本文關(guān)鍵詞： 原代培養(yǎng) 人垂體GH腺瘤細(xì)胞 PKCδ CREB ERK1/2 PMA Rottlerin PKCδ CREB ERK1/2 gsp癌基因 SSTA PKCδ CREB ERK1/2　出處：《華中科技大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分 PKCδ相關(guān)信號通路因子在體外原代培養(yǎng)垂體GH腺瘤胞內(nèi)的表達(dá) 目的探究原代培養(yǎng)人垂體GH腺瘤細(xì)胞的方法,證實(shí)PKCδ及其相關(guān)信號通路因子表達(dá)于人垂體GH腺瘤細(xì)胞胞內(nèi)。 方法收集5例垂體生長激素腺瘤患者術(shù)中切除腫瘤組織,同時行原代細(xì)胞培養(yǎng)及常規(guī)石蠟包埋切片,免疫熒光及免疫組化法檢測PKCδ、CREB、ERK1/2蛋白的表達(dá)情況。 結(jié)果靜置24小時后,可見觀察到原代細(xì)胞呈圓形透亮狀貼壁生長,可呈“鋪路石”樣外觀。免疫組化及免疫熒光法證實(shí)PKCδ及ERK1/2主要表達(dá)于胞質(zhì)。CREB主要表達(dá)于胞核,不同樣本之間表達(dá)水平存在個體差異。 結(jié)論成功獲得體外原代培養(yǎng)人垂體GH腺瘤細(xì)胞,PKCδ、CREB、ERK1/2均表達(dá)于人垂體GH腺瘤細(xì)胞內(nèi),且表達(dá)水平存在個體差異。第二部分 PKCδ相關(guān)信號通路因子在垂體GH腺瘤原代培養(yǎng)細(xì)胞模型中作用及調(diào)節(jié)機(jī)制研究 目的探究PKCδ相關(guān)信號通路因子在垂體GH腺瘤原代培養(yǎng)細(xì)胞模型中的作用及調(diào)節(jié)機(jī)制。 方法使用PMA、Rottlerin及兩者聯(lián)合分別干預(yù)原代培養(yǎng)的人垂體GH腺瘤細(xì)胞,GH-Elisa檢測各實(shí)驗(yàn)分組細(xì)胞分泌GH水平變化,CCK-8檢測各實(shí)驗(yàn)分組細(xì)胞活性變化,Western blot檢測CREδ、ERK1/2蛋白磷酸化水平變化 結(jié)果加入PMA后,細(xì)胞GH分泌及活化增殖水平上調(diào),p-CREB以及p-ERK1/2的磷酸化水平均呈不同程度上調(diào)趨勢；而加入Rottlerin后,細(xì)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趨勢,上述兩組與對照組相比p值均0.05,存在統(tǒng)計(jì)學(xué)差異。聯(lián)合使用PMA及Rottlerin后,較之單純使用PMA,細(xì)胞GH分泌及活化增殖水平下降,p-CREB以及p-ERK1/2均呈不同程度下降趨勢：但較之單純使用Rottlerin細(xì)胞GH分泌及活化增殖水平上調(diào),p-CREB以及p-ERK1/2均呈不同程度上調(diào)趨勢,上述p值均0.05,存在統(tǒng)計(jì)學(xué)差異。 結(jié)論在原代培養(yǎng)的人垂體GH腺瘤中,激活PKCδ不僅可輕微提高CREB的磷酸化水平并部分促進(jìn)GH分泌的效應(yīng),同時可提升ERK1/2的磷酸化水平并增強(qiáng)細(xì)胞的增殖效應(yīng)。但鑒于不同原代樣本間存在較大個體差異,上述結(jié)論尚需擴(kuò)充例數(shù)以便進(jìn)一步研究證實(shí)。 第三部分 SSTA療效與PKCδ相關(guān)信號通路的相互作用及其與gsp癌基因的關(guān)系 目的探究SSTA療效與PKCδ相關(guān)信號通路因子的關(guān)系,及其與gsp癌基因的聯(lián)系 方法使用SSTA干預(yù)原代培養(yǎng)的人垂體GH腺瘤細(xì)胞,GH-Elisa檢測各實(shí)驗(yàn)分組細(xì)胞分泌GH水平變化,CCK-8檢測各實(shí)驗(yàn)分組細(xì)胞活性變化,Western blot檢測PKCδ、CREB、ERK1/2蛋白磷酸化水平變化。同時篩選20例體內(nèi)及體外SSTA抑制效果一致的垂體GH腺瘤患者資料,通過PCR法檢測gsp突變情況。 結(jié)果20例樣本中,SSTA有效13例,其中g(shù)sp癌基因陽性6例,陰性7例；無效7例,均為gsp癌基因陰性。gsp癌基因(+)病例組GH分泌抑制效果優(yōu)于gsp癌基因(-)病例組,但進(jìn)一步行χ2檢驗(yàn)示p值0.05,提示gsp陽/陰性樣本間SSTA有效率差別無統(tǒng)計(jì)學(xué)差異。使用SSTA干預(yù)原代培養(yǎng)的人垂體GH腺瘤細(xì)胞后,細(xì)胞GH分泌及活化增殖水平下降,同時p-PKCδ、p-CREB以及p-ERK1/2均呈不同程度下降趨勢 結(jié)論SSTA作用于原代培養(yǎng)的人垂體GH腺瘤細(xì)胞后,細(xì)胞的GH分泌及增殖活性下降水平存在個體差異,同時p-PKCδ、p-CREB以及p-ERK1/2均呈不同程度下降趨勢。使用SSTA藥物后,gsp癌基因(+)病例組GH分泌抑制效果優(yōu)于gsp癌基因(-)病例組,但就本組病例而言,尚不能認(rèn)為組間差異具有統(tǒng)計(jì)學(xué)意義。鑒于不同原代樣本間存在較大個體差異,上述結(jié)論尚需擴(kuò)充例數(shù)以便進(jìn)一步研究證實(shí)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R736.4

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