胎盤免疫調(diào)節(jié)因子的分離純化及其對PC12細(xì)胞生長的影響
發(fā)布時間：2018-09-03 20:30
【摘要】： 第一部分胎盤免疫調(diào)節(jié)因子的分離純化及活性檢測 目的純化胎盤免疫調(diào)節(jié)因子(placenta factor PF)和篩選活性最顯著的組分。 方法將新鮮人胎盤去筋膜,生理鹽水沖凈、剪碎勻漿,反復(fù)凍融后,透析,取透析外液凍干,上樣于Sephadex G-25凝膠層析柱,洗脫,收集各組份。通過培養(yǎng)淋巴細(xì)胞,用MTT法鑒定PF純化產(chǎn)物的活性,反相高效液相色譜分析純產(chǎn)物的純度,基質(zhì)輔助激光解吸附電離串行飛行時間質(zhì)譜測定產(chǎn)物的分子量。 結(jié)果PF經(jīng)Sephadex G-25凝膠柱純化后,得到的第四峰具有明顯促進(jìn)淋巴細(xì)胞增殖活性,是胎盤免疫調(diào)節(jié)因子的主要活性成分,是分子量為6737.813Da的多肽,其純度為82%, 結(jié)論胎盤免疫調(diào)節(jié)因子經(jīng)Sephadex G-25凝膠柱純化得到的第四峰是胎盤免疫調(diào)節(jié)因子的主要活性成分,其純度為82%,分子量為6737.813Da。 第二部分胎盤免疫調(diào)節(jié)因子對PC12細(xì)胞生長的影響 目的:觀察胎盤免疫調(diào)節(jié)因子(placenta factor PF)對PC12細(xì)胞生長的影響。 方法:培養(yǎng)PC12細(xì)胞,用MTT法觀察PF對體外無血清培養(yǎng)PC12細(xì)胞存活率的影響及PF對低血清培養(yǎng)PC12細(xì)胞增殖的影響,普通光學(xué)顯微鏡觀察PC12細(xì)胞形態(tài)的變化。 結(jié)果:對無血清培養(yǎng)的PC12細(xì)胞加入不同濃度的PF培養(yǎng)44小時后, PF在濃度為50μg/ml、25μg/ml、12.5μg/ml和6.25μg/ml時可以顯著提高無血清培養(yǎng)PC12細(xì)胞的存活率(P0.05)。對低血清培養(yǎng)的PC12細(xì)胞加入不同濃度的PF培養(yǎng)68小時后, PF在濃度為12.5μg/ml和6.25μg/ml時可以顯著提高PC12細(xì)胞的數(shù)量(P0.01)。對低血清培養(yǎng)的PC12細(xì)胞加入不同濃度的PF培養(yǎng)10天, PF在濃度為3.13μg/ml和1.56μg/ml作用第6天時,細(xì)胞長出突起。 結(jié)論:PF能提高無血清培養(yǎng)PC12細(xì)胞的存活率,促進(jìn)PC12細(xì)胞增殖,誘導(dǎo)PC12細(xì)胞分化。 第三部分PF純化第四峰(PFIV)對PC12細(xì)胞生長的影響 目的:觀察胎盤免疫調(diào)節(jié)因子純化第四峰(PFIV)對PC12細(xì)胞生長的影響。 方法:培養(yǎng)PC12細(xì)胞,用MTT法觀察PFIV對體外無血清培養(yǎng)PC12細(xì)胞存活率的影響及PFIV對低血清培養(yǎng)PC12細(xì)胞增殖的影響,普通光學(xué)顯微鏡觀察PC12細(xì)胞形態(tài)的變化。 結(jié)果:對無血清培養(yǎng)的PC12細(xì)胞加入不同濃度的PFIV培養(yǎng)44小時后,在濃度為15μg/ml、7.5μg/ml、3.75μg/ml和1.88μg/ml時可以顯著提高無血清培養(yǎng)的PC12細(xì)胞的存活率(P0.05)。對低血清培養(yǎng)的PC12細(xì)胞加入不同濃度的PFIV培養(yǎng)68小時后,在濃度為0.47μg/ml和0.235μg/ml時可以顯著提高PC12細(xì)胞的數(shù)量(P0.01)。對低血清培養(yǎng)的PC12細(xì)胞加入不同濃度的PFIV培養(yǎng)10天, PFIV在濃度為0.118μg/ml和0.059μg/ml作用第6天時,細(xì)胞長出突起。 結(jié)論:胎盤免疫調(diào)節(jié)因子第四峰(PFIV)能提高無血清培養(yǎng)PC12細(xì)胞的存活率,促進(jìn)PC12細(xì)胞增殖,誘導(dǎo)PC12細(xì)胞分化。
[Abstract]:The first part: isolation and purification of placental immunomodulators objective to purify placental immunomodulatory factor (placenta factor PF) and to screen the most active components. Methods the fresh human placenta was removed from fascia, washed with normal saline, shredded and homogenized. After repeated freezing and thawing, dialysate was freeze-dried. The samples were eluted on Sephadex G-25 gel chromatography column, and the components were collected. The activity of the purified PF product was determined by MTT method, the purity of the purified product was analyzed by RP-HPLC, and the molecular weight of the purified product was determined by matrix assisted laser desorption ionization serial time-of-flight mass spectrometry. Results the fourth peak of PF was purified by Sephadex G-25 gel column. It was the main active component of placental immunomodulator and the polypeptide with molecular weight of 6737.813Da. Conclusion the fourth peak of placental immunomodulatory factor purified by Sephadex G-25 gel column is the main active component of placental immunomodulatory factor, its purity is 82 and its molecular weight is 6737.813 Da. Part two the effect of placental immunomodulator factor on the growth of PC12 cells objective: to observe the effect of placental immunomodulator (placenta factor PF) on the growth of PC12 cells. Methods: PC12 cells were cultured. The effect of PF on the survival rate of PC12 cells in serum-free culture and the effect of PF on the proliferation of PC12 cells cultured with low serum were observed by MTT method. The morphological changes of PC12 cells were observed by ordinary optical microscope. Results: the survival rate of serum-free PC12 cells was significantly increased by adding different concentrations of PF for 44 hours at the concentrations of 50 渭 g / ml, 25 渭 g / ml, 12.5 渭 g/ml and 6.25 渭 g/ml (P0.05). When PC12 cells cultured with low serum were cultured with different concentrations of PF for 68 hours, PF significantly increased the number of PC12 cells at the concentrations of 12.5 渭 g/ml and 6.25 渭 g/ml (P0.01). PC12 cells cultured with low serum were cultured with different concentrations of PF for 10 days. When the concentration of PF was 3.13 渭 g/ml and 1.56 渭 g/ml for 6 days, the cells grew processes. Conclusion:% PF can increase the survival rate of PC12 cells in serum-free culture, promote the proliferation of PC12 cells and induce the differentiation of PC12 cells. In the third part, the effect of the fourth peak (PFIV) purified by PF on the growth of PC12 cells was studied. Objective: to observe the effect of the purification of the fourth peak of placental immunoregulatory factor (PFIV) on the growth of PC12 cells. Methods: PC12 cells were cultured. The effect of PFIV on the survival rate of PC12 cells in serum-free culture and the effect of PFIV on the proliferation of PC12 cells cultured with low serum were observed by MTT method. The morphological changes of PC12 cells were observed by ordinary optical microscope. Results: the survival rate of serum-free PC12 cells cultured with different concentrations of PFIV for 44 hours was significantly increased at the concentration of 15 渭 g / ml (7.5 渭 g / ml) 3.75 渭 g/ml and 1.88 渭 g/ml (P0.05). When PC12 cells cultured with low serum were cultured with different concentrations of PFIV for 68 hours, the number of PC12 cells was significantly increased at the concentrations of 0.47 渭 g/ml and 0.235 渭 g/ml (P0.01). PC12 cells cultured with low serum were cultured with different concentrations of PFIV for 10 days. When the concentration of PFIV was 0.118 渭 g/ml and 0.059 渭 g/ml for 6 days, the cells developed protrusions. Conclusion: the fourth peak of placental immunoregulatory factor (PFIV) can increase the survival rate of PC12 cells cultured without serum, promote the proliferation of PC12 cells and induce the differentiation of PC12 cells.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
本文編號：2221086
[Abstract]:The first part: isolation and purification of placental immunomodulators objective to purify placental immunomodulatory factor (placenta factor PF) and to screen the most active components. Methods the fresh human placenta was removed from fascia, washed with normal saline, shredded and homogenized. After repeated freezing and thawing, dialysate was freeze-dried. The samples were eluted on Sephadex G-25 gel chromatography column, and the components were collected. The activity of the purified PF product was determined by MTT method, the purity of the purified product was analyzed by RP-HPLC, and the molecular weight of the purified product was determined by matrix assisted laser desorption ionization serial time-of-flight mass spectrometry. Results the fourth peak of PF was purified by Sephadex G-25 gel column. It was the main active component of placental immunomodulator and the polypeptide with molecular weight of 6737.813Da. Conclusion the fourth peak of placental immunomodulatory factor purified by Sephadex G-25 gel column is the main active component of placental immunomodulatory factor, its purity is 82 and its molecular weight is 6737.813 Da. Part two the effect of placental immunomodulator factor on the growth of PC12 cells objective: to observe the effect of placental immunomodulator (placenta factor PF) on the growth of PC12 cells. Methods: PC12 cells were cultured. The effect of PF on the survival rate of PC12 cells in serum-free culture and the effect of PF on the proliferation of PC12 cells cultured with low serum were observed by MTT method. The morphological changes of PC12 cells were observed by ordinary optical microscope. Results: the survival rate of serum-free PC12 cells was significantly increased by adding different concentrations of PF for 44 hours at the concentrations of 50 渭 g / ml, 25 渭 g / ml, 12.5 渭 g/ml and 6.25 渭 g/ml (P0.05). When PC12 cells cultured with low serum were cultured with different concentrations of PF for 68 hours, PF significantly increased the number of PC12 cells at the concentrations of 12.5 渭 g/ml and 6.25 渭 g/ml (P0.01). PC12 cells cultured with low serum were cultured with different concentrations of PF for 10 days. When the concentration of PF was 3.13 渭 g/ml and 1.56 渭 g/ml for 6 days, the cells grew processes. Conclusion:% PF can increase the survival rate of PC12 cells in serum-free culture, promote the proliferation of PC12 cells and induce the differentiation of PC12 cells. In the third part, the effect of the fourth peak (PFIV) purified by PF on the growth of PC12 cells was studied. Objective: to observe the effect of the purification of the fourth peak of placental immunoregulatory factor (PFIV) on the growth of PC12 cells. Methods: PC12 cells were cultured. The effect of PFIV on the survival rate of PC12 cells in serum-free culture and the effect of PFIV on the proliferation of PC12 cells cultured with low serum were observed by MTT method. The morphological changes of PC12 cells were observed by ordinary optical microscope. Results: the survival rate of serum-free PC12 cells cultured with different concentrations of PFIV for 44 hours was significantly increased at the concentration of 15 渭 g / ml (7.5 渭 g / ml) 3.75 渭 g/ml and 1.88 渭 g/ml (P0.05). When PC12 cells cultured with low serum were cultured with different concentrations of PFIV for 68 hours, the number of PC12 cells was significantly increased at the concentrations of 0.47 渭 g/ml and 0.235 渭 g/ml (P0.01). PC12 cells cultured with low serum were cultured with different concentrations of PFIV for 10 days. When the concentration of PFIV was 0.118 渭 g/ml and 0.059 渭 g/ml for 6 days, the cells developed protrusions. Conclusion: the fourth peak of placental immunoregulatory factor (PFIV) can increase the survival rate of PC12 cells cultured without serum, promote the proliferation of PC12 cells and induce the differentiation of PC12 cells.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳志華;吳文惠;王幸;張潔;包斌;;無肋馬尾藻多肽的制備及其促進(jìn)大鼠腎上腺嗜鉻細(xì)胞瘤細(xì)胞(PC12)分化特性的研究[J];中國海洋藥物;2011年01期
，本文編號：2221086
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