吳飛龍， 孔慶磊， 蔡松旺， 葉志強(qiáng)△

(中山大學(xué)附屬第三醫(yī)院 1急診科， 2心胸外科， 廣東 廣州 510630)

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CD147單抗介導(dǎo)的基因治療納米顆粒的肺癌細(xì)胞靶向性研究*

吳飛龍1， 孔慶磊1， 蔡松旺2， 葉志強(qiáng)1△

(中山大學(xué)附屬第三醫(yī)院1急診科，2心胸外科， 廣東 廣州 510630)

目的： 本研究采用靶向CD147的單克隆抗體對(duì)納米基因載體顆粒進(jìn)行靶向修飾后，進(jìn)行針對(duì)肺癌細(xì)胞的蛋白激酶Cε(protein kinase Cε，PKCε)小干擾RNA基因治療，觀察其對(duì)肺癌細(xì)胞增殖和遷移能力的抑制效果。方法: 制作可靶向CD147蛋白的磁性納米基因載體。激光掃描共聚焦顯微鏡觀察肺癌細(xì)胞CD147表達(dá)量。分別設(shè)立CP組、CN組和LP組復(fù)合物，按每6孔板孔質(zhì)?？偭?50 ng進(jìn)行細(xì)胞轉(zhuǎn)染。另設(shè)CD147靶向載體對(duì)照CA組和未轉(zhuǎn)染細(xì)胞的對(duì)照(control)組。激光掃描共聚焦顯微鏡觀察納米造影劑的細(xì)胞內(nèi)吞效果。實(shí)時(shí)熒光定量PCR檢測(cè)PKCε的mRNA表達(dá)。Western blot法檢測(cè)PKCε、Ki67、MMP3、Wnt1和GAPDH的蛋白表達(dá)。平板克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞的增殖能力。Transwell法檢測(cè)細(xì)胞的遷移能力。結(jié)果: 免疫熒光法染色觀察證實(shí)，人肺癌A549細(xì)胞的胞膜高表達(dá)CD147蛋白。CP組細(xì)胞中siRNA高效進(jìn)入A549細(xì)胞，質(zhì)粒內(nèi)吞效率大于CN組和LP組。CP組、CN組、LP組和CA組的A549細(xì)胞中PKCε的mRNA相對(duì)表達(dá)量分別為control組的(9.76±0.18)%、(98.51±0.32)%、(99.17±0.16)%和(99.68±0.11)%，CP組與control組間的差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，CN組、LP組與control組間的差異無(wú)統(tǒng)計(jì)學(xué)顯著性。CP組PKCε、Ki-67、MMP3及Wnt1蛋白的表達(dá)量明顯降低，CN組和LP組與對(duì)照組之間的蛋白表達(dá)量的差異無(wú)統(tǒng)計(jì)學(xué)顯著性。CP組的克隆形成數(shù)量明顯少于control組，差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.05)。CN組、LP組和CA組的有效克隆數(shù)量與control組相比差異沒(méi)有統(tǒng)計(jì)學(xué)顯著性。CP組的過(guò)膜細(xì)胞數(shù)量明顯少于control組，差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.05)。CN組、LP組和CA組的數(shù)量與control組相比差異沒(méi)有統(tǒng)計(jì)學(xué)顯著性。結(jié)論: 靶向CD147修飾的納米基因載體，可以對(duì)肺癌細(xì)胞進(jìn)行高效的PKCε-siRNA基因治療，實(shí)現(xiàn)對(duì)肺癌細(xì)胞增殖和遷移能力的高效抑制。

CD147； 納米載體； 基因治療； 蛋白激酶Cε

納米基因載體是可以結(jié)合目的基因、組成1～1 000 nm大小的納米級(jí)基因遞送系統(tǒng)。納米基因載體在遞送治療基因的過(guò)程中，可以避免基因損耗，增加病灶位置基因濃度，并促進(jìn)細(xì)胞內(nèi)吞，提高治療基因的生物利用率，經(jīng)過(guò)配體、抗體等表面修飾的納米載體可與目的器官和目的細(xì)胞表面的特異性受體或特異性抗原結(jié)合，實(shí)現(xiàn)在特定部位的濃集及高效釋放，進(jìn)一步提高基因傳遞的效率[1-2]。CD147是一種在肺癌、泌尿系腫瘤、乳腺癌等多種惡性腫瘤中高表達(dá)的跨膜糖蛋白，適合作為納米載體識(shí)別腫瘤細(xì)胞的靶向位點(diǎn)[3-5]。本研究采用CD147單抗，對(duì)納米載體顆粒進(jìn)行靶向化修飾，使其具備肺癌細(xì)胞特異性識(shí)別功能，進(jìn)而采用該納米載體負(fù)載本課題組前期報(bào)道的蛋白激酶Cε(protein kinase Cε，PKCε)小干擾RNA(small interfering RNA，siRNA)對(duì)肺癌細(xì)胞進(jìn)行基因治療，并研究其起效機(jī)制[6]。同時(shí)，對(duì)該納米載體與通用的新型脂質(zhì)體載體LipofectamineTM3000[7-8]的基因治療效果進(jìn)行了比較。本研究采用靶向CD147的單克隆抗體對(duì)納米基因載體顆粒進(jìn)行靶向修飾后，進(jìn)行針對(duì)肺癌細(xì)胞的蛋白激酶PKCε-siRNA基因治療，觀察其對(duì)肺癌細(xì)胞增殖和遷移能力的抑制效果，并初步探討其起效的分子生物學(xué)機(jī)制。

材 料 和 方 法

1 材料與試劑

人肺癌細(xì)胞株A549購(gòu)自ATCC，由華南師范大學(xué)分子生物學(xué)實(shí)驗(yàn)室保存；紅色熒光染料四乙基羅丹明(rhodamine)標(biāo)記的羊抗兔-IgG抗體、兔抗人CD147單克隆抗體(CD147-Ab)及其同型對(duì)照抗體購(gòu)自武漢博士德生物工程有限公司；免疫熒光染色試劑盒購(gòu)自廣州安邦生物科技有限公司；PKCε-siRNA和陰性對(duì)照siRNA (negative control siRNA， NC-siRNA)購(gòu)自Santa Cruz。紅色核酸熒光染料GelRed購(gòu)自Biotium[9]；TANBead?USPIO-101納米級(jí)顆粒購(gòu)自Advanced Nanotech；LipofectamineTM3000轉(zhuǎn)染試劑購(gòu)自Invitrogen；熒光染料Hoechst 33342購(gòu)自成都麥卡?；び邢薰?。

2 方法

2.1 納米顆粒靶向CD147蛋白識(shí)別功能的添加 按照產(chǎn)品說(shuō)明書要求，將CD147單克隆抗體配制為1 g/L溶液，與濃度為15 g/L的TANBead?USPIO-101納米顆粒混懸液等體積混懸重懸于含有5 g/L[1-乙基-(3-二甲基氨基丙基)]碳化二亞胺鹽酸鹽(EDC·HCl)的三蒸水中。4 ℃條件下振蕩8 h。于磁性分離器中收集含有磁性成分USPIO的納米顆粒，制成可靶向CD147蛋白的CD147-Ab-TANBead?USPIO-101磁性納米基因載體[10]。

2.2 肺癌細(xì)胞CD147表達(dá)量的鑒定 復(fù)蘇人肺癌A549細(xì)胞株，常規(guī)條件培養(yǎng)于37 ℃、5% CO2、飽和濕度的細(xì)胞培養(yǎng)箱中。PBS沖洗細(xì)胞3次后，4%中性甲醛固定液固定細(xì)胞。使用免疫熒光染色試劑盒，以CD147單抗或其同型抗體為 I 抗，RIB200標(biāo)記的羊抗兔-IgG抗體，進(jìn)行免疫熒光染色。免疫熒光染色后，藍(lán)色熒光染料Hoechst 33342避光孵育0.5 h染色細(xì)胞核。Olympus FV1200激光掃描共聚焦顯微鏡(confocal laser scanning microscope，CLSM)觀察染色結(jié)果。

2.3 基因載體與質(zhì)粒的復(fù)合 分別按照載體說(shuō)明書，采用靶向修飾的CD147-Ab-TANBead?USPIO-101、未靶向修飾的TANBead?USPIO-101 和脂質(zhì)體載體LipofectamineTM3000復(fù)合PKCε-siRNA，作為CP(CD147 targeted-PKCε-siRNA)組、CN(CD147-non-targeted-PKCε-siRNA)組和LP(LipofectamineTM3000-PKCε-siRNA)組復(fù)合物用于細(xì)胞轉(zhuǎn)染。另設(shè)不復(fù)合質(zhì)粒的靶向修飾的空載體CD147-Ab-TANBead?USPIO-101對(duì)照CA(CD147-Ab-TANBead?USPIO-101)組和未轉(zhuǎn)染細(xì)胞對(duì)照(control)組。

2.4 細(xì)胞培養(yǎng)和分組轉(zhuǎn)染 A549細(xì)胞培養(yǎng)于6孔板上，待細(xì)胞融合度達(dá)70%時(shí)，分為如上CP組、CN組、LP組、CA組或control組分別按照說(shuō)明操作步驟進(jìn)行轉(zhuǎn)染。轉(zhuǎn)染條件為L(zhǎng)ipofectamineTM3000說(shuō)明書推薦質(zhì)粒用量的1/30，每6孔板孔采用含250 ng質(zhì)粒的載體-siRNA復(fù)合物與細(xì)胞共孵育轉(zhuǎn)染。

2.5 激光掃描共聚焦顯微鏡觀察納米造影劑的細(xì)胞內(nèi)吞效果 采用紅色核酸熒光染料GelRed對(duì)載體-siRNA復(fù)合物進(jìn)行染色后轉(zhuǎn)染3 h，4%中性甲醛固定液固定細(xì)胞，Hoechst 33342避光孵育0.5 h染色細(xì)胞核。激光掃描共聚焦顯微鏡觀察。所有實(shí)驗(yàn)重復(fù)3次。

2.6 實(shí)時(shí)熒光定量PCR檢測(cè)PKCε的mRNA表達(dá) TRIzol法提取各組總細(xì)胞RNA，逆轉(zhuǎn)錄合成cDNA。PKCε的上游引物為5’-ATGGTAGTGTTCAATGGCCTTCT-3’，下游引物為5’-TCAGGGCATCAGGTCTTCAC-3’；內(nèi)參照GAPDH的上游引物為5’-TTGCATGCGACATAAGGTGTGA-3’，下游引物為5’-CTGGGAGATCTAGCAATGCATG-3’。PCR反應(yīng)條件為95 ℃ 35 s；95 ℃ 7 s、55 ℃ 32 s，62個(gè)循環(huán)，循環(huán)延伸末端收集熒光信號(hào)[6]。Real-time PCR數(shù)值分析采用2-ΔΔCt分析法。所有實(shí)驗(yàn)重復(fù)3次。

2.7 細(xì)胞功能蛋白表達(dá)鑒定 siRNA轉(zhuǎn)染肺癌細(xì)胞48 h。收集細(xì)胞后提取蛋白，按照常規(guī)方法進(jìn)行Western blot實(shí)驗(yàn),檢測(cè) PKCε、Ki67、MMP3、Wnt1和GAPDH的蛋白水平。

2.8 平板克隆形成實(shí)驗(yàn)檢測(cè)增殖能力 分別取5組轉(zhuǎn)染后細(xì)胞制備單細(xì)胞懸液，測(cè)定細(xì)胞密度后，分別取500個(gè)細(xì)胞，分別接種于6孔板中,每組細(xì)胞設(shè)3個(gè)復(fù)孔。于37 ℃、5% CO2、飽和濕度的細(xì)胞培養(yǎng)箱中培養(yǎng)14 d，PBS沖洗3次后，4%中性甲醛固定液固定細(xì)胞，結(jié)晶紫染色。光鏡下觀察每孔形成的細(xì)胞克隆數(shù)。設(shè)定每克隆的細(xì)胞數(shù)大于50個(gè)計(jì)作1個(gè)有效克隆。每組實(shí)驗(yàn)重復(fù)3次。

2.9 Transwell法檢測(cè)細(xì)胞的遷移能力 取Transwell小室，每室中加入50 μL預(yù)稀釋的Matrigel膠，37 ℃靜置2 h凝固后用于實(shí)驗(yàn)。分別取5組轉(zhuǎn)染后細(xì)胞制備單細(xì)胞懸液并測(cè)定細(xì)胞密度，分別取等量轉(zhuǎn)染后細(xì)胞接種入Transwell上室。下室中加入含胎牛血清培養(yǎng)基500 μL。37 ℃、5% CO2、飽和濕度的細(xì)胞培養(yǎng)箱中培養(yǎng)24 h。取出小室，去除小室底膜上面的細(xì)胞后，4%中性甲醛固定液固定小室底膜下面細(xì)胞。結(jié)晶紫染色，光鏡下計(jì)數(shù)穿過(guò)小室底膜的細(xì)胞數(shù)量。每組實(shí)驗(yàn)重復(fù)3次。

3 統(tǒng)計(jì)學(xué)處理

采用SPSS 13.0 統(tǒng)計(jì)學(xué)軟件分析數(shù)據(jù)。實(shí)驗(yàn)數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，多組數(shù)據(jù)比較采用單因素方差分析，各組均數(shù)間的兩兩比較采用SNK法。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 人肺癌A549細(xì)胞CD147表達(dá)的免疫熒光觀察

免疫熒光法染色觀察發(fā)現(xiàn)，采用CD147單抗作為 I 抗的A549細(xì)胞膜上出現(xiàn)RIB200免疫熒光標(biāo)記的紅色熒光染色，而使用同型對(duì)照抗體作為 I 抗的細(xì)胞膜上沒(méi)有出現(xiàn)紅色熒光染色，說(shuō)明人肺癌A549細(xì)胞的胞膜高表達(dá)CD147蛋白。所以可以嘗試在納米基因載體上連接CD147抗體后，實(shí)現(xiàn)對(duì)A549細(xì)胞進(jìn)行識(shí)別，見(jiàn)圖1。

Figure 1.The images of A549 cells with-CD147 immunofluorescence staining (laser confocal microscope, ×1 000). A: stained with an anti-CD147 antibody; B: using the same type of control antibody.

圖1 A549細(xì)胞的CD147免疫熒光染色結(jié)果

2 納米基因載體的細(xì)胞內(nèi)吞效果

如圖2所示，免疫熒光實(shí)驗(yàn)觀察發(fā)現(xiàn)，在經(jīng)過(guò)設(shè)定較低的載體-siRNA復(fù)合物用量轉(zhuǎn)染后，CP組腫瘤細(xì)胞中可以觀察明顯的大量紅色核酸熒光染料GelRed發(fā)出的紅色熒光，CN組和LP組僅可見(jiàn)極少的微弱紅色熒光，CA組和control組未見(jiàn)紅色熒光，說(shuō)明CP組細(xì)胞中siRNA通過(guò)載體的介導(dǎo)，高效進(jìn)入A549細(xì)胞，質(zhì)粒內(nèi)吞效率大于CN組和LP組。

3 5組細(xì)胞PKCε的mRNA表達(dá)

CP組、CN組、LP組和CA組的A549細(xì)胞中PKCε的mRNA相對(duì)表達(dá)量分別為control組的(9.76±0.18)%、(98.51±0.32)%、(99.17±0.16)%和(99.68±0.11)%，CP組與control組的差異有統(tǒng)計(jì)學(xué)顯著性(P<0.05)，CN組、LP組、CA組與control組的差異無(wú)統(tǒng)計(jì)學(xué)顯著性。這說(shuō)明在LipofectamineTM3000說(shuō)明書推薦質(zhì)粒用量的1/30進(jìn)行轉(zhuǎn)染時(shí)，僅有CD147靶向化的納米載體可以實(shí)現(xiàn)siRNA對(duì)PKCε基因的明顯沉默效果。

Figure 2.The effects of the gene vectors in 4 groups on the endocytosis of A549 cells (laser confocal microscope, ×1 000).

圖2 4組基因載體對(duì)A549細(xì)胞內(nèi)吞效果的觀察

4 5組細(xì)胞PKCε、Ki67、MMP3和Wnt1的蛋白表達(dá)

由圖3可見(jiàn)，在LipofectamineTM3000說(shuō)明書推薦質(zhì)粒用量的1/30進(jìn)行轉(zhuǎn)染時(shí)，僅有CP組PKCε、Ki-67、MMP3及Wnt1蛋白的表達(dá)量明顯降低，CN組、LP組和CA組與對(duì)照組之間的蛋白表達(dá)量差異無(wú)統(tǒng)計(jì)學(xué)顯著性。Western blot實(shí)驗(yàn)檢測(cè)的結(jié)果與前述RT-qPCR實(shí)驗(yàn)結(jié)果一致，說(shuō)明CD147靶向化的納米載體可以實(shí)現(xiàn)較高的siRNA抑制PKCε蛋白表達(dá)的效果，且同時(shí)對(duì)Ki-67、MMP9、Wnt1等功能蛋白的表達(dá)產(chǎn)生抑制。

5 5組細(xì)胞增殖能力的檢測(cè)

培養(yǎng)14 d后，分別計(jì)數(shù)5組細(xì)胞的有效克隆數(shù)量。發(fā)現(xiàn)CP組的克隆數(shù)量明顯少于control組，差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.05)。而CN組、LP組和CA組的有效克隆數(shù)量與control組相比差異沒(méi)有統(tǒng)計(jì)學(xué)顯著性，見(jiàn)圖4。

6 5組細(xì)胞細(xì)胞遷移能力的比較

Transwell小室培養(yǎng)24 h后，計(jì)數(shù)5組過(guò)膜細(xì)胞的數(shù)量，發(fā)現(xiàn)CP組的數(shù)量明顯少于control組，差異具有統(tǒng)計(jì)學(xué)顯著性(P<0.05)。而CN組、LP組和CA組的數(shù)量與control組相比差異沒(méi)有統(tǒng)計(jì)學(xué)顯著性，見(jiàn)圖5。

Figure 3.The protein expression levels in the A549 cells of 4 groups detected by Western blot. Mean±SD.n=3.*P<0.05vscontrol group.

圖3 4組A549細(xì)胞中蛋白表達(dá)水平的Western blot實(shí)驗(yàn)檢測(cè)

Figure 4.Comparison of the colony formation abilities of A549 cells in 5 groups. Mean±SD.n=3.*P<0.05vscontrol group.

圖4 5組細(xì)胞克隆形成能力的比較

Figure 5.Comparison of the invasion abilities of A549 cells in 5 groups. Mean±SD.n=3.*P<0.05vscontrol group.

圖5 5組細(xì)胞遷移能力的比較

討 論

本研究首先合成了一種CD147靶向的磁性納米基因載體CD147-Ab-TANBead?USPIO-101，并包裹PKCε-siRNA，實(shí)現(xiàn)對(duì)肺癌細(xì)胞A549的基因治療。磁性納米載體是一類粒徑在納米尺度的，由磁性金屬及金屬氧化物等原料組成的納米載體，可用于包裹核酸進(jìn)行基因治療。在本研究中，我們采用了被證明具有確切基因負(fù)載傳輸效果的TANBead?USPIO-101納米級(jí)顆粒進(jìn)行治療質(zhì)粒負(fù)載[11-12]。在治療基因方面，采用了研究團(tuán)隊(duì)前期研究中采用的可抑制細(xì)胞蛋白激酶Cε表達(dá)的商品化PKCε-siRNA。蛋白激酶Cε是蛋白激酶C的重要亞型之一，與腫瘤的增殖轉(zhuǎn)移關(guān)系密切。在我們的前期研究中已證實(shí)，利用該手段對(duì)蛋白激酶Cε表達(dá)進(jìn)行抑制可以通過(guò)NF-κB、AKT等信號(hào)通路對(duì)腫瘤細(xì)胞進(jìn)行基因治療[6]。磁性納米載體復(fù)合核酸后，可以通過(guò)靜電作用結(jié)合形成緊密的納米顆粒，可以有效地阻止核酸酶與核酸的接觸，并通過(guò)構(gòu)象變異，發(fā)揮酶切保護(hù)效果。在納米載體表面進(jìn)行適當(dāng)?shù)呐潴w或抗體靶向化修飾，可以有效提高特定部位納米復(fù)合物聚集的能力，進(jìn)而實(shí)現(xiàn)提高負(fù)載的治療基因的生物利用率的目的。在納米載體傳遞系統(tǒng)的設(shè)計(jì)中，靶向修飾位點(diǎn)的選擇非常重要。CD147是一種定位于19p13.3的跨膜糖蛋白，為免疫球蛋白超家族成員，其蛋白結(jié)構(gòu)包括細(xì)胞外2個(gè)免疫球蛋白區(qū)、跨膜區(qū)和胞內(nèi)區(qū)，其胞外區(qū)具有誘導(dǎo)臨近的成纖維細(xì)胞分泌MMP等功能[13]。目前已經(jīng)證實(shí)，在肺癌、乳腺癌、結(jié)直腸癌、膠質(zhì)瘤等腫瘤細(xì)胞中，CD147在細(xì)胞膜上的表達(dá)量均明顯升高，適合作為納米載體靶向識(shí)別上述腫瘤細(xì)胞的位點(diǎn)[14-15]。在本研究中，我們選用了在肺癌等腫瘤細(xì)胞表面特異性高表達(dá)的CD147作為靶點(diǎn)，對(duì)磁性納米載體進(jìn)行CD147抗體靶向修飾，期望使其具備肺癌細(xì)胞主動(dòng)靶向功能[16]。

本研究進(jìn)而通過(guò)免疫熒光法對(duì)肺癌A549細(xì)胞表面CD147的表達(dá)進(jìn)行了進(jìn)一步證明。通過(guò)免疫熒光染色發(fā)現(xiàn)，采用CD147單抗作為Ⅰ抗進(jìn)行染色后A549細(xì)胞膜上出現(xiàn)免疫熒光標(biāo)記的紅色熒光染色，而相對(duì)的同型對(duì)照抗體不能實(shí)現(xiàn)熒光染色，證明與國(guó)際上的報(bào)道相似，本研究采用的A549細(xì)胞膜上確實(shí)高表達(dá)CD147，且可以通過(guò)CD147單抗進(jìn)行A549細(xì)胞靶向。同時(shí)，我們以常用的脂質(zhì)體載體，和未經(jīng)靶向化修飾的磁性納米載體，對(duì)經(jīng)過(guò)靶向化修飾的納米載體的siRNA傳輸效率進(jìn)行了研究。為對(duì)不同載體轉(zhuǎn)染效率的差異進(jìn)行比較，本研究采用了通用脂質(zhì)體載體LipofectamineTM3000推薦質(zhì)粒量的1/30進(jìn)行轉(zhuǎn)染后，通過(guò)共聚焦顯微鏡直觀觀察發(fā)現(xiàn)，僅有CD147靶向化的納米載體轉(zhuǎn)染的A549細(xì)胞中可以觀察到明顯的核酸熒光染料聚集，而其它載體組僅可見(jiàn)極少的微弱紅色熒光，說(shuō)明本研究采用的CD147-Ab-TANBead?USPIO-101納米載體可以在極低的質(zhì)粒濃度下，實(shí)現(xiàn)明顯優(yōu)于LipofectamineTM3000的體外轉(zhuǎn)染效果。

經(jīng)過(guò)如上研究證明了CD147-Ab-TANBead?USPIO-101納米載體轉(zhuǎn)運(yùn)質(zhì)粒進(jìn)入細(xì)胞的效率后，我們對(duì)納米復(fù)合物進(jìn)行基因治療的效果進(jìn)行了研究。在RT-qPCR實(shí)驗(yàn)和Western blot實(shí)驗(yàn)中發(fā)現(xiàn)，靶向化的納米復(fù)合物可以有效降低PKCε的表達(dá)水平，并抑制肺癌A549細(xì)胞的增殖和遷移。對(duì)其分子生物學(xué)機(jī)制進(jìn)行考察后發(fā)現(xiàn)，Ki-67、MMP3及Wnt1蛋白的表達(dá)量與PKCε的表達(dá)量變化呈正相關(guān)。Ki67和MMP3分別是Wnt信號(hào)通路中與細(xì)胞增殖和遷移轉(zhuǎn)移密切相關(guān)的功能蛋白[17-19]，Wnt1蛋白是Wnt信號(hào)通路的關(guān)鍵蛋白[20-21]。國(guó)際上有類似研究表明，PKCε在腫瘤中的活化和功能與Wnt信號(hào)通路密切相關(guān)[22-23]。在研究中，我們同時(shí)采用了等量不復(fù)合質(zhì)粒的，單純CD147靶向化的納米載體對(duì)細(xì)胞進(jìn)行干預(yù)，發(fā)現(xiàn)沒(méi)有對(duì)細(xì)胞功能蛋白表達(dá)及遷移轉(zhuǎn)移能力產(chǎn)生影響，說(shuō)明本研究采用的納米載體在實(shí)現(xiàn)有效轉(zhuǎn)染和基因治療的劑量下，本身并不能產(chǎn)生對(duì)細(xì)胞生物學(xué)功能的影響。前述研究結(jié)果說(shuō)明，Ki-67、MMP3可能參與了CD147-Ab-TANBead?USPIO-101納米載體負(fù)載的PKCε-siRNA對(duì)肺癌A549細(xì)胞的增殖和遷移的抑制，這種抑制功能可能是通過(guò)wnt信號(hào)通路實(shí)現(xiàn)的。

綜上所述，靶向CD147修飾的納米基因復(fù)合物負(fù)載PKCε-siRNA后，可以對(duì)肺癌細(xì)胞進(jìn)行高效的基因治療，實(shí)現(xiàn)對(duì)肺癌細(xì)胞增殖和遷移能力的高效抑制。

[1] Guo Y, Wang J, Zhang L, et al. Theranostical nanosystem-mediated identification of an oncogene and highly effective therapy in hepatocellular carcinoma[J]. Hepatology, 2016, 63(4):1240-1255.

[2] Guo Y, Chen W, Wang W, et al. Simultaneous diagnosis and gene therapy of immuno-rejection in rat allogeneic heart transplantation model using a T-cell-targeted theranostic nanosystem[J]. ACS Nano, 2012, 6(12):10646-10657.

[3] Huang W, Luo WJ, Zhu P, et al. Modulation of CD147-induced matrix metalloproteinase activity: Role of CD147 N-glycosylation[J]. Biochem J, 2013, 449(2):437-448.

[4] Wang H, Zhuo Y, Hu X, et al. CD147 deficiency blocks IL-8 secretion and inhibits lung cancer-induced osteoclastogenesis[J]. Biochem Biophys Res Commun, 2015, 458(2):268-273.

[5] Grass GD, Toole BP. How, with whom and when: An overview of CD147-mediated regulatory networks influencing matrix metalloproteinase activity[J]. Biosci Rep, 2016, 36(1):e283.

[6] 葉志強(qiáng)，范 瑾，楊躍武，等. 蛋白激酶 Cε對(duì)肝癌 SK-H ep-1細(xì)胞生物學(xué)行為的影響[J]. 中國(guó)病理生理雜志, 2014,30(6):994-998.

[7] Rust A, Hassan HH, Sedelnikova S, et al. Two complementary approaches for intracellular delivery of exogenous enzymes[J]. Sci Rep, 2015, 5:12444.

[8] Miryala B, Feng Y, Omer A, et al. Quaternization enhances the transgene expression efficacy of aminoglycoside-derived polymers[J]. Int J Pharm, 2015, 489(1-2):18-29.

[9] Sonmezoglu OA, Ozkay K. A new organic dye-based staining for the detection of plant DNA in agarose gels[J]. Nucleosides Nucleotides Nucleic Acids, 2015, 34(7):515-522.

[10]Tong M, Xiong F, Shi Y, et al.Invitrostudy of SPIO-labeled human pancreatic cancer cell line BxPC-3[J]. Contrast Media Mol Imaging, 2013, 8(2):101-107.

[11]Lo YL, Chou HL, Liao ZX, et al. Chondroitin sulfate-polyethylenimine copolymer-coated superparamagnetic iron oxide nanoparticles as an efficient magneto-gene carrier for microRNA-encoding plasmid DNA delivery[J]. Nanoscale, 2015, 7(18):8554-8565.

[12]Namiki Y, Namiki T, Yoshida H, et al. A novel magnetic crystal-lipid nanostructure for magnetically guidedinvivogene delivery[J]. Nat Nanotechnol, 2009, 4(9):598-606.

[13]Cui HY, Wang SJ, Miao JY, et al. CD147 regulates cancer migration via direct interaction with Annexin A2 and DOCK3-beta-catenin-WAVE2 signaling[J]. Oncotarget, 2016, 7(5):5613-5629.

[14]Messeha SS, Zarmouh NO, Taka E, et al. The role of monocarboxylate transporters and their chaperone CD147 in lactate efflux inhibition and the anticancer effects of in neuroblastoma cell line N2-A[J]. Eur J Med Plants, 2016, 12(4): EJMP.23992.

[15]Kong X, Wang Y, Dai C, et al. Is CD147 a new biomarker reflecting histological malignancy of gliomas?[J]. Mol Neurobiol, 2016 May 20.[Epub ahead of print]

[16]Muramatsu T. Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners[J]. J Biochem, 2016, 159(5):481-490.

[17]Focke CM, Decker T, van Diest PJ. Intratumoral heterogeneity of Ki67 expression in early breast cancers exceeds variability between individual tumours[J]. Histopathology, 2016 Jun 6. [Epub ahead of print]

[18]Kim HJ, Kang GJ, Kim EJ, et al. Novel effects of sphingosylphosphorylcholine on invasion of breast cancer: involvement of matrix metalloproteinase-3 secretion leading to WNT activation[J]. Biochim Biophys Acta, 2016, 1862(9):1533-1543.

[19]Bleckmann A, Conradi LC, Menck K, et al. Beta-catenin-independent WNT signaling and Ki67 in contrast to the estrogen receptor status are prognostic and associated with poor prognosis in breast cancer liver metastases[J]. Clin Exp Metastasis, 2016, 33(4):309-323.

[20]Lindvall C, Evans NC, Zylstra CR, et al. The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis[J]. J Biol Chem, 2006, 281(46):35081-35087.

[21]Rota LM, Albanito L, Shin ME, et al. IGF1R inhibition in mammary epithelia promotes canonical Wnt signaling and Wnt1-driven tumors[J]. Cancer Res, 2014, 74(19):5668-5679.

[22]Dissanayake SK, Weeraratna AT. Detecting PKC phosphorylation as part of the Wnt/calcium pathway in cutaneous melanoma[J]. Methods Mol Biol, 2008, 468:157-172.

[23]Kim S, Chun SY, Kwon YS, et al. Crosstalk between Wnt signaling and Phorbol ester-mediated PKC signaling in MCF-7 human breast cancer cells[J]. Biomed Pharmacother, 2016, 77:114-119.

(責(zé)任編輯： 林白霜， 羅 森)

CD147 monoclonal antibody-mediated nanoparticles for gene therapy to target lung cancer cells

WU Fei-long1, KONG Qing-lei1, CAI Song-wang2, YE Zhi-qiang1

(1DepartmentofEmergency,2DepartmentofCardiothoracicSurgery,TheThirdAffiliatedHospitalofSunYat-senUniversity,Guangzhou510630,China.E-mail:zqye@163.com)

AIM: In this study, CD147 antibody was used to carry out targeted modification of nanoparticles for protein kinase Cε (PKCε)-siRNA gene therapy to target lung cancer cells. The inhibitory effects of the nanoparticles on the proliferation and invasion of the lung cancer cells were observed. METHODS: The magnetic nanoparticles targeting CD147 protein were assembled as gene vector. The expression of CD147 in the lung cancer cells was observed under laser scanning confocal microscope. The cells were divided into CP group, CN group and LP group as the experimental groups. Targeted nanoparticles were used as CA group. Non-transfected cells were used as control group. The cell transfection was carried out with 250 ng plasmids/well in 6-well plate. The effect of nanocontrast agent on the cell endocytosis was observed under laser scanning confocal microscope. The mRNA expression of PKCε was detected by RT-qPCR. The protein expression of Ki67, MMP3, PKCε, Wnt1 and GAPDH was determined by Western blot. The cell proliferation ability was detected with colony formation assay. The cell invasion ability was detected by Transwell method. RESULTS: The expression of CD147 protein in the human lung cancer A549 cells was confirmed by immunofluorescence staining. The endocytosis of siRNA into the A549 cells in CP group was observed with the highest efficiency as compared with CN group and LP group. The relative mRNA expression of PKCε in the A549 cells of CP group, CN group, LP group and CA group were (9.76±0.18)%, (98.51±0.32)%, (99.17±0.16)% and (99.68±0.11)%， respectively. The difference between CP group and control group was statistically significant (P<0.05). No significant difference among CN group, LP group and control group was observed. The protein expression of PKCε, Ki-67, MMP3 and Wnt1 in CP group was significantly reduced, and the protein expression levels among CN group， LP group and control group had no significant difference. The colony number in CP group was significantly smaller than that in control group (P<0.05). The effective colony numbers in CN group, LP group and CA group had no significant difference as compared with control group. The number of the invading cells in CP group was significantly less than that in control group (P<0.05). The numbers of the invading cells in CN group, LP group and CA group had no significant difference as compared with control group. CONCLUSION: Nanogene vector targeting CD147 can carry PKCε-siRNA to conduct gene therapy efficiently on the lung cancer cells to achieve effective inhibitory effects on the proliferation and invasion of the lung cancer cells.

CD147; Nanocarriers; Gene therapy; Protein kinase Cε

雜志網(wǎng)址： http://www.cjpp.net

1000- 4718(2016)09- 1562- 06

2016- 06- 21

2016- 08- 16

廣東省科技計(jì)劃 (No.2014A020212533)

△通訊作者 Tel： 020-85253333； E-mail： zqye@163.com

R730.23

A

10.3969/j.issn.1000- 4718.2016.09.005