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【摘要】目的研究神经肽内皮素(ET)和心钠素(ANP)在细胞培养及体内实验中对促甲状腺激素释放激素受体(TRHR)基因表达的影响。方法Northern印迹和S1核酸酶保护实验检测TRHR mRNA量，新生肽链试验测定TRHR基因的转录。结果ET-3 2h即增加胶质细胞TRHR mRNA量，6～8h达高峰，增加3倍以上(3.0±0.52)；相反，ANP 2h即降低TRHR mRNA，6～8h达最低值，并逆转60～80%因血浆所增高的TRHR mRNA。ET-3在6h时增加TRHR基因的转录达2倍(nuclear run on试验)，而ANP对TRHR基因的转录无明显变化。体内实验结果显示ET-3和ANP对TRHR mRNA影响与体外实验相似。结论ET-3可能通过调节TRHR基因的表达而影响TRHR mRNA，而ANP则可能影响TRHR mRNA的降解而降低mRNA的水平。
TRH receptor gene expression in central nervous system is regulated by endothelin and atrial natriuretic peptideHu Renming,Luo Bangyao,Ellis R Levin,et al.Depatment of Endocrinology,Rui Jin Hospital,Shanghai Second Medical University,Shanghai,200025
【Abstract】ObjectiveTo determine whether two neuropeptides, endothelin (ET) and atrial natriuretic peptide (ANP) regulate thyrotropin-releasing hormone receptor (TRHR) gene expression in astrocytes and hypothalamic tissues cultured both in vivo and in vitro.MethodsNorthern blot and S1 nuclease protection were used to determine steady-state mRNA. Nuclear run on transcription was adopted to examine the rate of transcription of the TRHR gene.ResultsET-3 increased steady-state mRNA levels by more than 3-fold (3.1±0.52) in cultured diencephalic astrocytes. Time-course studies indicated that the induction of TRHR mRNA began at 2 hours, peaked after 6-8h, and persisted above baseline for 24h. In contrast, ANP reduced basal and reversed plasma-induced TRHR mRNA by approximately 60-80%. Decrease in mRNA was seen after 2h, reached a nadir at 4-6h, and persisted for 24h. The rate of transcription of the TRHR gene, measured by nuclear run-on assay, was increased for about 2-fold in cells treated for 6 hours with ET-3 (100nmol/L), but was not significantly decreased in cells treated with ANP (100nmol/L). In vivo studies showed that ET-3 and ANP affectedTRHR mRNA in a similar way to their effects in cultured astrocytes.Conclusion The neuropeptide ET-3 stimulates TRHR gene expression both in vitro and in vivo, most likely by controlling the rate of transcription, while ANP inhibits steady-state TRHR mRNA levels, perhaps by increasing TRHR transcript degradation.
【Key words】Thyrotropin-releasing hormone receptorEndothelinAtrial natriuretic peptide Gene expression
(Chin J Endocrinol Metab, 1998, 14:318-321)
下丘脑神经元和胶质细胞产生和分泌血管活性肽心钠素(ANP)和内皮素(ET)。ANP和ET不仅为强大的血管活性物质，且为重要的神经递质。晚近我们已报告ANP和ET调节下丘脑胶质细胞和神经元基因的表达〔1，2〕，本文研究ET和ANP对促甲状腺激素释放激素受体(TRHR)基因表达的影响。
材料和方法
一、细胞培养：从妊娠16天胎鼠取出下丘脑区域的组织，分离细胞，经光镜及免疫化学染色证实95%分离的细胞为胶质细胞。99%第二代培养的细胞为胶质细胞。胶质细胞在ME/F12培养液(无血浆或含10% FBS)孵育或加入ANP(10-7mol/L)或ET-3(10-7mol/L)2-24h，然后抽提总RNA。
二、S1核酸酶保护实验(S1 nuclease protection)〔3，4〕：从经ANP、ET处理的胶质细胞或下丘脑组织中抽提总RNA(用Tri ReagentTM试剂盒Molecular Research Center, Inc. 美国)用Rsal酶解含有TRHR 430个核苷酸cDNA片段的质粒(P Blnescript质粒，Dr. Marrinc Gershergorn赠送)，用T3聚合酶转录Rsal酶解的质粒，制备32P标记的反义探针(TRHR-cRNA)。此标记物为与TRHR互补的170个核苷酸片段。10微克总RNA与TRHR-cRNA标记物在58℃杂交液中孵育20小时，S1核酸酶酶解未杂交的核酸，变性聚丙烯酰胺凝胶电泳分离游离的核酸。
三、Northern印迹〔5〕，变性的20微克总RNA，上样于1.2%凝胶，以10mmol/L磷酸缓冲液作为电泳缓冲液，约1小时后，当溴酚蓝染料指示剂至凝胶长度约3/4时停止电泳，并将分离的RNA转移至硝酸纤维膜(或尼龙膜)经预杂交后再在42℃孵育箱中与TRHR-cRNA标记物杂交24小时，在68℃中用2×SSC(含0.1% SDS)洗脱液洗膜，然后在室温中放射自显影2～3天。
四、新生肽链试验(Nuclear run on)〔1，3〕。培养的细胞经ANP或ET处理后，用PBS洗脱二次，然后用NP-40裂解液裂解细胞，离心收集细胞核并冷冻保存于甘油缓冲液待用。细胞核加入等量含有4mmol/L ATP、GTP和CTP及10μl 32 P-UTP的反应液，孵育30分钟后用DNase酶消化DNA及用蛋白激酶酶切蛋白质，测定并沉淀32P标记的RNA。将等量的TRHR及β-actin的cDNA转移至硝酸纤维膜(每个条状带含相同DNA量)。将含有TRHR及β-actin cDNA的膜分别与不同时间ET或ANP处理后制备的32P标记的RNA杂交。杂交36小时后，洗脱硝酸纤维膜，然后放射自显影。
五、体内研究：选用200～250克的雄性鼠，麻醉后仍具有自主呼吸，通过埋入第三脑室的导管，以10μl/h的速度注入ET-3(100nmol/L)、ANP(100nmol/L)或对照(0.9% NaCl)，6小时后，取鼠下丘脑组织，液氮处理后保存于-70℃，以便抽提RNA。
六、光密度测定：S1核酸酶保护实验及新生肽试验结果测定每个条带的光密度值，H-ras或β-actin显示每个样品的加样情况。TRHR测值/II-Ras或β-actin测值之比进行统计学处理。以上试验分别重复三次。
结果
S1核酸酶保护实验以及Northern印迹结果示：ET和ANP可影响TRHR mRNA的含量。ET-3(10-7mol/L)在处理胶质细胞2小时后即明显增加TRHR mRNA水平，6～8小时达高峰，增加三倍以上(3.1±0.52)，24小时后TRHR mRNA仍高于正常。相反，ANP(10-7mol/L)则在2小时后使TRHR mRNA量开始下降，4～6小时达最低值，与对照组相比下降60～80%(图1)，24小时后恢复正常。
图1S1核酸酶保护试验：A、经ANP或ET-3不同时间处理的细胞的总RNA与32P-标记的TRHR cRNA探针杂交，用S1核酸酶酶解后经聚丙烯酰胺凝胶电泳分离；B、与32P标记的H-ras杂交作为加样量的控制
Fig 1S1 nuclease protection: A:Each lane represents the signal from 30μg of extracted total RNA hybridized with a 32P-labeled cRNA probe for TRHR, determined after S1 nuclease digestion, separated on polyacrylamide gel, and autoradiographied, B:control hybridizatioin with a probe for H-ras
图2Northern印迹：A、经ANP或ET-3处理的细胞的总RNA与32P标记的TRHR cRNA探针杂交；B、电泳后经溴乙淀染色的RNA上样量
Fig 2Northern blot analysis of the time-course of serum and ANP on the expression of the mRNA for TRHR in cultured astrocytes. A:Total mRNA was extracted, denatured, electrophoresed in an agarose gel and transferred to nitrocellulose membranes. The blots were hybridized with 32P-labeled cRNA for TRHR. B: Loading control of total mRNA stained with ethidium bromide after electrophoresis
图3核酸酶保护试验：上排：ANP或ET-3处理的下丘脑组织的RNA与32P标记的TRHR cRNA探针杂交，用S1核酸酶酶解后，经6%聚丙烯酰胺凝胶电泳分离；下排：与32P标记的H-ras杂交，作为加样量的控制
Fig 3In vivo study (S1 nuclease protection): Top row: The effects of ANP and ET on expression of the mRNA extracted from hypothalamus treated with ANP or extracted total RNA hybridized with a 32 P-labeled cRNA probe for TRHR, determined after S1 nuclease digestion and separated on polyacrylamide gel. Bottom row: Control hybridization with a probe for H-ras
