發(fā)布時(shí)間：2018-06-16 12:30

  本文選題：精原干細(xì)胞 + 表皮生長(zhǎng)因子　； 參考：《江西醫(yī)學(xué)院》2005年碩士論文

【摘要】：目的:摸索完善精原干細(xì)胞分離、純化、培養(yǎng)的方法與技術(shù);研究EGF 對(duì)體外培養(yǎng)的精原干細(xì)胞自我更新增殖過(guò)程中所起的調(diào)控作用;研究EGF 對(duì)體外培養(yǎng)的精原干細(xì)胞的影響是否特異性地通過(guò)表皮生長(zhǎng)因子受體介導(dǎo);以及探討EGF 影響精原干細(xì)胞增殖中的可能的作用機(jī)制。建立完善的精原干細(xì)胞體外培養(yǎng)體系,為精原干細(xì)胞的體外大量擴(kuò)增提供技術(shù)和方法,為治療男性不育癥等提供相關(guān)技術(shù)。方法:不連續(xù)Percoll 梯度液分離純化精原干細(xì)胞,并應(yīng)用選擇性貼壁法進(jìn)一步純化精原干細(xì)胞;臺(tái)盼藍(lán)排斥實(shí)驗(yàn)確定精原干細(xì)胞的活力;c-kit 細(xì)胞免疫組化鑒定細(xì)胞類型;HE 染色法,根據(jù)細(xì)胞形態(tài)鏡下隨機(jī)取10 個(gè)視野細(xì)胞記數(shù)進(jìn)行細(xì)胞純度分析;細(xì)胞計(jì)數(shù)法研究不同濃度的表皮生長(zhǎng)因子對(duì)精原干細(xì)胞增殖的效應(yīng);MTT 法研究表皮生長(zhǎng)因子對(duì)精原干細(xì)胞增殖的效應(yīng):1、不同濃度表皮生長(zhǎng)因子對(duì)精原干細(xì)胞增殖的效應(yīng);2、表皮生長(zhǎng)因子對(duì)精原干細(xì)胞增殖的時(shí)間性效應(yīng);3、加入表皮生長(zhǎng)因子受體(epidermal growth factor receptor,EGFR)抑制劑AG1478 觀察表皮生長(zhǎng)因子受體的可能性作用;4、加入MAPK-ERK 信號(hào)通路特異性抑制劑PD98095JAK-STAT 信號(hào)通路特異性抑制劑AG490 及細(xì)胞內(nèi)Ca~(2+)螯合劑探討表皮生長(zhǎng)因子對(duì)精原干細(xì)胞增殖作用的可能機(jī)制。結(jié)果:(1)臺(tái)盼藍(lán)實(shí)驗(yàn)顯示細(xì)胞活力維持于88.73%±0.85%。(2)c-kit 細(xì)胞免疫組化結(jié)果顯示分離得到細(xì)胞為精原干細(xì)胞。(3)細(xì)胞純度為90.48%±1.78%。(4)細(xì)胞計(jì)數(shù)法結(jié)果顯示與對(duì)照組相比表皮生長(zhǎng)因子各劑量組對(duì)精原干細(xì)胞均有增殖作用(p㩳0.01), 20ng/ml 劑量組增殖作用最顯著。(5)MTT 結(jié)果也顯示各實(shí)驗(yàn)組比對(duì)照組細(xì)胞數(shù)量均有顯著增多(p㩳0.01),并且20ng/ml 劑量組的增殖作用最明顯;(6)20ng/ml 表皮生長(zhǎng)因子對(duì)精原干細(xì)胞的增殖效應(yīng)呈時(shí)間依賴性,在加入表皮生長(zhǎng)因子的第2d 到第4d 增殖作用最明顯;(7)加入表皮生長(zhǎng)因
[Abstract]:Objective: to explore the methods and techniques of isolation, purification and culture of spermatogonial stem cells, and to study the self-renewal of EGF on spermatogonial stem cells cultured in vitro. The effects of EGF on the proliferation of spermatogonial stem cells in vitro were studied. The effects of EGF on the proliferation of spermatogonial stem cells were investigated by epidermal growth factor receptor (EGF), and the possible mechanism of EGF on the proliferation of spermatogonial stem cells was discussed. To establish a perfect culture system of spermatogonial stem cells in vitro, to provide techniques and methods for the in vitro expansion of spermatogonial stem cells, and to provide related techniques for the treatment of male infertility. Methods: spermatogonial stem cells were isolated and purified by discontinuous Percoll gradient solution and further purified by selective adherent method, and the viability of spermatogonial stem cells was determined by trypan blue rejection assay. According to cell morphology, 10 visual field cells were randomly selected for cell purity analysis. Effects of different concentrations of EGF on proliferation of spermatogonial stem cells; MTT assay on proliferation of spermatogonial stem cells; effects of EGF on proliferation of spermatogonial stem cells: 1; EGF at different concentrations on proliferation of spermatogonial stem cells The temporal effect of EGF on the proliferation of spermatogonial stem cells was 3. The possibility of EG1478, an inhibitor of epidermal growth factor receptor, was used to observe the possibility of EGF receptor, and MAPK-ERK signaling pathway was added specifically to inhibit the proliferation of spermatogonial stem cells. Preparation of PD98095 / JAK-STAT signaling pathway specific inhibitor AG490 and intracellular CaFU 2) chelating agent to explore the possible mechanism of EGF on proliferation of spermatogonial stem cells. Results the cell viability was maintained at 88.73% 鹵0.85%.(2)c-kit cells by trypan blue assay. The results of immunohistochemistry showed that the cells isolated were spermatogonial stem cells. The purity of the cells was 90.48% 鹵1.78%. The proliferation of spermatogonial stem cells in the dose group was significantly higher than that in the control group, and the proliferative effect in the 20ng/ml group was the most significant. The results showed that the number of cells in each experimental group was significantly higher than that in the control group, and the proliferative effect of the 20ng/ml group was the most obvious in the 20ng/ml group with 20 ng / ml epidermal growth. The proliferation of spermatogonial stem cells was time-dependent. Epidermal growth factor was added on the 2nd to 4th day after adding epidermal growth factor (EGF) and epidermal growth factor (EGF) was added to the epidermal growth factor (EGF).
【學(xué)位授予單位】：江西醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R321

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳德寧;洪志明;周文彬;黃忠旺;黎杰運(yùn);;加味聚精食療方對(duì)少、弱精子癥大鼠EGF、EGFR睪丸內(nèi)表達(dá)的影響[J];世界中西醫(yī)結(jié)合雜志;2011年11期

相關(guān)碩士學(xué)位論文 前1條

1 洪志明;加味聚精食療方治療脾腎兩虛型少精子癥的臨床及實(shí)驗(yàn)研究[D];廣州中醫(yī)藥大學(xué);2010年

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本文編號(hào)：2026686