【作者】 张倩；

【导师】 陈靖； 郝传明；

【作者基本信息】 复旦大学 ， 内科学， 2010， 硕士

【摘要】 第一部分慢性肾衰竭时COX2下游通路mPGES1-EP2在增生的甲状旁腺组织中的异常表达目的：多种癌前病变及肿瘤组织中均发现COX2与mPGES1的协同高表达,该通路代谢产生的PGE2的受体EP2表达增加,而且表达阳性率越高,肿瘤恶性程度越高。慢性肾衰竭时在低钙、高磷或活性VitD缺乏等因素的持续刺激下,甲状旁腺由正常的低分化状态向增生状态转变,并逐渐呈自主性增殖,最终可导致单克隆结节、腺瘤甚至腺癌。本课题组既往研究发现在慢性肾衰竭患者及大鼠模型增生的甲状旁腺结节中均存在COX2的高表达,并且表达量与增生程度相关。为了证实COX2下游通路mPGES1-EP2在慢性肾衰竭甲状旁腺异常增生的作用,我们在本部分的研究中拟通过体内研究直接观察mPGES1-EP2是否参与慢性肾衰竭继发性甲旁亢的发生发展。(一)慢性肾衰竭继发性甲状旁腺功能亢进患者甲状旁腺组织中COX2下游通路mPGES1-EP2的表达方法：2009年4月至2010年3月在我院行甲状旁腺全切加自体前臂移植术的9名尿毒症患者的术后大、小结节共37个。制备石蜡和冰冻切片,HE染色区分甲状旁腺结节性与弥漫性增生,分别用免疫组化、免疫荧光和Western blot检测PGE2合成酶mPGES1, PGE2受体EP2以及增殖指标PCNA在甲状旁腺组织中的分布区域和表达量。结果：1.HE染色显示37个结节中有16个结节为轻度弥漫性增生,21个结节为结节性增生。2.免疫组化显示弥漫性增生的甲状旁腺组织中mPGES1表达较少,结节性增生的甲状旁腺mPGES1表达明显增加,其阳性染色主要分布于甲状旁腺细胞浆。采用免疫荧光的方法进一步证实mPGES1主要表达在甲状旁腺细胞的胞浆,尤其是细胞核的周围。3.免疫组化显示弥漫性增生的甲状旁腺组织中几乎未见EP2的表达,而结节性增生的甲状旁腺组织中EP2表达明显增加,散在性分布于细胞胞浆。免疫荧光的方法同样证实EP2主要表达在细胞的胞浆,尤其是细胞核的周围。4. Western blot检测证实EP2的蛋白表达水平在结节性增生部位明显高于弥漫性增生部位,增殖指标PCNA的蛋白表达也呈现同样的变化趋势。结论：慢性肾衰竭继发性甲状旁腺功能亢进患者增生的甲状旁腺组织中存在mPGES1-EP2的异常高表达,在结节性增生的部位更明显,可能与甲状旁腺的增生有关。(二)尿毒症继发性甲旁亢大鼠增生的甲状旁腺组织中COX2下游通路mPGES1-PGE2-EP2的表达方法：50只雄性SD大鼠,30只行5／6肾大部切除,20只行假手术。术后分2组：①假手术组(Sham)：假手术+正常饮食(P 0.8%,Ca 1.2%)；②肾大切组(Nx-HP)：5／6肾大部切除+高磷饮食(P 1.2%,Ca 1.2%)。3月后检测各组大鼠肾功能、血钙、血磷、iPTH水平；分离甲状旁腺,石蜡包埋连续切片进行HE染色,计算甲状旁腺最大面积反映腺体大小；Elisa方法检测PGE2水平；通过免疫组织化学,Western Blot方法或Real-time PCR方法检测组织中mPGES1、EP2、PCNA的表达,采用Real-time PCR方法检测EP1、EP3、EP4、IP、TP mRNA的表达。结果：1.成模后12周,5／6肾大部切除大鼠出现明显蛋白尿和肾功能严重损害。与Sham组大鼠相比,Nx-HP组大鼠iPTH水平显著升高,甲状旁腺腺体明显增大；PCNA阳性细胞显著增多,弥漫性分布于整个腺体；定量分析显示甲状旁腺组织中PCNA蛋白和mRNA水平明显升高；提示模型动物出现慢性肾衰竭继发性甲状旁腺功能亢进及腺体弥漫性增生。2.甲状旁腺局部的PGE2水平增加,免疫组织化学染色显示mPGES1, EP2在Sham组大鼠甲状旁腺组织中几乎未见表达,而Nx-HP组大鼠阳性细胞显著增多,弥漫性分布于腺体主细胞胞浆。Western Bolt和Real-time PCR进一步证实了这一点。应用Real-time PCR检测PGE2其他的受体表达,结果显示,Sham组与Nx-HP组两组大鼠甲状旁腺EP1、EP3、EP4 mRNA的表达均无差异；两组大鼠前列环素受体IP mRNA表达无明显差异；Nx-HP组大鼠TXA2受体TP mRNA较Sham组大鼠低下。结论：与正常对照组相比,5／6肾大部切除大鼠高磷饮食三个月后,出现明显肾功能损害和继发性甲旁亢伴甲状旁腺增生；在增生的腺体中COX2下游通路mPGES1和EP2的表达显著增加,与增生指标PCNA密切相关,提示mPGES1-EP2可能参与了尿毒症时甲状旁腺增生。第二部分体外研究观察COX2下游通路mPGESl-PGE2-EP2对甲状旁腺异常增生的影响目的：本课题第一部分在体研究显示COX2下游通路mPGESl-EP2在慢性肾衰竭异常增生的甲状旁腺组织中表达增加,并与增殖指标相关。为了排除众多体内因素的干扰,本部分研究将选择甲状旁腺全切加自体前臂移植术中所得的尿毒症患者甲状旁腺组织块进行体外孵育,旨在探讨阻断或促进COX2下游通路mPGESl-PGE2-EP2的作用能否直接影响PTH分泌和甲状旁腺的增生,从而揭示COX2下游通路mPGESl-PGE2-EP2在尿毒症时甲状旁腺异常增生中的确切作用。方法：体外37℃孵育新鲜获得的尿毒症患者甲状旁腺组织块,首先观察阻断mPGESl-PGE2-EP2对高磷刺激的影响,分组如下：(1)正常磷组(NP)：DMEM—F12培养液；(2)高磷组(HP)：高磷培养液(4mmol/L);(3)HP+NS398 (1μmol/L):NS398为COX2抑制剂；(4) HP+MK886 (1μmol/L):MK886为mPGESl抑制剂；(5)HP+AH6809 (1μmol/L):AH6809为EP2阻断剂。然后观察不同浓度EP2受体激动剂Butaprost的作用,分组如下：(1)对照组：DMEM—F12培养液；(2)10-7MButaprost; (3) 10-6MButaprost; (4) 10-5MButaprost。最后观察不同浓度PGE2的直接作用,分组如下：(1)对照组：DMEM—F12培养液；(2)10-7M PGE2；(3)10-6M PGE2；(4)10-5M PGE2。24小时后收集上清液和各组细胞,抽提总蛋白,总RNA。用免疫化学发光法检测iPTH水平,用Real-time PCR检测PreproPTH基因水平,Western blot和Real-time PCR检测PCNA蛋白和基因水平。结果：1.在体外孵育的尿毒症患者甲状旁腺组织块中,高磷刺激可导致上清液中iPTH水平明显升高(改变的倍数：NP1.00±0.49,HP4.54±2.11,p<0.05),分别给予COX2拮抗剂NS398(10-6M)、mPGES1拮抗剂MK886(10-6M)或EP2拮抗剂AH6089(10-6M)干预后,高磷的上述作用被明显抑制(改变的倍数：NS398+HP 2.00±1.24, MK886+HP 1.88±0.58, AH6809+HP 0.80±0.76,p<0.05)定量检测preproPTH mRNA的表达水平也呈现一致的变化趋势。另外,高磷刺激后甲状旁腺组织中PCNA的蛋白和基因表达水平也明显增加,给予各种拮抗剂干预后,高磷的上述作用被明显抑制,提示COX2-mPGES1-PGE2-EP2通路参与了高磷诱导的iPTH分泌和甲状旁腺增生。2.在体外孵育的甲状旁腺组织块中给予Butaprost浓度为10-7M时,上清液中PTH水平无明显变化；当浓度上升为10-6M时,PTH水平显著升高(1.74±0.28,p<0.05)；但Butaprost浓度为10-5M时,PTH水平反而下降(10-5M 0.42±0.18,p>0.)。preproPTH mRNA水平以及PCNA蛋白和基因的表达也有相似的变化趋势,提示EP2激动剂能直接促进iPTH合成分泌和甲状旁腺增生。3.直接给予体外孵育的甲状旁腺组织块PGE2刺激,当浓度为10-7M和10-6M时,上清液中PTH水平均无明显变化；当PGE2浓度上升为10-5M时,PTH水平显著升高(3.88±1.66,p<0.05)。组织块中preproPTH mRNA水平的升高在PGE2浓度为10-6M时已出现,当PGE2上升为10-5M时更明显(Control 14.78±3.59 vs. 10-5M 271.26±150.49,p<0.05).当PGE2浓度为10-7M时,组织块中PCNA蛋白表达无明显变化,但随着PGE2浓度上升,PCNA表达水平显著增加(Control 0.29±0.12 vs.10-6M 1.72±0.40, 10-5M 2.33±0.12,p<0.05)；PCNA基因表达的改变也与之相似。结论：COX2下游通路mPGES1-PGE2-EP2直接参与了尿毒症时甲状旁腺组织的增生,阻断该通路能明显纠正甲旁亢和和甲状旁腺的异常增生。更多还原

【Abstract】 PART I The expression of COX2 downstream mPGESl-PGE2-EP2 in the hyperplasia of parathyroid from chronic renal failure in vivoObjectiveSecondary hyperparathyroidism in patients with chronic kidney disease is characterized by hyperplasia of the parathyroid gland. In our previous work it was found that cyclooxygenase-2 (COX2) was detected in parathyroid gland in uremic patients and 5/6-nephrectomized rats and the expression of COX2 was associated with parathyroid gland cell proliferation. Since aberrant COX2 downstream mPGES1-EP2 expression has been shown to promote the epithelial cell proliferation in a number of tumors and precancerous lesion, the present studies examined potential role of mPGESl-EP2 in hyperplasia of the parathyroid gland in uremic patients and 5/6-nephrectomized (Nx) rats in vivo.1. The expression of mPGESl and EP2 in SHPT patientsMethodsParathyroid gland samples were collected from the uremic patients who accepted the parathyroidectomy with forearm autotransplation. According to the H.E staining the glands were divided into two groups including diffuse hyperplasia and nodular hyperplasia. mPGESl expression was examined by immunohistochemistry and immunofluorescence. EP2 and PCNA expression were determined by immunohistochemistry、immunofluorescence and immunoblot respectively.ResultsAmong all the glands, twenty one glands belonged to nodular hyperplasia and sixteen glands belonged to diffuse hyperplasia. The immunohistochemistry and immunofluorescence staining showed the expressions of mPGES1 and EP2 were more on the nodular hyperplasia than the diffuse hyperplasia glands. The cellular localization of mPGES1 and EP2 protein was cytoplasmic and preferentially perinuclear. The expression of PCNA, a marker of proliferation, was more on the nodular hyperplasia glands. The EP2 and PCNA expressions in PGs were further supported by immunoblot.ConclusionmPGES1 and EP2 exited on the glands of proliferated parathyroid cell. The expressions of mPGES1 and EP2 were associated with the PCNA.2. The expression of mPGESl-PGE2-EP2 in the parathyroid glands of chronic renal failure ratsMethodsThirty 5/6-nephrectomized and twenty sham operated rats were assigned to 2 groups:①Sham group:sham-operated+normal diet (P 0.8%, Ca 1.2%);②Nx-HP group: Nx+high phosphorus diet (P1.2%, Ca 1.2%). At the end of 3 month, blood, urine and parathyroid samples were collected. The expressions of mPGES1, EP2, PCNA were determined by immunohistochemistry, immunoblot and Real-time PCR. The level of PGE2 was assayed by Elisa. EP1, EP3, EP4, TP and IP were evaluated by Real-time PCR.ResultsNx-HP rats fed with high phosphorus diet for 3 months manifested progressively decreasing body weight and increasing proteinuria, serum creatinine, urea nitrogen as well as augmentation of parathyroid gland volume accompanied by up-regulation of PCNA expression, suggesting that secondary parathyroid hyperplasia animal model was established successfully. The level of PGE2 was increased in the Nx-HP group.Diffused positive staining of mPGES1 and EP2 were detected in the chief cells of parathyroid gland from Nx-HP rats, while nearly no positive cells were found in sham group, determined by immunohistochemistry. Western blot analysis showed that the protein levels of EP2 in parathyroid gland were greatly increased in Nx-HP group compared with that in sham group respectively. Real-time PCR analysis also demonstrated the same trends of mRNA expression of mPGES1 and EP2.There were no differences of EP1, EP3, EP4 mRNA expressions among two groups.ConclusionOur data in vivo suggested that mPGES1 and EP2 were detected in parathyroid gland of 5/6-nephrectomized rats. The expression of mPGES1 and EP2 were associated with parathyroid gland cell proliferation.PARTⅡEffect of mPGESl-PGE2-EP2 on the hyperplasia of parathyroid glands from Uremic Patients in vitroObjectiveOur data in vivo suggested that mPGES1 and EP2 were detected in parathyroid gland in uremic patients and 5/6-nephrectomized rats. The expression of mPGES1 and EP2 were associated with parathyroid gland cell proliferation. The second part of this study, therefore, is to investigate the effect of mPGESl-EP2 on the hyperplasia of parathyroid glands in vitro.MethodsWe established the method of parathyroid tissue slices culture according to literature. The tissue slices were cultured in different culture fluid for 24h. The tissues were divided as followings:Part a:NP group (phosphate concentration lmmol/L), HP group (phosphate concentration 4mmol/L), NS398+HP group (the COX2 inhibitor NS39810-6 mol/L were added), MK886+HP group (the mPGES1 inhibitor MK886 10-6 mol/L were added), AH6809+HP group (the antagonist of EP2 AH6809 10-6 mol/L were added); Part b:control group,10-7M Butaprost group (the agonist of EP2 Butaprost 10-7 mol/L were added),10-6M Butaprost group (Butaprost 10-6 mol/L were added),10-5M Butaprost group (Butaprost 10"5 mol/L were added); Part c: control group,10-7M PGE2 group (PGE2 10-7 mol/L were added),10-6M PGE2 group (PGE2 10-6 mol/L were added),10-5M PGE2 group (PGE2 10-5 mol/L were added). Incubation took place in an incubator at 37℃with the 10ml polypropylene tubes placed on a shaker. Each experiment was performed in triplicate. We detect the parathyroid hormone in the two groups. We observe the expression of PreproPTH and PCNA mRNA levels by Real-time PCR. And we also investigated PCNA protein levels by Western blot.ResultsPart a:After 24h exposure of parathyroid tissue slices to high phosphate (4mmol/L), PTH level was increased compared to NP group (1mmol/L) (p<0.05), the COX2 inhibitor NS398 (10-6 mol/L), the mPGES1 inhibitor MK886(10-6 mol/L) and the antagonist of EP2 AH6809 (10-6 mol/L) can inhibit this effect (p<0.05); The expression of PreproPTH mRNA and PCNA showed the similar result. Part b: Butaprost 10-7M and Butaprost 10-5M had no effect on PTH level. Butaprost 10-6M increased the PTH level (p<0.05). The expression of PreproPTH mRNA was also increased in Butaprost 10-6M group. Western blot showed the PCNA protein abundance of Butaprost 10-6M group was increased compared with the control group. The expression of PCNA mRNA showed the similar result. Part c:PGE2 10-7M and PGE2 10-6M had no effect on PTH level. PGE2 10-5M increased the PTH level (p<0.05). The expression of PreproPTH mRNA was increased in PGE2 10-6M group and PGE2 10-5M group. Western blot showed the PCNA protein abundance of Butaprost 10-6M and Butaprost 10-5M group were significantly increased compared with the control group. The expression of PCNA mRNA showed the similar result.ConclusionOur results therefore demonstrated that COX2 downstream mPGES1-EP2 antagonist can inhibitor PTH excretion and the hyperplasia of parathyroid tissue. EP2 agonist and PGE2 can stimulate PTH excretion and the hyperplasia of parathyroid tissue in vitro.更多还原