慢病毒載體介導(dǎo)下DJ- 1過表達(dá)細(xì)胞模型的建立

任 靜，李 毅，楊 慧，魯玲玲

(首都醫(yī)科大學(xué) 基礎(chǔ)醫(yī)學(xué)院 神經(jīng)生物學(xué)系 北京腦重大疾病研究院帕金森病研究所教育部神經(jīng)變性病重點實驗室，北京 100069)

研究論文

慢病毒載體介導(dǎo)下DJ- 1過表達(dá)細(xì)胞模型的建立

任 靜，李 毅，楊 慧*，魯玲玲*

(首都醫(yī)科大學(xué) 基礎(chǔ)醫(yī)學(xué)院 神經(jīng)生物學(xué)系 北京腦重大疾病研究院帕金森病研究所教育部神經(jīng)變性病重點實驗室，北京 100069)

目的構(gòu)建人野生型DJ- 1及其L166P突變體的慢病毒載體并探討慢病毒載體在構(gòu)建基因過表達(dá)細(xì)胞模型中的作用。方法分別構(gòu)建野生型DJ- 1與L166P突變型DJ- 1慢病毒載體質(zhì)粒。進(jìn)行測序確定比對正確后，進(jìn)行質(zhì)粒的大量擴增與制備并轉(zhuǎn)染包裝細(xì)胞系HEK293T細(xì)胞，熒光法和Western blot檢測野生型DJ- 1與L166P突變型DJ- 1在細(xì)胞系中的表達(dá)。在確定目的蛋白正確表達(dá)之后，大量轉(zhuǎn)染HEK293T細(xì)胞進(jìn)行包裝并生產(chǎn)攜帶目的基因的慢病毒顆粒。測定病毒上清滴度后感染PC12細(xì)胞，熒光顯微鏡和Western blot觀察GFP熒光強度以及目的蛋白的表達(dá)，確定病毒的感染效率。結(jié)果成功構(gòu)建攜帶DJ- 1野生型及其突變體的慢病毒載體。該病毒載體可以轉(zhuǎn)染進(jìn)入HEK293T細(xì)胞內(nèi)且目的蛋白能夠正確表達(dá)。LV-DJ- 1與LV-DJ- 1/L166P的病毒滴度分別為2×109TU/mL與2×108TU/mL。病毒上清可以高效感染PC12細(xì)胞，絕大多數(shù)細(xì)胞可表達(dá)目的蛋白。外源野生型DJ- 1和L166P突變體的蛋白表達(dá)量分別是內(nèi)源性含量的315%和285%。結(jié)論慢病毒感染細(xì)胞效率很高，是很好的制備基因過表達(dá)細(xì)胞的方法。通過慢病毒載體介導(dǎo)，本研究獲得了DJ- 1及其突變體的過表達(dá)細(xì)胞模型。該模型可以用于后續(xù)DJ- 1功能研究。

帕金森病；DJ- 1；PC12細(xì)胞；慢病毒載體

DJ- 1(PARK7)在細(xì)胞內(nèi)主要以可溶性二聚體的形式存在于細(xì)胞質(zhì)、細(xì)胞核及線粒體。DJ- 1的缺失和突變可以引起家族性常染色體隱性遺傳性帕金森病(Parkinson’s disease,PD)的早發(fā)及散發(fā)型PD的發(fā)生[1- 4]。2003年發(fā)現(xiàn)DJ- 1的缺失或者166位亮氨酸突變?yōu)楦彼岱謩e導(dǎo)致了荷蘭和意大利的2個家族型帕金森病[5]。DJ- 1可以作為分子伴侶抑制α-synuclein的聚合[6]。DJ- 1敲除可以破壞星形膠質(zhì)細(xì)胞介導(dǎo)的對魚藤酮損傷的神經(jīng)保護作用。因此建立DJ- 1的基因過表達(dá)模型對于研究DJ- 1的生理功能和相關(guān)發(fā)病機制有重要的意義。

慢病毒(lentivirus)載體[7]是以人類免疫缺陷型病毒(HIV)為基礎(chǔ)發(fā)展起來的基因治療載體,它對分裂細(xì)胞和非分裂細(xì)胞均具有感染能力,并可以在體內(nèi)較為長期的表達(dá)且安全性高[8]。因其獨特的生物學(xué)性狀顯示出不同于腺病毒載體的巨大潛力，例如慢病毒載體既能轉(zhuǎn)導(dǎo)分裂細(xì)胞又能轉(zhuǎn)導(dǎo)非分裂細(xì)胞;目的基因高效的整合于宿主細(xì)胞基因組內(nèi)可實現(xiàn)長期而穩(wěn)定的表達(dá)并可以傳代給子代細(xì)胞;去除了原病毒復(fù)制的關(guān)鍵位點,提高了安全性[9- 10]等一系列優(yōu)良特性。

因此，本研究擬分別構(gòu)建攜帶DJ- 1及其L166P突變體的慢病毒載體，通過包裝獲得高滴度病毒上清并以此感染小鼠腎上腺髓質(zhì)嗜鉻細(xì)胞瘤克隆化細(xì)胞系(pheochromocytoma, PC12細(xì)胞)以建立過表達(dá)DJ- 1及其L166P突變體的的細(xì)胞系。

1 材料和方法

1.1 材料

HEK293T與PC12細(xì)胞系(吉凱基因有限公司建立)；DMEM/F12培養(yǎng)基、胎牛血清(FBS)和胰蛋白酶和RIPA裂解(Gibco公司)；DJ- 1、β-actin抗體(Sigma公司)；Anti-GFP抗體、Anti-Mouse抗體(Santa-Cruz公司)；蛋白酶抑制劑、磷酸酶抑制劑(Merck公司)、Lipofectamine 2000和BCA試劑盒(Invitrogen公司)。

1.2 方法

1.2.1 細(xì)胞培養(yǎng)和轉(zhuǎn)染：將HEK293T細(xì)胞接種于含10%新生牛血清的F12培養(yǎng)基中，細(xì)胞在5% CO2、37 ℃培養(yǎng)箱中培養(yǎng)。選用對數(shù)增殖的細(xì)胞用于慢病毒轉(zhuǎn)染。按照Lipofectamine 2000轉(zhuǎn)染試劑盒使用說明共轉(zhuǎn)染HEK293T細(xì)胞，轉(zhuǎn)染8 h后更換為完全培養(yǎng)基。

1.2.2 慢病毒包裝與病毒滴度的檢測：以HEK293T細(xì)胞為包裝細(xì)胞，制備編碼慢病毒的重組病毒質(zhì)粒及其2種輔助包裝原件載體質(zhì)粒，3種質(zhì)粒分別進(jìn)行高純度無內(nèi)毒素抽提，按Lipofectamine 2000轉(zhuǎn)染試劑盒使用說明操作，分別收集含DJ- 1及L166P 基因慢病毒載體的細(xì)胞上清液，4 ℃、3 000 r/min離心15 min得到濃縮后的高滴度含DJ- 1及L166P基因慢病毒載體濃縮液，采用逐孔稀釋法測定病毒滴度(TU/mL)[11]。

病毒液進(jìn)行去RNA酶處理后按照試劑盒操作步驟要求提取病毒RNA，之后反轉(zhuǎn)錄為cDNA,繼而進(jìn)行實時定量PCR檢測，通過比較對照組與實驗組的Ct值差異可判斷滴度值。通常，認(rèn)為Ct值差異在2以上是存在顯著差異的。病毒滴度的計算公式為病毒滴度(TU/mL)=陽性細(xì)胞數(shù)×稀釋倍數(shù)×20×103。

1.2.3 慢病毒感染PC12細(xì)胞：向PC12細(xì)胞內(nèi)各加入上述包裝好的病毒上清(MOI=10)及polybrene(8 μg/mL)的完全培養(yǎng)基，PC12細(xì)胞經(jīng)過5次傳代，經(jīng)胰蛋白酶消化分散后制成5×105個/mL濃度的細(xì)胞懸液，接種于無菌培養(yǎng)皿中，每皿2 mL，置于37 ℃、10% CO2的培養(yǎng)箱內(nèi)進(jìn)行培養(yǎng)。24 h后更換為500 μL體系不含有病毒的完全培養(yǎng)基，待72 h觀察細(xì)胞熒光發(fā)光亮度[12]，以此判斷不同病毒對于PC12細(xì)胞的感染效率。

1.2.4 蛋白質(zhì)免疫印跡檢測蛋白表達(dá)：在HEK293T細(xì)胞系轉(zhuǎn)染后24和48 h時間點分別提取全細(xì)胞蛋白，選用RIPA蛋白裂解液裂解蛋白后使用BCA法測定蛋白濃度，隨后進(jìn)行SDS-PAGE電泳，濕轉(zhuǎn)法將蛋白轉(zhuǎn)移至PVDF膜，5%脫脂牛奶封閉1 h，分別入Anti-GFP抗體 (1∶2 000),DJ- 1兔單克隆抗體(1∶10 000)，β-actin兔抗體(1∶1 000)，4 ℃過夜。0.1% TBST洗膜4次，加入相應(yīng)兔二抗(1∶10 000),Anti-Mouse抗體(1∶5 000)，室溫孵育1 h，洗膜方法同前。加入化學(xué)發(fā)光液，于暗室化學(xué)發(fā)光及顯影和定影。

1.3 統(tǒng)計學(xué)分析

2 結(jié)果

2.1 成功構(gòu)建攜帶DJ- 1/WT與DJ- 1/L166P的慢病毒載體pGC-LV-DJ- 1/WT與pGC-LV-DJ- 1/L166P

通過亞克隆的方法，將已有DJ- 1/WT與 DJ- 1/L166P序列插入慢病毒載體pGC-LV。PCR結(jié)果(圖1)顯示條帶為774 bp，與目標(biāo)條帶位置相一致, 經(jīng)PCR實驗初步證實目的片段嵌入位置正確，且測序證明插入的目的片段(DJ- 1/WT和DJ- 1/L166P基因)序列完全正確。將pGC-LV-DJ- 1/WT與pGC-LV-DJ- 1/L166P分別轉(zhuǎn)染HEK293T細(xì)胞后，熒光顯微鏡下可以看到大量綠色GFP熒光的表達(dá)(圖2A)。將上述細(xì)胞裂解并收獲蛋白后，進(jìn)行Western blot檢測，可以在49 ku處看到DJ- 1與GFP融合蛋白的條帶(圖2B)。證明插入的目的基因與GFP形成融合蛋白，且此帶有目的基因的融合蛋白可以很好地在真核細(xì)胞中表達(dá)。在后續(xù)的實驗中，可以通過檢測GFP的綠色熒光指示外源基因DJ- 1/WT和DJ- 1/L166P的表達(dá)。

1.negative control,ddH2O; 2.negative control,no-load; 3.positive control,GAPDH; 4.marker; 6～8.pGC-LV-DJ- 1/WT group; 9～12.pGC-LV-DJ- 1/L166P group; PCR product size: positive transformants obtained 774 bp bands, consistent with the size of the target band圖1 PCR鑒定慢病毒載體pGC-LV-DJ- 1/WT與pGC-LV-DJ- 1/L166PFig 1 PCR identified DJ- 1 carrying lentiviral vectors pGC-LV-DJ- 1/WT and pGC- LV-DJ- 1/L166P

2.2 成功包裝并獲得了高滴度的假病毒顆粒

將攜帶有外源基因的pGC-LV質(zhì)粒(pGC-LV-DJ- 1/WT或pGC-LV-DJ- 1/L166P)，pHelper1.0與pHelper2.0，3個質(zhì)粒共轉(zhuǎn)HEK293T細(xì)胞，將富含慢病毒顆粒的細(xì)胞上清濃縮后得到高滴度的慢病毒濃縮液，在HKE293T細(xì)胞中測定并標(biāo)定病毒滴度。經(jīng)檢測在1×10-5μL組樣品和對照組樣品的Ct值間存在2個左右差異，認(rèn)為在1×10-5μL組樣品中存在病毒顆粒。假定該組樣品含有至少有1個病毒顆粒，則攜帶DJ- 1/WT基因的假病毒顆粒滴度為1/(1×10-5) ×20=2×106TU/μL=2×109TU/mL(表1)，同樣測定攜帶DJ- 1/L166P基因的假病毒顆粒滴度為2×108TU/Ml(表2)。證明獲得了高滴度的慢病毒假病毒顆粒。

2.3 攜帶pGC-LV-DJ- 1/WT和pGC-LV-DJ- 1/L166P的慢病毒感染PC12細(xì)胞

將濃縮后得到高滴度的慢病毒濃縮液分別感染PC12細(xì)胞，熒光顯微鏡下觀察其熒光強度，確立其分別對兩種慢病毒對于PC12細(xì)胞的感染效率。同樣，用Western blot方法檢測用收集到高滴度的野生型DJ- 1和L166P突變型病毒感染PC12細(xì)胞后，RIPA裂解液提取細(xì)胞蛋白，進(jìn)行蛋白電泳實驗，結(jié)果顯示，WT組和L166P組與對照組相比，DJ- 1的蛋白表達(dá)量均很高，分別是內(nèi)源性對照組含量的315%和285%(圖3)。

A.GFP fluorescence (green) was detected under the fluorescent microscope both in the DJ- 1/WT and DJ- 1/L166P transfected 293T cells(scale bar=50 μm); B.Western blot to detect the fused protein, as shown in the above figure, an immuno-positive band was detected at the site of 49 ku which was consistent with the molecular weight of DJ- 1 and GFP fusion protein in the cells transfected with DJ- 1/WT or DJ- 1/L166P

圖2 構(gòu)建質(zhì)粒pGC-LV-DJ- 1/WT與pGC-LV-DJ- 1/L166P中外源基因表達(dá)的檢測Fig 2 Protein DJ- 1/WT or DJ- 1/L166P fused with GFP is successfully overexpressed in HEK293T cells

There were about 2 differences in the Ct values between the 1×10-5μL group and control group, and the virus particles were found in the 1×10-5group; assuming that the sample group contained at least one virus particle, the titer of the virus was 1/(1×10-5) ×20=2×106TU/μL=2×109TU/mL.

3 討論

DJ- 1具有廣泛的生理學(xué)功能,在信號傳導(dǎo)、細(xì)胞凋亡和氧化應(yīng)激等多種生物學(xué)功能中發(fā)揮重要作用， 建立DJ- 1的基因過表達(dá)模型對于研究DJ- 1的生理功能和相關(guān)發(fā)病機制有重要的意義。

表2 GFP-DJ- 1/L166P慢病毒顆粒滴度測定Table 2 Determination of particle titer of GFP-DJ- 1/L166P lentivirus

There were about 2 differences in the Ct values between the 1×10-4μL group and the control group, and the virus particles were found in the 1×10-4μL group; assuming that the sample group contains one virus particle at least; the titer of the virus was 1/(1×10-4) ×20=2×105TU/μL =2×108TU/mL.

A.GFP fluorescence (green) was detected under the fluorescent microscope both in the DJ- 1/WT and DJ- 1/L166P transfected 293T cells(scale bar=50 μm); B.Western blot to detect the fused protein, an immuno-positive band was detected at the site of 55 ku which was the molecular weight of DJ- 1; C.statistical analysis, as is shown above, both the WT and L166P groups were more effective in infecting PC12 cells than the control group about 315% and 285%;*P<0.05 compared with normal control group

目前基因治療中將目的基因轉(zhuǎn)移的工具多采用病毒載體，其中以反轉(zhuǎn)錄病毒載體和腺病毒載體最常用。但是反轉(zhuǎn)錄病毒載體只能感染分裂期的細(xì)胞, 容納外源基因DNA片段長度不超過8 kb。而腺病毒載體與之不同，在感染細(xì)胞時, 病毒DNA 游離在細(xì)胞核內(nèi)，且并不整合到染色體上，在體內(nèi)不能實現(xiàn)穩(wěn)定的長期表達(dá)，且反復(fù)應(yīng)用容易引起免疫反應(yīng)。因此人類免疫缺陷病毒21 (HIV 21) 來源的慢病毒載體越來越受到人們的重視[13- 14]。研究發(fā)現(xiàn)慢病毒載體最大的特點是可以感染非分裂期細(xì)胞。截至目前，已成功地應(yīng)用慢病毒載體感染了神經(jīng)細(xì)胞、肝細(xì)胞、造血干細(xì)胞、胰島細(xì)胞、肌肉細(xì)胞、視網(wǎng)膜色素上皮細(xì)胞和氣道上皮細(xì)胞等。此外, 慢病毒載體容納外源性目的基因的片段大, 可以在體內(nèi)較長期的表達(dá), 免疫反應(yīng)小, 安全性較好。

慢病毒載體因其傳導(dǎo)效率高和表達(dá)外源性基因效果好成為常用研究工具。與其他反轉(zhuǎn)錄病毒相比,慢病毒有其獨特優(yōu)點，例如有更廣泛的宿主；可增加目的基因整合到宿主細(xì)胞基因組的幾率；對轉(zhuǎn)錄沉默有一定的抵抗能作用；可以插入組織細(xì)胞特異性的啟動子或增強子,提高轉(zhuǎn)入基因的轉(zhuǎn)錄靶向性等。其眾多優(yōu)勢使慢病毒載體成為一種能實現(xiàn)外源基因高效導(dǎo)入及疾病基因治療方面的有效工具,且具有良好應(yīng)用前景。本實驗以pGC-FU-DJ- 1為載體，KL1337- 2，- 3為目的質(zhì)粒轉(zhuǎn)染HEK293T細(xì)胞，經(jīng)過收集到濃縮的細(xì)胞上清感染PC12細(xì)胞，所得結(jié)果顯示成功建立了過表達(dá)DJ- 1的野生型和L166P突變型的小鼠細(xì)胞模型，無論是WT還是L166P，熒光觀察結(jié)果均顯示其感染效率較高，且Western blot證明其蛋白表達(dá)量也均較高。成功建立過表達(dá)野生型DJ- 1和突變型L166P型細(xì)胞系模型，對于今后研究DJ- 1的功能以及DJ- 1對于帕金森病都提供了條件，具有一定的意義。

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Preparation of DJ- 1 overexpressing cell model by using lentiviral vector

REN Jing, LI Yi, YANG Hui*, LU Ling-ling*

(Dept. of Neurobiology, School of Basic Medical Sciences, Capital Medical University, Center for Parkinson’s Disease,Beijing Institute for Brain Disorders, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing 100069, China)

ObjectiveTo prepare gene overexpressing cell model of human wild-type DJ- 1 and its L166P mutant, and to investigate the role of lentiviral vector in gene overexpressing cell model.MethodsWild type DJ- 1 and L166P mutant DJ- 1 lentiviral vector plasmids were respectively constructed. After sequencing and comparing correctly, the plasmid was amplified and transfected into HEK293T cell line. Expression of WT DJ- 1 and L166P mutant DJ- 1 in cell lines was detected by fluorescence and Western blot.After determining the accurate expression of the target protein, a large amount of HEK293T cells was transfected and packaged to produce lentiviral particles. The PC12 cells were infected with the titer of virus supernatant. The fluorescence intensity of GFP and the expression of target protein were observed by fluorescence microscope and Western blot method,and the infection effi-ciency of the virus was determined.ResultsLentiviral vectors carrying wild type DJ- 1 and its mutants were successfully constructed. The virus vector can be transfected into HEK293T cells and the target protein can be correctly expressed. The viral titers of LV-DJ- 1 and LV-DJ- 1/L166P were 2×109TU/mL and 2×108TU/mL, respectively. Virus supernatant can efficiently infect PC12 cells, and most cells can express target proteins. The protein expressions of exogenous wild-type DJ- 1 and L166P mutants were 315% and 285% of endogenous content,respectively.ConclusionsLentivirus vector can infect cells efficiently, and it is a good way to prepare gene over expressing cell model. A cell model overexpressing DJ- 1 or its L166P mutant is successfully prepared. The model can be used for subsequent DJ- 1 function research.

Parkinson’s disease; DJ- 1；PC12 cell；lentivirus vector

2017- 10- 17

2017- 11- 20

國家重點研究發(fā)展計劃(2016YFC1306002);國家自然科學(xué)基金(81371398, 81371200);北京市自然科學(xué)基金(7131001);北京市創(chuàng)新團隊建設(shè)提升計劃(IDHT20140514);北京市教育委員會科技發(fā)展計劃(KM201710025001)

*通信作者(correspondingauthor)：lllu@ccmu.edu.cn; huiyang@ccmu.edu.cn

1001-6325(2018)01-0001-06

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