發(fā)布時(shí)間：2018-01-05 21:20

  本文關(guān)鍵詞：羊水來源干細(xì)胞的分離培養(yǎng)及其生物學(xué)特征的研究　出處：《泰山醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人羊水來源干細(xì)胞 細(xì)胞培養(yǎng) 多能性

【摘要】：目的 研究羊水來源干細(xì)胞(amniotic fluid-drived stem cells,AFS)的確切來源。目前,鑒定羊水來源干細(xì)胞沒有什么好方法,只是通過細(xì)胞表面干細(xì)胞因子的表達(dá)情況和生物學(xué)特性來確定其是否為AFS。本實(shí)驗(yàn)在分離培養(yǎng)干細(xì)胞的基礎(chǔ)上,研究其生物學(xué)特性;通過細(xì)胞形態(tài)及細(xì)胞原位免疫組化來明確羊水干細(xì)胞的確切來源,為下一步的臨床治療提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 研究方法 在知情同意的情況下,通過B超引導(dǎo)下腹腔穿刺抽取人孕16-24周的羊水18-20ml,原位培養(yǎng)2周后,待其達(dá)到50%融合后,將一部分P1培養(yǎng)瓶中的細(xì)胞分為兩部分,倒置顯微鏡下,將培養(yǎng)瓶一分為二,用進(jìn)口刮刀將其分別刮下,倒入新的培養(yǎng)瓶中,進(jìn)行傳代,記為P21、P22。由于刮刀對(duì)細(xì)胞的損傷較大,故繼續(xù)傳代用胰酶消化的方法,一瓶用來倒置顯微鏡下觀察各種細(xì)胞的形態(tài),繪制生長(zhǎng)曲線、構(gòu)建類胚胎及分析染色體核型;另一瓶,通過細(xì)胞免疫組化研究OCT-4、CD117的表達(dá)情況;另一部分P1,倒置顯微鏡下觀察其形態(tài)并通過細(xì)胞原位免疫組化來研究其表面分子的表達(dá)情況,檢測(cè)各種標(biāo)志物如波形蛋白Vimen、廣譜角蛋白CK、CD44、CD29、CD34、CD45的表達(dá)情況。 結(jié)果 1、通過細(xì)胞的原位培養(yǎng),根據(jù)倒置顯微鏡下細(xì)胞形態(tài)的不同,將羊水中的細(xì)胞分為四類,為小島樣細(xì)胞、E樣細(xì)胞、羊水特異細(xì)胞及成纖維樣細(xì)胞。 2、多次傳代后,細(xì)胞不再呈集落樣生長(zhǎng),而呈重疊樣生長(zhǎng),以梭形為主,排列緊密,相互間界限不清。形態(tài)比原代更均勻,生長(zhǎng)旺盛�？梢赃B續(xù)傳10代,沒有發(fā)現(xiàn)細(xì)胞老化現(xiàn)象。 3、羊水細(xì)胞生長(zhǎng)曲線呈S形,P10比P3、P5生長(zhǎng)緩慢。生長(zhǎng)曲線的特征為:接種后2天左右為潛伏適應(yīng)期,大約從第3天起為對(duì)數(shù)生長(zhǎng)期,第8天達(dá)到高峰期,之后就進(jìn)入了平臺(tái)期,說明其在體外具有很強(qiáng)的增殖能力。 4、將P7羊水細(xì)胞倒置懸浮培養(yǎng)3天,顯微鏡下可見類胚體樣結(jié)構(gòu)。證明了其具有向三個(gè)胚層分化的潛能,有待進(jìn)一步的研究。 5、取每代羊水細(xì)胞做染色體核型分析,都為正常二倍體核型,說明了其具有穩(wěn)定遺傳的特性。 6、取P10做細(xì)胞免疫組化,結(jié)果顯示其表達(dá)OCT-4、CD117,提示羊水中含有干細(xì)胞。 7、小島樣細(xì)胞表達(dá)CK、Vimen、CD44、OCT-4,不表達(dá)CD117、CD34、CD45;E樣細(xì)胞表達(dá)CK、Vimen、CD44、Oct-4、CD117,不表達(dá)CD34、CD45;羊水特異細(xì)胞表CK、Vimen、CD44、OCT-4,不表達(dá)CD117、CD34、CD45;成纖維樣細(xì)胞表達(dá)Vimen、CD44、Oct-4、CD117,不表達(dá)CD34、CD45。 結(jié)論及意義 1、本實(shí)驗(yàn)證明上皮樣細(xì)胞和成纖維樣細(xì)胞都為羊水干細(xì)胞的確切來源。 2、本實(shí)驗(yàn)根據(jù)其形態(tài)的不同,將羊水細(xì)胞分為小島樣細(xì)胞、E樣細(xì)胞、羊水特異細(xì)胞及成纖維樣細(xì)胞。 3、本實(shí)驗(yàn)證明羊水中含有表達(dá)CD44、Vimen的間充質(zhì)干細(xì)胞、表達(dá)干細(xì)胞因子CD117的干細(xì)胞、表達(dá)OCT-4的多能干細(xì)胞。 4、本實(shí)驗(yàn)證明了羊水來源的干細(xì)胞在體外具有很強(qiáng)的增殖能力、分化潛能及穩(wěn)定遺傳的特性。
[Abstract]:Purpose To study the exact origin of amniotic fluid-drived stem cells (AFSs) derived from amniotic fluid. Identification of amniotic fluid derived stem cells is not a good method, only through the cell surface stem cell factor expression and biological characteristics to determine whether it is AFS. this experiment is based on the isolation and culture of stem cells. To study its biological characteristics; The exact origin of amniotic fluid stem cells was determined by cell morphology and in situ immunohistochemistry to provide experimental and theoretical basis for the next clinical treatment. Research method In the case of informed consent, the amniotic fluid of 16-24 weeks gestation was extracted by abdominal puncture guided by B-ultrasound. After in situ culture for 2 weeks, it reached 50% fusion. The cells in the P1 culture bottle were divided into two parts. Under the inverted microscope, the culture bottle was divided into two parts. The culture bottle was scraped off separately with an imported scraper, then poured into the new culture bottle and subcultured as P21. P22. because of the large damage to cells caused by scraper, the method of trypsin digestion was continued. A bottle was used to observe the morphology of various cells under inverted microscope and to draw the growth curve. Construction of embryoid and karyotype analysis; In the other bottle, the expression of OCT-4 CD117 was studied by immunohistochemistry. In the other part, the morphology of P1 was observed under inverted microscope, and the expression of surface molecules was studied by in situ immunohistochemistry, and various markers such as vimentin Vimenin and broad-spectrum keratin CK were detected. Expression of CD44, CD29, CD34, CD45. Results 1. The cells in amniotic fluid were divided into four groups by in situ culture. According to the different morphology of the cells under inverted microscope, the cells in amniotic fluid were divided into small island like cells, amniotic fluid specific cells and fibroblast cells. (2) after repeated passages, the cells were no longer colony like growth, but overlapped like growth, mainly fusiform, closely arranged, with unclear boundaries between each other. The morphology was more uniform than the original generation, and the growth was exuberant. The cells could be passed on for 10 generations in succession. No cell aging was found. 3. The growth curve of amniotic fluid cells showed S-shaped P10 was slower than P3P5. The characteristic of the growth curve was that the incubation period was about 2 days after inoculation and the logarithmic growth period was about 3 days after inoculation. On the 8th day, it reached its peak and then entered the platform stage, which indicated that it had strong proliferative ability in vitro. (4) when the P7 amniotic fluid cells were cultured in reverse suspension for 3 days, the embryoid structure could be seen under microscope. It was proved that P7 amniotic fluid cells had the potential to differentiate into three endosperms, which should be further studied. 5. The chromosome karyotype analysis of every generation of amniotic fluid cells is normal diploid karyotype, which shows that it has stable genetic characteristics. 6. P10 was taken as cell immunohistochemistry. The results showed that OCT-4 and CD117 were expressed in amniotic fluid, suggesting that there were stem cells in amniotic fluid. (7) the small island like cells expressed CK-Vimenus CD44-OCT-4, and did not express CD117- CD34- CD45; E-like cells expressed CK-Vimenn ~ (4) -Oct-4 + CD117, but not CD34 ~ (4) ~ (+) CD45; In amniotic fluid specific cell surface, Vimenus CD4and OCT-4 did not express CD117, CD34 and CD45; Fibroblasts expressed Vimenin CD44, Oct-4, CD117, but not CD34, CD45. Conclusion and significance 1. It was proved that both epithelioid cells and fibroblasts were the exact sources of amniotic fluid stem cells. 2. According to its morphology, amniotic fluid cells were divided into small island like cells, amniotic fluid specific cells and fibroblasts. 3. The results showed that the amniotic fluid contained mesenchymal stem cells expressing CD44-Vimen, stem cells expressing stem cell factor CD117 and pluripotent stem cells expressing OCT-4. 4. The results showed that the stem cells derived from amniotic fluid had strong proliferative ability, differentiation potential and stable heredity in vitro.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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