粒細(xì)胞-巨噬細(xì)胞集落刺激因子轉(zhuǎn)染對(duì)白血病細(xì)胞K562形態(tài)及增殖的影響

陳蓉，常晉霞，李雪璐，彭麗娟，朱紅

(川北醫(yī)學(xué)院醫(yī)學(xué)微生物學(xué)與免疫學(xué)教研室，四川南充 637000)

粒細(xì)胞-巨噬細(xì)胞集落刺激因子轉(zhuǎn)染對(duì)白血病細(xì)胞K562形態(tài)及增殖的影響

陳蓉，常晉霞，李雪璐，彭麗娟，朱紅

(川北醫(yī)學(xué)院醫(yī)學(xué)微生物學(xué)與免疫學(xué)教研室，四川南充 637000)

目的 觀察重組PcDNA3.1-粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)、外源性GM-CSF對(duì)髓系白血病細(xì)胞(K562)形態(tài)及增殖的影響。方法 將K562細(xì)胞分為4組，重組質(zhì)粒組、外源性組、空質(zhì)粒組細(xì)胞分別以重組PcDNA3.1-GM-CSF、外源性GM-CSF、PcDNA3.1空質(zhì)粒轉(zhuǎn)染，細(xì)胞對(duì)照組細(xì)胞未轉(zhuǎn)染任何質(zhì)粒，觀察各組細(xì)胞形態(tài)學(xué)變化及細(xì)胞增殖情況。結(jié)果 光學(xué)顯微鏡下，細(xì)胞對(duì)照組、空質(zhì)粒組及外源性組細(xì)胞形態(tài)相似，細(xì)胞著色呈深藍(lán)色，細(xì)胞質(zhì)和細(xì)胞核不易區(qū)分；重組質(zhì)粒組細(xì)胞形態(tài)和其余3組明顯不同，細(xì)胞質(zhì)呈淺藍(lán)色，細(xì)胞核呈淡粉紅色，分界清楚，部分細(xì)胞內(nèi)可見腎形、馬蹄形細(xì)胞核。顯微鏡油鏡下，細(xì)胞對(duì)照組、外源性組及空質(zhì)粒組結(jié)果相似，細(xì)胞著色深，呈深藍(lán)色，細(xì)胞內(nèi)未見任何顆粒沉積；重組質(zhì)粒組部分細(xì)胞著色呈深藍(lán)色，部分細(xì)胞著色呈淺粉色，呈淺粉色的部分細(xì)胞內(nèi)可見有斑點(diǎn)狀甲臜顆粒沉積。與其余2組比較，重組質(zhì)粒組第3、5、7、9天細(xì)胞增殖抑制率升高(P均<0.05)；與空質(zhì)粒組比較，外源性組第3、5、7天細(xì)胞增殖抑制率升高(P均<0.05)。結(jié)論 重組PcDNA3.1-GM-CSF及外源性GM-CSF均能使K562細(xì)胞形態(tài)發(fā)生改變、增殖受到抑制，作用強(qiáng)度隨作用時(shí)間延長(zhǎng)而增加，且重組PcDNA3.1-GM-CSF抑制作用強(qiáng)于外源性GM-CSF。

粒細(xì)胞-巨噬細(xì)胞集落刺激因子；白血病細(xì)胞；細(xì)胞形態(tài)；細(xì)胞增殖

粒細(xì)胞-巨噬細(xì)胞集落刺激因子(GM-CSF)是一種能調(diào)節(jié)造血祖細(xì)胞增殖、分化的細(xì)胞因子[1]，亦可刺激巨噬系、紅系、嗜酸系、粒系等多系造血細(xì)胞的前體細(xì)胞增殖和分化[2]。GM-CSF的生物學(xué)作用較為廣泛，能增加巨噬細(xì)胞和單核細(xì)胞抗體依賴細(xì)胞介導(dǎo)細(xì)胞毒性作用，可調(diào)整機(jī)體免疫細(xì)胞對(duì)腫瘤細(xì)胞的攻擊殺傷能力，具有抗腫瘤的作用[3]。研究[4～6]表明，GM-CSF在輔助治療惡性腫瘤方面有其不可替代的作用。因GM-CSF在抗瘤治療方面的優(yōu)越性，使其受到基礎(chǔ)研究者及臨床工作者的廣泛關(guān)注。本研究應(yīng)用外源性GM-CSF及已構(gòu)建的PcDNA3.1-GM-CSF真核表達(dá)質(zhì)粒[7]轉(zhuǎn)染髓系白血病細(xì)胞(K562)，觀察兩者對(duì)細(xì)胞形態(tài)及增殖的影響，旨在為進(jìn)一步探索GM-CSF治療白血病提供理論依據(jù)。

1 材料與方法

1.1 質(zhì)粒及主要試劑 重組質(zhì)粒PcDNA3.1-GM-CSF(本研究團(tuán)隊(duì)自己構(gòu)建)；脂質(zhì)體轉(zhuǎn)染試劑(天根生化科技有限公司)；K562細(xì)胞(NTCC典型培養(yǎng)物保藏中心-BioVector質(zhì)粒載體菌種細(xì)胞基因保藏中心)；人源性GM-CSF(美國(guó)派普泰克公司，用1640培養(yǎng)液配制，過(guò)濾除菌后-20 ℃保存)；1640培養(yǎng)基、新生牛血清(塞默飛世爾科技公司)；NBT染液(NBT 0.28 g加入生理鹽水100 mL，濾器過(guò)濾，4 ℃保存)；NBT培養(yǎng)液(正常人血清0.5 mL加入生理鹽水0.3 mL和NBT染液0.6 mL)。

1.2 粒細(xì)胞-巨噬細(xì)胞集落刺激因子轉(zhuǎn)染對(duì)K562細(xì)胞形態(tài)的影響觀察 ① 光學(xué)顯微鏡下觀察：常規(guī)復(fù)蘇K562細(xì)胞，待細(xì)胞生長(zhǎng)良好，用1640培養(yǎng)液配制K562細(xì)胞懸液，將K562細(xì)胞接種于24孔板中，每孔細(xì)胞數(shù)約5×104個(gè)。于細(xì)胞培養(yǎng)箱37 ℃、5%CO2環(huán)境下培養(yǎng)24 h，離心，去上清。將培養(yǎng)細(xì)胞分為4組，每組六孔。細(xì)胞對(duì)照組：只有K562細(xì)胞和細(xì)胞培養(yǎng)液，即只在該組細(xì)胞培養(yǎng)孔中加入含20%新生牛血清1640培養(yǎng)液500 μL，培養(yǎng)72 h?？召|(zhì)粒組：先配置脂質(zhì)體-空質(zhì)粒混合物(將2 μL脂質(zhì)體轉(zhuǎn)染試劑加入48 μL無(wú)血清無(wú)抗生素的1640培養(yǎng)液中)，混勻，室溫放置5 min，同時(shí)將2 μL的空質(zhì)粒(0.5 μg/μL)和48 μL無(wú)血清無(wú)抗生素的1640培養(yǎng)液混勻，再將上述兩種液體放在一起混勻，室溫放置20 min。在每孔細(xì)胞培養(yǎng)孔中先加入400 μL的無(wú)血清的1640培養(yǎng)液，然后將脂質(zhì)體-空質(zhì)?；旌衔镏鸬渭尤肟字?，每孔100 μL，輕輕搖動(dòng)，使其均勻分布(方法來(lái)源于脂質(zhì)體轉(zhuǎn)染試劑盒使用說(shuō)明)，37 ℃、5%CO2環(huán)境下培養(yǎng)6 h后，離心，去上清，每孔加入500 μL含20%新生牛血清的1640培養(yǎng)液，在細(xì)胞培養(yǎng)箱中(37 ℃、5%CO2)培養(yǎng)72 h。重組質(zhì)粒組：具體操作方法及質(zhì)粒濃度同空質(zhì)粒組，只是將轉(zhuǎn)染的空質(zhì)粒換成了重組質(zhì)粒。外源性組：先在每孔細(xì)胞培養(yǎng)孔中加入含20%新生牛血清1640培養(yǎng)液400 μL，再在每孔中加入外源性GM-CSF 100 μL(800 ng/mL)，輕輕搖動(dòng)，混勻，在細(xì)胞培養(yǎng)箱中(37 ℃、5%CO2)培養(yǎng)72 h。分別收集各組培養(yǎng)細(xì)胞，離心，取沉淀細(xì)胞涂片于載玻片上，自然干燥，Wright-Giemsa染色，水洗，干后低、高倍鏡鏡檢，觀察各組細(xì)胞染色情況、形態(tài)及細(xì)胞核的變化，攝片保存。該實(shí)驗(yàn)重復(fù)3次。②顯微鏡油鏡下觀察：采用硝基四氮唑藍(lán)還原試驗(yàn)(NBT還原試驗(yàn))。常規(guī)復(fù)蘇K562細(xì)胞，待細(xì)胞生長(zhǎng)良好，用1640培養(yǎng)液配制K562細(xì)胞懸液，將K562細(xì)胞接種于24孔板中，每孔細(xì)胞數(shù)約5×104個(gè)。于細(xì)胞培養(yǎng)箱37 ℃、5%CO2培養(yǎng)24 h，離心，去上清。將培養(yǎng)細(xì)胞分為4組，每組六孔，分組及各組細(xì)胞處理方法同①。在細(xì)胞培養(yǎng)箱中37 ℃、5%CO2培養(yǎng)72 h。72 h后，分別收集各組細(xì)胞，離心，棄上清，取細(xì)胞沉淀0.1 mL和NBT培養(yǎng)液0.1 mL加入凹玻片孔中混勻，置濕盒37 ℃、20 min，置室溫15 min，中間振動(dòng)1次。取1滴混懸液于防脫載玻片一端推成薄片，空氣中快速干燥。甲醇固定1 min，Wright-Giemsa染色，水洗，干后油鏡鏡檢，觀察細(xì)胞內(nèi)是否有異常顆粒，攝片保存。該實(shí)驗(yàn)重復(fù)3次。

1.3 粒細(xì)胞-巨噬細(xì)胞集落刺激因子轉(zhuǎn)染對(duì)K562細(xì)胞增殖的影響觀察 按1.2方法采用脂質(zhì)體轉(zhuǎn)染試劑轉(zhuǎn)染質(zhì)粒方式，分別轉(zhuǎn)染PcDNA3.1空質(zhì)粒于K562細(xì)胞及重組質(zhì)粒PcDNA3.1-GM-CSF于K562細(xì)胞，37 ℃、5%CO2培養(yǎng)72 h后，離心，去上清備用。用1640培養(yǎng)液分別配制K562細(xì)胞懸液(分3種細(xì)胞配制：K562細(xì)胞、轉(zhuǎn)染PcDNA3.1空質(zhì)粒細(xì)胞、轉(zhuǎn)染重組質(zhì)粒PcDNA3.1-GM-CSF細(xì)胞)。實(shí)驗(yàn)分組如下：①細(xì)胞對(duì)照組：?jiǎn)渭僈562細(xì)胞；②空質(zhì)粒組：細(xì)胞轉(zhuǎn)染PcDNA3.1空質(zhì)粒；③重組質(zhì)粒組：細(xì)胞轉(zhuǎn)染重組質(zhì)粒PcDNA3.1-GM-CSF；④外源性組：細(xì)胞轉(zhuǎn)染外源性GM-CSF。將上述細(xì)胞懸液按上述分組分別接種于96孔板中，使每孔含5×104個(gè)細(xì)胞，每組設(shè)3個(gè)平行孔。用細(xì)胞培養(yǎng)液將每孔終體積補(bǔ)足為200 μL，置37 ℃、5%CO2培養(yǎng)箱中培養(yǎng)72 h，離心，棄上清，每孔加入MTT溶液(5 mg/mL)20 μL和1640培養(yǎng)液180 μL，37 ℃繼續(xù)孵育4 h終止培養(yǎng)。離心，棄去培養(yǎng)上清，每孔加入150 μL二甲基亞砜，低速震搖10 min，酶標(biāo)儀測(cè)定570 nm吸光度OD值。細(xì)胞增殖抑制率=(1-實(shí)驗(yàn)組OD值/對(duì)照組OD值)×100%。觀察第3、5、7、9天細(xì)胞增殖抑制率。實(shí)驗(yàn)重復(fù)3次。

2 結(jié)果

2.1 K562細(xì)胞形態(tài)學(xué)變化 光學(xué)顯微鏡下，細(xì)胞對(duì)照組、空質(zhì)粒組及外源性組的形態(tài)相似，細(xì)胞著色呈深藍(lán)色，細(xì)胞質(zhì)和細(xì)胞核不易區(qū)分(圖1A)；重組質(zhì)粒組細(xì)胞形態(tài)和細(xì)胞對(duì)照組、外源性組細(xì)胞明顯不同，細(xì)胞質(zhì)呈淺藍(lán)色，細(xì)胞核呈淡粉紅色，分界清楚，部分細(xì)胞內(nèi)可見腎形、馬蹄形細(xì)胞核(圖1B)。顯微鏡油鏡下可見，對(duì)照組、外源性組組及空質(zhì)粒組結(jié)果相似，細(xì)胞著色深，呈深藍(lán)色，細(xì)胞內(nèi)未見任何顆粒沉積(圖2A)；重組質(zhì)粒組鏡下可見部分細(xì)胞著色呈深藍(lán)色，部分細(xì)胞著色呈淺粉色，呈淺粉色的部分細(xì)胞內(nèi)可見有斑點(diǎn)狀甲臜顆粒沉積(圖2B)。

注：A為細(xì)胞對(duì)照組，B為重組質(zhì)粒組。

圖1 光學(xué)顯微鏡下細(xì)胞形態(tài)學(xué)變化

注：A為細(xì)胞對(duì)照組，B為重組質(zhì)粒組。

圖2 顯微鏡油鏡下細(xì)胞形態(tài)學(xué)變化(×1 000)

2.2 K562細(xì)胞增殖抑制率比較 結(jié)果見表1。

表1 各組不同時(shí)點(diǎn)細(xì)胞增殖抑制率比較

注：與空質(zhì)粒組比較，△P<0.05；與外源性組比較，﹟P<0.05。

3 討論

白血病是一類好發(fā)于青少年的惡性腫瘤，是造血干細(xì)胞惡性克隆性疾病，患者可出現(xiàn)不同程度的貧血、出血、感染及肝、脾、淋巴結(jié)腫大和骨骼疼痛。據(jù)文獻(xiàn)報(bào)道，我國(guó)各地區(qū)白血病發(fā)病率在各種腫瘤中居第6位。目前，白血病的臨床治療以化療、放療、靶向治療、免疫治療、干細(xì)胞移植等為主。免疫療法可特異性殺傷腫瘤細(xì)胞，對(duì)正常細(xì)胞危害較小，或有望成為治療惡性腫瘤包括白血病的重要方法。研究[8]發(fā)現(xiàn)，白血病患者外周血、淋巴結(jié)、脾臟、骨髓中可檢測(cè)到抑制正常造血干細(xì)胞增殖的惡性克隆B細(xì)胞。白血病患者的T細(xì)胞通過(guò)抗原提呈細(xì)胞上調(diào)免疫反應(yīng)，降低免疫突觸的形成[9]。因此，白血病的治療或可從裂解白血病患者惡性克隆的細(xì)胞入手，如抗CD20單克隆抗體通過(guò)激活補(bǔ)體系統(tǒng)，形成攻膜復(fù)合物，從而裂解白血病患者惡性克隆的B細(xì)胞，或以集落刺激因子來(lái)刺激惡性克隆的細(xì)胞，以達(dá)到治療效果[10]。K562細(xì)胞為人類髓性白血病人工培養(yǎng)的第一個(gè)細(xì)胞，同時(shí)也具有多向分化潛能，因此被廣泛用于研究腫瘤如白血病的治療、藥物靶標(biāo)等領(lǐng)域中。

已有研究[11,12]證實(shí)，GM-CSF不僅可作用于骨髓造血細(xì)胞分化的較早階段，同時(shí)還可刺激多能干細(xì)胞及早期紅細(xì)胞的增殖、分化和促進(jìn)成熟細(xì)胞向外周血釋放，從而提高機(jī)體抗腫瘤和抗感染免疫力。Stripecke等[13]發(fā)現(xiàn)，將B7.1基因和GM-CSF基因修飾的聯(lián)合瘤苗應(yīng)用于白血病的治療，抑瘤率比單一基因更顯著。將GM-CSF注射到小鼠皮下，會(huì)增加白細(xì)胞的吞噬功能，還會(huì)促進(jìn)巨噬細(xì)胞的成熟及影響巨噬細(xì)胞在組織中的數(shù)量[14]。GM-CSF作為細(xì)胞因子，能夠調(diào)節(jié)機(jī)體免疫細(xì)胞對(duì)腫瘤細(xì)胞的攻擊殺傷能力，具有抗腫瘤的作用[15]。目前，GM-CSF主要用于惡性腫瘤放化療后所致白細(xì)胞減少癥及某些免疫缺陷性疾病的造血、再生性障礙貧血和免疫功能重建等治療中[16]。GM-CSF對(duì)治療癌癥放化療、骨髓移植、艾滋病等各種原因引起的白細(xì)胞減少癥，提高患者抗感染能力亦有較好療效[17]。研究[18]發(fā)現(xiàn)，GM-CSF可通過(guò)調(diào)節(jié)糖蛋白或其自分泌產(chǎn)物，而保護(hù)粒細(xì)胞與巨噬細(xì)胞的功能免受白血病細(xì)胞攻擊，還可提高癌癥患者化療后白細(xì)胞水平。GM-CSF可刺激造血干細(xì)胞釋放入外周血，釋放入血的造血干細(xì)胞很大程度上取代了骨髓移植手術(shù)植入的造血干細(xì)胞，骨髓移植手術(shù)還易給癌癥患者造成醫(yī)源性骨髓移植損傷，故GM-CSF或?qū)⒊蔀橹委煇盒阅[瘤的新途徑。

本研究顯示，與細(xì)胞對(duì)照組及空質(zhì)粒組比較，重組質(zhì)粒組及外源性組第3、5、7天細(xì)胞增殖抑制率均降低(P均<0.05)；與外源性組比較，重組質(zhì)粒組第3、5、7天細(xì)胞增殖抑制率均降低(P均<0.05)。提示重組PcDNA3.1-GM-CSF及外源性GM-CSF均能使K562細(xì)胞增殖受到抑制，作用強(qiáng)度隨作用時(shí)間延長(zhǎng)而增加，且重組PcDNA3.1-GM-CSF抑制作用強(qiáng)于外源性GM-CSF。本研究還顯示，與外源性組、空質(zhì)粒組、細(xì)胞對(duì)照組比較，重組質(zhì)粒組第9天細(xì)胞增殖抑制率均降低(P均<0.05)；而外源性組與其余3組比較均無(wú)統(tǒng)計(jì)學(xué)差異。說(shuō)明外源性GM-CSF對(duì)K562細(xì)胞的抑制作用隨著時(shí)間的延長(zhǎng)出現(xiàn)衰減現(xiàn)象。綜上，重組PcDNA3.1-GM-CSF抑制作用強(qiáng)于外源性GM-CSF，而且使用內(nèi)源性的重組PcDNA3.1-GM-CSF作用于腫瘤細(xì)胞，可以在一定程度上避免在使用外源性GM-CSF時(shí)可能出現(xiàn)的污染，也在一定程度上彌補(bǔ)了外源性GM-CSF作用時(shí)間短暫的缺陷，具有一定的優(yōu)越性。本研究還發(fā)現(xiàn)，轉(zhuǎn)染重組質(zhì)粒PcDNA3.1-GM-CSF后，部分K562細(xì)胞轉(zhuǎn)變成NBT陽(yáng)性細(xì)胞，NBT陽(yáng)性細(xì)胞具有一定吞噬功能。轉(zhuǎn)染PcDNA3.1-GM-CSF的K562細(xì)胞形態(tài)發(fā)生了明顯改變，部分細(xì)胞內(nèi)出現(xiàn)腎形馬蹄形核，這與吞噬細(xì)胞的形態(tài)學(xué)特點(diǎn)有相似之處。如果將重組質(zhì)粒PcDNA3.1-GM-CSF轉(zhuǎn)染到K562細(xì)胞內(nèi)，能使K562細(xì)胞轉(zhuǎn)為具備吞噬白血病細(xì)胞功能的細(xì)胞，有望用其治愈白血病。

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Effects of granulocyte-macrophage colony-stimulating factor transfection on morphology and proliferation of leukemia cells K562

CHENRong,CHANGJinxia,LIXuelu,PENGLijuan,ZHUHong

(DepartmentofMicrobiologyandImmunologyofNorthernSichuanMedicalCollege,Nanchong637000,China)

Objective To observe the effects of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant PcDNA3.1-GM-CSF on the morphology and proliferation of myeloid leukemia cells K562. Methods K562 cells were randomly divided into four groups. Recombinant PcDNA3.1- GM-CSF, exogenous GM-CSF, and PcDNA3.1 null plasmids were transfected into K562 cells as the recombinant plasmid group, the exogenous group and the empty plasmid group, respectively, and the non-transfected plasmid was set as the control group. The morphological changes of cells were observed under the optical microscope by using high power lens and oil lens, respectively, and the cell proliferation was measured by MTT. Results Morphological changes seen under high power lens: the control group, the empty plasmid group and exogenous group were similar in morphology, cells were stained dark blue, whereas no clear boundary was seen between the cytoplasm and nucleus; compared with these three groups, the cells of the recombinant plasmid group showed a significant difference, the cytoplasm was stained light blue, and the nuclei were pale pink along with a clear bounds between them. Kidney or horseshoe shaped nuclei could be seen in some of the cells. Morphological changes seen under oil lens: the control group, the exogenous group, and empty plasmid group were similar, the cells were stained dark blue, and granules deposition wasn't seen in the cells; as to the recombinant plasmid group, some cells were stained dark blue, while some were pale pink in which dotted formazan particles were seen. Results of cell proliferation assay: Compared with the other two groups, the inhibition rate of cell proliferation in the recombinant plasmid group showed an increase on the 3th , 5th, 7th and 9th days (P<0.05). Compared with the empty plasmid group, the inhibition rate in the exogeneous group displayed an increase on the 3th, 5th and 7th days (P<0.05). Conclusion Both recombinant PcDNA3.1-GM-CSF and exogenous GM-CSF could induce morphological change and lead to proliferation inhibition in K562 cells, the effects increase along with time, and the inhibitory effect of recombinant PcDNA3.1-GM-CSF is stronger than that of the exogenous GM-CSF.

granulocyte-macrophage colony-stimulating factor; leukemic cells; cell morphology; cell proliferation

四川省教育廳自然科學(xué)基金資助項(xiàng)目(12ZB214)。

陳蓉(1982-)，女，碩士，講師，主要研究方向?yàn)獒t(yī)學(xué)免疫學(xué)和微生物學(xué)。E-mail： chenr1232@163.com

朱紅(1975-)，女，碩士，副教授，主要研究方向?yàn)獒t(yī)學(xué)免疫學(xué)和微生物學(xué)。E-mail： zhuhongyuxia@163.com

10.3969/j.issn.1002-266X.2017.31.002

R730.5

A

1002-266X(2017)31-0005-04

2017-03-06)