PKCδ在垂体GH腺瘤细胞内异常信号通路中的调控机制及其与SSTA疗效的关系

发布时间：2018-02-24 08:03

本文关键词： 原代培养人垂体GH腺瘤细胞 PKCδ CREB ERK1/2 PMA Rottlerin PKCδ CREB ERK1/2 gsp癌基因 SSTA PKCδ CREB ERK1/2　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分 PKCδ相关信号通路因子在体外原代培养垂体GH腺瘤胞内的表达目的探究原代培养人垂体GH腺瘤细胞的方法,证实PKCδ及其相关信号通路因子表达于人垂体GH腺瘤细胞胞内。方法收集5例垂体生长激素腺瘤患者术中切除肿瘤组织,同时行原代细胞培养及常规石蜡包埋切片,免疫荧光及免疫组化法检测PKCδ、CREB、ERK1/2蛋白的表达情况。结果静置24小时后,可见观察到原代细胞呈圆形透亮状贴壁生长,可呈“铺路石”样外观。免疫组化及免疫荧光法证实PKCδ及ERK1/2主要表达于胞质。CREB主要表达于胞核,不同样本之间表达水平存在个体差异。结论成功获得体外原代培养人垂体GH腺瘤细胞,PKCδ、CREB、ERK1/2均表达于人垂体GH腺瘤细胞内,且表达水平存在个体差异。第二部分 PKCδ相关信号通路因子在垂体GH腺瘤原代培养细胞模型中作用及调节机制研究目的探究PKCδ相关信号通路因子在垂体GH腺瘤原代培养细胞模型中的作用及调节机制。方法使用PMA、Rottlerin及两者联合分别干预原代培养的人垂体GH腺瘤细胞,GH-Elisa检测各实验分组细胞分泌GH水平变化,CCK-8检测各实验分组细胞活性变化,Western blot检测CREδ、ERK1/2蛋白磷酸化水平变化结果加入PMA后,细胞GH分泌及活化增殖水平上调,p-CREB以及p-ERK1/2的磷酸化水平均呈不同程度上调趋势；而加入Rottlerin后,细胞GH分泌及活化增殖水平下降,p-PKCδ、p-CREB以及p-ERK1/2的磷酸化水平均呈不同程度下降趋势,上述两组与对照组相比p值均0.05,存在统计学差异。联合使用PMA及Rottlerin后,较之单纯使用PMA,细胞GH分泌及活化增殖水平下降,p-CREB以及p-ERK1/2均呈不同程度下降趋势：但较之单纯使用Rottlerin细胞GH分泌及活化增殖水平上调,p-CREB以及p-ERK1/2均呈不同程度上调趋势,上述p值均0.05,存在统计学差异。结论在原代培养的人垂体GH腺瘤中,激活PKCδ不仅可轻微提高CREB的磷酸化水平并部分促进GH分泌的效应,同时可提升ERK1/2的磷酸化水平并增强细胞的增殖效应。但鉴于不同原代样本间存在较大个体差异,上述结论尚需扩充例数以便进一步研究证实。第三部分 SSTA疗效与PKCδ相关信号通路的相互作用及其与gsp癌基因的关系目的探究SSTA疗效与PKCδ相关信号通路因子的关系,及其与gsp癌基因的联系方法使用SSTA干预原代培养的人垂体GH腺瘤细胞,GH-Elisa检测各实验分组细胞分泌GH水平变化,CCK-8检测各实验分组细胞活性变化,Western blot检测PKCδ、CREB、ERK1/2蛋白磷酸化水平变化。同时筛选20例体内及体外SSTA抑制效果一致的垂体GH腺瘤患者资料,通过PCR法检测gsp突变情况。结果20例样本中,SSTA有效13例,其中gsp癌基因阳性6例,阴性7例；无效7例,均为gsp癌基因阴性。gsp癌基因(+)病例组GH分泌抑制效果优于gsp癌基因(-)病例组,但进一步行χ2检验示p值0.05,提示gsp阳/阴性样本间SSTA有效率差别无统计学差异。使用SSTA干预原代培养的人垂体GH腺瘤细胞后,细胞GH分泌及活化增殖水平下降,同时p-PKCδ、p-CREB以及p-ERK1/2均呈不同程度下降趋势结论SSTA作用于原代培养的人垂体GH腺瘤细胞后,细胞的GH分泌及增殖活性下降水平存在个体差异,同时p-PKCδ、p-CREB以及p-ERK1/2均呈不同程度下降趋势。使用SSTA药物后,gsp癌基因(+)病例组GH分泌抑制效果优于gsp癌基因(-)病例组,但就本组病例而言,尚不能认为组间差异具有统计学意义。鉴于不同原代样本间存在较大个体差异,上述结论尚需扩充例数以便进一步研究证实。
[Abstract]:Part one
Expression of PKC delta related signal pathway factor in primary culture of pituitary GH adenoma in vitro
Objective to explore the primary culture of human pituitary GH adenoma cells, and to confirm that PKC Delta and its related signaling pathway factors are expressed in the cell of human pituitary GH adenoma cells.
Methods a total of 5 patients with pituitary growth hormone adenoma were resected, and primary tumor cell culture and conventional paraffin embedded sections were performed. Immunofluorescence and immunohistochemistry were used to detect the expression of PKC Delta, CREB and ERK1/2 protein.
The results of static after 24 hours, observed visible primary cells were round and bright like adherent growth, can be a paving stone like appearance. Immunohistochemistry and immunofluorescence confirmed that PKC and ERK1/2 mainly expressed in the cytoplasm of delta.CREB expressed mainly in the nucleus. There are individual differences in expression level between different samples.
Conclusion the primary cultured human pituitary GH adenoma cells were successfully obtained. PKC Delta, CREB and ERK1/2 were expressed in human pituitary adenoma cells, and there were individual differences in the expression level. The second part is the difference.
The role of PKC delta related signal pathway factor in the primary cultured cell model of pituitary GH adenoma and its regulatory mechanism
Objective to explore the role and regulation mechanism of PKC delta related signal pathway factor in the primary culture cell model of pituitary GH adenoma.
Methods PMA, Rottlerin and their combination were used to intervene the primary cultured human pituitary GH adenoma cells. GH-Elisa was used to detect the secretion of GH in each experimental group. CCK-8 was used to detect cell activity changes in each experimental group. Western blot was used to detect CRE Delta and the phosphorylation level of ERK1/2 protein was changed.
Results after joining PMA, GH cell secretion and activation and proliferation of p-CREB and upregulated the phosphorylation level of p-ERK1/2 were up-regulated; and after joining the Rottlerin, the GH cell activation and proliferation and secretion levels decreased, p-PKC Delta, p-CREB and p-ERK1/2 phosphorylation level showed a decreased trend, the two group and control compared with group P value was 0.05, there was no significant difference. The combined use of PMA and Rottlerin, compared with PMA, GH cell activation and proliferation and secretion levels decreased, p-CREB and p-ERK1/2 were decreased: but compared with the single use of Rottlerin cell activation and proliferation of GH secretion and increase the level of p-CREB and p-ERK1/2 were different. The degree of upward trend, the p value was 0.05, there was no significant difference.
Conclusion in primary cultured human pituitary adenoma GH, activation of PKC delta not only can slightly increase the phosphorylation level of CREB and promote GH secretion, also can improve the level of ERK1/2 phosphorylation and enhanced proliferation of effector cells. But in view of the fact that there exist great individual differences in different primary samples, this conclusion still needs to expand the number of cases in order to further study.
The third part
Interaction between SSTA effect and PKC delta related signal pathway and its relationship with GSP oncogene
Objective to explore the relationship between the effect of SSTA and the PKC delta signal pathway factor and its association with the GSP oncogene
Methods using the SSTA intervention in primary cultured human pituitary adenoma cells GH, GH levels in each experimental group to detect secretion of GH-Elisa cells, changes in all experimental groups CCK-8 cell activity detection, Western detection of blot PKC Delta, CREB, the phosphorylation of ERK1/2. At the same time, a total of 20 cases of in vivo and in vitro inhibitory effect of SSTA was consistent with pituitary GH adenomas were detected by GSP method, PCR mutation.
Results in 20 cases, SSTA 13 cases were effective, the GSP gene was positive in 6 cases, 7 cases were negative; 7 cases were invalid, GSP gene negative.Gsp gene (+) group GH secretion inhibition effect is better than that of cancer gene GSP (-) patients, but further 2 test shows the p value 0.05, suggesting that GSP positive / negative samples SSTA was not statistically significant difference. The use of SSTA intervention in primary cultured human pituitary adenoma cells GH, GH secretory cell proliferation and activation level decreased, while the p-PKC Delta, p-CREB and p-ERK1/2 were decreased
Conclusion the effect of SSTA on primary cultured human pituitary adenoma cells GH, cell GH secretion and proliferation activity of individual differences in the level of p-CREB and p-PKC Delta, and p-ERK1/2 were decreased. The use of SSTA drugs, cancer gene GSP (+) group GH secretion inhibition effect is better than that of GSP gene (-) the case group, but this group of cases, it is not considered a statistically significant difference between the groups. In view of the fact that there exist great individual differences in different primary samples, this conclusion still needs to expand the number of cases confirmed by further studies.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R736.4

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