蕨麻多糖對(duì)小鼠脾淋巴細(xì)胞上清液IFN-γ和可溶性LAG-3分子水平的影響

魏 慧, 何多誼, 白德成*, 程 菊, 沈 蓉, 張倩倩

1.蘭州大學(xué)基礎(chǔ)醫(yī)學(xué)院, 蘭州 730000;

2.甘肅省武威職業(yè)學(xué)院, 甘肅 武威 733000

蕨麻多糖對(duì)小鼠脾淋巴細(xì)胞上清液IFN-γ和可溶性LAG-3分子水平的影響

魏 慧1, 何多誼2, 白德成1*, 程 菊1, 沈 蓉1, 張倩倩1

1.蘭州大學(xué)基礎(chǔ)醫(yī)學(xué)院, 蘭州 730000;

2.甘肅省武威職業(yè)學(xué)院, 甘肅 武威 733000

為了觀察蕨麻多糖對(duì)急性低劑量鎘染毒小鼠脾淋巴細(xì)胞上清液中IFN-γ和可溶性LAG-3(soluble LAG-3，sLAG-3)分子水平的影響。將試驗(yàn)小鼠一次性腹腔注射氯化鎘溶液(1 mg/kg)染毒，觀察24h即造模成功后，腹腔注射低、中、高劑量蕨麻多糖治療一周，于實(shí)驗(yàn)終末測(cè)各組小鼠脾臟、胸腺指數(shù)，ELISA法測(cè)IFN-γ、sLAG-3含量；免疫熒光法觀察CD3+、CD4+、CD8+T淋巴細(xì)胞。結(jié)果表明蕨麻多糖低、中、高劑量組中，IFN-γ、sLAG-3的含量較空白對(duì)照組和單純鎘染陽(yáng)性對(duì)照組均增高，以高濃度組增高明顯(P<0.05)；CD3+、CD4+T淋巴細(xì)胞較空白對(duì)照組和單純鎘染陽(yáng)性對(duì)照組均增多，仍以高濃度組增多明顯,CD8+T變化不顯著。蕨麻多糖能促進(jìn)急性低劑量鎘染毒小鼠脾淋巴細(xì)胞分泌IFN-γ和sLAG-3，CD3+、CD4+T淋巴細(xì)胞亦相應(yīng)增加。

蕨麻多糖；IFN-γ；sLAG-3

鎘是一種廣泛存在的工業(yè)級(jí)環(huán)境毒物，主要通過(guò)職業(yè)接觸和被污染的空氣、水、食物等進(jìn)入人和動(dòng)物體內(nèi)并蓄積，導(dǎo)致機(jī)體急慢性中毒和多器官受損[1]。它能產(chǎn)生細(xì)胞、體液等的免疫抑制, 抑制T淋巴細(xì)胞增殖從而引起其亞群的改變[2]，并有強(qiáng)致癌性。

蕨麻多糖(PAP)是藏藥蕨麻的活性成分，具有提高機(jī)體免疫力、抗疲勞和耐缺氧等作用[3]。LAG-3是廣泛存在于動(dòng)物和人體內(nèi)的免疫負(fù)調(diào)節(jié)分子, 具有維持內(nèi)環(huán)境穩(wěn)定和參與免疫調(diào)節(jié)的功能，其表達(dá)與IFN-γ相關(guān)[4]。PAP可通過(guò)改變IFN-γ等的水平，增強(qiáng)機(jī)體的免疫功能[5]；因此PAP對(duì)機(jī)體中的IFN-γ和LAG-3有直接影響，但PAP對(duì)鎘抑制的影響尚缺乏資料。本實(shí)驗(yàn)旨在通過(guò)小鼠急性低劑量腹腔注射鎘，觀察蕨麻多糖對(duì)脾淋巴細(xì)胞IFN-γ和sLAG-3分子的影響，以期為研究蕨麻多糖干預(yù)鎘引起的免疫抑制提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 材料

清潔級(jí)昆明系小鼠，雌雄各半，體重20 g左右，由蘭州大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物科提供；蕨麻多糖，由本試驗(yàn)室經(jīng)乙醇提取保存，采用硫酸-苯酚法[5]測(cè)得蕨麻多糖含量2%，用蒸餾水分別配成所需濃度的蕨麻多糖溶液，無(wú)菌濾器過(guò)濾備用；免疫熒光抗體(CD4-Phycoerythrin、CD8-Phycoery-thrin、CD3-FITC)、LAG-3酶聯(lián)免疫試劑盒、IFN-γ酶聯(lián)免疫試劑盒購(gòu)于南京建成生物公司；小鼠淋巴細(xì)胞分離液購(gòu)自深圳達(dá)科為公司；氯化鎘(CdCl2)、臺(tái)盼蘭染色劑等試劑均為國(guó)產(chǎn)分析純。

1.2 方法

1.2.1 動(dòng)物分組及給藥 清潔級(jí)昆明系小鼠，8周齡，60只，雌雄各半，隨機(jī)分為6組，具體操作：第1組為空白對(duì)照組(C1)，每只小鼠分別于第1 d、3 d、5 d、7 d腹腔注射0.9%的生理鹽水；第2組為PAP陽(yáng)性對(duì)照組(C2)，分別于第1 d、3 d、5 d、7 d腹腔注射PAP 50 mg/kg；第3組為鎘染毒模型組(C3)，每只小鼠實(shí)驗(yàn)前一天一次性腹腔注射氯化鎘1 mg/kg，24 h后按第1 d、3 d、5 d、7 d腹腔注射同體積的生理鹽水；第4、5、6組為PAP低(L)、中(M)、高(H)治療組，每只小鼠實(shí)驗(yàn)前一天一次性腹腔注射氯化鎘1 mg/kg，24 h后各組分別按50 mg/kg、100 mg/kg、200 mg/kg劑量于第1 d、3 d、5 d、7 d腹腔注射PAP；治療后觀察48 h,所有供試小鼠斷椎處死。

1.2.2 測(cè)脾臟指數(shù)和胸腺指數(shù)、脾淋巴細(xì)胞上清液IFN-γ、sLAG-3及CD3+、CD4+、CD8+T淋巴細(xì)胞[6,7]各組小鼠撲殺后稱重，分別取脾臟、胸腺稱重，計(jì)算脾臟指數(shù)和胸腺指數(shù)(mg/g)。無(wú)菌取脾臟，小鼠淋巴細(xì)胞分離液分離脾淋巴細(xì)胞，加5 mL RPMI-1640培養(yǎng)液，用吸管吹打均勻，細(xì)胞計(jì)數(shù)調(diào)節(jié)細(xì)胞密度至2×106～5×106/mL，臺(tái)盼蘭染色檢測(cè)細(xì)胞存活率大于85%，RPMI-1640完全培養(yǎng)基鋪板，將細(xì)胞懸液加到96孔細(xì)胞板中，100 μL/孔，5% CO2、37℃、95% 濕度孵箱中,培養(yǎng)48 h，并隨時(shí)觀察細(xì)胞情況。48 h后，用毛細(xì)玻璃管將培養(yǎng)物移入無(wú)菌離心管中，2 500 r/min離心20 min，仔細(xì)收集上清，按照ELISA檢測(cè)試劑盒說(shuō)明書(shū)操作，進(jìn)行脾臟淋巴細(xì)胞上清液中IFN-γ和sLAG-3分子水平的測(cè)定(測(cè)定時(shí)間不超過(guò)24 h)[8]。收集完脾臟淋巴細(xì)胞上清液，將細(xì)胞用PBS稀釋成細(xì)胞懸液，細(xì)胞濃度達(dá)到2×106～5×106/mL，24孔細(xì)胞板鋪板，1 mL/孔，再分別加入稀釋的Anti-Mouse CD3c-FITC、Anti-Mouse CD4-Phycoerythrin、Anti-Mouse CD8-Phycoerythrin熒光素標(biāo)記抗體，室溫避光反應(yīng)1 h，熒光顯微鏡拍照觀測(cè)。

采用三色直接免疫熒光標(biāo)記法，觀察PAP對(duì)小鼠脾臟CD3+、CD4+、CD8+T淋巴細(xì)胞的影響[7]。

2 結(jié)果與分析

2.1 不同劑量 PAP 對(duì)小鼠胸腺指數(shù)及脾臟指數(shù)的影響

經(jīng)方差分析，結(jié)果顯示：PAP陽(yáng)性對(duì)照組胸腺、脾臟指數(shù)高于空白對(duì)照組，說(shuō)明PAP能提高正常小鼠的脾臟指數(shù)和胸腺指數(shù)；中、高劑量組脾臟指數(shù)高于鎘染毒模型組(P<0.05)，低劑量組差異不顯著；低、中、高劑量組胸腺指數(shù)與生理鹽水組和鎘染毒模型組相比均無(wú)統(tǒng)計(jì)學(xué)差異(P>0.05)；不同劑量PAP組的脾臟指數(shù)均高于生理鹽水組和鎘染毒模型組(P<0.05)，表明PAP能提高急性低劑量鎘染毒小鼠的脾臟指數(shù)，說(shuō)明造模成功,但PAP對(duì)急性低劑量鎘染毒小鼠的胸腺指數(shù)無(wú)明顯恢復(fù)和提升作用，具體結(jié)果見(jiàn)表1。

注: 同一列內(nèi)不同小寫(xiě)字母表示差異顯著(P<0.05)。

2.2 不同劑量PAP對(duì)小鼠脾臟淋巴細(xì)胞培養(yǎng)上清液中IFN-γ水平和sLAG-3分子水平的影響

經(jīng)方差分析，結(jié)果顯示：PAP陽(yáng)性組IFN-γ、sLAG-3高于空白對(duì)照組和鎘染毒模型組(P<0.05)；低劑量組IFN-γ、sLAG-3與PAP陽(yáng)性組相比差異不顯著(P>0.05)；中、高劑量組IFN-γ、sLAG-3高于空白對(duì)照組和鎘染毒模型組，以高劑量組差異顯著(P<0.05)。表明PAP能夠促進(jìn)正常小鼠和急性低劑量鎘染毒小鼠脾淋巴細(xì)胞分泌IFN-γ和sLAG-3，且與劑量相關(guān)，具體結(jié)果見(jiàn)表2。

表2 不同劑量PAP對(duì)小鼠脾臟淋巴細(xì)胞培養(yǎng)上清液中IFN-γ水平和sLAG-3分子水平的影響

注: 同一列內(nèi)不同小寫(xiě)字母表示差異顯著(P<0.05)。

2.3 不同劑量PAP對(duì)小鼠脾臟CD3+、CD4+、CD8+ T淋巴細(xì)胞的影響

熒光顯微鏡結(jié)果顯示(圖1，彩圖見(jiàn)封三圖版)：高劑量組中CD3+、CD4+T細(xì)胞熒光標(biāo)記數(shù)目較空白對(duì)照組和鎘染毒模型組有增高趨勢(shì)；低、中劑量組與空白對(duì)照組和鎘染毒模型組相比增多不顯著，表明PAP能增加正常小鼠和急性低劑量鎘染毒小鼠CD3+、CD4+T細(xì)胞數(shù)目，以高劑量200 mg/kg組明顯；CD8+T細(xì)胞變化不顯著。

圖1 不同劑量PAP對(duì)小鼠脾臟CD3+、CD4+ T淋巴細(xì)胞影響的熒光顯微鏡圖片F(xiàn)ig.1 Effect of PAP with different content on CD3+、CD4+ T in spleen lymphocytes.(彩圖見(jiàn)封三圖版)

3 討論

研究認(rèn)為重金屬鎘暴露可引起人類(lèi)的免疫抑制，目前許多急、慢性鎘中毒仍只能采取對(duì)癥支持治療[1]，為此研究者不斷探究鎘損傷的保護(hù)劑,尋找既能排鎘,又能提高機(jī)體免疫力的藥物，故中藥對(duì)抗鎘抑制的研究變得很有意義。隨著PAP的免疫研究不斷深入，它對(duì)鎘抑制的干預(yù)機(jī)制將引起越來(lái)越多的學(xué)者的重視。本實(shí)驗(yàn)通過(guò)對(duì)急性低劑量鎘染毒小鼠采用PAP治療后,中、高濃度組的IFN-γ和sLAG-3水平,與鎘染毒模型組比較有所上升,表明PAP可以增強(qiáng)急性低劑量鎘染毒小鼠的免疫力。

免疫系統(tǒng)中IFN-γ和LAG-3由機(jī)體免疫器官活化后的T細(xì)胞等產(chǎn)生，當(dāng)抗原、PHA或ConA刺激后T細(xì)胞分泌IFN-γ[9]，IFN-γ能誘導(dǎo)單核細(xì)胞、樹(shù)突狀細(xì)胞等MHC-Ⅱ類(lèi)抗原的表達(dá)，MHC-Ⅱ與LAG-3結(jié)合后負(fù)調(diào)控T細(xì)胞功能[10]。LAG-3參與調(diào)控記憶性T細(xì)胞池和樹(shù)突狀細(xì)胞[11]，它只表達(dá)于活化的淋巴細(xì)胞和類(lèi)漿細(xì)胞性樹(shù)突狀細(xì)胞(pDC)[9]。在金屬蛋白酶的作用下，LAG-3分子的連接肽發(fā)生斷裂，形成可溶性的部分(包括D1-D3域)和跨膜-胞質(zhì)部分；LAG-3的可溶性單體最先在炎癥性疾病患者體內(nèi)發(fā)現(xiàn)，治療有效時(shí)，其表達(dá)水平增高，sLAG-3分子與MHC-Ⅱ分子作用可誘導(dǎo)DC成熟并遷移到次級(jí)淋巴器官[5]。另外sLAG-3分子可以明顯抑制腫瘤細(xì)胞的生長(zhǎng),人LAG-3抑制作用更加顯著[12]。

實(shí)驗(yàn)表明蕨麻多糖能增強(qiáng)急性低劑量鎘染毒小鼠的免疫力，使活化T淋巴細(xì)胞產(chǎn)生IFN-γ增多；它能直接作用于脾淋巴細(xì)胞，促進(jìn)其分泌IFN-γ，使sLAG-3的水平發(fā)生相應(yīng)變化；本 研 究 中 得 出 的IFN-γ和sLAG-3均低于以往文獻(xiàn)[6]報(bào)道中的數(shù)據(jù)，考慮可能與標(biāo)本量較少有關(guān)，下一步需擴(kuò)大樣本量加以佐證；蕨麻多糖可以干預(yù)鎘的抑制作用，其具體機(jī)制有待進(jìn)一步研究證實(shí)。

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[8] 王少敏，張 潔，馬 麗. 30例女性SLE患者淋巴細(xì)胞培養(yǎng)上清液中IL-2、 TNF-α、IFN-γ、IL-4水平測(cè)定[J].中國(guó)婦幼保健，2008， 23 (7) : 982-983.

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研究發(fā)現(xiàn)控制水稻粒形和稻米品質(zhì)的重要基因

近日，中國(guó)科學(xué)院遺傳與發(fā)育生物學(xué)研究所傅向東研究員領(lǐng)導(dǎo)的團(tuán)隊(duì)從優(yōu)質(zhì)雜交水稻不育系泰豐A中成功分離并克隆了一個(gè)控制水稻粒形和提升稻米品質(zhì)的重要基因GW7。這一基因能通過(guò)改變細(xì)胞分裂模式，讓稻米變得更為細(xì)長(zhǎng)，有效地減少堊白率和堊白面積，從而提高稻米在外觀、口感等方面的品質(zhì)。該研究還表明將GW7和GS3基因的優(yōu)異等位變異聚合并應(yīng)用到我國(guó)高產(chǎn)秈稻中，可明顯提高稻米品質(zhì)，同時(shí)還可提高產(chǎn)量。該項(xiàng)研究為水稻高產(chǎn)優(yōu)質(zhì)分子模塊設(shè)計(jì)育種提供了具有重要應(yīng)用價(jià)值的新基因，也為揭示水稻品質(zhì)和產(chǎn)量協(xié)同遺傳改良的分子奧秘提供了新線索。

論文鏈接: Wang S，etal.. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nature Genetics, 2015, doi: 10.1038/ng.3352. Published online: 06 July, 2015.

Abstract: The deployment of heterosis in the form of hybrid rice varieties has boosted grain yield, but grain quality improvement still remains a challenge. Here we show that a quantitative trait locus for rice grain quality, qGW7, reflects allelic variation of GW7, a gene encoding a TONNEAU1-recruiting motif protein with similarity to C-terminal motifs of the human centrosomal protein CAP350. Upregulation of GW7 expression was correlated with the production of more slender grains, as a result of increased cell division in the longitudinal direction and decreased cell division in the transverse direction. OsSPL16 (GW8), an SBP-domain transcription factor that regulates grain width, bound directly to the GW7 promoter and repressed its expression. The presence of a semidominant GW7TFAallele from tropical japonica rice was associated with higher grain quality without the yield penalty imposed by the Basmati gw8 allele. Manipulation of the OsSPL16-GW7 module thus represents a new strategy to simultaneously improve rice yield and grain quality.

水稻氮利用效率改良研究取得重大突破

植物主要以銨態(tài)氮和硝態(tài)氮為主要氮源，中國(guó)科學(xué)院遺傳與發(fā)育生物學(xué)研究所儲(chǔ)成才研究員領(lǐng)導(dǎo)的團(tuán)隊(duì)研究表明，秈稻品種利用硝酸鹽的能力顯著高于粳稻品種，該團(tuán)隊(duì)通過(guò)圖位克隆技術(shù)從秈稻中克隆出高氮利用效率基因NRT1.1B，NRT1.1B編碼一個(gè)硝酸鹽轉(zhuǎn)運(yùn)蛋白，在秈、粳稻間只有一個(gè)氨基酸的差別，且秈稻與粳稻呈現(xiàn)出顯著的分化，各種證據(jù)表明，秈稻型具有更高的硝酸鹽吸收及轉(zhuǎn)運(yùn)活性。尤為重要的是，秈稻中的硝酸鹽同化過(guò)程的關(guān)鍵基因也被顯著上調(diào)，這種結(jié)果導(dǎo)致秈稻具有更高的氮肥利用能力，說(shuō)明NRT1.1B在粳稻氮肥利用效率改良上具有巨大應(yīng)用價(jià)值。

論文鏈接: Hu B，etal.. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nature Genetics, 2015,47(7):834-838. doi: 10.1038/ng.3337. Published online: 08 June, 2015.

Abstract: Asian cultivated rice (Oryza sativa L.) consists of two main subspecies, indica and japonica. Indica has higher nitrate-absorption activity than japonica, but the molecular mechanisms underlying that activity remain elusive. Here we show that variation in a nitrate-transporter gene, NRT1.1B (OsNPF6.5), may contribute to this divergence in nitrate use. Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica. NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes. The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication. Notably, field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency (NUE) compared to the variety without that allele. Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica.

科學(xué)家發(fā)明單堿基分辨率測(cè)序技術(shù)：CeU-Seq

近日，北京大學(xué)生命科學(xué)學(xué)院伊成器研究組報(bào)道了一種通過(guò)化學(xué)標(biāo)記和富集手段實(shí)現(xiàn)全轉(zhuǎn)錄組水平上假尿嘧啶RNA修飾的單堿基分辨率測(cè)序技術(shù)CeU-Seq，并繪制了人和小鼠細(xì)胞轉(zhuǎn)錄組中假尿嘧啶RNA修飾的譜圖。該研究進(jìn)一步確定了多個(gè)可以作用于mRNA上的假尿嘧啶合成酶(其中PUS1、DKC1兩種酶之前被發(fā)現(xiàn)與線粒體肌病、先天性角化不良等人類(lèi)疾病相關(guān))，并且發(fā)現(xiàn)轉(zhuǎn)錄組中假尿嘧啶的含量與分布均會(huì)受到各種環(huán)境刺激的調(diào)控，呈現(xiàn)出“刺激條件特異性”的誘導(dǎo)修飾。該研究為假尿嘧啶轉(zhuǎn)錄后修飾參與基因表達(dá)調(diào)控的研究提供了重要工具，為近年來(lái)興起的“RNA表觀遺傳學(xué)”領(lǐng)域提供了嶄新的研究方向。

論文鏈接: Li X，etal.. Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome. Nature Chemical Biology, 2015, doi: 10.1038/nchembio.1836. Published online: 15 June, 2015.

Abstract: Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification, yet little is known about its prevalence, mechanism and function in mRNA. Here, we performed quantitative MS analysis and show that Ψ is much more prevalent (Ψ/U ratio～0.2～0.6%) in mammalian mRNA than previously believed. We developed N3-CMC-enriched pseudouridine sequencing (CeU-Seq), a selective chemical labeling and pulldown method, to identify 2 084 Ψ sites within 1 929 human transcripts, of which four (in ribosomal RNA and EEF1A1 mRNA) are biochemically verified. We show that hPUS1, a known Ψ synthase, acts on human mRNA; under stress, CeU-Seq demonstrates inducible and stress-specific mRNA pseudouridylation. Applying CeU-Seq to the mouse transcriptome revealed conserved and tissue-specific pseudouridylation. Collectively, our approaches allow comprehensive analysis of transcriptome-wide pseudouridylation and provide tools for functional studies of Ψ-mediated epigenetic regulation.

Crispr-Cas9技術(shù)獲重大新成果

來(lái)自麻省總醫(yī)院的一個(gè)研究小組找到了一種新方法來(lái)擴(kuò)大強(qiáng)大基因編輯工具——Crispr-Cas9 RNA引導(dǎo)核酸酶的使用及提高其精確性。相比于迄今為止使用的自然形式的Cas9，演化版本的Cas9能夠識(shí)別前者無(wú)法靶向的不同范圍的核酸序列。采用該小組設(shè)計(jì)改進(jìn)的新的Cas9變體，可以靶向過(guò)去用野生型Cas9無(wú)法改造的人類(lèi)和斑馬魚(yú)基因。這將使得研究人員能夠靶向各種基因組中更大范圍內(nèi)的一些位點(diǎn)，這個(gè)新方法可以用于需要高度精確靶向DNA序列的研究。該研究第一次證實(shí)了可以通過(guò)定向的蛋白質(zhì)演化來(lái)改變SpCas9的活性，通過(guò)相似的方法還可以改變Cas9酶其他的有用特性，使得定制化一些重要特性成為可能。

論文鏈接: Kleinstiver B P，etal.. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 2015,doi: 10.1038/nature14592. Published online: 22 June, 2015.

Abstract: Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

科學(xué)家開(kāi)發(fā)出簡(jiǎn)單便宜的組織蛋白分析新技術(shù)

近日，來(lái)自瑞典烏普薩拉大學(xué)的研究人員開(kāi)發(fā)了一種蛋白質(zhì)分析技術(shù)，利用這種技術(shù)不需要高級(jí)設(shè)備、專(zhuān)門(mén)的實(shí)驗(yàn)室以及昂貴的試劑就可對(duì)組織蛋白進(jìn)行分析。該技術(shù)以兩個(gè)抗體對(duì)同一蛋白兩個(gè)不同位點(diǎn)或兩個(gè)定位很近的不同蛋白的結(jié)合為基礎(chǔ)，將兩個(gè)抗體分別與一段含發(fā)卡結(jié)構(gòu)的DNA鏈連接，當(dāng)兩個(gè)抗體靠得非常近的時(shí)候，其帶有的DNA鏈會(huì)結(jié)合形成一個(gè)起始序列。當(dāng)這種情況發(fā)生時(shí)，通過(guò)雜交鏈?zhǔn)椒磻?yīng)(hybridization chain reaction)將帶有熒光標(biāo)記并具有發(fā)卡結(jié)構(gòu)的寡聚核苷酸連接到起始序列上，這樣擴(kuò)增出的每一條DNA鏈上都連接有熒光底物，實(shí)現(xiàn)了信號(hào)擴(kuò)增，當(dāng)使用特定波長(zhǎng)的光進(jìn)行激發(fā)，DNA鏈上連接的熒光底物會(huì)發(fā)射出熒光。當(dāng)鏈?zhǔn)椒磻?yīng)進(jìn)行到一定程度，便可以在熒光顯微鏡下觀察到明亮的點(diǎn)狀熒光，點(diǎn)狀熒光越多，蛋白就越多。除此之外，這種鏈?zhǔn)椒磻?yīng)不需要任何酶的催化，在室溫情況下就可進(jìn)行反應(yīng)。這項(xiàng)技術(shù)將給基礎(chǔ)研究和醫(yī)學(xué)診斷帶來(lái)極大便利。

論文鏈接: Koos B，etal.. Proximity-dependent initiation of hybridization chain reaction. Nature Communications, 2015, 6: 7294. doi:10.1038/ncomms8294.

Abstract: Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

研究發(fā)現(xiàn)檢測(cè)血液中miRNA的簡(jiǎn)便技術(shù)

最近，密歇根大學(xué)的研究人員開(kāi)發(fā)出一種有效的方法，能在血液中檢測(cè)到癌變腫瘤脫落的microRNAs。這種方法可通過(guò)一種廉價(jià)的血液測(cè)試，同時(shí)對(duì)多種類(lèi)型的癌癥進(jìn)行篩選——最終可能超過(guò)100種不同的類(lèi)型。在實(shí)驗(yàn)中，研究人員用稱為“捕獲探針” 的分子包覆一個(gè)載玻片，這些探針?lè)肿涌删o緊抓住在其附近的microRNAs。這種方法的獨(dú)特之處在于，DNA和RNA結(jié)合太弱，因此它們不能停留。雖然之前有研究人員已經(jīng)在血清中檢測(cè)到microRNA，但是本研究中的這種方法更為直接，并且?guī)缀鯖](méi)有假陽(yáng)性。

論文鏈接: Johnson-Buck A,etal.. Kinetic fingerprinting to identify and count single nucleic acids. Nature Biotechnology, 2015, 33(7):730-732. doi: 10.1038/nbt.3246. Published online: 22 June, 2015.

Abstract: MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers. We introduce a kinetic fingerprinting approach calledsingle-molecule recognition through equilibrium Poisson sampling (SiMREPS) for the amplification-free counting of singleunlabeled miRNA molecules, which circumvents thermodynamic limits of specificity and virtually eliminates false positives. We demonstrate high-confidence, single-molecule detection of synthetic and endogenous miRNAs in both buffer and minimally treated biofluids, as well as >500-fold discrimination between single nucleotide polymorphisms.

Influence of PAP on the IFN-γ and Soluble LAG-3 Molecule Level of Spleen Lymphocyte Supernatant in Mice

WEI Hui1, HE Duo-yi2, BAI De-cheng1*, CHENG Ju1, SHEN Rong1, ZHANG Qian-qian1

1.SchoolofBasicMedicalSciences,LanzhouUniversity,Lanzhou730000,China;

2.WuweiOccupationalCollege,GansuWuwei733000,China

To observe the influence of PAP on the IFN-γ and soluble LAG-3 molecule level of spleen lymphocyte supernatant in mice which acute exposured to low doses of cadmium. The solution of cadmium chloride were injected into the intraperitoneal of the test mice with 1 mg/kg. After 24 h, the model had been successfully made, PAP of low, medium and high doses were injected into the intraperitoneal for a week, spleen index and thymus index were measured at the end of the experiment, the content of IFN-γ and sLAG-3 were measured by ELISA. The cells of CD3+,CD4+,CD8+in spleen lymphocytes was observed by immunofluorescence. The results showed that, compared with the blank control and the cadmium stained positive control, the content of IFN-γ and sLAG-3 were increased among the different doses of PAP groups. The high concentration group increased the most obvious(P<0.05), the amount of CD3+, CD4+cells were also correspond to the number of rising but CD8+cells were not increased significantly. It was still the most significant increase in the high dose group. PAP could increase the content of IFN-γ and soluble LAG-3 of the mice acute exposuring to low doses of cadmium, and CD3+,CD4+lymphocytes were in a corresponding increase.

PAP; IFN-γ; sLAG-3

2015-03-27; 接受日期：2015-04-09

魏慧,碩士研究生，主要從事中西醫(yī)結(jié)合基礎(chǔ)研究。E-mail:1159300949@qq.com。*通信作者：白德成，教授，研究生導(dǎo)師，主要從事基礎(chǔ)醫(yī)學(xué)研究。E-mail：bdc@lzu.edu.cn

10.3969/j.issn.2095-2341.2015.04.12