本文關(guān)鍵詞：不同培養(yǎng)方法和細胞因子對小鼠生精細胞的增殖分化效應(yīng)　出處：《安徽醫(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:比較曲細精管片段培養(yǎng)和混合細胞共培養(yǎng)兩種不同方法對小鼠生精細胞的增殖分化效應(yīng),旨在建立一個高效、穩(wěn)定的小鼠生精細胞體外培養(yǎng)方法,獲得更多接近成熟的單倍體精子細胞。方法:對7~8天齡小鼠生精細胞分別進行曲細精管片段培養(yǎng)和混合細胞共培養(yǎng),通過細胞形態(tài)學(xué)觀察、存活率和粗線期精母細胞特異性基因P19、單倍體精子細胞特異性基因TP1檢測及染色體倍性分析,比較兩種培養(yǎng)方法生精細胞的存活、增殖以及分化情況。結(jié)果:曲細精管片段培養(yǎng)法第10~12天可見到圓形精子細胞,13~14天出現(xiàn)少量帶鞭毛的精子細胞或長形精子,第10天可檢測出單倍體峰和單倍體精子細胞特異性基因TP1表達;混合細胞培養(yǎng)法5~7天可見圓形精子細胞,第5天即可檢測出單倍體峰和單倍體精子細胞特異性基因TP1表達,8~10天少量精子細胞長出鞭毛或胞體漸變成長形。混合細胞培養(yǎng)法所獲細胞數(shù)、存活率及單倍體精子細胞分化率均顯著高于曲細精管片段培養(yǎng)法(P0.05)。培養(yǎng)過程中,各級生精細胞隨培養(yǎng)時間延長,存活率逐漸降低,細胞數(shù)減少,曲細精管片段培養(yǎng)和混合細胞培養(yǎng)分別在第3周和第4周出現(xiàn)大部分生精細胞和支持細胞脫落死亡。結(jié)論:曲細精管片段培養(yǎng)和生精細胞-支持細胞共培養(yǎng)均可獲得單倍體精子細胞,較之曲細精管片段培養(yǎng)法,混合細胞共培養(yǎng)法存活率更高,可更早獲得更多單倍體精子細胞。 目的:通過在生精細胞體外培養(yǎng)過程中添加不同濃度表皮生長因子(epidermal growth factor,EGF)和干細胞因子(stem cell factor,SCF),探討兩種細胞因子對生精細胞增殖、分化的最佳作用濃度以及它們之間的最佳組合濃度。方法:對7~8天齡小鼠生精細胞進行混合細胞體外培養(yǎng),在培養(yǎng)基中分別添加不同濃度的EGF和SCF(分別為5 ng/ml、10 ng/ml、20 ng/ml、40 ng/ml、100 ng/ml),并進行EGF和SCF的交互實驗,通過細胞形態(tài)學(xué)觀察、存活率和粗線期精母細胞特異性基因P19、單倍體精子細胞特異性基因TP1檢測及染色體倍性分析,探討EGF和SCF對生精細胞的體外增殖分化效應(yīng)。結(jié)果:加入EGF或SCF2-4天,各濃度組中可見不同程度增殖呈團或鏈狀的細胞團緊密連接,以EGF 20ng/ml組和SCF 40ng/ml組較為明顯,第5-7天出現(xiàn)圓形精子細胞,8-10天少部分精子細胞長出鞭毛或漸變成長形精子細胞,第4周出現(xiàn)大部分生精細胞和支持細胞脫落死亡。培養(yǎng)第7天,EGF濃度為20ng/ml、SCF濃度為40ng/ml時生精細胞數(shù)和存活率顯著高于與其它各濃度組(P0.05),且濃度為40ng/ml的SCF可顯著提高單倍體峰并降低P19/TP1比值(P0.05)。EGF與SCF配伍時,EGF濃度為12 ng/ml時生精細胞增殖率最高,而SCF的作用效果呈線性遞增趨勢。結(jié)論:在混合生精細胞體外培養(yǎng)體系中,添加一定濃度的EGF和SCF可顯著提高生精細胞數(shù)和存活率,而且SCF可提高體單倍體精子的形成率,兩者交互實驗時,對細胞增殖有相互疊加效應(yīng)。
[Abstract]:Objective: to compare the proliferation and differentiation effects of seminiferous tubule fragment culture and mixed cell co-culture on mouse spermatogenic cells in order to establish an efficient and stable culture method of mouse spermatogenic cells in vitro. More haploid sperm cells were obtained. Methods: the spermatogenic cells of 7-day-old mice were cultured with seminiferous tubules and mixed cells, and the morphology of spermatogenic cells was observed. Survival rate, spermatocyte specific gene P19, haploid spermatocyte-specific gene TP1 and chromosome ploidy analysis were compared. Results: on the 10th and 12th day of seminiferous tubule culture, a small amount of sperm cells with flagellated or long sperm could be found in the round spermatozoa on day 1314. The haploid peak and haploid sperm cell specific gene TP1 expression could be detected on the 10th day. Round spermatocytes could be found in mixed cell culture method for 5 days. Haploid peak and haploid sperm cell specific gene TP1 expression could be detected on the 5th day. After 10 days, a small number of spermatozoa cells grew flagella or the cell body gradually formed. The number of cells obtained by mixed cell culture method. The survival rate and the differentiation rate of haploid sperm cells were significantly higher than those of seminiferous tubule culture (P0.05). In the process of culture, the survival rate and the number of spermatogenic cells decreased with the increase of culture time. The majority of spermatogenic cells and Sertoli cells died in the third and fourth weeks of seminiferous tubule culture and mixed cell culture, respectively. Haploid sperm cells could be obtained in seminiferous tubule fragment culture and spermatogenic cell-Sertoli cell co-culture. The survival rate of mixed cell coculture was higher than that of seminiferous tubule culture, and more haploid sperm cells could be obtained earlier. Objective: to add epidermal growth factor of different concentrations to spermatogenic cells in vitro. To investigate the proliferation of spermatogenic cells by two cytokines: EGF) and stem cell factor (SCF). Methods: mixed spermatogenic cells of 7-day old mice were cultured in vitro. Different concentrations of EGF and SCF were added to the medium (5 ng / ml, 10 ng / ml, 20 ng / ml, 40 ng/ml, respectively). 100ng 路ml ~ (-1) EGF and SCF were used to study the cell morphology, survival rate and spermatocyte specific gene P19 in coarse-line phase. The effects of EGF and SCF on the proliferation and differentiation of spermatogenic cells in vitro were studied by TP1 detection and chromosome ploidy analysis. Results: EGF or SCF2-4 were added to the spermatogenic cells for days. In each concentration group, the cell clusters with different degrees of proliferation were closely connected, especially in the EGF 20ng / ml group and the SCF 40ng / ml group. On the 5th to 7th day, a few of the spermatozoa cells grew out of flagella or maturation. At the 4th week, most of the spermatogenic cells and Sertoli cells died, and on the 7th day of culture, most of the spermatogenic cells and Sertoli cells died. The number and survival rate of spermatogenic cells at EGF concentration of 20 ng / ml were significantly higher than those of other groups (P 0.05). When the concentration of SCF was 40ng / ml, the haploid peak was significantly increased and the ratio of P19 / TP1 was decreased when P0.05N 路EGF was compatible with SCF. When the concentration of EGF was 12 ng/ml, the proliferation rate of spermatogenic cells was the highest, while the effect of SCF showed a linear increasing trend. Conclusion: in the culture system of mixed spermatogenic cells in vitro. Adding a certain concentration of EGF and SCF could significantly increase the number and survival rate of spermatogenic cells, and SCF could increase the formation rate of haploid spermatozoa. In the experiment of interaction between the two groups, there was a superposition effect on the proliferation of spermatogenic cells.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻】

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本文編號：1370882