負(fù)責(zé)我們認(rèn)知、意識(shí)的神經(jīng)活動(dòng)主要發(fā)生于我們大腦內(nèi)部,負(fù)責(zé)體表的一系列感覺有外周神經(jīng)通路,而對(duì)于我們體腔內(nèi)的內(nèi)臟器官的感覺傳入和神經(jīng)支配,則主要由迷走神經(jīng)完成。迷走神經(jīng)負(fù)責(zé)的組織器官多樣,功能也各不相同,但迷走神經(jīng)核團(tuán)是如何對(duì)各個(gè)內(nèi)臟的傳入信息進(jìn)行區(qū)分和編碼,目前了解的不甚清晰。不同器官傳入的神經(jīng)元涉及種類繁多,對(duì)不同刺激產(chǎn)生應(yīng)答,了解不同類型神經(jīng)元的空間分布及分類特點(diǎn),對(duì)于理解信息如何編碼匯聚非常重要。2022年3月,美國(guó)耶魯大學(xué)醫(yī)學(xué)院的常瑞實(shí)驗(yàn)室與張樂(lè)實(shí)驗(yàn)室合作在Nature上發(fā)表了一篇研究,通過(guò)使用RNAscope HiPlex結(jié)合單細(xì)胞測(cè)序、活體鈣成像等技術(shù),對(duì)迷走神經(jīng)元進(jìn)行了空間及細(xì)胞活動(dòng)特征的研究[1]。

作者先在小鼠不同器官內(nèi)分別注射帶有熒光標(biāo)記和不同barcode的逆行AAV病毒,以此標(biāo)記不同器官來(lái)源的迷走神經(jīng)。然后分離出標(biāo)記好的迷走神經(jīng)元做單細(xì)胞測(cè)序,篩選總結(jié)出大量靶標(biāo),及不同靶標(biāo)在每類神經(jīng)元中的相對(duì)表達(dá)量。隨后作者使用RNAscope HiPlex技術(shù)對(duì)篩選出的不同器官來(lái)源的迷走神經(jīng)元中感興趣靶標(biāo)進(jìn)行了原位驗(yàn)證,并將此方法稱之為“Projection-seq” (圖1)。在多個(gè)組織器官來(lái)源的迷走神經(jīng)元中原位驗(yàn)證結(jié)果與單細(xì)胞測(cè)序結(jié)果匹配良好,且各靶標(biāo)相對(duì)表達(dá)量與單細(xì)胞測(cè)序接近。

圖1. Projection-seq驗(yàn)證肺臟投射來(lái)源不同亞群迷走神經(jīng)元的靶標(biāo)分布。
隨后,作者在迷走神經(jīng)元中泛表達(dá)鈣指示劑GCamP6s,并在Gpr65陽(yáng)性神經(jīng)元中表達(dá)紅色熒光蛋白指示劑,用于對(duì)神經(jīng)元進(jìn)行定位。通過(guò)對(duì)小鼠肺、食管、胃腸道等不同內(nèi)臟器官或不同節(jié)段分別進(jìn)行機(jī)械刺激,同時(shí)記錄迷走神經(jīng)元的鈣反應(yīng)情況,之后,再結(jié)合RNAscope HiPlex技術(shù),對(duì)迷走神經(jīng)元進(jìn)行多靶標(biāo)染色,通過(guò)紅色熒光蛋白定位,將RNA原位信息與鈣活動(dòng)信息整合到一起。作者將此方法命名為“vCatFISH”。通過(guò)vCatFISH,得以讓我們看到對(duì)于相同器官的同一刺激,會(huì)有表達(dá)不同基因的神經(jīng)元產(chǎn)生反應(yīng),說(shuō)明對(duì)于同樣的刺激,在迷走神經(jīng)元中也存在多個(gè)維度的編碼。神經(jīng)系統(tǒng)通過(guò)對(duì)不同維度信息進(jìn)行整合,最終幫助我們實(shí)現(xiàn)對(duì)刺激強(qiáng)度、時(shí)間、位置等具體細(xì)節(jié)的區(qū)分,從而使機(jī)體對(duì)不同情況進(jìn)行應(yīng)對(duì)。而本篇文章的工作幫助我們未來(lái)對(duì)神經(jīng)活動(dòng)進(jìn)行解碼提供了一種新的選擇和啟發(fā)。

圖2. vCatFISH技術(shù)揭示了對(duì)于機(jī)體不同刺激反應(yīng)的神經(jīng)元的基因特征。
RNAscope技術(shù)是由Bio-Techne旗下Advanced Cell Diagnostics (ACD)公司研發(fā)的RNA原位雜交產(chǎn)品,在近年來(lái)的生物檢測(cè)領(lǐng)域發(fā)展迅速。與傳統(tǒng)的RNA原位雜交相比,RNAscope技術(shù)屬于新一代RNA原位雜交技術(shù),其特異性的雙Z探針設(shè)計(jì)避免了傳統(tǒng)長(zhǎng)鏈RNA探針的弊端,配以自身級(jí)聯(lián)放大檢測(cè)原理,可以高效敏感地檢測(cè)到目標(biāo)RNA。該方法的具體優(yōu)勢(shì)如下:
使用RNAscope技術(shù),靶點(diǎn)RNA為大于等于300個(gè)堿基的特異序列,即可進(jìn)行探針設(shè)計(jì)。因而RNAscope技術(shù)可以應(yīng)用于幾乎所有物種,所有組織以及所有基因的檢測(cè)。
RNAscope獨(dú)特的雙Z(ZZ) 探針設(shè)計(jì)有效的防止了探針的非特異性結(jié)合,同時(shí)降低了背景干擾。由于結(jié)合在非特異性位點(diǎn)的單個(gè)的Z 探針不會(huì)產(chǎn)生完整的信號(hào)放大分子結(jié)合位點(diǎn),并會(huì)在雜交過(guò)程中被洗脫掉,從而防止非特異性信號(hào)的放大,使得探針的信號(hào)具有高度特異性。探針設(shè)計(jì)合成需要2~4周時(shí)間即可完成。
RNAscope方法檢測(cè)每個(gè)RNA 分子時(shí),只需三對(duì)雙Z(ZZ)探針即可完成雜交和信號(hào)的可視化。
使用RNAscope技術(shù)雜交上三對(duì)及以上雙Z 探針即可在標(biāo)準(zhǔn)的顯微鏡下呈現(xiàn)可觀察到的點(diǎn)狀信號(hào)。ACD公司提供的分析軟件更可以定量每一個(gè)單細(xì)胞內(nèi)RNA 的表達(dá)水平。
由于RNAscope三對(duì)雙Z探針即可檢測(cè)到目標(biāo)RNA,而通常針對(duì)靶點(diǎn)RNA設(shè)計(jì)的探針為20對(duì)雙Z 探針,因而即使目標(biāo)RNA發(fā)生部分降解,仍可以穩(wěn)定有效地檢測(cè)到靶點(diǎn)RNA。
RNAscope技術(shù)用到的探針以及所有檢測(cè)試劑全部由工業(yè)化合成,且該技術(shù)針對(duì)不同樣本類型(冰凍切片, 石蠟切片,懸浮細(xì)胞,貼壁細(xì)胞等)已經(jīng)有成熟的實(shí)驗(yàn)操作流程,故使得RNAscope技術(shù)檢測(cè)結(jié)果具有穩(wěn)定性和一致性。除了可以在實(shí)驗(yàn)室進(jìn)行手工操作外,該產(chǎn)品也可以在Leica以及羅氏Ventana自動(dòng)平臺(tái)上運(yùn)行,為結(jié)果的一致性和穩(wěn)定性提供了可靠的依據(jù)。
多通道多靶點(diǎn)同時(shí)檢測(cè)
由于RNAscope技術(shù)可以同時(shí)進(jìn)行多通道探針雜交以及信號(hào)放大,故在可見光檢測(cè)中可以在同一張切片上同時(shí)檢測(cè)兩個(gè)靶點(diǎn);而在熒光檢測(cè)過(guò)程中,可以在同一張切片上檢測(cè)三個(gè)或三個(gè)以上靶點(diǎn)RNA。
在RNAscope技術(shù)的基礎(chǔ)上,ACD公司旗下的Basescope技術(shù)可以檢測(cè)RNA水平的外顯子跳躍,點(diǎn)突變,環(huán)狀RNA等;miRNAscope可以原位對(duì)小RNA,包括miRNA, siRNA, ASO進(jìn)行追蹤定位;而近年來(lái)推出的RNAscope HiPlex技術(shù)則可以在一張石蠟切片上同時(shí)檢測(cè)最多12個(gè)RNA靶標(biāo)的分布定位,成為神經(jīng)科學(xué),腫瘤微環(huán)境研究以及單細(xì)胞測(cè)序后驗(yàn)證等領(lǐng)域的常用檢測(cè)方法。
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- 請(qǐng)求信息 : 2026-05-07 19:23:31 HTTP/1.1 GET : m.haolingkeji.com/news_detail/3686315.html
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