各組細(xì)胞隨著培養(yǎng)時(shí)間的延長(zhǎng)細(xì)胞凋亡有增加的趨勢(shì)，各組細(xì)胞凋亡率比較存在顯著性差異，a、b、c、d、e組無(wú)顯著性差異，f組同其他各組比較存在顯著性差異(P＜0.001)，說(shuō)明FE-PLL復(fù)合物在25μg／ml以下時(shí)，對(duì)細(xì)胞活力無(wú)顯著影響。 7．標(biāo)記BMSCs的細(xì)胞MRI：在T_2及T_2~*序列，隨FE濃度的增加，標(biāo)記細(xì)胞信號(hào)降低的越明顯，未標(biāo)記的細(xì)胞呈顯著高信號(hào)。 結(jié)論： 1．不同濃度FE-PLL復(fù)合物可以用來(lái)體外標(biāo)記新西蘭兔BMSCs。 2．隨FE濃度的增高，其標(biāo)記效率亦增高，且標(biāo)記后T_2 WI或T_2~* WI信號(hào)改變愈明顯。濃度在25μg／ml以下時(shí)，應(yīng)用FE-PLL復(fù)合物標(biāo)記新西蘭兔BMSCs安全、有效。 3．25μg／ml是FE標(biāo)記細(xì)胞適宜的濃度。
[Abstract]:At present, the purpose of the treatment of central nervous system (CNS) disease is to alleviate the symptoms and control the development of the disease. In recent years, it has been found that there are neural stem thin (neural stem cells, NSCs) in adult mammalian brain tissue. These NSCs can be differentiated into nerve tissue, which indicates that CNS also has the ability to regenerate and repair, which is the research of CNS. The repair of CNS damage is hopeful. Stem cells (SCs) is a cell with a variety of differentiation potential and self renewal capacity. This multipotential cell exists in many tissues and embryos of adult individuals. In recent years, with the in-depth study of SCs, the bone marrow stromal cells (bone marrow derived stroma cells, BMSCs) have been proved to be directed to N. SCs has the potential of differentiation, and BMSCs also has the advantages of convenience, wide source, low immunogenicity, stable genetic character and so on. So it has become one of the most ideal seed cell sources for the replacement therapy of SCs transplantation. The application of NSCs, especially the BMSCs derived NSCs differentiation and regulation and the transplantation of CNS function, has also become the focus of research.
So far, whether it is CNS or peripheral nervous system (PNS) disease, most of them have been treated with NSCs transplantation to determine whether the improvement of the clinical symptoms is caused by the transplanted cells, and the identification of the regeneration of the nerve after transplantation is mainly detected by the immunohistochemical method. It is possible to carry out in vivo evaluation. In clinical and scientific research, it is eager to carry out in vivo studies on the survival, migration and functional differentiation of NSCs after noninvasive transplantation.
With the development of superparamagnetic magnetic resonance imaging (magnetic resonance imaging, MRI) contrast agent and the rapid development of MRI equipment and methods, it is possible to dynamically monitor the survival, migration and survival of bone marrow stromal cells after transplantation in vivo. At present, MRI can provide a resolution of 25~50 mu m. The level of a single cell can be used in theory to trace the transplanted cells in vivo. The superparamagnetic iron oxide (superparamagnetic iron oxide, SPIO) and ultra small paramagnetic iron oxide (ultrasmall superparamagnetic iron oxide, USPIO) are ideal MRI tracers in the iron oxide class, which have been successfully used in vitro. Feridex (FE) is one of the SPIO class MRI contrast agents approved by the food and Drug Administration (FDA) by the United States Food and Drug Administration (FDA), which has been successfully labeled with polylysine (poly-L-lysine, PLL) with the aid of the transfection reagent. The experimental study of FE was carried out. The FE marker BMSCs of rats, rabbits and Ganges RIver monkeys was cultured and transformed into NSCs. The transplantation of the traumatic animal model to the traumatic animal model was studied. The survival and migration of NSCs in the brain was marked with MRI imaging location, and the growth of NSCs was observed by using the techniques of immunofluorescence and transmission electron microscopy. The formation of synapses and synapses and the integration of brain tissue have obtained positive results. But at present, the research on the detection of biological activity of BMSCs before and after FE markers is not perfect, and there is no series of reports on the related research. This subject is intended to mark BMSCs with different concentration of FE specificity at the cell level and to mark in vitro. The activity, proliferation, differentiation, and apoptosis of BMSCs before and after are studied, and the effects of different concentrations of FE on labeled BMSCs and the optimum concentration of labeled BMSCs are explored, which will provide a technical reference for further research.
Chapter one: isolation, culture and induction of differentiation of New Zealand rabbit BMSCs in vitro
Objective: To observe the in vitro culture, expansion and differentiation of BMSCs into NSCs, and to familiarise and master cell culture techniques, and lay the foundation for further experiments.
Method錛

本文編號(hào)：1937437