本文關(guān)鍵詞：支持細(xì)胞對體外培養(yǎng)精原干細(xì)胞的作用途徑研究　出處：《重慶醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的 通過不同培養(yǎng)條件對精原干細(xì)胞(Spermatogonial Stem Cells,SSCs)進(jìn)行體外培養(yǎng),觀察每種培養(yǎng)條件下SSCs24h貼壁率,增殖特性并繪制其增殖曲線,比較上述指標(biāo)在不同培養(yǎng)條件下的差異,從而探討支持細(xì)胞對體外培養(yǎng)精原干細(xì)胞的作用途徑。 方法 選用7d齡雄性昆明種小鼠,兩步酶消化法獲得睪丸組織細(xì)胞懸液,差異時(shí)間貼壁法分離精原干細(xì)胞和支持細(xì)胞,免疫熒光法和油紅O染色法分別對其進(jìn)行生物學(xué)鑒定,流式細(xì)胞儀對精原干細(xì)胞進(jìn)行純度分析。按培養(yǎng)條件的不同將實(shí)驗(yàn)分為3組:精原干細(xì)胞與支持細(xì)胞共培養(yǎng)組(A組),條件培養(yǎng)基組(B組),常規(guī)培養(yǎng)基組(C組)。其中B組所用條件培養(yǎng)基按單純支持細(xì)胞培養(yǎng)上清液:雙倍濃縮的DMEM/F12:胎牛血清=4.5:4.5:1的比例配置;A、C組所用常規(guī)培養(yǎng)基即含體積分?jǐn)?shù)10%胎牛血清的DMEM/F12。臺盼蘭法測定各組貼壁率,四甲基偶氮唑鹽(MTT)法測定各組精原干細(xì)胞的吸光度并繪制增值曲線。倒置顯微鏡下觀察各組精原干細(xì)胞增殖特點(diǎn)及集落形成情況。比較各組精原干細(xì)胞24h貼壁率、增值曲線及存活時(shí)間。 結(jié)果 A組精原干細(xì)胞的24h貼壁率大于B組及C組(P0.05),而B、C組間無差異(P0.05);A組精原干細(xì)胞接種起即穩(wěn)定增殖,于(7-10d)形成穩(wěn)定集落并維持約30d的特性。B組和C組均表現(xiàn)為精原干細(xì)胞經(jīng)短暫的增殖后呈現(xiàn)快速減少的趨勢,培養(yǎng)一周后,精原干細(xì)胞數(shù)量明顯減少。 結(jié)論 支持細(xì)胞對體外培養(yǎng)精原干細(xì)胞的作用是依靠兩者之間的直接聯(lián)系和支持細(xì)胞的旁分泌兩種途徑;僅依靠支持細(xì)胞的旁分泌作用不能促進(jìn)精原干細(xì)胞的貼壁和增殖。
[Abstract]:Purpose Spermatogonial Stem cells (SSCs) were cultured in vitro under different culture conditions. The adherent rate and proliferation characteristics of SSCs24h were observed under each culture condition and the proliferation curve was plotted to compare the differences of the above indexes under different culture conditions. In order to explore the role of Sertoli cells on cultured spermatogonial stem cells in vitro. Method Spermatogonial stem cells and Sertoli cells were isolated from male Kunming mice by two-step enzyme digestion. They were identified by immunofluorescence and oil red O staining. The purity of spermatogonial stem cells was analyzed by flow cytometry. According to the different culture conditions, the experiment was divided into three groups: spermatogonial stem cells and Sertoli cells co-culture group (group A), conditioned medium group (group B). The conditioned medium used in group B was as follows: supernatant of Sertoli cell culture: double concentration of DMEM / F12: fetal bovine serum of 4.5: 4.5: 1; DMEM / F12 containing 10% fetal bovine serum was used in the conventional medium used in group A (C). The adherent rate of each group was determined by Trypan blue method. Tetramethylazolium (MTT). The proliferation characteristics and colony formation of spermatogonial stem cells in each group were observed under inverted microscope. 24 h adherent rate of spermatogonial stem cells was compared. Increment curve and survival time. Results The 24 h adherent rate of spermatogonial stem cells in group A was higher than that in group B and group C (P 0.05), but there was no difference between group B and C (P 0.05). In group A, spermatogonial stem cells proliferated stably as soon as they were inoculated. Group B and group C showed a rapid decrease of spermatogonial stem cells after a short period of proliferation, and cultured for one week. The number of spermatogonial stem cells decreased significantly. Conclusion The effect of Sertoli cells on the culture of spermatogonial stem cells in vitro depends on the direct connection between them and the paracrine of Sertoli cells. Paracrine function of Sertoli cells alone can not promote adherent and proliferation of spermatogonial stem cells.
【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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本文編號：1382106