【摘要】： 本實驗室利用臨床多余的囊胚經(jīng)患者知情同意進行人胚胎干細胞(human embryonic stem cells, hES)建系研究,首先用鏈蛋白酶消化以去除囊胚的透明帶,然后直接機械分離出內(nèi)細胞團,接種到飼養(yǎng)層細胞(feeders)上,在hES細胞培養(yǎng)液中進行培養(yǎng)。一共成功建立了5個hES細胞系,全部來自體外受精(in vitro fertilization,IVF)囊胚,分別命名為J6、J13、J14、J30和J32。 根據(jù)hES細胞系公認的鑒定標(biāo)準(zhǔn),我們對其中新建立的2株細胞系的各項特征進行了檢測鑒定。這些hES細胞在體外能長期穩(wěn)定增殖;表達堿性磷酸酶活性;表達hES細胞特異的標(biāo)志物Oct-4、SSEA-3、SSEA-4、TRA-1-60、TRA-1-81;在體外能形成包含三個胚層來源分化細胞的類胚體(embryoid body,EB);在免疫缺陷小鼠(SCID mouse)體內(nèi)能形成包含三胚層來源組織的畸胎瘤;經(jīng)過細胞的單克隆化,產(chǎn)生兩個hES細胞亞系(J6-1和J13-1),并且在體外仍能長期穩(wěn)定增殖;這兩個細胞株的群體倍增時間為31.43小時(J6)和21.38小時(J13)。到目前為止,這5個細胞系在體外已經(jīng)分別培養(yǎng)485(J6)、478(J13)、259(J14)、120(J30)、121(J32)天,培養(yǎng)代數(shù)達46(J6)、55(J13)、19(J14)、25(J30)、24(J32)。
[Abstract]:Human embryonic stem cells (human embryonic stem cells, hES) were established by using clinical superfluous blastocysts with the informed consent of patients. Firstly, the blastocysts were digested with strepsin to remove the pellucida of blastocysts, and then the inner cell masses were mechanically isolated. (feeders) was inoculated into feeder layer cells and cultured in hES cell culture medium. Five hES cell lines, all from (in vitro fertilization,IVF blastocyst, were successfully established and named J6 J13, J14, J30 and J32, respectively. According to the generally accepted criteria for the identification of hES cell lines, we detected and identified the characteristics of two newly established cell lines. These hES cells can proliferate steadily for a long time in vitro, express alkaline phosphatase activity and express Oct-4,SSEA-3,SSEA-4,TRA-1-60,TRA-1-81;, a specific marker of hES cells. In vitro, embryoid bodies (embryoid body,EB) containing three embryo-derived differentiated cells were formed in vitro, teratoma containing three-layer derived tissue was formed in (SCID mouse) of immunodeficient mice. Two hES cell sublines (J6-1 and J13-1) were produced by cell monoclonal transformation, and the population doubling time of these two cell lines was 31.43 hours (J6) and 21.38 hours (J13). So far, 485 (J6), 478 (J13), 259 (J14), 120 (J30), 121 (J32) days have been cultured in vitro, and the cultured algebras are 46 (J6), 55 (J13), 19 (J14), 25 (J30), 24 (J32).
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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