大鼠消化道促性腺激素釋放激素受體mRNA的原位雜交研究

1、    大鼠消化道促性腺激素釋放激素受體mRNA 的原位雜交研究        【摘要】目的探討大鼠消化道是否存在GnRH受體mRNA及其定位。方法用原位雜交組織化學(xué)法。結(jié)果胃底腺壁細(xì)胞、胃小凹上皮細(xì)胞可檢測(cè)到較強(qiáng)的GnRH受體mRNA雜交信號(hào)。3段小腸絨毛上皮細(xì)胞、小腸腺細(xì)胞可檢測(cè)到較強(qiáng)的GnRH受體mRNA雜交信號(hào)。盲腸、結(jié)腸和直腸的粘膜上皮細(xì)胞、腸腺上皮細(xì)胞也能檢測(cè)到GnRH受體mRNA雜交信號(hào)。信號(hào)物質(zhì)均分布在胞質(zhì)內(nèi)，胞核呈陰性。結(jié)論大鼠消化道能合成GnRH

2、受體。GnRH也是一種胃腸激素，它由消化系統(tǒng)自身合成，又作用于胃腸道參與胃腸功能的調(diào)節(jié)?！娟P(guān)鍵詞】GnRH受體mRNA；原位雜交；消化道；大鼠【中分類號(hào)】R322.4【文獻(xiàn)標(biāo)識(shí)碼】A【文章編號(hào)】0529-1356(2000)03-250 In situ HYBRIDIZATION STUDY OF GONADOTROPIN RELEASING HORMONE RECEPTOR mRNA IN RAT DIGESTIVE TRACTYAO BingHUANG Wei-quanPU Ruo-leiSUN LanZHANG Rong-qingWANG LeiDepartment of Histolo

3、gy and Embryology, the Fourth Military Medical University, Xi'an 710032,ChinaDepartment of Biology, Tsinghua University, Beijing 100084,China【Abstract】ObjectiveTo study whether gonadotropin releasing hormone receptor(GnRHR) mRNA exists in rat digestive tract and it's cellular localization. M

4、ethodsIn situ hybridization using digoxigenin-labeled oligonucleotide probes was performed. ResultsParietal cells of gastric gland and epithelium of gastric pit were detected to have GnRHR mRNA hybridization signal. In small intestine, the villou' s epithelial cells and glandular cells could be

5、detected to have strong GnRHR mRNA hybridization signal. In caecum、colon、rectum,very strong GnRHR mRNA hybridization signal was also detected in mucosa epithelium and glandular epithelium.The signals distributed in cytoplasm of all positive cells with negative nuclei. ConclusionThe GnRHR mRNA was wi

6、dely distributed in rat digestive tract with distributive pattern similar to those of GnRHR in pituitary. It suggests that the GnRH is produced by digestive system and response for modulating the digestive function as a gastrointestinal hormone.【Key words】GnRHR mRNA;In situ hybridization;Digestive t

7、ract;RatGnRH及其受體可在下丘腦-垂體軸外表達(dá)已被公認(rèn)1，我們先前的研究已證明，胃腸胰系統(tǒng)能自身合成GnRH2，而且證實(shí)胃腸胰系統(tǒng)及頜下腺均含有GnRH受體免疫反應(yīng)性物質(zhì)3，4。本實(shí)驗(yàn)采用原位雜交法，觀察大鼠消化道能否表達(dá)GnRH受體mRNA，為進(jìn)一步的功能性研究提供形態(tài)學(xué)依據(jù)。材料和方法1. 試劑地高辛標(biāo)記的GnRH受體cDNA寡核苷酸探針由大連寶生物工程有限公司合成。敏感性加強(qiáng)型原位雜交檢測(cè)試劑盒(堿性磷酸酶)為武漢博士德生物工程公司產(chǎn)品。2.組織材料雄性SD大鼠5只，體重230250g，斷頸處死打開腹腔，分別取出胃、十二指腸、空腸、回腸、盲腸、結(jié)腸和直腸，置Bouin液中室溫固

8、定過液，按石蠟包埋程序?qū)⑸鲜鼋M織包埋于同一蠟塊內(nèi)，在嚴(yán)格限制RNA酶污染的條件下切成6m厚的切片，貼于涂有3-Amino propyltriethoxy silane(APES)的載玻片上。3.探針的制備參照大鼠垂體GnRH受體cDNA序列設(shè)計(jì)而成5，序列如下：5-ATGTATGCCCCAGCCTTCATGATGGTGGTGATTAGCCTGGATC-3，經(jīng)大連寶生物公司合成并在5端作地高辛標(biāo)記。4.原位雜交組織化學(xué)程序切片脫蠟水化后，經(jīng)0.3%H2O2甲醇室溫處理30min，蒸餾水洗滌3次。0.5mol/L TBS(pH7.3)1200稀釋的蛋白酶K37消化15min，4%多聚甲醛5min，

9、蒸餾水洗5min×3次，切片空氣干燥后，每張切片滴加含2.0mg/L標(biāo)記探針的原位雜交液20l，濕盒中42雜交18h，2×SSC洗滌5min×2次(37)，0.1×SSC洗滌10min 1次(37)，滴加封閉液室溫20min，不洗，滴加小鼠抗地高辛抗體372h，0.5mol/L TBS洗2min×3次滴加生物素化羊抗小鼠IgG371h，0.5mol/L TBS洗2min×3次，滴加SABC-AP3730min，0.5mol/L TBS洗5 min×4次，最后用BCIP/NBT顯色液顯色，室溫3050min，水洗終止反應(yīng)，梯度

10、乙醇脫水，中性樹膠封片。陰性對(duì)照作如下設(shè)置：(1)將切片用RNA酶進(jìn)行預(yù)處理后雜交；(2)以不含探針的雜交液進(jìn)行雜交；(3)用正常小鼠血清取代小鼠抗地高辛抗體進(jìn)行雜交。結(jié)果GnRH受體mRNA雜交信號(hào)陽性反應(yīng)性細(xì)胞呈藍(lán)色，背景不著色，反差明顯，陽性細(xì)胞易于識(shí)別。3種陰性對(duì)照試驗(yàn)均呈陰性反應(yīng)。胃小凹的上皮細(xì)胞(1)、大鼠胃底腺的壁細(xì)胞(2)可檢測(cè)到GnRH受體mRNA雜交反應(yīng)信號(hào)，信號(hào)物質(zhì)分布于胞質(zhì)，核呈陰性。3段小腸絨毛上皮細(xì)胞均檢到較強(qiáng)GnRH受體mRNA雜交信號(hào)，小腸腺細(xì)胞也有GnRH受體mRNA雜交信號(hào)，信號(hào)物質(zhì)分布在胞質(zhì)，胞核為陰性反應(yīng)(3)。盲腸、結(jié)腸和直腸的粘膜上皮及大腸腺上皮細(xì)胞

11、也都檢到較強(qiáng)GnRH受體mRNA雜交信號(hào)，信號(hào)物質(zhì)也分布在胞質(zhì)內(nèi)，胞核為陰性反應(yīng)(4)。討論我們先前的研究已證明大鼠消化道從胃到直腸的一些粘膜上皮、腺上皮及肌間神經(jīng)叢的副交感神經(jīng)節(jié)細(xì)胞呈GnRH受體免疫反應(yīng)性2，本實(shí)驗(yàn)觀察到在胃腸上皮細(xì)胞及腺細(xì)胞均可檢測(cè)到GnRH受體mRNA，說明消化道的這些細(xì)胞能合成GnRH受體。GnRH受體由327個(gè)氨基酸構(gòu)成，有7個(gè)跨膜結(jié)構(gòu)域，屬G蛋白偶聯(lián)受體，哺乳類垂體促性腺細(xì)胞上的GnRH受體和GnRH的結(jié)合有高度特異性，光親和標(biāo)記、層析電泳證明GnRH受體為分子量60 000道爾頓的糖蛋白，有3個(gè)N-糖基化位點(diǎn)，在90、98和291位的酸性氨基酸殘基可能和GnRH

12、的Arg相互作用，而發(fā)揮GnRH的生理調(diào)節(jié)功能6，7。GnRH在下丘腦-垂體軸外產(chǎn)生并在下丘腦-垂體軸外發(fā)揮生物學(xué)效應(yīng)已有一些報(bào)道。GnRH受體mRNA在生殖器官如卵巢、睪丸、子宮肌層以及胎盤等處均有表達(dá)，提示GnRH在生殖調(diào)控中除刺激垂體分泌LH和FSH外，尚可直接和生殖器官發(fā)生作用，調(diào)節(jié)這些器官和細(xì)胞的功能812。在腫瘤細(xì)胞如乳腺癌、垂體促性腺細(xì)胞瘤、肝癌、肺癌等處也發(fā)現(xiàn)GnRH受體mRNA的表達(dá)，GnRH的類似物對(duì)肝癌等多種腫瘤細(xì)胞的增殖有抑制作用，提示GnRH參與腫瘤發(fā)生過程的調(diào)節(jié)1315。GnRH對(duì)消化系統(tǒng)是否有功能意義，未見報(bào)道。我們先前的研究已經(jīng)證實(shí)大鼠消化系統(tǒng)能自身合成GnRH

13、3，4。本實(shí)驗(yàn)又進(jìn)一步證實(shí)了消化道能合成GnRH受體。由此我們認(rèn)為GnRH也是一種胃腸激素，它可由消化系統(tǒng)產(chǎn)生并作用于消化道粘膜上皮、腺上皮細(xì)胞，參與胃腸功能的調(diào)節(jié)。胃小凹上皮的主要功能是分泌粘液。胃底腺壁細(xì)胞主要功能是產(chǎn)生胃酸，小腸絨毛上皮細(xì)胞的主要功能是參與食物的消化及營養(yǎng)物質(zhì)吸收，大腸各段的粘膜上皮及腺上皮主要功能是分泌粘液。本實(shí)驗(yàn)發(fā)現(xiàn)以上細(xì)胞均能表達(dá)GnRH受體mRNA，提示GnRH對(duì)胃腸系統(tǒng)的功能有廣泛的調(diào)節(jié)作用，但是具體對(duì)那些功能起調(diào)節(jié)作用和如何調(diào)節(jié)有待功能性實(shí)驗(yàn)加以進(jìn)一步證實(shí)。    版說明1胃小凹粘膜上皮細(xì)胞可檢測(cè)到GnRH受體mRNA陽

14、性雜交信號(hào)，陽性信號(hào)物質(zhì)分布在胞質(zhì)內(nèi)，胞核陰性。×2002胃底腺壁細(xì)胞有GnRH受體mRNA陽性雜交信號(hào)，信號(hào)物質(zhì)分布在胞質(zhì)內(nèi)，胞核陰性。×4003小腸絨毛上皮細(xì)胞和小腸腺細(xì)胞有GnRH受體mRNA陽性雜交信號(hào)，信號(hào)物質(zhì)分布在胞質(zhì)內(nèi)，胞核陰性。×2004結(jié)腸粘膜上皮和腺上皮細(xì)胞有GnRH受體mRNA陽性雜交信號(hào)，信號(hào)物質(zhì)分布在胞質(zhì)內(nèi)，胞核陰性。×200Explanation of figuresFig.1GnRHR mRNA hybridization signals were detected in epithelial cells in gastri

15、c pit, the signals were distributed in cytoplasm with negative nuclei.×200Fig.2GnRHR mRNA hybridization signals were detected in parietal cells in stomach gland, the signals were distributed in cytoplasm with negative nuclei.×400Fig.3GnRHR mRNA hybridization signals were detected in epithe

16、lial cells in villi and small intestinal gland, the signals were distributed in cytoplasm with negative nuclei.×200Fig.4GnRHR mRNA hybridization signals were detected in mucosa epithelial cells and glandular epithelial cells in colon, the signals were distributed in cytoplasm with negative nucl

17、ei.×200【基金項(xiàng)目】國家自然科學(xué)基金資助項(xiàng)目(39770388)【作者簡介】姚兵(1969)，男(漢族)，江蘇人，醫(yī)學(xué)博士，主治軍醫(yī)通訊作者(To whom correspondence should be addressed)姚兵（第四軍醫(yī)大學(xué)組織學(xué)胚胎學(xué)教研室，西安710032）黃威權(quán)（第四軍醫(yī)大學(xué)組織學(xué)胚胎學(xué)教研室，西安710032）蒲若蕾（第四軍醫(yī)大學(xué)組織學(xué)胚胎學(xué)教研室，西安710032）孫嵐（第四軍醫(yī)大學(xué)組織學(xué)胚胎學(xué)教研室，西安710032）張榮慶（清華大學(xué)生物系，北京100084）王雷（清華大學(xué)生物系，北京100084）參考文獻(xiàn)1韓新兵，曹永清，莊臨之.促性腺激素釋

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