發(fā)布時(shí)間：2018-05-19 14:16

  本文選題：內(nèi)皮祖細(xì)胞 + 體外擴(kuò)增　； 參考：《湖南師范大學(xué)》2009年碩士論文

【摘要】： 目的:用含小鼠骨髓內(nèi)皮細(xì)胞系條件培養(yǎng)液(BMEC-CM)的培養(yǎng)體系,進(jìn)行骨髓高增殖潛能內(nèi)皮祖細(xì)胞(HPP-EPC)及其后代細(xì)胞的培養(yǎng)和純化,建立這些細(xì)胞的一種擴(kuò)增培養(yǎng)方法。檢測小鼠骨髓內(nèi)皮細(xì)胞系細(xì)胞的血管內(nèi)皮細(xì)胞生長因子(VEGF)、堿性成纖維細(xì)胞生長因子(bFGF)、胰島素樣生長因子-1(IGF-1)mRNA表達(dá)水平,分析BMEC-CM促進(jìn)EPC增殖的分子機(jī)理。 方法和結(jié)果:本實(shí)驗(yàn)分為以下三個(gè)部分: 1.總EPC集落形成實(shí)驗(yàn)制備小鼠骨髓單細(xì)胞懸液,骨髓細(xì)胞預(yù)貼壁培養(yǎng)6h后,吸出含未貼壁細(xì)胞的上懸液,移入新的培養(yǎng)瓶,并添加BMEC-CM,進(jìn)行二次貼壁培養(yǎng)。培養(yǎng)5d后用胰酶法將貼壁細(xì)胞消化下來,洗滌后計(jì)數(shù)。以每mm~2孔底面積1個(gè)細(xì)胞(1/mm~2)的密度,將這些細(xì)胞種入培養(yǎng)板,培養(yǎng)體系中仍添加BMEC-CM。觀察貼壁細(xì)胞集落的形成,計(jì)數(shù)集落數(shù)和計(jì)算集落形成率。數(shù)碼顯微拍攝集落,計(jì)數(shù)集落細(xì)胞數(shù),用集落平均細(xì)胞數(shù)繪制細(xì)胞生長曲線,并計(jì)算細(xì)胞群體倍增時(shí)間(DT)。 按原代骨髓細(xì)胞種植后的天數(shù)計(jì),9～12d可見EPC細(xì)胞簇,2～3d內(nèi)形成集落(細(xì)胞數(shù)≥50),集落形成率為每10~6骨髓有核細(xì)胞生成600.0±52.4個(gè)集落(個(gè)/10~6NBMCs)。集落細(xì)胞大多呈橢圓形、圓形和紡錘形。大部分集落約在1周內(nèi)生長較快,以后生長減慢,至30d左右,集落平均細(xì)胞數(shù)為504.6±126.0。在原代骨髓細(xì)胞種植后10～13d、14～17d、18～21d、22～25d四個(gè)時(shí)間段的細(xì)胞群體倍增時(shí)間依次是58.0h、74.4h、131.2h、358.2h。 2.HPP-EPC集落形成實(shí)驗(yàn)及單集落源細(xì)胞的擴(kuò)增培養(yǎng)和鑒定用上述消化和洗滌的貼壁細(xì)胞,以每mm~2孔底面積50個(gè)細(xì)胞(50/mm~2)的密度種入培養(yǎng)板,培養(yǎng)體系同前,每周換液。觀察貼壁細(xì)胞集落的形成,計(jì)數(shù)細(xì)胞數(shù)達(dá)5000左右的集落,計(jì)算集落形成率。進(jìn)行單集落細(xì)胞擴(kuò)增培養(yǎng),每4d做細(xì)胞計(jì)數(shù),按單個(gè)集落繪制細(xì)胞生長曲線和計(jì)算DT。用擴(kuò)增細(xì)胞做CD31和vWF免疫熒光染色、FITC-UEA-1結(jié)合實(shí)驗(yàn)和Dil-Ac-LDL攝取實(shí)驗(yàn)。 按原代骨髓細(xì)胞種植后的天數(shù)計(jì),28～35d時(shí)細(xì)胞數(shù)5000左右的HPP-EPC集落形成,其細(xì)胞緊密聚集,細(xì)胞形態(tài)大多為橢圓形和圓形,集落形成率為3.0±0.6個(gè)/10~6NBMCs。單集落源細(xì)胞傳代擴(kuò)增培養(yǎng)顯示,細(xì)胞在低密度時(shí)可形成索狀和網(wǎng)狀排列,高密度時(shí)細(xì)胞呈鋪路石狀排列。在原代骨髓細(xì)胞種植后50～53d,DT最短,平均為51.2h;在74～77d時(shí)間段,DT延長至324.2h。至原代骨髓細(xì)胞種植后80d左右,集落的平均細(xì)胞數(shù)達(dá)8.0×10~6±1.2×10~5個(gè),這些細(xì)胞為CD31、vWF免疫熒光染色陽性,能結(jié)合FITC-UEA-1和吞噬Dil-Ac-LDL。 3.mBMEC細(xì)胞中三種細(xì)胞因子mRNA水平的檢測Trizol法提取小鼠骨髓內(nèi)皮細(xì)胞系細(xì)胞RNA,進(jìn)行熒光實(shí)時(shí)定量逆轉(zhuǎn)錄PCR,檢測VEGF、bFGF、IGF-1 mRNA的表達(dá)水平。PCR結(jié)果顯示mBMEC細(xì)胞高水平表達(dá)VEGF mRNA和bFGF mRNA,而IGF-1 mRNA的表達(dá)水平較低。 結(jié)論:添加BMEC-CM的培養(yǎng)體系十分適合小鼠骨髓EPC的生長,用這種體系進(jìn)行HPP-EPC單集落源細(xì)胞培養(yǎng)能大量獲得其后代細(xì)胞。骨髓內(nèi)皮細(xì)胞系細(xì)胞高水平表達(dá)VEGF和bFGF,這可能是BMEC-CM促進(jìn)EPC增殖的重要分子基礎(chǔ)。這些結(jié)果還為內(nèi)皮細(xì)胞與EPC之間的反饋調(diào)控機(jī)制提供了新的實(shí)驗(yàn)證據(jù)。
[Abstract]:Objective : To investigate the expression of vascular endothelial growth factor ( VEGF ) , basic fibroblast growth factor ( bFGF ) and insulin - like growth factor - 1 ( IGF - 1 ) mRNA in bone marrow endothelial cells of mice and to analyze the molecular mechanism of BMEC - CM .



Methods and Results : This experiment is divided into the following three parts :



1 . The mouse bone marrow mononuclear cell suspension was prepared by EPC colony formation experiment . After 6 hours of pre - adherent culture of bone marrow cells , the supernatant containing unadherent cells was aspirated into the new culture flask , and BMEC - CM was added to culture the cells . After 5 days of culture , BMEC - CM was added . The colony formation , colony count and colony formation rate were counted .



The cell population doubling time was 54.6 鹵 126.0 . The doubling time of colony was 54.6 鹵 126.0 . The doubling time of colony was 58.0h , 74.4 h , 131.2 h , 358.2 h after the first generation of bone marrow cells .



2 . The colony forming experiment and the amplification culture of single colony source cells were carried out and the adherent cells were cultured and identified with the above digested and washed adherent cells . The colony formation rate was calculated by using the density of 50 cells ( 50 / mm ~ 2 ) per mm ~ 2 hole bottom area . The colony formation rate was calculated . The colony formation rate was calculated . The cell growth curve and the calculation DT were drawn on the basis of single colony . The cells were used as CD31 and von Wilms immunofluorescence staining , FITC - UEA - 1 binding experiment and Dil - Ac - LDL uptake experiment .



The colony formation rate was 3.0 鹵 0.6 / 10 ~ 6NB12.The average number of cells was 8.0 脳 10 ~ 6 鹵 1.2 脳 10 ~ 5 after the growth of primary bone marrow cells . The cells were CD31 and VWF immunofluoresence staining , which could bind to FITC - UEA - 1 and phagocytose Dil - Ac - LDL .



3 . The mRNA levels of three cytokines mRNA in mBMEC cells were detected by Trizol method . The expression of VEGF , bFGF and IGF - 1 mRNA was detected by real - time quantitative reverse transcription polymerase chain reaction ( RT - PCR ) , and the expression level of VEGF , bFGF and IGF - 1 mRNA was detected in mBMEC cells .



Conclusion : The culture system of BMEC - CM is very suitable for the growth of mouse bone marrow EPC , and its progeny cells can be obtained by using this system . The high level expression of VEGF and bFGF in bone marrow endothelial cell line cells may be an important molecular basis for BMEC - CM to promote EPC proliferation . These results also provide new experimental evidence for the feedback regulation mechanism between endothelial cells and EPC .
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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