本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：納米再生蜘蛛絲　出處：《蘇州大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)是骨髓中的非造血干細(xì)胞,能夠自我更新和多向分化。在不同的誘導(dǎo)條件下可以分化為多種細(xì)胞,包括成骨細(xì)胞、成纖維細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞、肌肉細(xì)胞、內(nèi)皮細(xì)胞及神經(jīng)細(xì)胞等。骨髓間充質(zhì)干細(xì)胞還具有取材方便,對(duì)機(jī)體損傷小,體外培養(yǎng)增殖能力強(qiáng)等優(yōu)點(diǎn),選用自體骨髓間充質(zhì)干細(xì)胞進(jìn)行組織再生或移植,不僅沒(méi)有移植排異反應(yīng),還避免了倫理紛爭(zhēng)。因此,對(duì)骨髓間充質(zhì)干細(xì)胞的研究成為當(dāng)今生物醫(yī)學(xué)的熱點(diǎn),骨髓間充質(zhì)干細(xì)胞也被認(rèn)為是最有前途的組織工程的種子細(xì)胞。 1骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng) 為了建立一個(gè)有效的分離培養(yǎng),體外擴(kuò)增的方法,我們采用Percoll淋巴細(xì)胞分離液進(jìn)行不連續(xù)密度梯度離心法分離培養(yǎng)BMSCs,實(shí)現(xiàn)在較短的時(shí)間內(nèi)體外擴(kuò)增和純化骨髓間充質(zhì)干細(xì)胞。免疫組織細(xì)胞化學(xué)檢測(cè)骨髓間充質(zhì)干細(xì)胞表面抗原,結(jié)果顯示細(xì)胞表達(dá)CD29、CD44、CD90、CD106,不表達(dá)CD34、CD45。 2納米再生蜘蛛絲與骨髓間充質(zhì)干細(xì)胞生物相容性研究 為了研究納米再生蜘蛛絲(SHSH)與骨髓間充質(zhì)干細(xì)胞(BMSCs)之間的生物相容性,我們將BMSCs培養(yǎng)在SHSH和多聚賴氨酸(PLL)上。通過(guò)死活試劑染色我們對(duì)培養(yǎng)在SHSH上24 h、48 h、72 h后的BMSCs進(jìn)行存活分析;同時(shí)以BrdU作為標(biāo)記物,觀察這些細(xì)胞在0-24 h、24-48 h和48-72 h的增殖狀況;利用油紅O染色檢測(cè)細(xì)胞的定向成脂分化能力。結(jié)果顯示SHSH和常用的細(xì)胞培養(yǎng)基質(zhì)PLL一樣能夠很好地支持BMSCs的生長(zhǎng)。BMSCs在SHSH上表現(xiàn)出正常的粘附、鋪展和生長(zhǎng)行為。接種后的48 h內(nèi),與PLL相比較,細(xì)胞呈現(xiàn)較低的增殖速度;之后恢復(fù)為與PLL相同的增殖。經(jīng)過(guò)定向成脂誘導(dǎo),BMSCs在SHSH上能夠分化為脂肪細(xì)胞。這些數(shù)據(jù)說(shuō)明SHSH與BMSCs之間具有良好的生物相容性,同時(shí)考慮到其自身優(yōu)秀的力學(xué)性能,SHSH將成為組織工程與再生醫(yī)學(xué)中極具應(yīng)用潛力的生物支架。 3骨髓間充質(zhì)干細(xì)胞向脂肪細(xì)胞分化過(guò)程中關(guān)鍵點(diǎn)的研究 為了研究BMSCs向脂肪細(xì)胞分化過(guò)程中間關(guān)鍵點(diǎn),我們加入脂肪誘導(dǎo)劑對(duì)BMSCs進(jìn)行成脂誘導(dǎo),分別誘導(dǎo)12 h、18 h、1 d、2 d、3 d后撤去誘導(dǎo)劑,換入生長(zhǎng)培養(yǎng)(L-DMEM + 10%FCS),在第6 d,第9 d時(shí)油紅O染色。結(jié)果顯示BMSCs在加入脂肪誘導(dǎo)劑誘導(dǎo)12 h、18 h后,換入生長(zhǎng)培養(yǎng)基,第6 d,第9 d油紅O染色成陰性;誘導(dǎo)1 d、2 d、3 d后,換入生長(zhǎng)培養(yǎng)基,第6 d,第9 d油紅O染色成陽(yáng)性。這些數(shù)據(jù)說(shuō)明BMSCs在向脂肪細(xì)胞分化過(guò)程中非�？赡艽嬖谝粋€(gè)關(guān)鍵點(diǎn),這個(gè)關(guān)鍵點(diǎn)是細(xì)胞的分化決定點(diǎn),關(guān)鍵點(diǎn)之前的細(xì)胞仍然未分化,關(guān)鍵點(diǎn)之后的細(xì)胞已經(jīng)被決定了命運(yùn),即使不存在分化誘導(dǎo)劑,仍然能夠向脂肪細(xì)胞分化。在BMSCs向脂肪細(xì)胞分化過(guò)程中它的關(guān)鍵作用時(shí)間點(diǎn)是BMSCs成脂肪誘導(dǎo)24 h。 同時(shí)我們檢測(cè)了關(guān)鍵點(diǎn)前后時(shí)間點(diǎn)Rac1及相關(guān)蛋白的表達(dá)變化:通過(guò)western blotting檢測(cè)BMSCs向脂肪細(xì)胞分化早期12 h、18 h、24 h、48 h的Rac1及相關(guān)蛋白的表達(dá)變化;Pull-down assay分析Rac1-GTP及相關(guān)蛋白活性變化。結(jié)果顯示Rac1及其相關(guān)蛋白總蛋白的表達(dá)量發(fā)生了變化;并且Rac1-GTP蛋白隨著誘導(dǎo)時(shí)間的延長(zhǎng),在誘導(dǎo)24 h內(nèi)蛋白表達(dá)不斷升高,誘導(dǎo)24 h時(shí)達(dá)到最大,隨后的24 h蛋白表達(dá)下降,低于誘導(dǎo)12 h時(shí)蛋白表達(dá)。RhoA-GTP蛋白表達(dá)則與Rac1-GTP相反,隨著誘導(dǎo)時(shí)間的延長(zhǎng),RhoA-GTP蛋白表達(dá)逐漸降低,誘導(dǎo)24 h時(shí)降到最低,在隨后的24 h中,RhoA-GTP蛋白表達(dá)則明顯升高,高于誘導(dǎo)12 h時(shí)RhoA的蛋白表達(dá)水平。Cdc42-GTP蛋白表達(dá)在誘導(dǎo)24 h內(nèi)保持穩(wěn)定,誘導(dǎo)24 h后顯示出與Rac1-GTP蛋白相同的趨勢(shì),表達(dá)下降且低于誘導(dǎo)12 h時(shí)的水平。 總的來(lái)說(shuō),BMSCs在向脂肪細(xì)胞分化的過(guò)程中存在一個(gè)關(guān)鍵點(diǎn),這個(gè)關(guān)鍵點(diǎn)的作用時(shí)間為BMSCs成脂肪誘導(dǎo)24 h時(shí),在這個(gè)關(guān)鍵點(diǎn)前后細(xì)胞內(nèi)Rac1及其相關(guān)蛋白的表達(dá)量發(fā)生了變化。
[Abstract]:Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) in the bone marrow non hematopoietic stem cells capable of self-renewal and multilineage differentiation. Differentiation into a variety of cells in different conditions can be induced, including osteoblasts, fibroblasts, cartilage cells, fat cells, muscle cells, and endothelial cells nerve cells. Bone marrow mesenchymal stem cells also has convenient, small injury to the body, has the advantages of strong proliferation ability and in vitro selection of autologous bone marrow mesenchymal stem cells for tissue regeneration or transplantation, not only did the graft rejection, but also avoid the ethical disputes. Therefore, the study of bone marrow mesenchymal stem the cell has become the hotspot of biomedical, bone marrow mesenchymal stem cells are also considered to be the most promising seed cells for tissue engineering.
Isolation and culture of 1 bone marrow mesenchymal stem cells
In order to establish an effective method of isolation and culture, in vitro, we used Percoll lymphocyte separation medium by discontinuous density gradient centrifugation method BMSCs, achieved in a relatively short time in vitro amplification and purification of bone marrow mesenchymal stem cells. Immunohistochemical detection of bone marrow mesenchymal stem cell surface antigen results show CD29 expression, CD44, CD90, CD106, the expression of CD34, CD45.
Biocompatibility of 2 nanoscale regenerated spider silk and bone marrow mesenchymal stem cells
In order to study the nano regenerated spider silk (SHSH) and bone marrow mesenchymal stem cells (BMSCs) compatibility between organisms, we will BMSCs cultured in SHSH and poly-L-lysine (PLL). The death of our reagent staining cultured in SHSH 24 h, 48 h, 72 h after BMSCs survival analysis; at the same time using BrdU as a marker to observe these cells in 0-24 h, 24-48 h and 48-72 h proliferation; directed by oil red O staining was used to detect the cell adipogenic differentiation ability. The results showed that SHSH and PLL as the common cell culture matrix can support the growth of BMSCs.BMSCs show normal adhesion on SHSH, spreading and growth behavior. After inoculation of 48 h, compared with PLL cells showed a lower proliferation rate; after restoration with the same PLL proliferation. After adipogenesis induction, BMSCs SHSH can differentiate into fat cells. These data suggest that SHSH and BM SCs has good biocompatibility and takes account of its excellent mechanical properties. SHSH will become a potential scaffold for tissue engineering and regenerative medicine.
3 key points in the differentiation of bone marrow mesenchymal stem cells into adipocytes
涓轟簡(jiǎn)鐮旂┒BMSCs鍚戣剛鑲粏鑳?yōu)鍒嗗寲杩嚱E嬩腑闂村叧閿偣,鎴戜滑鍔犲叆鑴傝偑璇卞鍓傚BMSCs榪涜鎴愯剛璇卞,鍒嗗埆璇卞12 h,18 h,1 d,2 d,3 d鍚庢挙鍘昏瀵煎墏,鎹㈠叆鐢熼暱鍩瑰吇(L-DMEM + 10%FCS),鍦ㄧ6 d,絎，

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