丙戊酸對雙峰駝成纖維細(xì)胞重編程影響

張啟冉，李宗帥，馬甜，李怡娜，趙興緒,，張勇,

丙戊酸對雙峰駝成纖維細(xì)胞重編程影響

1甘肅農(nóng)業(yè)大學(xué)生命科學(xué)技術(shù)學(xué)院/甘肅農(nóng)業(yè)大學(xué)甘肅省動物生殖生理及繁殖調(diào)控重點(diǎn)實(shí)驗(yàn)室，蘭州 730070；2甘肅農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院，蘭州 730070

【目的】探討提高雙峰駝成纖維細(xì)胞重編程效率，減少因原癌基因引入造成致瘤風(fēng)險。試驗(yàn)在成纖維細(xì)胞重編程過程中添加丙戊酸（valproic acid，VPA），探索小分子物質(zhì)對雙峰駝成纖維細(xì)胞重編程的影響，為雙峰駝發(fā)病機(jī)制研究提供參考依據(jù)?！痉椒ā坎捎?月齡雙峰駝胎兒成纖維細(xì)胞，結(jié)合經(jīng)典誘導(dǎo)組合OSKM（Oct4、Sox2、Klf4和c-Myc及治療）和EGFP等5種逆轉(zhuǎn)錄病毒對雙峰駝成纖維細(xì)胞進(jìn)行重編程（OSKM組），并在第二次病毒感染后添加VPA處理7 d（OSKM+VPA組）收集細(xì)胞。利用PCR技術(shù)對所得細(xì)胞進(jìn)行內(nèi)、外源基因檢測以證實(shí)逆轉(zhuǎn)錄病毒對雙峰駝成纖維細(xì)胞的修飾作用，根據(jù)RNA-seq數(shù)據(jù)從VPA影響較為明顯的基因中隨機(jī)挑選8個基因，檢驗(yàn)其在添加VPA前后的變化趨勢是否與RNA-seq數(shù)據(jù)趨勢一致，以驗(yàn)證RNA-seq數(shù)據(jù)的準(zhǔn)確性。通過GO分析對轉(zhuǎn)錄組樣本基因進(jìn)行分類，并使用KEGG通路富集分析和超幾何驗(yàn)證分析明確目的基因顯著富集通路。對收集到的細(xì)胞進(jìn)行總RNA提取并結(jié)合RNA-seq技術(shù)和實(shí)時熒光定量PCR（Real time-quantitative interpretation，RT-qPCR）技術(shù)檢測VPA對雙峰駝成纖維細(xì)胞重編程的影響。【結(jié)果】使用PCR技術(shù)檢測內(nèi)、外源基因在不同分組的表達(dá)，結(jié)果顯示、、、、在OSKM和OSKM+VPA組中均有表達(dá)，且OSKM+VPA組表達(dá)量高于OSKM組，而其在BCEFs組不表達(dá)。隨機(jī)挑選8個基因檢測，結(jié)果表明與細(xì)胞周期信號通路有關(guān)的、、這3個基因在添加VPA后表達(dá)量下調(diào)；與癌癥表型特征相關(guān)的、、、表達(dá)下調(diào)；PI3k-Akt信號通路中表達(dá)上調(diào)；此表達(dá)趨勢與組學(xué)數(shù)據(jù)趨勢一致。結(jié)果顯示在添加VPA后，增殖基因和表達(dá)下調(diào)，而凋亡基因表達(dá)上調(diào)。對轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行KEGG和超幾何驗(yàn)證分析，根據(jù)分析結(jié)果篩選出959個差異表達(dá)基因，富集在276個信號通路中，其中Q值小于0.05的信號通路有八條：類固醇生物合成、細(xì)胞周期、PPAR信號通路、孕酮介導(dǎo)的卵母細(xì)胞成熟、脂肪酸代謝、ECM-受體相互作用、細(xì)胞黏附分子和膽固醇代謝。經(jīng)篩選獲得與細(xì)胞周期、脂肪酸代謝、細(xì)胞黏附分子和膽固醇代謝等相關(guān)的26個差異表達(dá)基因，并隨機(jī)選取其中四個基因進(jìn)行檢測，結(jié)果表明：VPA可使雙峰駝成纖維細(xì)胞黏附分子信號通路中、、三個基因表達(dá)上調(diào)，細(xì)胞間互作增強(qiáng)，同時上調(diào)脂肪酸信號通路中基因的表達(dá)?！窘Y(jié)論】VPA可將細(xì)胞阻滯在分裂期之前，以減少重編程過程中分化幾率。同時，VPA對雙峰駝成纖維細(xì)胞重編程過程中多條信號通路均具有影響，調(diào)節(jié)信號通路中相關(guān)基因表達(dá)趨勢，有效提高細(xì)胞重編程效率，在雙峰駝成纖維細(xì)胞重編程中發(fā)揮重要作用。

誘導(dǎo)多能干細(xì)胞；雙峰駝；丙戊酸；細(xì)胞重編程

0 引言

【研究意義】雙峰駝是荒漠特有物種，主要生活在我國西北地區(qū)，近年來由于人口數(shù)量不斷增長，開墾范圍進(jìn)一步擴(kuò)大，導(dǎo)致野生雙峰駝的水源地被人為破壞，造成其數(shù)量急劇下降，現(xiàn)已被列為瀕危動物。雙峰駝不僅在歷史文化中有著重要作用，在科研方面也具有很好的研究前景。因此，運(yùn)用科學(xué)技術(shù)手段對其種質(zhì)資源進(jìn)行保護(hù)具有重要意義?！厩叭搜芯窟M(jìn)展】日本京都大學(xué)Takahashi等[1]成功將與細(xì)胞多能性相關(guān)的4個基因、、和隨機(jī)整合到小鼠胚胎成纖維細(xì)胞中，從而將其重編程為一類具有類似于胚胎干細(xì)胞的誘導(dǎo)多能干細(xì)胞（induced pluripotent stem cells，iPSCs），此過程被稱為iPS經(jīng)典誘導(dǎo)途徑。iPSCs在細(xì)胞形態(tài)、增殖能力、分化能力及形成畸胎瘤等方面均與胚胎干細(xì)胞相似，且可以避免因使用胚胎干細(xì)胞而造成的倫理問題，這對于干細(xì)胞、表觀遺傳學(xué)及生物醫(yī)學(xué)等研究領(lǐng)域具有深遠(yuǎn)影響。此外，研究人員還利用此技術(shù)成功得到人類和多種動物的iPSCs[2-10]?！颈狙芯壳腥朦c(diǎn)】然而，到目前為止有關(guān)雙峰駝iPSCs的報道屈指可數(shù)，獲得雙峰駝iPSCs對其種質(zhì)資源保護(hù)顯得尤為重要。在經(jīng)典誘導(dǎo)途徑中外源誘導(dǎo)因子參與調(diào)控多條癌癥信號通路，且其易與細(xì)胞基因組發(fā)生整合，因此減少細(xì)胞重編程中外源誘導(dǎo)因子的使用，可提高細(xì)胞重編程的安全性。隨著不斷探索，研究人員發(fā)現(xiàn)許多小分子化合物，如丙戊酸（valproic acid，VPA）、5-氮雜氧胞苷（5-Azadeoxycytidine，Aza）、維生素C等可有效提高細(xì)胞重編程效率[11-13]。其中，丙戊酸是一種組蛋白去乙酰化酶抑制劑（histone deacetylase inhibitors，HDACi），可代替小鼠成纖維細(xì)胞重編程中的c-Myc誘導(dǎo)因子[14]，甚至可以在人成纖維細(xì)胞重編程中代替c-Myc和Klf4完成細(xì)胞重編程過程[12]。不僅如此，VPA還可將iPSCs的誘導(dǎo)效率提高100倍以上，并保持其穩(wěn)定性[14-16]?！緮M解決的關(guān)鍵問題】本試驗(yàn)通過在雙峰駝成纖維細(xì)胞重編程過程中添加VPA，并運(yùn)用RNA-seq、RT-qPCR技術(shù)對收集到的細(xì)胞進(jìn)行分析，以闡明VPA在雙峰駝成纖維細(xì)胞重編程過程中的作用及iPSCs早期誘導(dǎo)過程中的分子機(jī)制，為雙峰駝發(fā)病機(jī)制研究及細(xì)胞替代性治療、新藥篩選等提供參考依據(jù)。

1 材料與方法

本研究于2019年8月至2021年6月在甘肅省甘肅農(nóng)業(yè)大學(xué)甘肅省動物生殖生理及繁殖調(diào)控重點(diǎn)實(shí)驗(yàn)室完成。

1.1 質(zhì)粒的制備

制備經(jīng)典誘導(dǎo)途徑中PMXS-Oct4、PMXS-Sox2、PMXS-Klf4、PMXS-cMyc和PMXS-EGFP質(zhì)粒，同時制備用于病毒包裝的pCMV-VSV-G和PumV質(zhì)粒（甘肅省動物生殖生理及繁殖調(diào)控重點(diǎn)實(shí)驗(yàn)室）。

1.2 細(xì)胞系和細(xì)胞培養(yǎng)條件

雙峰駝胎兒成纖維細(xì)胞（BCFFs）來源于三月齡雙峰駝胎兒（甘肅省張掖市駱駝養(yǎng)殖場），組織塊貼壁法消化分離后培養(yǎng)，凍存F1—F3細(xì)胞。高轉(zhuǎn)染293細(xì)胞由中國科學(xué)院蘭州獸醫(yī)研究所捐贈。兩種細(xì)胞都在含10%胎牛血清（BI，Israel）的DMEM/F12葡萄糖（Hyclone，USA）培養(yǎng)基中，并置于37 ℃、5% CO2培養(yǎng)箱中進(jìn)行培養(yǎng)。

1.3 逆轉(zhuǎn)錄病毒感染和VPA處理

293T包裝細(xì)胞以每60 mm培養(yǎng)皿1×106個細(xì)胞接種，孵育過夜。次日，根據(jù)產(chǎn)品說明書，使用 Lipofectamine 2000(Invitrogen，USA)用5 μg pMXs載體(OSKMG)和5 μg逆轉(zhuǎn)錄病毒包裝混合物轉(zhuǎn)染細(xì)胞。48 h后，收集培養(yǎng)基作為第一支含病毒上清液并為細(xì)胞更換新培養(yǎng)基，24 h后再次收集培養(yǎng)基作為第二支含病毒上清液。

轉(zhuǎn)導(dǎo)前一天，將BCFFS接種至六孔板（每孔2×105個細(xì)胞）。轉(zhuǎn)導(dǎo)過程中，將含病毒的上清液通過0.45 mm孔徑濾器過濾，濾液等比例混合并補(bǔ)充8 mg·mL-1聚凝胺，置于成纖維細(xì)胞培養(yǎng)皿中孵育過夜。轉(zhuǎn)導(dǎo)24 h后，用第二支含病毒上清液替換已有培養(yǎng)基。

細(xì)胞隨機(jī)分為對照組和處理組，每組重復(fù)3次。兩次轉(zhuǎn)導(dǎo)后，將培養(yǎng)基更換為iPSC誘導(dǎo)培養(yǎng)基，該培養(yǎng)基由DMEM/F12培養(yǎng)基（Gibco，USA）、20%胎牛血清（Gibco，USA）、2 mmol·L-1非必需氨基酸（Gibco，USA）、0.1 mmol·L-1β-巰基乙醇(Sigma- Aldrich，USA)、20 ng·mL-1FGF2 (Thermo，USA)和10 ng·mL-1白血病抑制因子（Thermo，USA）組成；處理組細(xì)胞額外補(bǔ)充1 mmol·L-1丙戊酸(Sigma，USA)。7 d后，通過胰蛋白酶消化處理收集細(xì)胞樣品[17]，加入TRIzol試劑（Sigma，USA）重懸，液氮速凍后儲存于-80 ℃。

1.4 RNA提取、文庫構(gòu)建和RNA測序

對照組（n=3）隨機(jī)命名為CK-1、CK-2、CK-3；處理組（n=3）隨機(jī)命名為T-1、T-2、T-3。根據(jù)產(chǎn)品說明，使用TRIzol試劑從6個樣品中提取總RNA。通過Agilent 2100生物分析儀，在去除rRNA后進(jìn)行無RNase瓊脂糖凝膠電泳檢測RNA質(zhì)量和濃度。隨后，使用oligo-dT珠子分離Poly (A) mRNA，并通過超聲波中斷mRNA，以片段化mRNA為模板，隨機(jī)寡核苷酸為引物，采用M-MuLV逆轉(zhuǎn)錄酶系統(tǒng)合成第一條cDNA鏈，緊接著使用RNaseH降解RNA鏈，在DNA聚合酶I的作用下，以dNTPs為模板合成第二條cDNA鏈。純化的雙鏈cDNA在末端被修復(fù)，連接至poly A尾并連接測序接頭。使用AMPure XP微珠篩選200 bp的cDNA并進(jìn)行PCR擴(kuò)增。最后，使用AMPure XP珠對PCR產(chǎn)物進(jìn)行純化和富集，以構(gòu)建最終的cDNA文庫。

使用雙末端技術(shù)，在Genedenovo Biotechnology Co., Ltd（中國廣州）Illumina測序平臺（Illumina HiSeq? 2500）上對cDNA文庫進(jìn)行測序。用fastp[18]程序刪除低質(zhì)量序列（一個序列中有超過50%的堿基質(zhì)量低于20）、N堿基超過10%的讀數(shù)（堿基未知）和包含適配器的序列（表1）。

表1 引物序列及參數(shù)

、、、：轉(zhuǎn)錄因子；：腫瘤蛋白p53；：細(xì)胞周期蛋白B1；：基質(zhì)金屬肽酶9；：細(xì)胞分裂周期20；：S100鈣結(jié)合蛋白A4；：血管內(nèi)皮生長因子C；：紋狀蛋白；：CDC28蛋白激酶調(diào)節(jié)亞基2；：單克隆抗體Ki 67鑒定抗原；：增殖的細(xì)胞核抗原；：半胱天冬酶7；：脂肪酸轉(zhuǎn)位酶；：神經(jīng)纖維素；：L1細(xì)胞黏附分子；：接觸蛋白1

,,,: Transcription factors;: tumor protein p53;: cyclin B1;: matrix metallopeptidase 9;: cell division cycle 20;: S100 calcium binding protein A4;: vascular endothelial growth factor C;: vimentin;: CDC28 protein kinase regulatory subunit 2;: antigen identified by monoclonal antibody ki 67;: proliferating cell nuclear antigen;: caspase7;: fatty acid translocation enzyme;: neurofascin;: L1 cell adhesion molecule;: contactin 1

1.5 轉(zhuǎn)錄本組裝和表達(dá)值估計

利用HISAT2[19]對RNA測序讀數(shù)和雙曲線Camelus bactrianus Ca_bactrianus_MBC_1.0基因組組裝進(jìn)行對比。

已知雙峰駝基因（https://www.ncbi.nlm.nih.gov/ genome/?term=Camelus+bactrianus）作為基因組參照組。在HISAT2比較的基礎(chǔ)上，使用Stringtie[20]重建轉(zhuǎn)錄本并計算每個樣本中全部基因的表達(dá)水平，校正測序深度以及基因或轉(zhuǎn)錄本長度，以確保后續(xù)分析的準(zhǔn)確性。在此之前，計算得到每個基因的FPKM值。

基于基因表達(dá)信息，使用R（http://www.r-project. org/）對主成分進(jìn)行分析（PCA，Principal Component Analysis）并計算皮爾遜相關(guān)系數(shù)。隨后使用DESeq2[21]軟件進(jìn)行差異基因表達(dá)分析，結(jié)合KEGG（//www. kegg.jp/）和GO（//geneontology.org/）研究分析相關(guān)途徑。

1.6 引物設(shè)計及實(shí)時熒光定量PCR

利用NCBI和primer 3.0網(wǎng)站設(shè)計特異性引物，以作為內(nèi)參基因?qū)λx目的基因進(jìn)行RT- qPCR分析，以檢驗(yàn)測序結(jié)果的準(zhǔn)確性并進(jìn)一步分析轉(zhuǎn)錄組數(shù)據(jù)。

使用RevertAid? First Strand cDNA Synthesis Kit (Thermo FisherScientific Inc., USA) 產(chǎn)品說明制備cDNA。RT-qPCR反應(yīng)體積為20 μL，其中包含10 μLReal Master Mix SYBR、8 μL ddH2O、正向和反向引物各1 μL，以及1 μL cDNA，使用ΔΔCT方法計算所有基因的相對表達(dá)量。

1.7 重編程進(jìn)度檢測

為區(qū)分對照組與處理組，將未添加VPA組命名為OSKM，將添加VPA組命名為OSKM+VPA。通過PCR技術(shù)測試BCFFs、OSKM和OSKM+VPA此3組中的特定基因，其中包含3組外源基因、干細(xì)胞特異性基因和3個胚層標(biāo)記基因。檢測成纖維細(xì)胞中特定基因的表達(dá)水平以驗(yàn)證外源基因?qū)Τ衫w維細(xì)胞的修飾作用及重編程進(jìn)度。

2 結(jié)果

2.1 VPA處理

使用293T細(xì)胞包裝含有經(jīng)典組合Oct3、c-Myc、Klf4、Sox2和EGFP逆轉(zhuǎn)錄病毒載體，在病毒轉(zhuǎn)導(dǎo)后72 h，觀察到EGFP逆轉(zhuǎn)錄病毒包裝結(jié)果（圖1-A），利用EGFP預(yù)測其余4種逆轉(zhuǎn)錄病毒的轉(zhuǎn)錄效率。第二次逆轉(zhuǎn)錄病毒感染后，在iPSC培養(yǎng)基中添加1 mmol·L-1VPA對細(xì)胞進(jìn)行處理，第二次病毒感染后第8天觀察到轉(zhuǎn)染效果（圖1-B），光學(xué)顯微鏡觀察第二次病毒感染后8 d的細(xì)胞形態(tài)（圖1-D），及未處理的空白對照組細(xì)胞形態(tài)（圖1-C）。光學(xué)顯微鏡觀察VPA處理7 d后的細(xì)胞狀態(tài)（圖1-F）及其對照組（圖1-E）。

A. 轉(zhuǎn)導(dǎo)后72 h的EGFP逆轉(zhuǎn)錄病毒包裝；B. 第二次病毒感染后8 d；C. 培養(yǎng)8 d的成纖維細(xì)胞；D. 第二次病毒感染后8 d的細(xì)胞形態(tài)；E. 培養(yǎng)基中未添加VPA處理7 d的細(xì)胞形態(tài)；F. 培養(yǎng)基中添加VPA并處理7 d的細(xì)胞形態(tài)

2.2 基因轉(zhuǎn)移驗(yàn)證和組學(xué)數(shù)據(jù)驗(yàn)證

重編程后雙峰駝成纖維細(xì)胞中內(nèi)、外源基因的表達(dá)規(guī)律如圖2所示，、、、、在OSKM和OSKM+VPA組中均有表達(dá)（圖2-A），且OSKM+VPA組表達(dá)量高于OSKM組，而其在BCEFs組不表達(dá)。為了驗(yàn)證組學(xué)數(shù)據(jù)的可靠性，隨機(jī)選取8個基因進(jìn)行檢測，結(jié)果顯示、、、、、在OSKM+VPA組中表達(dá)下調(diào)，且差異極顯著（＜0.01）；基因表達(dá)呈上調(diào)趨勢，且差異極顯著（＜0.01）。在OSKM+VPA組中表達(dá)量無明顯差異（圖2-B），8個基因變化趨勢與RNA-seq組學(xué)數(shù)據(jù)一致，表明組學(xué)數(shù)據(jù)準(zhǔn)確可信，可用于后續(xù)分析。

A. 半定量PCR檢測BCFFs、OSKM和OSKM＋VPA組中內(nèi)、外源基因mRNA表達(dá)水平；B. TP53、CCNB1、MMP9、CDC20、S100A4、VEGFC、VIM和CKS2 mRNA表達(dá)水平（內(nèi)參基因：β-actin）。**表示差異極顯著（P＜0.01）

2.3 轉(zhuǎn)錄組測序分析

為確定VPA在成纖維細(xì)胞重編程中的作用，通過RNA-seq技術(shù)對處理組和對照組的6個樣本進(jìn)行轉(zhuǎn)錄組分析。從6個樣本中分別獲得了61 256 236、65 376 850、66 062 928、69 282 900、68 014 520和67 418 570個clean reads（表2）。其中90.40%、90.12%、90.32%、90.39%、90.12%和90.75%的clean reads被比對到參考基因組（Table 2），并注釋了14 377、14 332、14 399、14 367、14 427和14 427個基因（表3）。

根據(jù)每個樣本基因表達(dá)譜，使用PCA（圖3-A）對樣本進(jìn)行分析，了解樣本重復(fù)性并排除異常值。此外，通過小提琴圖將每個樣本中基因豐度的表達(dá)可視化（圖3-B）?；蚩倲?shù)是19 748個，根據(jù)基因豐度≥1進(jìn)行篩選后，基因數(shù)是11 780個，其中處理組獨(dú)有的318個基因、對照組獨(dú)有的260個基因，兩者共11 202個基因。如圖3-C所示?；诓町惙治鼋Y(jié)果，在添加VPA處理7d后，F(xiàn)DR＜0.05和| log2FC |＞1為顯著差異表達(dá)基因。因此，根據(jù)每個對照組的差異表達(dá)基因（469個上調(diào)基因和490個下調(diào)基因）建立火山圖；基因越接近兩端，差異程度越大（圖3-D）。并且假設(shè)具有相似表達(dá)模式的基因有共同功能或參與共同代謝途徑和信號傳導(dǎo)途徑，以此為基礎(chǔ)進(jìn)行了進(jìn)一步分析。

利用GO分析對樣本基因進(jìn)行功能分類?；谛蛄型葱?，unigenes分為三大功能類別：生物學(xué)過程，細(xì)胞組分和分子功能（圖3-E）。為確定目的基因顯著富集通路，使用KEGG通路富集分析和超幾何檢驗(yàn)分析。篩選獲得959個差異表達(dá)基因，富集在276個信號通路中。其中值小于0.05的信號通路有42條，值小于0.05的信號通路有8條。這8條信號通路分別為類固醇生物合成、細(xì)胞周期、PPAR信號通路、孕酮介導(dǎo)的卵母細(xì)胞成熟、脂肪酸代謝、ECM-受體相互作用、細(xì)胞黏附分子和膽固醇代謝（圖3-F）。

表2 計數(shù)據(jù)比較

Sample：樣本名；Total：過濾核糖體后reads數(shù)量，稱為有效reads；Unmapped (%)：未比對上參考基因組的reads數(shù)及占有效reads比例；Unique_Mapped (%)：唯一對比上參考基因組的reads數(shù)及占有效reads比例；Multiple_Mapped (%)：多處比對上參考基因組的reads數(shù)及占有效reads比例；All_Mapped：全部的可以定位到基因組上的reads數(shù)量及占有效reads比例

Sample: sample name; Total: number of reads after filtering ribosomes, called valid reads; Unmapped (%): number of reads that are not matched to the reference genome and the proportion of valid reads; Unique_Mapped (%): number of reads that are uniquely matched to the reference genome and the proportion of valid reads; Multiple_Mapped (%): number and proportion of effective reads in the reference genome compared at multiple locations; All_Mapped: the number of reads that can be localized to the genome and the proportion of valid reads

表3 基因檢測統(tǒng)計

Sample：樣本名；Refer genes：參考基因組（或參考基因集合）的基因總數(shù)，當(dāng)不存在新基因時，無該列結(jié)果；Sequenced refer genes (%)：測序結(jié)果所檢測到的Refer genes總數(shù)及占Refer genes的百分比，當(dāng)不存在新基因時，無該列結(jié)果；Novel genes：項(xiàng)目發(fā)現(xiàn)的新基因數(shù)目，當(dāng)不存在新基因時，無該列結(jié)果；Sequenced novel genes (%)：測序結(jié)果檢測到的Novel genes總數(shù)及占Novel genes的百分比，當(dāng)不存在新基因時，無該列結(jié)果；Total genes：所有基因總數(shù)，包含參考基因組及新基因；Sequenced total genes (%)：測序結(jié)果所檢測到的基因總數(shù)及占All genes的百分比

Sample: sample name; Refer genes: total number of genes in the reference genome (or reference gene collection), no result in this column when no new genes are present; sequenced Refer genes (%): total number and percentage of Refer genes detected by sequencing results, no result in this column when no new genes are present; Novel genes: number of new genes discovered by the project, no result in this column when no new genes are present; sequenced_Novel genes (%): total number of Novel genes detected by sequencing results and percentage of Novel genes , when no new genes are present, no result in this column; Total_Genes: total number of all genes, when no new genes are present, no result in this column; Total_Genes: total number of all genes, including reference genome and novel genes; sequenced_Total genes (%): total number of genes detected by sequencing results and percentage of All_Genes

2.4 增殖和凋亡基因的影響

增殖和凋亡基因在BCEF、OSKM和OSKM+VPA組的表達(dá)規(guī)律如圖4所示。iPSC培養(yǎng)基中加入VPA后，增殖基因和表達(dá)呈下降趨勢，差異極顯著（＜0.01）；而凋亡基因表達(dá)極顯著上調(diào)（＜0.01）。

A. 主成分分析：PC1坐標(biāo)代表第一主成分；PC2坐標(biāo)代表第二主成分；圖中的彩色點(diǎn)代表每個樣本；B. 樣本的相關(guān)熱圖：圖中橫坐標(biāo)和縱坐標(biāo)分別為樣本，顏色深度（強(qiáng)度）表示兩個樣本之間的相關(guān)系數(shù)；C. 樣本或組間基因豐度表達(dá)的可視化；D. 維恩圖顯示了兩組的共同和獨(dú)特基因；E. 橫坐標(biāo)代表兩組差異倍數(shù)的對數(shù)，縱坐標(biāo)代表兩組差異FDR的負(fù)Log10值，紅色（group_2向上，相對于group_1的調(diào)節(jié)表達(dá)）和藍(lán)色（下調(diào)表達(dá)）表示基因表達(dá)水平的差異；F. 用不同顏色表示不同樣本基因，顏色越紅，表達(dá)越高；顏色越藍(lán)，表達(dá)越低

A. Principal Component Analysis: the PC1 coordinate represents the first principal component; the PC2 coordinate represents the second principal component; while, the colored dots in the figure represent each sample; B. Correlation heat map of the samples: In the figure, the abscissa and ordinate are the respective samples and the color depth (intensity) indicates the correlation coefficient between the two samples; C. Visualization of gene abundance expression between samples or groups; D. Venn diagram showing the common and unique genes of the two groups; E. The abscissa represents the logarithm of the multiple of the difference between the two groups, the ordinate represents the negative Log10 value of the FDR of the difference between the two groups, and the red (group_2 up-regulated expression relative to group_1) and blue (down-regulated expression) indicate a difference in the gene expression levels; whereas, the black dots represent no difference; F. Genes from different samples are expressed in different colors; the redder the color, the higher the expression; whereas, the bluer the color, the lower the expression

圖3 轉(zhuǎn)錄組測序、相關(guān)性的基本分析和組間差異

Fig. 3 Transcriptomic sequencing, basic analysis of correlations, and differences between groups

2.5 VPA處理影響的路徑分析

在本研究中，根據(jù)值小于0.05的8個KEGG通路，篩選出與細(xì)胞周期、脂肪酸代謝、細(xì)胞黏附分子和膽固醇代謝等相關(guān)的26個差異表達(dá)基因，根據(jù)這些基因構(gòu)建熱圖，分析其在不同分組的表達(dá)差異。結(jié)果顯示，在OSKM和OSKM+VPA組之間這些差異表達(dá)基因在重編程細(xì)胞中表達(dá)具有顯著性差異，尤其是脂肪酸合酶和細(xì)胞黏附分子（圖5-A）。在OSKM組中表達(dá)量顯著下調(diào)（＜0.01），經(jīng)VPA處理后（OSKM+VPA組）較OSKM組表達(dá)量上調(diào)，且差異顯著（＜0.01）（圖5-B）；在OSKM組中表達(dá)量下調(diào)，且差異顯著（＜0.01），其在VPA處理后（OSKM+VPA組）表達(dá)量無明顯差異（圖5-C）；和，在OSKM+VPA組中表達(dá)量顯著上調(diào)（＜0.01），而在BCEF組中不表達(dá)（圖5-D、E）；和基因在OSKM和OSKM+VPA組均有表達(dá)，且OSKM+VPA組表達(dá)量高于OSKM組，而其在BCEFs組不表達(dá)（圖5-F）。

A. Mki67 mRNA表達(dá)水平（內(nèi)參基因：β-actin）；B. PCNA mRNA表達(dá)水平（內(nèi)參基因：β-actin）；C. CASP7 mRNA表達(dá)水平（內(nèi)參基因：β-actin）。**表示差異極顯著（P＜0.01）

3 討論

細(xì)胞重編程技術(shù)在種質(zhì)資源保護(hù)、再生醫(yī)學(xué)和新藥篩選等方向都具有潛在的應(yīng)用價值。但因其重編程效率較低，使得該技術(shù)很難由實(shí)驗(yàn)室向臨床應(yīng)用發(fā)展，導(dǎo)致重編過程中因原癌基因引入造成的安全問題限制了其在臨床上的應(yīng)用[22]。隨著對重編程技術(shù)的探索，研究人員發(fā)現(xiàn)小分子物質(zhì)可以提高重編程的效率以及減少原癌基因的使用。本研究以雙峰駝成纖維細(xì)胞為研究對象，結(jié)合RNA-seq、RT-qPCR和半定量PCR等方法，探討小分子物質(zhì)丙戊酸對雙峰駝成纖維細(xì)胞重編程的影響。結(jié)果表明，丙戊酸可抑制細(xì)胞周期信號通路，阻滯細(xì)胞分化，并通過影響細(xì)胞黏附分子信號通路增強(qiáng)細(xì)胞間互作，在脂肪酸代謝途徑中發(fā)揮調(diào)控作用。

3.1 丙戊酸對細(xì)胞周期信號通路的影響

細(xì)胞衰老是OSKM因子感染細(xì)胞使其進(jìn)入重編程的阻礙之一，而VPA可通過將細(xì)胞阻滯在G2/M期來提高細(xì)胞重編程的效率[23-24]。本研究檢測了與細(xì)胞周期信號通路相關(guān)的、、、、和等基因，其中效應(yīng)凋亡蛋白是參與細(xì)胞凋亡的重要成員之一[25]，有研究表明的缺乏會減弱大鼠細(xì)胞凋亡程度[26]；在細(xì)胞復(fù)制和DNA修復(fù)過程中表達(dá)，從細(xì)胞G1期開始合成，S期達(dá)到峰值，G2和M期表達(dá)量開始下降[27]。研究發(fā)現(xiàn)在添加VPA后增殖相關(guān)基因、、和表達(dá)下調(diào)，而凋亡基因則呈上調(diào)趨勢。這一結(jié)果提示VPA通過調(diào)控重編程中增殖和凋亡基因的表達(dá)趨勢將細(xì)胞阻滯在未分化階段，以促進(jìn)雙峰駝成纖維細(xì)胞重編程效率。

3.2 丙戊酸對脂肪酸代謝相關(guān)基因的影響

脂肪酸作為脂質(zhì)組成中最基礎(chǔ)同時也是最重要的組成部分，是機(jī)體主要能量來源之一，其合成對于人類多能干細(xì)胞的生存有重要意義[28]。Abumrad等[29]通過克隆大鼠脂肪細(xì)胞膜蛋白，發(fā)現(xiàn)大鼠脂肪酸轉(zhuǎn)位酶（fatty acid translocase，F(xiàn)AT）與人有同源性，證明了的FAT活性，從而明確了其在脂肪酸攝取中的作用。本研究結(jié)果顯示VPA可使脂肪酸代謝途徑中的表達(dá)量上調(diào)，提示VPA可上調(diào)表達(dá)從而促進(jìn)長鏈脂肪酸的吸收，調(diào)節(jié)脂肪酸代謝，以期獲得質(zhì)量更優(yōu)的重編程細(xì)胞。

A. 26個差異表達(dá)基因的熱圖；B. CD36 mRNA表達(dá)水平（內(nèi)參基因：β-actin）；C. NFASC mRNA表達(dá)水平（內(nèi)參基因：β-actin）；D. L1CAM mRNA表達(dá)水平（內(nèi)參基因：β-actin）；E. CNTN1 mRNA表達(dá)水平（內(nèi)參基因：β-actin）；F. 半定量PCR檢測BCFFs、OSKM和OSKM＋VPA三組中L1CAM、CNTN1 mRNA表達(dá)水平。**表示差異極顯著（P＜0.01）

3.3 丙戊酸對細(xì)胞黏附分子信號通路的影響

細(xì)胞黏附分子介導(dǎo)的細(xì)胞間互作，對人類多能干細(xì)胞的自我更新和多能干細(xì)胞狀態(tài)有重要貢獻(xiàn)[30]。細(xì)胞黏附分子一般分為5類，包括整合素、選擇素、鈣黏素、免疫球蛋白超家族（IgSF）和黏蛋白血管地址素[31]。研究發(fā)現(xiàn)，人類干細(xì)胞表面存在很多CAM家族成員，它們對干細(xì)胞的自身更新和多能性具有調(diào)節(jié)作用[1,32-36]。試驗(yàn)檢測了、和這3個與細(xì)胞黏附分子信號通路相關(guān)的基因，其中和屬于免疫球蛋白超家族成員（IgSF）且已被證明由未分化的人類胚胎干細(xì)胞表達(dá)，但其作用機(jī)制尚不完全清楚[36]。結(jié)果表明，丙戊酸可以促進(jìn)、和的表達(dá)，使其表達(dá)量上調(diào)，這一結(jié)果提示丙戊酸可以促進(jìn)與細(xì)胞黏附分子信號通路相關(guān)的基因表達(dá)，增強(qiáng)細(xì)胞重編程過程中互作作用，從而促進(jìn)重編程過程中細(xì)胞的自我更新。

4 結(jié)論

丙戊酸可抑制細(xì)胞周期信號通路，減少重編程中細(xì)胞分化幾率；同時在脂肪酸代謝中發(fā)揮調(diào)控作用；上調(diào)細(xì)胞黏附分子信號通路中相關(guān)基因表達(dá)，增強(qiáng)細(xì)胞間互作。丙戊酸在雙峰駝成纖維細(xì)胞重編程中通過調(diào)控相關(guān)信號通路從而對重編程起到促進(jìn)作用。這將為丙戊酸促進(jìn)雙峰駝成纖維細(xì)胞重編程作用機(jī)制的研究提供試驗(yàn)基礎(chǔ)，同時為雙峰駝種質(zhì)資源保護(hù)提供理論參考。

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Effect of Valproic Acid on Reprogramming of Bactrian Camel Fibroblasts

1College of Life Science and Technology/Gansu Key Laboratory of Animal Reproductive Physiology and Reproductive Regulation, Gansu Agricultural University, Lanzhou 730070;2College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070

【Objective】To improve the efficiency of the reprogramming process of Bactrian camel fibroblasts and to reduce the risk of tumorigenesis caused by the introduction of proto-oncogenes. In this experiment, valproic acid (VPA) was added to the fibroblast reprogramming process to explore the effect of small molecules on the reprogramming of Bactrian camel fibroblasts. 【Method】Given this, March-aged Bactrian camel fetal fibroblasts were used as test materials, combined with classic induction combination OSKM (Oct4, Sox2, Klf4, and c-Myc) and EGFP five retrovirus reprogramming of Bactrian camel fibroblasts (OSKM group), and the cells by adding VPA treatment for 7 days after the second viral infection (OSKM+VPA group) were collected. Endogenous and exogenous genes were examined by using PCR to confirm the modification effect of retrovirus on Bactrian camel fibroblasts. Eight genes were randomly selected from those more significantly affected by VPA. According to RNA-seq data, whether their trends before and after VPA addition were consistent with the trends of RNA-seq data was checked to verify the accuracy of RNA-seq data. The transcriptome sample genes were classified by GO analysis and significant enrichment pathways for target genes were clarified by using KEGG pathway enrichment analysis and hypergeometric validation analysis. Total RNA was extracted from the collected cells, and then, combined with RNA-seq and Real time-quantitative interpretation (RT-qPCR) techniques to detect the effect of VPA on the reprogramming of Bactrian camel fibroblasts. 【Result】 It was detected by using PCR that the expression of endogenous and exogenous genes in different groups. The results showed that,,,, andgenes were expressed in both OSKM and OSKM+VPA groups, and the expression in OSKM+VPA group was higher than that in the OSKM group, while they were not expressed in BCEFs group. Eight genes were randomly selected for testing, and the results showed that: three genes of,, andwere down-regulated in expression after the addition of VPA, which were related to the cell cycle signaling pathway.,,, andgenes signaling were down-regulated in expression, which was related to the phenotypic characteristics of cancer;gene expression was up-regulated in the PI3k-Akt signaling pathway. This expression trend was consistent with the trend of the histological data. The results showed that the expression of proliferation genesandwere down-regulated, while the expression of apoptosis genewas up-regulated after the addition of VPA. KEGG and hypergeometric validation analyses of the transcriptome data were performed, and 959 differentially expressed genes were screened according to the analysis results, which were enriched in 276 signaling pathways, including eight signaling pathways with Q values less than 0.05: steroid biosynthesis, cell cycle, PPAR signaling pathway, progesterone-mediated oocyte maturation, fatty acid metabolism, ECM-receptor interactions, cell adhesion molecules, and cholesterol metabolism. The 26 differentially expressed genes related to cell cycle, fatty acid metabolism, cell adhesion molecule, and cholesterol metabolism were screened, and four of which were randomly selected for testing, showing that VPA upregulated the expression of,andgenes in the Bactrian camel fibroblast adhesion molecule signalling pathway and enhanced intercellular interactions. It was also upregulated that the expression ofgene in the fatty acid signaling pathway. 【Conclusion】 The results showed that the VPA blocked cell before the split phase to reduce risk differentiation during the process of reprogramming. Meanwhile, VPA affected several signaling pathways in the reprogramming process of Bactrian camel fibroblasts, and regulated the expression trend of related genes in the signaling pathways, which effectively improved the reprogramming efficiency of the cells and played an important role in the reprogramming of Bactrian camel fibroblasts.

induced pluripotent stem cells; Bactrian camel; valproic acid; cell reprogramming

10.3864/j.issn.0578-1752.2023.12.014

2022-03-14；

2022-05-19

國家自然科學(xué)基金（31560638，31960725）

張啟冉，E-mail：1074671622@qq.com。通信作者張勇，E-mail：zhychy@163.com

（責(zé)任編輯 林鑒非）