本文選題：內(nèi)皮祖細胞 + 體外擴增��； 參考：《湖南師范大學》2009年碩士論文

【摘要】： 目的:用含小鼠骨髓內(nèi)皮細胞系條件培養(yǎng)液(BMEC-CM)的培養(yǎng)體系,進行骨髓高增殖潛能內(nèi)皮祖細胞(HPP-EPC)及其后代細胞的培養(yǎng)和純化,建立這些細胞的一種擴增培養(yǎng)方法。檢測小鼠骨髓內(nèi)皮細胞系細胞的血管內(nèi)皮細胞生長因子(VEGF)、堿性成纖維細胞生長因子(bFGF)、胰島素樣生長因子-1(IGF-1)mRNA表達水平,分析BMEC-CM促進EPC增殖的分子機理。 方法和結(jié)果:本實驗分為以下三個部分: 1.總EPC集落形成實驗制備小鼠骨髓單細胞懸液,骨髓細胞預貼壁培養(yǎng)6h后,吸出含未貼壁細胞的上懸液,移入新的培養(yǎng)瓶,并添加BMEC-CM,進行二次貼壁培養(yǎng)。培養(yǎng)5d后用胰酶法將貼壁細胞消化下來,洗滌后計數(shù)。以每mm~2孔底面積1個細胞(1/mm~2)的密度,將這些細胞種入培養(yǎng)板,培養(yǎng)體系中仍添加BMEC-CM。觀察貼壁細胞集落的形成,計數(shù)集落數(shù)和計算集落形成率。數(shù)碼顯微拍攝集落,計數(shù)集落細胞數(shù),用集落平均細胞數(shù)繪制細胞生長曲線,并計算細胞群體倍增時間(DT)。 按原代骨髓細胞種植后的天數(shù)計,9～12d可見EPC細胞簇,2～3d內(nèi)形成集落(細胞數(shù)≥50),集落形成率為每10~6骨髓有核細胞生成600.0±52.4個集落(個/10~6NBMCs)。集落細胞大多呈橢圓形、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,集落平均細胞數(shù)為504.6±126.0。在原代骨髓細胞種植后10～13d、14～17d、18～21d、22～25d四個時間段的細胞群體倍增時間依次是58.0h、74.4h、131.2h、358.2h。 2.HPP-EPC集落形成實驗及單集落源細胞的擴增培養(yǎng)和鑒定用上述消化和洗滌的貼壁細胞,以每mm~2孔底面積50個細胞(50/mm~2)的密度種入培養(yǎng)板,培養(yǎng)體系同前,每周換液。觀察貼壁細胞集落的形成,計數(shù)細胞數(shù)達5000左右的集落,計算集落形成率。進行單集落細胞擴增培養(yǎng),每4d做細胞計數(shù),按單個集落繪制細胞生長曲線和計算DT。用擴增細胞做CD31和vWF免疫熒光染色、FITC-UEA-1結(jié)合實驗和Dil-Ac-LDL攝取實驗。 按原代骨髓細胞種植后的天數(shù)計,28～35d時細胞數(shù)5000左右的HPP-EPC集落形成,其細胞緊密聚集,細胞形態(tài)大多為橢圓形和圓形,集落形成率為3.0±0.6個/10~6NBMCs。單集落源細胞傳代擴增培養(yǎng)顯示,細胞在低密度時可形成索狀和網(wǎng)狀排列,高密度時細胞呈鋪路石狀排列。在原代骨髓細胞種植后50～53d,DT最短,平均為51.2h;在74～77d時間段,DT延長至324.2h。至原代骨髓細胞種植后80d左右,集落的平均細胞數(shù)達8.0×10~6±1.2×10~5個,這些細胞為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細胞中三種細胞因子mRNA水平的檢測Trizol法提取小鼠骨髓內(nèi)皮細胞系細胞RNA,進行熒光實時定量逆轉(zhuǎn)錄PCR,檢測VEGF、bFGF、IGF-1 mRNA的表達水平。PCR結(jié)果顯示mBMEC細胞高水平表達VEGF mRNA和bFGF mRNA,而IGF-1 mRNA的表達水平較低。 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,用這種體系進行HPP-EPC單集落源細胞培養(yǎng)能大量獲得其后代細胞。骨髓內(nèi)皮細胞系細胞高水平表達VEGF和bFGF,這可能是BMEC-CM促進EPC增殖的重要分子基礎(chǔ)。這些結(jié)果還為內(nèi)皮細胞與EPC之間的反饋調(diào)控機制提供了新的實驗證據(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 .
【學位授予單位】：湖南師范大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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