胎盘免疫调节因子的分离纯化及其对PC12细胞生长的影响
发布时间：2018-09-03 20:30
【摘要】： 第一部分胎盘免疫调节因子的分离纯化及活性检测 目的纯化胎盘免疫调节因子(placenta factor PF)和筛选活性最显著的组分。 方法将新鲜人胎盘去筋膜,生理盐水冲净、剪碎匀浆,反复冻融后,透析,取透析外液冻干,上样于Sephadex G-25凝胶层析柱,洗脱,收集各组份。通过培养淋巴细胞,用MTT法鉴定PF纯化产物的活性,反相高效液相色谱分析纯产物的纯度,基质辅助激光解吸附电离串行飞行时间质谱测定产物的分子量。 结果PF经Sephadex G-25凝胶柱纯化后,得到的第四峰具有明显促进淋巴细胞增殖活性,是胎盘免疫调节因子的主要活性成分,是分子量为6737.813Da的多肽,其纯度为82%, 结论胎盘免疫调节因子经Sephadex G-25凝胶柱纯化得到的第四峰是胎盘免疫调节因子的主要活性成分,其纯度为82%,分子量为6737.813Da。 第二部分胎盘免疫调节因子对PC12细胞生长的影响 目的:观察胎盘免疫调节因子(placenta factor PF)对PC12细胞生长的影响。 方法:培养PC12细胞,用MTT法观察PF对体外无血清培养PC12细胞存活率的影响及PF对低血清培养PC12细胞增殖的影响,普通光学显微镜观察PC12细胞形态的变化。 结果:对无血清培养的PC12细胞加入不同浓度的PF培养44小时后, PF在浓度为50μg/ml、25μg/ml、12.5μg/ml和6.25μg/ml时可以显著提高无血清培养PC12细胞的存活率(P0.05)。对低血清培养的PC12细胞加入不同浓度的PF培养68小时后, PF在浓度为12.5μg/ml和6.25μg/ml时可以显著提高PC12细胞的数量(P0.01)。对低血清培养的PC12细胞加入不同浓度的PF培养10天, PF在浓度为3.13μg/ml和1.56μg/ml作用第6天时,细胞长出突起。 结论:PF能提高无血清培养PC12细胞的存活率,促进PC12细胞增殖,诱导PC12细胞分化。 第三部分PF纯化第四峰(PFIV)对PC12细胞生长的影响 目的:观察胎盘免疫调节因子纯化第四峰(PFIV)对PC12细胞生长的影响。 方法:培养PC12细胞,用MTT法观察PFIV对体外无血清培养PC12细胞存活率的影响及PFIV对低血清培养PC12细胞增殖的影响,普通光学显微镜观察PC12细胞形态的变化。 结果:对无血清培养的PC12细胞加入不同浓度的PFIV培养44小时后,在浓度为15μg/ml、7.5μg/ml、3.75μg/ml和1.88μg/ml时可以显著提高无血清培养的PC12细胞的存活率(P0.05)。对低血清培养的PC12细胞加入不同浓度的PFIV培养68小时后,在浓度为0.47μg/ml和0.235μg/ml时可以显著提高PC12细胞的数量(P0.01)。对低血清培养的PC12细胞加入不同浓度的PFIV培养10天, PFIV在浓度为0.118μg/ml和0.059μg/ml作用第6天时,细胞长出突起。 结论:胎盘免疫调节因子第四峰(PFIV)能提高无血清培养PC12细胞的存活率,促进PC12细胞增殖,诱导PC12细胞分化。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 陈志华;吴文惠;王幸;张洁;包斌;;无肋马尾藻多肽的制备及其促进大鼠肾上腺嗜铬细胞瘤细胞(PC12)分化特性的研究[J];中国海洋药物;2011年01期
，本文编号：2221086
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