【摘要】：目的 采用兩種酶兩步法分離純化人睪丸精原干細胞,探索精原干細胞體外培養(yǎng)方法,在體外建立穩(wěn)定的精原干細胞培養(yǎng)體系。 方法 先后應(yīng)用I型膠原酶和胰蛋白酶消化法,Percoll密度梯度離心,差異貼壁法,進行人精原干細胞的分離及培養(yǎng);細胞免疫熒光法行人精原干細胞的鑒定;流式細胞免疫熒光法對Oct-4標(biāo)記陽性的細胞行篩選,并同時檢測所獲精原干細胞的純度,建立精原干細胞和支持細胞共培養(yǎng)體系。 結(jié)果 兩種酶兩步法消化的人睪丸細胞懸液活細胞率為91.07%,Percoll密度梯度離心結(jié)合差異貼壁離心純化后的細胞經(jīng)Oct-4細胞免疫熒光法檢測表達陽性,流式細胞技術(shù)檢測陽性細胞所占比例為86.7%,該陽性細胞能夠在支持細胞飼養(yǎng)層上穩(wěn)定生長30 d。 結(jié)論 所獲Oct-4表達陽性細胞為精原干細胞。I型膠原酶和胰蛋白酶兩步酶消化法是一種經(jīng)濟的、簡單易行的、對細胞污染損傷少的分離培養(yǎng)人精原干細胞的方法,利用此方法分離的人精原干細胞能夠在支持細胞層上形成穩(wěn)定集落。
[Abstract]:Objective to isolate and purify human testicular spermatogonial stem cells by two enzyme two-step method, to explore the culture method of spermatogonial stem cells in vitro, and to establish a stable spermatogonial stem cell culture system in vitro. Methods Human spermatogonia stem cells were isolated and cultured by type I collagenase and trypsin digestion, Percoll density gradient centrifugation and differential adherent method, and the spermatogonia stem cells were identified by cellular immunofluorescence assay. Oct-4 positive cells were screened by flow cytometry, and the purity of spermatogonia stem cells was detected at the same time, and the co-culture system of spermatogonia stem cells and Sertoli cells was established. Results the living cell rate of human testicular cell suspension digested by two-step digestion was 21.07%. The expression of human testicular cell suspension purified by Percoll density gradient centrifugation combined with differential adherent centrifugation was detected by Oct-4 cell immunofluorescence assay. The proportion of positive cells detected by flow cytometry was 86.7%. The positive cells could grow stably on the feeder layer of Sertoli cells for 30 days. Conclusion the positive cells of Oct-4 expression are spermatogonial stem cells. Type I collagenase and trypsin two-step enzyme digestion is an economical, simple and easy method to isolate and culture human spermatogonial stem cells with less cell pollution. Human spermatogonia stem cells isolated by this method can form stable colonies on the supporting cell layer.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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