本文選題：骨髓細(xì)胞 + 原代培養(yǎng)��； 參考：《華中科技大學(xué)》2010年碩士論文

【摘要】：第一部分BALB/c小鼠多能成體祖細(xì)胞的分離培養(yǎng) 目的建立獲得大量BALB/c小鼠多能成體祖細(xì)胞(MAPC)的細(xì)胞分離和原代培養(yǎng)方法,為下一步獲得純化的MAPC奠定基礎(chǔ)。 方法從BALB/c小鼠脛骨和股骨中分離得到骨髓細(xì)胞懸液,采用全骨髓差異貼壁法,在含EGF、PDGF、LIF和低血清的培養(yǎng)基中培養(yǎng),2周后以一定比例傳代,繼續(xù)培養(yǎng),將不同時(shí)期細(xì)胞進(jìn)行Giemsa染色,觀察細(xì)胞形態(tài),通過MTT比色法比較不同周齡小鼠、原代接種密度和傳代接種密度對(duì)原代MAPC生長(zhǎng)的影響。 結(jié)果取材自3周小鼠所獲得原代細(xì)胞均一性和增殖情況最好,原代接種密度在(6～12)×10~5/cm~2,傳代接種密度在(20-30)×10~3/cm~2最有利于原代MAPC的生長(zhǎng)。Giemsa染色可見梭形和三角形細(xì)胞。 結(jié)論在本實(shí)驗(yàn)條件下,BALB/c小鼠的原代MAPC能較穩(wěn)定生長(zhǎng)和擴(kuò)增,給后續(xù)MACS分選提供了大量的細(xì)胞來源。 第二部分BALB/c小鼠多能成體祖細(xì)胞的純化和擴(kuò)增 目的獲得純化的MAPC并探索MAPC體外穩(wěn)定傳代擴(kuò)增的培養(yǎng)條件,建立MAPC細(xì)胞系,為探索MAPC向生殖細(xì)胞的誘導(dǎo)分化潛能打下基礎(chǔ)。 方法取傳2代的骨髓細(xì)胞,磁珠陰性分選去除CD45+和Ter119+細(xì)胞,將所獲目的細(xì)胞以一定密度接種,觀察細(xì)胞生長(zhǎng)情況,并繪制MAPC生長(zhǎng)曲線。臺(tái)盼藍(lán)檢測(cè)MACS分選前后細(xì)胞活力有無差異,MTT比色法比較不同接種密度對(duì)MAPC生長(zhǎng)的影響。 結(jié)果MACS分選前后細(xì)胞活性無顯著性差異(P㧐0.05),MAPC的最適接種密度為(6-10)×10~3/cm~2,MAPC接種后1-2天處于生長(zhǎng)停滯期,第3天進(jìn)入對(duì)數(shù)增長(zhǎng)期,約持續(xù)3天,以后進(jìn)入平臺(tái)期。分選后MAPC核大,胞質(zhì)少,呈梭形或三角形。 結(jié)論MACS陰性分選能獲得相對(duì)單一并具有良好增殖活性的MAPC。 第三部分BALB/c小鼠多能成體祖細(xì)胞的鑒定和冷凍保存 目的鑒定本實(shí)驗(yàn)培養(yǎng)的MAPC,并對(duì)MAPC的冷凍保存方法的效果進(jìn)行了評(píng)價(jià),為誘導(dǎo)分化實(shí)驗(yàn)提供足夠的細(xì)胞來源。 方法流式細(xì)胞術(shù)檢測(cè)MAPC表面標(biāo)記CD 13、CD44和CD34的表達(dá)情況。采用文獻(xiàn)報(bào)道的方法對(duì)MAPC進(jìn)行冷凍保存,對(duì)溫度稍作調(diào)整,比較凍存前后細(xì)胞活力。 結(jié)果MAPC表面CD13~+、CD44~-和CD34~-。細(xì)胞凍存前后活力無顯著性差異(P㧐0.05)。 結(jié)論全骨髓貼壁結(jié)合磁性分選是獲得MAPC的有效方法,采用文獻(xiàn)報(bào)道的冷凍方法能較好的保存MAPC,保持其生長(zhǎng)活性。
[Abstract]:The first part: isolation and culture of BALB/c mouse pluripotent adult progenitor cells Objective to establish a method for isolation and primary culture of a large number of BALB/c mouse multipotent adult progenitor cells (MAPCs) so as to lay a foundation for the further purification of MAPC. Methods Bone marrow cell suspensions were isolated from the tibia and femur of BALB/c mice. The bone marrow cells were cultured in a certain proportion for 2 weeks in the culture medium containing EGFN PDGFGF-LIF and low serum. The cells were stained with Giemsa at different stages. The effects of primary inoculation density and passage inoculation density on the growth of primary MAPC were compared by MTT colorimetry. Results the homogeneity and proliferation of primary cells were the best obtained from 3 weeks old mice. The primary inoculation density was 612 脳 10 ~ (5) / cm ~ (-2), and the inoculation density was 20 ~ 30 脳 10~3/cm~2, which was most favorable to the growth of primary MAPC. Giemsa staining showed fusiform cells and triangular cells. Conclusion the primary MAPC of BALB / c mice can grow and amplify stably under the condition of this experiment, which provides a large number of cell sources for subsequent MACS sorting. The second part: purification and amplification of BALB/c mouse pluripotent adult progenitor cells Objective to obtain purified MAPC and explore the culture conditions of stable passage and amplification of MAPC in vitro, and to establish MAPC cell line, which will lay a foundation for exploring the potential of inducing and differentiating MAPC into germ cells. Methods CD45 and Ter119 cells were removed by magnetic bead negative sorting. The target cells were inoculated with a certain density to observe the growth of the cells and draw the MAPC growth curve. Trypan blue was used to detect the difference of cell viability before and after MACS sorting. The effects of different inoculation densities on the growth of MAPC were compared by MTT colorimetry. Results there was no significant difference in cell activity before and after MACS sorting. The optimum inoculation density of MACS was 6-10) 脳 10 ~ (-3) / cm ~ (2) C ~ (2) C ~ (2) C ~ (2) ~ (-1) ~ (-1) ~ (-1) days after inoculation, and 3 days entered logarithmic growth period, which lasted for 3 days, and then entered the plateau phase. After sorting, the nucleus of MAPC is large and the cytoplasm is less, which is fusiform or triangular. Conclusion MACS negative sorting can obtain relatively single MAPCs with good proliferative activity. The third part: identification and cryopreservation of BALB/c mouse pluripotent adult progenitor cells Objective to identify MAPCs cultured in this experiment and evaluate the effect of cryopreservation method of MAPC in order to provide sufficient cell source for inducing differentiation experiment. Methods flow cytometry was used to detect the expression of CD 13 CD 44 and CD34 on MAPC surface. The cryopreservation of MAPC was carried out by using the method reported in the literature, and the temperature was adjusted slightly to compare the cell viability before and after cryopreservation. Results on the surface of MAPC, CD13 ~ + CD44 ~-and CD _ 34 ~ +-were observed. There was no significant difference in cell viability before and after cryopreservation. Conclusion\\\
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 米美玲;周玲;鄒挺;楊蓓;秦海燕;王晶磊;熊明悌;徐斯凡;萬秋華;;睪丸支持細(xì)胞和全反式視黃酸誘導(dǎo)骨髓干細(xì)胞向精原細(xì)胞分化的研究[J];江西醫(yī)學(xué)院學(xué)報(bào);2007年06期

2 劉風(fēng)華,楊冬梓,王沂峰,梁曉萍,彭文明,曹長(zhǎng)安,陳系古,郭忠敏;骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及在精原細(xì)胞培養(yǎng)條件下生物學(xué)特征的觀察[J];中華男科學(xué)雜志;2005年05期

3 陳黎紅,程樺,閔軍,吳木潮,嚴(yán)勵(lì),傅祖植;小鼠骨髓多能成體祖細(xì)胞的分離、培養(yǎng)及體外誘導(dǎo)分化為胰島素分泌細(xì)胞的研究[J];中國病理生理雜志;2005年09期

4 冀凱宏;熊俊;向正華;胡凱猛;王英;劉厚奇;;成年大鼠骨髓多能成體祖細(xì)胞的克隆化培養(yǎng)及其鑒定[J];中華血液學(xué)雜志;2006年07期

，

本文編號(hào)：1802992