本文選題：肺 + 脂肪��； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:分離小鼠脂肪微血管內(nèi)皮細(xì)胞(AMECs)及肺微血管內(nèi)皮細(xì)胞(PMECs),建立微血管內(nèi)皮細(xì)胞(MECs)的培養(yǎng)方法。比較兩種MECs生物學(xué)特性的差別。為我們下一步研究MECs對(duì)造血干細(xì)胞(HSCs)體外擴(kuò)增的影響奠定基礎(chǔ),同時(shí)也給MECs的相關(guān)實(shí)驗(yàn)研究及臨床應(yīng)用提供依據(jù)。方法:1、微血管內(nèi)皮細(xì)胞分離培養(yǎng)方法的建立:(1)提取C57小鼠的附睪旁脂肪組織和肺組織,用0.1%I型膠原酶在37℃條件下?lián)u床消化,用70um濾器過(guò)濾細(xì)胞懸液。(2)尋找分離附睪旁脂肪組織的最佳鼠齡,分組:(1)4w,(2)6w,(3)8w,(4)10w,判斷標(biāo)準(zhǔn):每ml脂肪組織所含種子細(xì)胞數(shù)量。(3)探索最佳首次換液的時(shí)間,分組:(1)24h,(2)48h,(3)72h,判斷標(biāo)準(zhǔn):能否獲得原代MECs。(4)尋找原代培養(yǎng)的最佳接種密度,分組:(1)5×105/cm2,(2)1×106/cm2,(3)2×106/cm2,判斷標(biāo)準(zhǔn):培養(yǎng)至第8天的細(xì)胞形態(tài)、數(shù)量。(5)探索明膠包被培養(yǎng)皿的最適宜濃度,分組:(1)0,(2)0.1%,(3)0.5%,(4)1%,判斷標(biāo)準(zhǔn):收獲的原代MECs細(xì)胞數(shù)。2、對(duì)AMECs與PMECs的生物學(xué)特性進(jìn)行比較:(1)比較平均每只小鼠獲得的脂肪微血管內(nèi)皮細(xì)胞(AMECs)和肺微血管內(nèi)皮細(xì)胞(PMECs)數(shù)量。(2)倒置顯微鏡觀察AMECs及PMECs的生長(zhǎng)、形態(tài)變化。(3)流式細(xì)胞分析技術(shù)及免疫熒光技術(shù)測(cè)得AMECs和PMECs的CD31、CD34、CD45、vWF的表達(dá)。(4)繪制生長(zhǎng)曲線比較AMECs和PMECs的增殖能力。(5)將獲得的原代AMECs和PMECs按3:1進(jìn)行傳代,比較兩種細(xì)胞的傳代能力及傳代細(xì)胞形態(tài)。(6)采用10ng/mlVEGF培養(yǎng)兩種MECs觀察形態(tài)特征。結(jié)果:1.微血管內(nèi)皮細(xì)胞分離培養(yǎng)的最佳方法:(1)采用酶消法能夠成功分離兩種組織獲得種子細(xì)胞。(2)4周齡小鼠單位體積脂肪獲得的種子細(xì)胞數(shù)為4.67±0.58(106),6周組為10.18±0.28(106),8周組為9.94±0.35(106),10周組為6.72±0.39(106);6周組及8周組較4周組、10周組多(p0.05),6周組與8周組比較無(wú)差異(p0.05)。(3)24h首次換液可獲得原代amecs和pmecs;48h及72h首次換液,雜細(xì)胞生長(zhǎng)占優(yōu)勢(shì),無(wú)法獲得原代mecs。(4)1×106/cm2為原代最佳接種密度;5×105/cm2細(xì)胞稀疏,mecs增殖不良;2×106/cm2細(xì)胞接觸抑制,死亡脫壁。(5)培養(yǎng)至第8天,amecs的未包被明膠組的200×鏡下細(xì)胞數(shù)為148.33±13.54,0.1%組為201.83±12.97,0.5%組為191.50±11.52,1%組為193.00±8.67;明膠包被的3組得到的amecs多于未包被組(p0.05),而3種濃度間無(wú)差異(p0.05)。培養(yǎng)至第10天,pmecs的未包被明膠組的200×鏡下細(xì)胞數(shù)為182.33±11.03,0.1%組為219.50±11.4,0.5%組為212.00±12.39,1%組為213.00±12.12;明膠包被的3組得到的pmecs多于未包被組(p0.05),而3種濃度間無(wú)差異(p0.05)。2.amecs與pmecs的生物學(xué)特性比較的結(jié)果:(1)原代培養(yǎng)的amecs及pmecs均在第48h-72h形成細(xì)胞團(tuán),第4天左右細(xì)胞呈多角形、卵圓形,amecs在第10天、pmecs在第14天形成“鋪路石樣”改變,細(xì)胞表面可觀察到微絨毛。(2)每只小鼠獲得的amecs種子細(xì)胞為2.08±0.57(×106),pmecs種子細(xì)胞為19.07±0.72(×106),二者有差異(p0.05)。每只小鼠獲得的amecs原代細(xì)胞數(shù)為0.26±0.05(×106),pmecs原代細(xì)胞數(shù)為3.37±0.18(×106),二者有差異(p0.05)。(3)amecs及pmecs均不表達(dá)cd45。amecs的cd31、cd34、vwf表達(dá)率分別為44.28%、30.15%、76.1%,pmecs的cd31、cd34、vwf表達(dá)率分別為45.8%、57.48%、81.39%。(4)傳代的兩種細(xì)胞生長(zhǎng)均快于原代細(xì)胞,與原代細(xì)胞形態(tài)相比無(wú)明顯差異。(5)生長(zhǎng)曲線顯示PMECs緩慢增殖期較AMECs長(zhǎng);AMECs生長(zhǎng)高峰為接種后3-6天,PMECs生長(zhǎng)高峰為接種后4-7天。(6)10ng/mlVEGF培養(yǎng)兩種細(xì)胞,第6天可見(jiàn)細(xì)胞伸展、遷移融合形成細(xì)胞索,細(xì)胞索相互之間連接溝通。結(jié)論:1.本研究建立了經(jīng)濟(jì)有效、簡(jiǎn)便、可重復(fù)的分離培養(yǎng)AMECs及PMECs的最佳方案:24h首次換液、1×106/cm2接種密度、0.1%明膠、2ng/mlVEGF、6-8周齡小鼠。2.AMECs的細(xì)胞形態(tài)、特異性標(biāo)志、細(xì)胞傳代、生長(zhǎng)曲線與PMECs類似;每只小鼠收獲的AMECs和PMECs的數(shù)量有差異。
[Abstract]:Objective: to isolate the mouse fat microvascular endothelial cells (AMECs) and pulmonary microvascular endothelial cells (PMECs), to establish the culture method of microvascular endothelial cells (MECs), to compare the differences between the two kinds of MECs biological characteristics, and to lay the foundation for the next step to study the effect of MECs on the amplification of hematopoietic stem cells (HSCs) and to study the related experimental research of MECs. Methods: 1, 1, the isolation and culture of microvascular endothelial cells were established: (1) the epididymal adipose tissue and lung tissue of C57 mice were extracted and digested with 0.1%I collagenase at 37 degrees centigrade, and cell suspension was filtered by 70UM filter. (2) the optimal age of para epididymal adipose tissue was found. (1) 4W, (2) 6W, (3) ) 8W, (4) 10W, determine the standard: the number of seed cells in each ml fat tissue. (3) explore the best time for the first change of liquid, grouping: (1) 24h, (2) 48h, (3) 72h, judging whether the original MECs. (4) can find the best inoculation density for primary culture, grouping: (1) 5 x 105/cm2, (2) 1 * 106/cm2, (3) 2 * 106/cm2, criteria: cells cultivated to eighth days Form, quantity. (5) explore the optimum concentration of gelatine coated Petri dish, grouping: (1) 0, (2) 0.1%, (3) 0.5%, (4) 1%, judging standard: the number of primary MECs cells harvested.2, compared with the biological characteristics of AMECs and PMECs: (1) compared the average number of fat microvascular endothelial cells (AMECs) and pulmonary microvascular endothelial cells (PMECs) obtained in each mouse. (2) the growth and morphological changes of AMECs and PMECs were observed by inverted microscope. (3) flow cytometry and immunofluorescence techniques were used to detect the expression of CD31, CD34, CD45, vWF of AMECs and PMECs. (4) the growth curve was plotted to compare the proliferation ability of AMECs and PMECs. (5) the primary AMECs and PMECs were subpassable and the passages of the two cells were compared. Force and subcellular morphology. (6) the morphological characteristics of two kinds of MECs were observed by 10ng/mlVEGF. Results: the best method of isolation and culture of 1. microvascular endothelial cells: (1) the seed cells were successfully isolated from two tissues by enzyme digestion. (2) the number of seed cells obtained by unit volume fat of 4 week old mice was 4.67 + 0.58 (106) and 6 weeks was 10.18. 0.28 (106), 8 weeks group was 9.94 + 0.35 (106), 10 week group was 6.72 + 0.39 (106), and 6 week group and 8 week group were more than 4 week group (P0.05), and there was no difference (P0.05) in 8 weeks group. The density of 5 x 105/cm2 cells was sparse and MECs was poorly proliferating; 2 x 106/cm2 cells were exposed to contact inhibition, and death was removed. (5) culture to eighth days. The number of cells in the 200 x group of the unwrapped group of AMECS was 201.83 + 12.97,0.5% and 191.50 + 11.52,1% in group 201.83 was 193 + 8.67, and the AMECS of gelatin bag was more than that of the unwrapped group. P0.05), and there was no difference between the 3 concentrations (P0.05). For tenth days, the number of cells in the 200 x mirror group of the unwrapped gelatin group of PMECs was 182.33 + 11.03,0.1% and 213 + 12.12 in the 219.50 + 11.4,0.5% group, and the 3 groups of the gelatin bag were more than the unwrapped group (P0.05), and 3 concentrations were no difference (P0.05).2.amecs and pmec. The results of biological characteristics of s were compared: (1) the primary culture of AMECS and PMECs formed cell clusters in 48h-72h, fourth days or so, the cells were polygonal, oval, AMECS was tenth days, PMECs formed the "pave stone like" change on the fourteenth day, and the cell surface could be observed microvilli. (2) the AMECS seed cells of each mouse were 2.08 + 0.57 (x 106). The PMECs seed cells were 19.07 + 0.72 (x 106) and two were different (P0.05). The number of primary AMECS cells in each mouse was 0.26 + 0.05 (x 106), the number of primary cells of PMECs was 3.37 + 0.18 (x 106), and there was a difference between them (P0.05). (3) AMECS and PMECs did not express cd45.amecs's CD31, CD34, vWF expression rate, respectively, PMECs The expression rate of vWF was 45.8%, 57.48%, and 81.39%. (4) of the two cells were faster than the original cells. (5) the growth curve showed that the slow proliferation period of PMECs was longer than that of AMECs; the peak of AMECs growth was 3-6 days after inoculation, and the peak of PMECs growth was 4-7 days after inoculation. (6) 10ng/mlVEGF culture two cells sixth. Sixth Days can be seen that cell extension, migration and fusion form cell cables, and cell lines connect and communicate with each other. Conclusion: 1. this study established the best economic, simple, repeatable isolation and culture of AMECs and PMECs: 24h first liquid, 1 x 106/cm2 inoculation density, 0.1% gelatin, 2ng /mlVEGF, 6-8 week old mouse.2.AMECs cell morphology, specific markers The cell growth curve was similar to that of PMECs, and the number of AMECs and PMECs harvested per mouse was different.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R457.7

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