干細(xì)胞轉(zhuǎn)染CMV-Luciferase-PGK-Puro慢病毒后化學(xué)發(fā)光成像

阮光萍，劉菊芬，李自安，王金祥，呂燕波，龐榮清，潘興華

干細(xì)胞轉(zhuǎn)染CMV-Luciferase-PGK-Puro慢病毒后化學(xué)發(fā)光成像

阮光萍，劉菊芬，李自安，王金祥，呂燕波，龐榮清，潘興華

目的研究用化學(xué)發(fā)光成像的方法觀察臍帶間充質(zhì)干細(xì)胞轉(zhuǎn)染CMV-Luciferase-PGK-Puro慢病毒后的情況，代替活體成像儀的可行性。方法用CMV-Luciferase-PGK-Puro慢病毒轉(zhuǎn)染樹鼩臍帶間充質(zhì)干細(xì)胞，用最適濃度的嘌呤霉素篩選，存活的細(xì)胞用6孔板的3個孔培養(yǎng)，貼壁后，3個孔依次加入底物D-熒光素鉀鹽，用化學(xué)發(fā)光成像儀拍照，再用軟件進(jìn)行活體成像轉(zhuǎn)換。將轉(zhuǎn)染成功的細(xì)胞注入麻醉后的樹鼩皮下，樹鼩靜脈注射底物。結(jié)果細(xì)胞加入底物后有生物發(fā)光，發(fā)光強度隨底物作用時間延長而減弱。樹鼩皮下也觀察到發(fā)光細(xì)胞。結(jié)論CMV-Luciferase-PGK-Puro慢病毒能成功轉(zhuǎn)染樹鼩臍帶間充質(zhì)干細(xì)胞，轉(zhuǎn)染成功的細(xì)胞用于動物模型治療后，可進(jìn)行活體成像，觀察細(xì)胞在動物體內(nèi)的分布。

樹鼩；臍帶；間充質(zhì)干細(xì)胞；CMV-Luciferase-PGK-Puro慢病毒；轉(zhuǎn)染；化學(xué)發(fā)光成像

近年來，小動物活體成像技術(shù)得到了較大推廣，廣泛應(yīng)用于動物模型、腫瘤機制等研究[1-4]。各種細(xì)胞在小動物體內(nèi)的定位示蹤進(jìn)一步推廣了這一技術(shù)的應(yīng)用。細(xì)胞通常用CMV-Luciferase-PGK-Puro慢病毒轉(zhuǎn)染，細(xì)胞回輸后在動物體內(nèi)定位、擴(kuò)增后，可通過給動物注射底物D-熒光素鉀鹽后，帶有CMV-Luciferase-PGK-Puro基因的細(xì)胞就會與底物作用而發(fā)光，通過活體成像儀可檢測到發(fā)光現(xiàn)象。但活體成像儀價格昂貴，只有少數(shù)單位能夠購買。本實驗室有化學(xué)發(fā)光成像儀，可以觀察到生物發(fā)光。于是本研究用化學(xué)發(fā)光成像儀觀察CMV-Luciferase-PGK-Puro慢病毒轉(zhuǎn)染的樹鼩臍帶間充質(zhì)干細(xì)胞的情況，探討其代替活體成像儀的可行性，為下一步的樹鼩疾病模型研究奠定基礎(chǔ)。

1 材料與方法

1.1 試劑 CMV-Luciferase-PGK-Puro慢病毒和底物D-熒光素鉀鹽購自上海吉滿生物技術(shù)有限公司，化學(xué)發(fā)光成像儀為上海天能科學(xué)儀器廠生產(chǎn) （型號：Tanon 5200）；樹鼩購自中科院昆明動物所，實驗動物生產(chǎn)許可證號為SCXK（滇）K2013-0005，實驗動物使用許可證號為SYXK（滇）K2013-0012。

1.2 樹鼩臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng) 收集進(jìn)行剖腹產(chǎn)的樹鼩的臍帶，用鹽水沖洗，用青、鏈霉素雙抗浸泡，用小剪刀盡量將臍帶剪碎，放入培養(yǎng)瓶中貼壁培養(yǎng)。第2 d可見單個細(xì)胞從組織塊爬出貼壁，第7 d左右細(xì)胞長滿可傳代；傳代后為第1代細(xì)胞，第7 d左右細(xì)胞再次長滿，可傳代為第2代細(xì)胞；第3代細(xì)胞用于CMV-Luciferase-PGK-Puro慢病毒轉(zhuǎn)染。

1.3 CMV-Luciferase-PGK-Puro慢病毒轉(zhuǎn)染樹鼩臍帶間充質(zhì)干細(xì)胞 200 μl CMV-Luciferase-PGK-Puro慢病毒，滴度2×107TU/ml，每瓶4×106個樹鼩臍帶間充質(zhì)干細(xì)胞，加入50 μl（1×106TU）CMV-Luciferase-PGK-Puro慢病毒，轉(zhuǎn)染 3 d后，加入嘌呤霉素2～3 μg/m l。加入嘌呤霉素后，只有轉(zhuǎn)染成功的細(xì)胞能夠存活。待細(xì)胞篩選成功后，取5×105個細(xì)胞接種6孔板，共接種3個孔。

1.4 化學(xué)發(fā)光儀成像觀察慢病毒轉(zhuǎn)染干細(xì)胞結(jié)果

接種后第2 d、細(xì)胞貼壁后，6孔板每孔有2 ml培養(yǎng)基，3個孔依次加20 μl D-熒光素鉀鹽（15 mg/ml），先在A1孔加入20 μl底物D-熒光素鉀鹽，化學(xué)發(fā)光儀檢測生物發(fā)光度；再在A2孔加入20 μl底物D-熒光素鉀鹽，化學(xué)發(fā)光儀檢測生物發(fā)光度；最后在A3孔加入20 μl底物D-熒光素鉀鹽，化學(xué)發(fā)光儀檢測生物發(fā)光度。3個孔依次加入底物，可以證明生物發(fā)光的特異性。用儀器自帶軟件進(jìn)行活體成像合成，最后可觀察到6孔板中的發(fā)光現(xiàn)象。

1.5 慢病毒轉(zhuǎn)染成功的樹鼩臍帶間充質(zhì)干細(xì)胞的體內(nèi)實驗 將一只10 w的樹鼩背部用脫毛膏脫毛，將樹鼩麻醉后，背部皮下注入3.7×106個慢病毒轉(zhuǎn)染成功的樹鼩臍帶間充質(zhì)干細(xì)胞，同時尾靜脈注射1.3 ml底物D-熒光素鉀鹽，化學(xué)發(fā)光儀成像拍照檢測樹鼩背部皮下的生物發(fā)光，用儀器自帶軟件進(jìn)行活體成像合成。樹鼩的背部用脫毛膏脫毛，方便觀察結(jié)果。

2 結(jié)果

2.1 樹鼩臍帶間充質(zhì)干細(xì)胞的生長與形態(tài) 原代培養(yǎng)的樹鼩臍帶間充質(zhì)干細(xì)胞從剪碎的組織塊爬出，貼壁生長（圖1A）。隨著培養(yǎng)時間的延長，貼壁生長的細(xì)胞越來越多，呈長梭形，長滿后可傳代（圖1B）。

圖1 樹鼩臍帶間充質(zhì)干細(xì)胞的生長與形態(tài)

2.2 細(xì)胞生物發(fā)光檢測結(jié)果 A1孔加入20 μl底物后，化學(xué)發(fā)光成像儀拍照觀察到生物發(fā)光（圖2）。

圖2 細(xì)胞生物發(fā)光檢測結(jié)果

A2孔加入20 μl底物后，化學(xué)發(fā)光成像儀觀察到A1和A2孔均發(fā)光，新加入底物的A2孔發(fā)光更強（圖3）。

圖3 化學(xué)發(fā)光成像儀觀察結(jié)果

A3孔加入20 μl底物后，化學(xué)發(fā)光成像儀觀察到A1、A2和A3孔均發(fā)光，新加入底物的A3孔發(fā)光更強，而A2孔發(fā)光強于A1孔（圖4）。

圖4 化學(xué)發(fā)光成像儀觀察結(jié)果

通過平行3個孔的研究，證明細(xì)胞生物發(fā)光是特異的，并且細(xì)胞發(fā)光強弱與D-熒光素鉀鹽底物的加入時間有關(guān)，隨加入時間延長，細(xì)胞生物發(fā)光逐漸減弱。

2.3 樹鼩皮下化學(xué)發(fā)光成像 在樹鼩皮下注射慢病毒轉(zhuǎn)染成功的樹鼩臍帶間充質(zhì)干細(xì)胞、靜脈注射底物后，化學(xué)發(fā)光成像儀觀察到生物發(fā)光，用軟件合成后類似活體成像（圖5）。

3 討論

本實驗通過用普通的化學(xué)發(fā)光成像儀觀察到細(xì)胞轉(zhuǎn)染CMV-Luciferase-PGK-Puro慢病毒是成功的，通過平行3個孔的研究，證明細(xì)胞生物發(fā)光是特異的，并且細(xì)胞發(fā)光強弱與D-熒光素鉀鹽底物的加入時間有關(guān)，隨加入時間延長，細(xì)胞生物發(fā)光逐漸減弱。通過本實驗研究，證明CMV-Luciferase-PGK-Puro慢病毒可以成功轉(zhuǎn)染樹鼩臍帶間充質(zhì)干細(xì)胞，轉(zhuǎn)染成功的細(xì)胞可用于治療各種動物疾病模型。近年來，越來越多的文獻(xiàn)報道了臍帶間充質(zhì)干細(xì)胞可用于多種人類疾病的治療，包括系統(tǒng)性紅斑狼瘡、糖尿病、代謝綜合征等[5-8]。由于臍帶間充質(zhì)干細(xì)胞來源于生產(chǎn)后廢棄的臍帶，沒有倫理學(xué)爭論，培養(yǎng)容易，又具有干細(xì)胞多向分化與修復(fù)損傷的作用，已越來越受到研究者的重視[9-12]。

圖5 樹鼩皮下活體成像結(jié)果

轉(zhuǎn)染CMV-Luciferase-PGK-Puro慢病毒后的樹鼩臍帶間充質(zhì)干細(xì)胞，通過嘌呤霉素的篩選，轉(zhuǎn)染成功的細(xì)胞存活下來，并可以繼續(xù)傳代擴(kuò)增。有了種子細(xì)胞，今后用于各種樹鼩動物模型治療，可以更好地通過活體成像儀追蹤細(xì)胞在體內(nèi)的分布與定位，為臍帶間充質(zhì)干細(xì)胞治療各種動物模型的療效與機制研究打下了良好的基礎(chǔ)。

通過軟件轉(zhuǎn)換，生物發(fā)光效果圖可轉(zhuǎn)化為活體成像效果圖，轉(zhuǎn)化后的發(fā)光圖片與小動物活體成像儀觀察到的效果一致。所以，用化學(xué)發(fā)光成像儀可以對細(xì)胞的轉(zhuǎn)染效果進(jìn)行觀察，可在本單位完成實驗，而不需要聯(lián)系到外單位的小動物活體成像儀，避免細(xì)胞運輸過程培養(yǎng)基逸出，細(xì)胞活性受到影響，使化學(xué)發(fā)光成像儀發(fā)揮更大的作用與價值。

[1] Vicidomini C,Panico M,Greco A,et al.In vivo imaging and characterization of[(18)F]DPA-714,a potential new TSPO ligand, in mouse brain and peripheral tissues using small-animal PET[J]. Nucl Med Biol,2015,42(3):309-316.

[2] Lee O,Lee G,Kim M,et al.Multimodal evaluation of xenograft tumors in mice with an in-vivo stereo imaging system and small-animal PET/CT[J].Melanoma Res,2013.

[3] Hag AM,Ripa RS,Pedersen SF,et al.Small animal positron emission tomography imaging and in vivo studies of atherosclerosis[J].Clin Physiol Funct Imaging,2013,33(3):173-185.

[4] Fuchs K,Kohlhofer U,Quintanilla-Martinez L,et al.In vivo imaging of cell proliferation enables the detection of the extent of experimental rheumatoid arthritis by 3'-deoxy-3'-18ffluorothymidine and small-animal PET[J].J Nucl Med,2013,54 (1):151-158.

[5] Zhuang Y,Li D,Fu J,et al.Comparison of biological properties of umbilical cord-derived mesenchymal stem cells from early and late passages:immunomodulatory ability is enhanced in aged cells[J].Mol Med Rep,2015,11(1):166-174.

[6] Zhu L,Ruan Z,Yin Y,et al.Expression and significance of DLL4--Notch signaling pathway in the differentiation of human umbilical cord derived mesenchymal stem cells into cardiomyocytes induced by 5-azacytidine [J].Cell Biochem Biophys,2015,71(1):249-253.

[7] Zhu JQ,Lu HK,Cui ZQ,et al.Therapeutic potential of human umbilical cord blood mesenchymal stem cells on erectile function in rats with cavernous nerve injury[J].Biotechnol Lett,2015，37 (7):1-11.

[8] Zhou X,Gu J,Gu Y,et al.Human umbilical cord-derived mesenchymal stem cells improve learning and memory function in hypoxic-ischemic brain-damaged rats via an IL-8-mediated secretion mechanism rather than differentiation pattern induction[J].Cell Physiol Biochem,2015,35(6):2383-2401.

[9] Todeschi MR,Backly R El,Capelli C,et al.Transplanted umbilical cord mesenchymal stem cells modify the in vivo microenvironment enhancing angiogenesis and leading to bone regeneration[J].Stem Cells Dev,2015,24(13):1570-1581.

[10] Shuai H,Shi C,Lan J,et al.Double labelling of human umbilical cord mesenchymal stem cells with Gd-DTPA and PKH26 and the influence on biological characteristics of hUCMSCs[J].Int J Exp Pathol,2015,96(1):63-72.

[11] Xie L,Lin L,Tang Q,et al.Sertoli cell-mediated differentiation of male germ cell-like cells from human umbilical cord Wharton's jelly-derived mesenchymal stem cells in an in vitro co-culture system[J].Eur J Med Res,2015,20(1):9.

[12] Ding DC,Chang YH,Shyu WC,et al.Human umbilical cord mesenchymal stem cells:a new era for stem cell therapy[J].Cell Transplant,2015,24(3):339-347.

Chemiluminescence imaging of conditions after tree shrews UC-MSCs are transfected with CMV-Luciferase-PGK-Puro lentivirus

Ruan Guangping,Liu Jufen,Li Zi'an,Wang Jinxiang,Lv Yanbo,Pang Rongqing,Pan Xinghua Cell Biological Therapy Center, Kunming General Hospital of Chengdu Military Command,Kunming,Yunnan,650032,China;National Joint Engineering Laboratory of Stem Cells and Immune Cells and Biological Medicine Technology,Kunming,Yunnan,650032,China;Key Laboratory of Cell Therapy Technology and Translational Medicine of Yunnan Province,Kunming,Yunnan,650032,China;Yunnan Stem Cell Engineering Laboratory,Kunming,Yunnan,650032,China;Key Laboratory of Stem Cell and Regenerative Medicine of Kunming, Kunming,Yunnan,650032,China

Objective To study the feasibility of chemiluminescence imaging instead of living imaging for observing the conditions after tree shrews umbilical cord mesenchymal stem cells(UC-MSCs)are transfected with CMV-Luciferase-PGK-Puro lentivirus.MethodsThe tree shrew UC-MSCs transfected with CMV-Luciferase-PGK-Puro lentivirus were screened with puromycin of optimum concentration.The survival cells were cultured with three holes of six-hole plate.Upon adherence,the substrate d-luciferin potassium salt was successively added to the three holes.A picture was taken by chemiluminescence imager,after which living imaging conversion was conducted.The cells successfully transfected were injected under the skin of postanesthetic tree shrew,which were injected with substrate by intravenous injection.ResultsUpon the addition of substrate,the cells had bioluminescence;the luminousintensity weakened with the extension of substrate action time.Subcutaneous luminous cells were also observed under the skin of tree shrew.ConclusionCMV-Luciferase-PGK-Puro lentivirus may successfully transfect tree shrews UC-MSCs.Successfully transfected cells used for the treatment of animal models can undergo living imaging to observe the distribution of cells in the animal body.

tree shrew;UC;MSC;CMV-Luciferase-PGK-Puro lentivirus;transfection;chemiluminescence imaging

R 318.1

A

1004-0188（2016）12-1378-04

10.3969/j.issn.1004-0188.2016.12.008

2016-06-28)

國家科技支撐計劃項目（2014BI01B0）；國家973計劃項目（2012CB5181060）；云南省科技計劃重點項目(2013CA005）

650032昆明,成都軍區(qū)昆明總醫(yī)院細(xì)胞生物治療中心，干細(xì)胞與免疫細(xì)胞生物醫(yī)藥技術(shù)國家地方聯(lián)合工程實驗室,云南省細(xì)胞治療技術(shù)轉(zhuǎn)化醫(yī)學(xué)重點實驗室，云南省干細(xì)胞工程實驗室，昆明市干細(xì)胞與再生醫(yī)學(xué)研究重點實驗室

潘興華，E-mail:xinghuapan@aliyun.com;龐榮清,E-mail:pangrq2000@aliyun.com