【作者】 何跃；

【导师】 雷霆；

【作者基本信息】 华中科技大学 ， 外科学， 2006， 博士

【摘要】 第一部分神经生长因子受体(NGFR)在垂体泌乳素腺瘤中的表达及与肿瘤生物学特点的相关性研究目的探讨垂体泌乳素腺瘤中神经生长因子受体及增殖细胞核抗原(PCNA)表达与PRL腺瘤生物学特性之间的相关性。方法应用免疫组化方法检测38例垂体泌乳素腺瘤中两种神经生长因子受体及增殖细胞核抗原的表达情况,并根据肿瘤的生物学特点即侵袭性、多巴胺激动剂敏感性、性别、泌乳素水平、肿瘤大小进行分类。结果P75-NGFR的表达在侵袭性垂体腺瘤、高泌乳素水平,大腺瘤、多巴胺激动剂耐受的垂体腺瘤的表达明显要低于非侵袭性垂体腺瘤、低泌乳素水平,小腺瘤、多巴胺激动剂敏感的垂体腺瘤,而PCNA表达则明显要高,两者在各组中比较均具有显著性差异(p<0.05),而TrkA的表达在各组间没有明显差异;男性垂体泌乳素腺瘤PCNA表达明显要高于女性垂体腺瘤(p<0.05),而P75-NGFR及TrkA的表达在男性与女性垂体泌乳素腺瘤之间没有明显差异。结论P75NGFR的表达水平能间接反映垂体泌乳素腺瘤的增殖活性及对多巴胺受体激动剂的敏感性,P75NGFR有可能是某些腺瘤新的肿瘤生长抑制基因。第二部分神经生长因子(NGF)对GH3细胞增殖活性及细胞周期的影响目的通过观察NGF对GH3细胞增殖及细胞周期及其受体表达的影响,探讨其诱导GH3细胞分化的机制。方法应用MTT法、流式细胞仪、免疫组化法及western blot分别检测不同浓度NGF(10ng/ml、20ng/ml、50ng/ml和100ng/ml)对GH3细胞增殖的抑制情况、细胞周期的影响及两种多巴胺受体的表达情况。结果不同浓度的NGF作用下均能抑制GH3细胞的增殖,使细胞S期明显减少,G0/G1比例增高,P75NGFR受体的表达明显增高,与对照组相比,差异有显著性,而TrkA受体在对照组中的表达与各实验组比较,无显著性差别。当NGF浓度为50ng/ml时,其抑制增殖效应,P75NGFR受体的表达量最高。结论NGF可诱导GH3细胞分化,降低其增殖活性,S期比例降低,这可能与NGF诱导其P75NGFR受体的表达上调有关。第三部分神经生长因子(NGF)对GH3细胞PRL激素分泌活性及D2受体表达的影响目的通过检测NGF应用后GH3细胞激素分泌活性及D2受体表达的改变,以更深入了解NGF对PRL腺瘤的作用机制。方法应用酶联免疫吸附实验检测了NGF应用前后对GH3细胞PRL激素分泌的改变,通过RT-PCR方法检测了NGF应用前后GH3细胞膜上D2受体的表达。结果单独应用NGF对GH3细胞PRL激素分泌抑制作用较小,联合应用NGF及溴隐停较各单用组对PRL激素分泌抑制作用更加明显,(p<0.05)。结论NGF可增强溴隐停对GH3细胞PRL激素分泌抑制作用,作用机制可能与其诱导GH3细胞多巴胺D2受体的重新表达有关。更多还原

【Abstract】 Part I Study of correlativity between the NGFR expression and thebiological characters in prolactin-secreting pituitary adenomaObjective To explore the relationship between the expression of two receptors of nerve growth factor(NGF) , proliferation nuclear antigen (PCNA) and the biological characters of Prolactinomas. Methods The tumor specimens of 38 patients with prolactinoma was obtained by transsphenoid approach from 2003 to 2005.The expression of two receptors of nerve growth factor(NGF), Proliferation nuclear antigen (PCNA) was detected by immunohistochemistry methods. The patients were classified according to the invasion of surrounding structures or not, the volume of tumor, sex, the level of Prolactin secretion and the sensitivity to the dopamine agonist therapy. Results The expression of P75-NGFR was lower in invasive Prolactinomas , the tumor with high level of prolactin , macroadenomas , dopamine agonist -resistance adenomas than non-invasive Prolactinomas, the tumor with low level of prolactin , microadenomas, dopamine agonist-responsive adenomas respectively(p<0.05). The expression of was PCNA relatively higher, but the expression of another receptor of NGF (TrkA) is no differences between the each group. The PCNA in male prolactinomas was higher than that in female prolactinomas (p<0.05) and the two receptors of NGF were no difference between the male and female groups. Conclusions The expression of P75-NGFR could indirectly reflect the proliferation activity and the sensitivity to dopamine agonist therapy of the tumor. P75NGFR could be a novel tumor suppression gene of certain adenomas.Part II The effect of proliferation activity and cell cycle inGH3 cell treated by NGFObjective To explore the mechanism of differentiation induced of by NGF administration in GH3 cell by observing the changes of proliferation, cell-cycle and the expression of its receptor. Methods The GH3 cell was treated by NGF with different concentrations, MTT was detected the proliferation of GH3 by NGF, and Flow cytometry was used for evaluating the cell cycle and the expression of two NGF receptors was detected by immunohistochemistry and western blot after NGF administration. Results The proliferating ability of GH3 cell was decreased by NGF, The GH3 cell showed the reduction of proportion of S phase and cell was arrested at G0/G1 phase. The expression of P75NGFR was up-regulated after NGF treatment. Comparing with the control group, All the effect at other groups were significantly different. The maximal inhibition effect of proliferation and the expression value of P75NGFR were achieved at 50ng/ml NGF. Conclusions NGF could induce the differentiation of GH3 cell into the more benign phenotype, the mechanism of reduction of proliferation and proportion of S phase after NGF treatment were probably due to the up-regulation the P75NGFR. Part III The effect of secretion activity of prolactin and expression of dopamine receptor-2 in GH3 cell treated by NGFObjective To further investigate the mechanism treated by NGF through observing the changes of PRL secretion activity and the expression of dopamine receptor-2. Methods ELISA was measured for PRL basal secreting level and changes of its secreting level after administration the NGF and bromocriptine alone and combination, in GH3 cell. RT-PCR methods was detected the dopamine receptor-2 mRNA before and after administration the NGF. Results The inhibition effect of NGF on PRL secretion was little, and combination with bromocriptine could decrease the PRL secreting level obviously. Comparing with the other groups, the difference was statistically significant (p<0.05). The reexpression of dopamine receptor-2 mRNA was observed in GH3 cell treated by NGF and this effect was blocked by anti-NGF antibody. Conclusions NGF could enhance the inhibition effect of bromocriptine on PRL secretion, this action may be related to induction of the reexpression of dopamine receptor-2.更多还原