本文關(guān)鍵詞：胚胎干細(xì)胞體外培養(yǎng)體系的初步探討　出處：《廣西醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胚胎干細(xì)胞 飼養(yǎng)層 胚胎成纖維細(xì)胞 內(nèi)細(xì)胞團(tuán)

【摘要】： 目的:在前期課題組研究的基礎(chǔ)之上,優(yōu)化飼養(yǎng)層體系,穩(wěn)定培養(yǎng)條件,探索適合自身實驗室條件的胚胎干細(xì)胞體外培養(yǎng)體系,對實驗細(xì)胞的描述記錄實行標(biāo)準(zhǔn)化和規(guī)范化。 方法:(1).使用胰酶消化法體外分離培養(yǎng)MEF,MTT法測定P3、P8和復(fù)蘇P3 MEF生長活力,優(yōu)化原代取材、傳代、凍存和復(fù)蘇細(xì)胞的條件,免疫組化檢測MEF特異性標(biāo)志——波形蛋白的表達(dá),使用絲裂霉素C處理MEF,建立飼養(yǎng)層培養(yǎng)體系。(2).自然受孕法和超排卵法獲取昆明小鼠3.5dpc胚胎,體外培養(yǎng)發(fā)育至囊胚,全胚培養(yǎng)法獲取ICM,在飼養(yǎng)層上增殖,然后離散ICM種于新鮮的飼養(yǎng)層上,觀察ESC集落的形成,探索ESC體外培養(yǎng)體系。(3).收集生殖中心IVF-ET后剩余無冷凍價值的胚胎,醫(yī)院倫理委員會討論通過,簽署患者知情同意書后用于體外培養(yǎng),探索其體外培養(yǎng)條件。(4).依據(jù)《實驗細(xì)胞資源的描述標(biāo)準(zhǔn)與管理規(guī)范》,整理實驗數(shù)據(jù),對實驗細(xì)胞的描述記錄實行標(biāo)準(zhǔn)化和規(guī)范化。 結(jié)果:(1).使用胰酶消化法可從10~18dpc小鼠胚胎中獲取MEF,倒置顯微鏡下觀察,13~15dpc小鼠胚胎獲得的MEF質(zhì)量較佳;第五代之后MEF逐漸呈衰老相;MEF生長曲線顯示:在第3天時三組細(xì)胞均進(jìn)入對數(shù)增長期,P8組細(xì)胞的增殖速度明顯低于其他兩組(P0.05)。SABC免疫組化染色鑒定MEF,90%以上細(xì)胞中間絲波形蛋白呈棕色顆�；蜃厣�,即為陽性。絲裂霉素C處理MEF,終濃度為10μg/ml,作用時間2h,實驗顯示在抑制細(xì)胞有絲分裂的同時又不引起大量細(xì)胞死亡。(2).自然受孕法每只昆明小鼠可獲取8~12個3.5dpc胚胎,質(zhì)量較好的胚胎在飼養(yǎng)層體系中可逐漸發(fā)育至囊胚擴(kuò)張期,自然從透明帶孵出,ICM附著于飼養(yǎng)層上快速增殖。(3).整理了近三年課題組培養(yǎng)和凍存實驗細(xì)胞數(shù)據(jù),制定了規(guī)范化的實驗細(xì)胞記錄表格,標(biāo)準(zhǔn)化了實驗細(xì)胞的描述。 結(jié)論:(1).優(yōu)化穩(wěn)定了飼養(yǎng)層培養(yǎng)體系并鑒定飼養(yǎng)層細(xì)胞。(2).初步探索了胚胎培養(yǎng)及ICM集落形成的條件。
[Abstract]:Objective: to optimize the feeding layer system, to stabilize the culture conditions, and to explore an in vitro culture system of embryonic stem cells suitable for their laboratory conditions. The description of experimental cells is standardized and standardized. Methods the growth activities of P3P8 and P3 MEF were determined by trypsin digestion in vitro, and the primary material was optimized and subcultured. Under the condition of cryopreservation and resuscitation, the expression of vimentin, a specific marker of MEF, was detected by immunohistochemistry. Mitomycin C was used to treat MEF. The 3.5dpc embryos of Kunming mice were obtained by natural fertilization and superovulation. The embryos were cultured to blastocysts in vitro, and ICMs were obtained from the whole embryo culture method and proliferated on the feeder layer. Then discrete ICM was seeded on fresh feeder layer to observe the formation of ESC colony and to explore the culture system of ESC in vitro. The remaining embryos without freezing value after IVF-ET in reproductive center were collected. The hospital ethics committee discussed and approved, signed the informed consent of patients and used it for in vitro culture, and explored its culture condition in vitro. According to the description Standard and Management Standard of Experimental Cell Resources. Collate the experimental data and standardize the description and record of experimental cells. Results using trypsin digestion method, MEF could be obtained from 10 ~ 18dpc mouse embryos. The MEF obtained from 13 ~ (13) D ~ (PC) mouse embryos was better under inverted microscope. After the fifth generation, MEF gradually showed senescence phase. The growth curve of MEF showed that the proliferation rate of P8 cells in the three groups was significantly lower than that in the other two groups by immunohistochemical staining on the third day. More than 90% cells showed brown or brown staining of vimentin, which was positive. The final concentration of mitomycin C was 10 渭 g / ml for 2 h. The experiment showed that while inhibiting cell mitosis without causing a large number of cell death, 8 ~ 12 3.5dpc embryos could be obtained from each Kunming mouse by natural conception. The embryos with good quality can gradually develop into blastocyst dilation in the feeder layer system, and naturally hatched from the pellucida. ICM was attached to the feeder layer to proliferate rapidly. In the last three years, the experimental cell data were collected and frozen by our group, and a standardized record table of experimental cells was established, and the description of experimental cells was standardized. Conclusion the culture system of the feeder layer was optimized and stable, and the cells of the feeder layer were identified. The conditions of embryo culture and colony formation of ICM were preliminarily explored.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 秦茂林,蔡文琴,姚忠祥;昆明種系小鼠胚胎干細(xì)胞的分離培養(yǎng)及特性鑒定[J];第三軍醫(yī)大學(xué)學(xué)報;2001年09期

2 金潔;周金玲;史小林;;小鼠睪丸支持細(xì)胞對大腦皮質(zhì)細(xì)胞的體外營養(yǎng)作用[J];基礎(chǔ)醫(yī)學(xué)與臨床;2006年08期

3 蔡霞,高學(xué)軍,張鵬,唐勝建,鞠曉燕;體外培養(yǎng)不同代次人皮膚成纖維細(xì)胞超微結(jié)構(gòu)的研究[J];解剖科學(xué)進(jìn)展;2005年03期

4 陳東玲;邵文釗;王秀琴;吳e，

本文編號：1411461