畢 涌, 洪 娟, 李力群, 李曉莉, 魏 鵬, 施鎮(zhèn)江, 王廷華, 張 旭
(溫州醫(yī)科大學(xué) 1神經(jīng)生物學(xué)重點(diǎn)學(xué)科, 2附屬第一醫(yī)院神經(jīng)內(nèi)科,浙江 溫州 325000;3昆明醫(yī)學(xué)院神經(jīng)科學(xué)研究所,云南 昆明 650031)
·短篇論著·
重組質(zhì)粒pcDNA3-β-NGF的構(gòu)建及其轉(zhuǎn)染小鼠骨髓間充質(zhì)干細(xì)胞生物學(xué)活性的研究*
畢 涌1,2, 洪 娟1, 李力群1, 李曉莉1,2, 魏 鵬3, 施鎮(zhèn)江3, 王廷華3, 張 旭1,2
(溫州醫(yī)科大學(xué)1神經(jīng)生物學(xué)重點(diǎn)學(xué)科,2附屬第一醫(yī)院神經(jīng)內(nèi)科,浙江 溫州 325000;3昆明醫(yī)學(xué)院神經(jīng)科學(xué)研究所,云南 昆明 650031)
目的構(gòu)建人神經(jīng)生長因子β亞基(β-NGF)真核表達(dá)載體,轉(zhuǎn)染熒光小鼠骨髓間充質(zhì)干細(xì)胞(MSCs)并研究其生物學(xué)活性。方法全骨髓貼壁培養(yǎng)法分離、培養(yǎng)和純化熒光小鼠MSCs,構(gòu)建真核表達(dá)載體pcDNA3-β-NGF,并轉(zhuǎn)染MSCs;免疫組織化學(xué)染色檢測β-NGF的表達(dá),觀察β-NGF陽性的MSCs對(duì)小鼠海馬神經(jīng)元生長的作用。結(jié)果熒光小鼠MSCs可發(fā)出明亮的綠色熒光,且熒光不隨培養(yǎng)時(shí)間延長和傳代次數(shù)增加而衰減。重組質(zhì)粒pcDNA3-β-NGF轉(zhuǎn)染MSCs后β-NGF陽性率為(37.12±2.14)%,高于MSCs組[(2.36±0.62)%]和空白對(duì)照組[(1.43±0.76)%],差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。pcDNA3-β-NGF轉(zhuǎn)染MSCs上清液培養(yǎng)的新生小鼠海馬神經(jīng)元的突起長度[(31±3)μm]較pcDNA3轉(zhuǎn)染MSCs組[(23±4)μm]明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P<0.05),表明β-NGF基因轉(zhuǎn)染MSCs培養(yǎng)上清液中的產(chǎn)物能促進(jìn)小鼠海馬神經(jīng)元的突起生長,具有明顯的生物學(xué)活性。結(jié)論成功構(gòu)建了pcDNA3-β-NGF真核表達(dá)載體,其轉(zhuǎn)染的熒光小鼠MSCs能正確、穩(wěn)定表達(dá)和分泌有生物學(xué)活性的β-NGF。
神經(jīng)生長因子; 間充質(zhì)干細(xì)胞; 基因轉(zhuǎn)染
骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)具有免疫原性弱、體外基因轉(zhuǎn)染率高、能穩(wěn)定高效表達(dá)外源基因等優(yōu)點(diǎn),是作為細(xì)胞和基因治療種子細(xì)胞的理想選擇[5]。然而,干細(xì)胞移植后在體內(nèi)的遷移、定植、分布和分化等有賴于對(duì)干細(xì)胞的示蹤和定量技術(shù)。綠色熒光蛋白(green fluorescent protein, GFP)轉(zhuǎn)基因小鼠MSCs能穩(wěn)定表達(dá)GFP,是MSCs研究中良好的示蹤工具[6]。β亞單位是NGF完整的生物活性單位,分子量較小,相對(duì)于完整的NGF分子則較易透過血腦屏障。因此,本研究將構(gòu)建人β-NGF真核細(xì)胞表達(dá)載體并轉(zhuǎn)染GFP轉(zhuǎn)基因小鼠MSCs,觀察β-NGF的表達(dá)及其對(duì)小鼠海馬神經(jīng)元的影響,為β-NGF的基因治療探索一種新的方法。
1主要試劑和儀器
DMEM/F12培養(yǎng)基和胰蛋白酶購自Gibco;胎牛血清(fetal bovine serum, FBS)購自HyClone。TRIzol RNA提取試劑盒、G418和β-NGF抗體購Sigma;IPTG、X-Gal、One Step RNA PCR Kit(AMV)、T4 DNA連接酶、限制性內(nèi)切酶HindIII及XhoI、DNA凝膠純化試劑盒和DNA marker(DL2000和λ-EcoT14 I digest)購自TaKaRa;DH5α大腸桿菌由本實(shí)驗(yàn)室保存;pcDNA3載體購自Invitrogen;脂質(zhì)體LipofectamineTM2000購自Gibco;二步法免疫組化試劑盒購自北京中山生物技術(shù)有限公司;PCR引物由上海博亞生物技術(shù)有限公司合成。超凈工作臺(tái);25 cm2培養(yǎng)瓶(HyClone);SHEL LAB TC2323 CO2Incubator;Leica DM IRB熒光顯微鏡;Olympus倒置相差顯微鏡。
2方法
2.1GFP轉(zhuǎn)基因小鼠MSCs的分離培養(yǎng)和鑒定 GFP轉(zhuǎn)基因小鼠由昆明醫(yī)學(xué)院神經(jīng)科學(xué)研究所提供。參照Phinney等[7]方法,脫頸法處死GFP轉(zhuǎn)基因小鼠,分離雙側(cè)脛股骨中全骨髓,采用全骨髓貼壁培養(yǎng)法獲取MSCs。按1×109/L接種于含10%胎牛血清的DMEM/F12混合培養(yǎng)液中,在37 ℃、5%CO2、飽和濕度的恒溫培養(yǎng)箱中培養(yǎng)。8 h后首次全量換液,此后每隔3 d全量換液。當(dāng)細(xì)胞貼壁達(dá)90%融合時(shí)即可進(jìn)行傳代。采用CD44、CD45和CD54抗體進(jìn)行免疫組織化學(xué)染色,通過DAB顯色后光鏡下觀察并計(jì)算陽性細(xì)胞的百分比進(jìn)行鑒定。
2.2真核表達(dá)載體pcDNA3-β-NGF的構(gòu)建及鑒定
溫室栽培番茄育苗管理的關(guān)鍵時(shí)期,此時(shí)期如果管理不當(dāng),會(huì)對(duì)幼苗生產(chǎn)造成很大影響,稍有疏忽就會(huì)造成嚴(yán)重的經(jīng)濟(jì)損失。那么應(yīng)注意哪些問題呢?
2.2.1引物的設(shè)計(jì)與合成 根據(jù)GenBank公布的人β-NGFcDNA(No. X52599)序列設(shè)計(jì)PCR引物。上游引物為5’-CGAAGCTTAGCGTAATGTCCATGTTG-3’,下游引物為5’- GCCTCGAGGGCAGGTCAGGCTCTTCT-3’,下劃線為HindIII和XhoI的酶切位點(diǎn)。
2.2.2RNA的提取和RT-PCR反應(yīng)擴(kuò)增目的基因 取本實(shí)驗(yàn)室保存的人腦瘤旁組織約50 mg,按TRIzol RNA提取試劑盒操作說明提取RNA,甲醛變性凝膠電泳檢測其完整性。取RNA 1 μg,濃度為20 μmol/L的上、下游引物各1 μL,參考TaKaRa One Step RNA PCR Kit(AMV)試劑盒說明,按下列條件在PCR自動(dòng)擴(kuò)增儀上進(jìn)行RT-PCR。50 ℃、30 min變性,94 ℃ 30 s,60 ℃ 30 s,72 ℃ 1 min,共30個(gè)循環(huán)。瓊脂糖凝膠電泳檢測RT-PCR反應(yīng)結(jié)果,用DNA凝膠純化試劑盒提取目的DNA的PCR產(chǎn)物。
2.2.3構(gòu)建pcDNA3-β-NGF質(zhì)粒并鑒定 RT-PCR反應(yīng)產(chǎn)物目的DNA片段和pcDNA3分別進(jìn)行HindIII和XhoI雙酶切,瓊脂糖凝膠電泳檢測并線性化載體,用DNA凝膠純化試劑盒回收酶切DNA片段。酶切后的pcDNA3線性質(zhì)粒和目的DNA片段以T4DNA連接酶建立連接反應(yīng),16 ℃連接20 h。取連接反應(yīng)的產(chǎn)物轉(zhuǎn)化大腸桿菌DH5α,接種在含有X-Gal和IPTG并帶有氨芐青霉素的培養(yǎng)皿上,倒置培養(yǎng)12~16 h。根據(jù)藍(lán)白斑進(jìn)行篩選,取陽性克隆(白斑)用SDS堿裂解法提取質(zhì)粒。HindIII和XhoI雙酶切鑒定重組體及其插入方向,并送測序鑒定。
2.3重組pcDNA3-β-NGF轉(zhuǎn)染GFP轉(zhuǎn)基因小鼠MSCs 用含10%胎牛血清的DMEM/F12培養(yǎng)液將MSCs輕柔吹打成密度為1×109/L的細(xì)胞懸液,常規(guī)培養(yǎng)后用于轉(zhuǎn)染,轉(zhuǎn)染前全量換液1次。將MSCs以1×105/well的密度接種在24孔板中,分別加入不同濃度的G418培養(yǎng)基各1 mL置于37 ℃、5%CO2、95%濕度條件下常規(guī)培養(yǎng)并觀察細(xì)胞生長情況,確定抑制細(xì)胞生長的最低G418濃度。
按脂質(zhì)體LipofectamineTM2000的操作說明書,分別取純化的pcDNA3-β-NGF及空載體pcDNA3各4 μg,溶于100 μL DMEM/F12培養(yǎng)液中;加入LipofectamineTM2000和DMEM/F12培養(yǎng)液,室溫放置15 min后加入0.8 mL含10%胎牛血清的DMEM/F12培養(yǎng)液,于37℃、50%CO2培養(yǎng)箱中培養(yǎng)。轉(zhuǎn)染后72~96 h,待細(xì)胞生長至接近融合時(shí),收集各組細(xì)胞的上清凍存?zhèn)溆?,?xì)胞傳代培養(yǎng)。繼續(xù)培養(yǎng)至細(xì)胞密度達(dá)培養(yǎng)皿底面積的60%~70%時(shí),棄去培養(yǎng)液,更換含濃度為700 mg/L(參照最小致死濃度為600 mg/L的測定結(jié)果)的G418培養(yǎng)液進(jìn)行篩選,對(duì)篩選形成的陽性克隆繼續(xù)擴(kuò)增培養(yǎng)。陰性對(duì)照組以含10%胎牛血清的DMEM/F12培養(yǎng)液同法培養(yǎng)、篩選。
2.4重組pcDNA3-β-NGF轉(zhuǎn)染MSCs后β-NGF的表達(dá) 收集轉(zhuǎn)染后經(jīng)G418篩選形成的陽性細(xì)胞克隆,以β-NGF抗體進(jìn)行免疫組織化學(xué)染色檢測β-NGF的表達(dá)。先用4%多聚甲醛固定細(xì)胞20 min,再用0.01 mol/L PBS液洗去固定液,參考二步法免疫組織化學(xué)檢測試劑盒的使用說明進(jìn)行操作,DAB顯色后光鏡下觀察。其中I抗稀釋度為1∶1 000,陰性對(duì)照組以0.01 mol/L PBS液代替I抗。
ELISA檢測β-NGF蛋白的表達(dá):分別收集MSCs和各組轉(zhuǎn)染細(xì)胞培養(yǎng)48~72 h后的上清液,4 ℃保存,48 h內(nèi)進(jìn)行人β-NGF的ELISA檢測,每組設(shè)6個(gè)復(fù)孔;靈敏度為15 ng/L。
2.5重組pcDNA3-β-NGF轉(zhuǎn)染MSCs的生物學(xué)活性鑒定 將本實(shí)驗(yàn)室保存的新生小鼠海馬神經(jīng)元按每個(gè)培養(yǎng)培皿1×106個(gè)細(xì)胞接種在涂有多聚賴氨酸的35 mm培養(yǎng)皿中,用含10%胎牛血清的DMEM/F12培養(yǎng)液培養(yǎng)。48 h后將已收集備用的各組轉(zhuǎn)染細(xì)胞上清液與含10%胎牛血清的DMEM/F12培養(yǎng)液按1∶1的比例加入新生小鼠海馬元培養(yǎng)皿中,觀察細(xì)胞生長情況,測定神經(jīng)突起長度并進(jìn)行統(tǒng)計(jì)學(xué)分析。
3統(tǒng)計(jì)學(xué)處理
計(jì)量數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,采用SPSS 17.0進(jìn)行單因素方差分析,以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
1MSCs生長情況的形態(tài)學(xué)觀察和鑒定
骨髓細(xì)胞接種于培養(yǎng)瓶,4 h后可見到細(xì)胞貼壁生長,2 d后呈現(xiàn)勻一梭形成纖維細(xì)胞樣形態(tài),呈集落生長,增殖迅速,見圖1A。原代細(xì)胞基本融合時(shí)進(jìn)行傳代培養(yǎng),傳至第5代時(shí),MSCs形成形態(tài)均一、呈漩渦狀或放射狀排列生長的梭形細(xì)胞,見圖1B。CD44、CD54和CD45抗體對(duì)原代培養(yǎng)3 d及傳至5代的細(xì)胞進(jìn)行免疫組織化學(xué)染色,第5代細(xì)胞的CD44(95.8%±0.8%)、CD54(91.4%±1.3%)陽性率較高,而CD45(2.1%±1.8%)陽性率較低,說明經(jīng)全骨髓貼壁分離法傳代培養(yǎng),MSCs的純度得到了提高,見圖1C、D。
Figure 1. Culture and identification of MSCs from GFP transgenic mouse.A: primary cells; B: fifth-passage cells after culture; C: CD44 immunopositivity; D: CD54 immunopositivity. Scale bar=100 μm.
圖1GFP轉(zhuǎn)基因小鼠MSCs的培養(yǎng)和鑒定
2RNA的提取和RT-PCR
RNA提取物經(jīng)甲醛變性凝膠電泳檢測可見18S及28S 2個(gè)條帶,見圖2A;RT-PCR反應(yīng)產(chǎn)物經(jīng)瓊脂糖凝膠電泳顯示為大小為750 bp的特異性擴(kuò)增條帶,見圖2B。將重組質(zhì)粒pcDNA3-β-NGF進(jìn)行限制性內(nèi)切酶HindIII和XhoI雙酶切,可見酶切片段與插入基因長度750 bp相符,見圖2C。
Figure 2. Construction and identification of recombinant plasmid pcDNA3-β-NGF. A: formaldehyde-denaturing gel electrophoresis of total RNA from human brain tumor adjacent tissues. B: agarose gel electrophoresis of RT-PCR products. M: DL2000 DNA marker; 1~3: RT-PCR products (about 750 bp). C: agarose gel electrophoresis of digestion products. M: λ-EcoT14 I digest DNA marker; 1: pc-DNA3 digested byHindIII; 2: pcDNA3-β-NGF digested byHindIII andXhoI; 3: pcDNA3-β-NGF digested byHindIII.
圖2重組質(zhì)粒pcDNA3-β-NGF的構(gòu)建與雙酶切鑒定
3重組質(zhì)粒pcDNA3-β-NGF測序鑒定
將重組質(zhì)粒pcDNA3-β-NGF進(jìn)行限制性內(nèi)切酶HindIII和XhoI雙酶切,送上海申友公司測序,測序結(jié)果與GenBank公布的人β-NGFcDNA完全一致,見圖3、4。
Figure 3. Sequence analysis of pcDNA3-β-NGF beginning with the sequence of BGH poly(A).
圖3重組質(zhì)粒pcDNA3-β-NGF以BGHpoly(A)序列為起點(diǎn)的測序圖
4重組質(zhì)粒pcDNA3-β-NGF轉(zhuǎn)染MSCs后β-NGF的表達(dá)
對(duì)轉(zhuǎn)染后經(jīng)G418篩選的陽性克隆細(xì)胞進(jìn)行二步法免疫組織化學(xué)檢測,DAB顯色后光鏡下觀察,G418篩選的重組質(zhì)粒轉(zhuǎn)染陽性克隆細(xì)胞(37.12±2.14)%呈棕色的陽性反應(yīng),明顯高于MSCs組[(2.36±0.62)%]和空白對(duì)照組[(1.43±0.76)%],差異有統(tǒng)計(jì)學(xué)意義(P<0.05),說明pcDNA3-β-NGF可以轉(zhuǎn)染MSCs,并使β-NGF表達(dá)顯著增加,見圖5A、B。
Figure 4. Sequence analysis of pcDNA3-β-NGF beginning with the sequence of T7 promoter.
圖4重組質(zhì)粒pcDNA3-β-NGF以T7啟動(dòng)子為起點(diǎn)的測序圖
轉(zhuǎn)染pcDNA3-β-NGF的3個(gè)單克隆MSCs擴(kuò)增培養(yǎng)48 h后上清液中β-NGF的含量分別為(96±6.2)ng/L、(81±6.8)ng/L和(74±5.9)ng/L;轉(zhuǎn)染pcDNA3空質(zhì)粒的2個(gè)單克隆MSCs和正常培養(yǎng)的MSCs培養(yǎng)48 h后上清液均未檢測到β-NGF蛋白。
5重組質(zhì)粒pcDNA3-β-NGF轉(zhuǎn)染MSCs后生物學(xué)活性的鑒定
重組質(zhì)粒pcDNA3-β-NGF轉(zhuǎn)染MSCs上清液培養(yǎng)的新生小鼠海馬神經(jīng)元于3 d后在倒置相差顯微鏡下觀察見細(xì)胞存活良好,胞體長出明顯的突起;pcDNA3轉(zhuǎn)染MSCs組新生小鼠海馬神經(jīng)元的胞體呈圓形,沒有明顯的突起。pcDNA3-β-NGF轉(zhuǎn)染MSCs組神經(jīng)元的突起長度[(31±3) μm]較pcDNA3轉(zhuǎn)染MSCs組[(23±4) μm]明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P<0.05),表明陽性轉(zhuǎn)染細(xì)胞培養(yǎng)上清液中的產(chǎn)物能促進(jìn)新生小鼠海馬神經(jīng)元的突起生長,有明顯的生物學(xué)活性,見圖5C、D。
Figure 5. Biological characteristics of MSCs transfected with recombinant plasmid pcDNA3-β-NGF. A: immunopositive expression of β-NGF in the MSCs transfected with pcDNA3-β-NGF; B: negative expression of β-NGF in the MSCs transfected with pcDNA3; C: neonatal mouse hippocampal neurons cultured with culture supernatant from the MSCs transfected with pcDNA3-β-NGF; D: neonatal mouse hippocampal neurons cultured with culture supernatant from the MSCs transfected with pcDNA3. Scale bar=100 μm.
圖5重組質(zhì)粒pcDNA3-β-NGF轉(zhuǎn)染MSCs后生物學(xué)活性的鑒定
NGF主要由α、β和γ 3個(gè)亞單位組成,對(duì)神經(jīng)細(xì)胞的生長、發(fā)育、分化、再生及功能發(fā)揮起著重要的調(diào)節(jié)作用,與神經(jīng)系統(tǒng)的發(fā)育、功能維持和損傷修復(fù)有密切關(guān)系[1]。然而,NGF不能有效通過血腦屏障,在腦內(nèi)很快降解,需要持續(xù)給藥維持效果,且由于人體廣泛存在NGF受體,易出現(xiàn)明顯不良反應(yīng)[3]。因此,選擇合適的給藥方式向靶部位連續(xù)不斷給藥顯得十分重要。通過基因轉(zhuǎn)染技術(shù)將細(xì)胞替代治療和NGF的神經(jīng)營養(yǎng)能力聯(lián)合應(yīng)用,使受損部位在一段時(shí)間內(nèi)持續(xù)大量表達(dá)NGF可能是比較理想的給藥方式。
MSCs是一種具有自我復(fù)制、多向分化潛能的成體干細(xì)胞,具有免疫原性弱,體外基因轉(zhuǎn)染率高,能穩(wěn)定高效表達(dá)外源基因等優(yōu)點(diǎn),已成為基因治療研究中理想的種子細(xì)胞。β亞單位是NGF完整的生物活性單位,不僅對(duì)神經(jīng)細(xì)胞分化和生長具有有效的神經(jīng)營養(yǎng)作用,還可誘導(dǎo)MSCs向神經(jīng)元分化和生長[1-2,8]。因此,本研究通過重組質(zhì)粒pcDNA3-β-NGF轉(zhuǎn)染成功實(shí)現(xiàn)了β-NGF在MSCs中的高效表達(dá);轉(zhuǎn)染的MSCs擴(kuò)增培養(yǎng)后上清液中β-NGF的含量明顯增多,細(xì)胞免疫組織化學(xué)染色結(jié)果表明,轉(zhuǎn)染組β-NGF陽性細(xì)胞明顯增多,染色更深,提示重組質(zhì)粒β-NGF轉(zhuǎn)染MSCs可使β-NGF表達(dá)顯著增加。
NGF在腦部主要由皮質(zhì)和海馬區(qū)產(chǎn)生,保護(hù)基底前腦膽堿能神經(jīng)元以及紋狀體和海馬區(qū)神經(jīng)元。汪軍兵等[9]研究顯示,NGF可抑制c-Jun氨基端激酶通路的活化,拮抗6-羥基多巴胺所誘導(dǎo)的大鼠腎上腺嗜鉻細(xì)胞瘤細(xì)胞的凋亡。本研究中,pcDNA3-β-NGF轉(zhuǎn)染的MSCs上清液中β-NGF蛋白表達(dá)明顯增加;新生小鼠海馬神經(jīng)元與pcDNA3-β-NGF轉(zhuǎn)染的MSCs上清液共培養(yǎng)后,胞體長出明顯的突起,其長度較pcDNA3轉(zhuǎn)染的MSCs明顯增加,說明β-NGF基因轉(zhuǎn)染后的MSCs具有理想的生物學(xué)活性。
此外,本研究采用全骨髓貼壁培養(yǎng)法,成功分離、培養(yǎng)了GFP轉(zhuǎn)基因小鼠的MSCs,在原代及傳代細(xì)胞中均持續(xù)表達(dá)GFP,發(fā)出明亮的綠色熒光,為干細(xì)胞移植后在體內(nèi)的遷移、定植、分布、分化等研究提供了成熟、有效的示蹤技術(shù)。本研究還采用目前認(rèn)為特異性相對(duì)較高的標(biāo)記分子CD44和CD54抗體對(duì)原代培養(yǎng)3 d及傳至5代的細(xì)胞進(jìn)行免疫組織化學(xué)染色進(jìn)行鑒定,利用造血細(xì)胞的特異性標(biāo)記CD45作為對(duì)照。結(jié)果顯示,傳代培養(yǎng)第5代細(xì)胞的CD44和CD54陽性率較高,而CD45陽性率較低,說明經(jīng)貼壁分離傳代培養(yǎng)后,MSCs的純度得到了明顯提高,與Colter等[10]研究一致 。
綜上所述,本研究成功構(gòu)建了人β-NGF真核細(xì)胞表達(dá)載體并轉(zhuǎn)染GFP轉(zhuǎn)基因小鼠MSCs,應(yīng)用ELISA和免疫組織化學(xué)染色檢測等方法證實(shí)了構(gòu)建的成功,并在體外培養(yǎng)條件下,驗(yàn)證了β-NGF的生物學(xué)活性,為進(jìn)一步的在體移植研究奠定了一定的基礎(chǔ)。
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BiologicalcharacteristicsofbonemarrowmesenchymalstemcellsfromGFPtransgenicmicetransfectedwithhumanβ-nervegrowthfactorgene
BI Yong1,2, HONG Juan1, LI Li-qun1, LI Xiao-li1,2, WEI Peng3, SHI Zhen-jiang3, WANG Ting-hua3, ZHANG Xu1,2
(1DepartmentofNeurobiology,2DepartmentofNeurology,theFirstAffiliatedHospital,WenzhouMedicalUniversity,Wenzhou325000,China;3InstituteofNeuroscience,KunmingMedicalCollege,Kunming650031,China.E-mail:drzhangxu@live.cn)
AIM: To investigate the changes of biological characteristics of GFP transgenic mouse bone marrow mesenchymal stem cells (MSCs), which were transfected with human β-nerve growth factor (β-NGF) gene in a recombinant eukaryotic expression vector.METHODSMSCs obtained from GFP transgenic mice were isolated, cultured and purified by the whole bone marrow adherence methods. Humanβ-NGFgene was transfected into the MSCs by a recombinant eukaryotic expression vector. The β-NGF expression in the MSCs was detected by the method of immunocytochemistry. Hippocampal neurons from neonatal mice were cultured with culture supernatant of the MSCs transfected with pcDNA3-β-NGF and the biological characteristics of the MSCs were investigated 3 d after culture under inverted phase-contrast microscope.RESULTSThe β-NGF positive rate of MSCs in pcDNA3-β-NGF transfection group [(37.12±2.14)%] was significantly higher than that in MSCs control group [(2.36±0.62)%] and blank control group [(1.43±0.76)%].The neurite length of neonatal mouse hippocampal neurons cultured with culture supernatant from pcDNA3-β-NGF-transfected MSCs [(31±3)μm] was significantly longer than that in negative control group [(23±4)μm], suggesting that MSCs transfected withβ-NGFgene maintained better biological characteristics.CONCLUSIONThe constructed recombinant eukaryotic expression vector of humanβ-NGFgene can be transfected into MSCs efficiently and NGF can be effectively expressed in MSCs. MSCs transfected withβ-NGFgene are capable of stable expression and secretion of β-NGF, and maintenance of better biological characteristics.
Nerve growth factor; Mesenchymal stem cells; Gene transfection
R741.05; R329.28
A
10.3969/j.issn.1000- 4718.2013.11.030
1000- 4718(2013)11- 2082- 06
2013- 04- 08
2013- 10- 16
浙江省高校“十二五”神經(jīng)生物學(xué)重點(diǎn)學(xué)科資助項(xiàng)目(No.204-071006);浙江省自然科學(xué)基金資助項(xiàng)目(No.Y2101091);溫州市科技計(jì)劃項(xiàng)目(No.Y2013S0212;No.Y2013S0239)
△通訊作者 Tel: 0577-55579371; E-mail: drzhangxu@live.cn