外磁場誘導下SPIO、hNIS、EGFP共標記人臍帶間充質(zhì)干細胞的靶向遷移及活體示蹤
發(fā)布時間：2018-08-04 15:03
【摘要】：目的： 采用納米磁性靶向技術和細胞生物學等技術，將人鈉/碘同向轉(zhuǎn)運體（hNIS）報告基因、增強型綠色熒光蛋白（EGFP）報告基因和超順磁氧化鐵顆粒（SPIO）對人臍帶間充質(zhì)干細胞共標記，建立間充質(zhì)干細胞活體單光子發(fā)射計算機斷層成像(SPECT)、核磁共振成像（MRI）、活體小動物熒光擬合成像活體示蹤系統(tǒng)，利用多模式顯像機理，精確區(qū)分局部器官干細胞聚集量；外加磁場使干細胞在獲得靶向移動能力的同時，用MRI及SPECT99mTc顯像、活體小動物熒光擬合成像進行示蹤，探討建立干細胞靶向遷移能力與示蹤的多模式分子影像技術的可行性。 方法： 1.共標記干細胞系的建立：分離鑒定人臍帶間充質(zhì)干細胞，選擇EGFP報告基因來間接反應目的基因hNIS的表達，PCR擴增后將目的基因hNIS克隆到pcDNA3.1中，再將EGFP連接到hNIS的下游，，然后與入門載體通過BP反應同源重組得到入門克隆,最后將入門克隆與表達載體同源重組產(chǎn)生表達克隆,經(jīng)酶切、PCR及測序證實成功構建重組質(zhì)粒pCMV-NIS-EF1-GFP-PGK-puro,再感染人臍帶間充質(zhì)干細胞。獲得穩(wěn)定表達hNIS及EGFP的干細胞系后，進行125I內(nèi)流及125I外流實驗鑒定hNIS蛋白功能。將適當濃度的SPIO與細胞系孵育獲得hNIS及EGFP、SPIO共同標記的干細胞，并驗證其活性及干性。 2.不同外磁場暴露時間篩選的體外研究：對照組為未標記的hUCMSCs，實驗組為SPIO-hNIS-EGFP-hUCMSCs細胞并在培養(yǎng)板下方放置磁場（200mT）連續(xù)3天，按每天接受的磁場暴露時間分為1小時組、6小時組、12小時組、24小時組。臺盼蘭染色檢測細胞活力，MTT法進行細胞增殖能力測試，Transwell實驗測磁場干預下的遷移能力、成脂成骨誘導分化實驗測分化能力，Western blot測BCL-1、Bax、Caspase-3、Caspase-9等凋亡因子的表達的影響，篩選合適的暴露時間為活體體內(nèi)研究提供依據(jù)。 3.外磁場作用下干細胞的靶向遷移及活體示蹤：建立全層皮膚缺損裸鼠模型，在細胞移植后0小時、24小時、48小時及7天進行核磁共振顯像外磁場MRI、SPECT、活體小動物熒光成像示蹤。分別測量MRI圖像參數(shù)原位干細胞團的面積減少率、信噪比、載噪比、細胞團位移。普魯士藍染色及冰凍熒光染色鑒定皮膚創(chuàng)面干細胞數(shù)量。比較兩組間皮膚創(chuàng)面愈合時間。 結果： 1.成功分離培養(yǎng)人臍帶間充質(zhì)干細胞，并鑒定其分化增殖能力及干性。第三代慢病毒包裝系統(tǒng)介導下完成構建包含目的基因hNIS的真核質(zhì)粒并獲得穩(wěn)定表達的hNIS-EGFP-hUCMSCs細胞系，經(jīng)Western blot鑒定hNIS表達良好。hNIS-EGFP-hUCMSCs實驗組細胞攝取125I活性125I內(nèi)流實驗及外流實驗證實人臍帶間充質(zhì)干細胞表達hNIS后的功能良好。成功建立SPIO、hNIS EGFP共標記的人臍帶間充質(zhì)干細胞系命名為SPIO-hNIS-EGFP-hUCMSCs，普魯士藍染色證實SPIO進入細胞內(nèi),細胞標記率達98%。SPIO-hNIS-EGFP-hUCMSCs在生物活性、細胞干性、增殖活性、分化能力與原始hUCMSCs無顯著差別，可以作為動物模型的治療細胞，并為下一步SPECT、MRI、活體小動物熒光顯像示蹤干細胞做好準備。 2.表面磁力200mT的永磁鐵作用下，連續(xù)三天每天磁場暴露時間6小時在細胞活性，增殖能力，分化能力方面與空白對照組無差異，磁場暴露時間達12小時以上細胞生物活性及分化能力得到全面抑制。Transwell細胞遷移實驗證明磁場暴露時間達到6小時后，SPIO-hNIS-EGFP-hUCMSCs細胞遷移能力不會隨著暴露時間發(fā)生進一步增加。外磁場暴露時間不超過6小時/天，不會誘發(fā)過多的促凋亡基因表達。磁場暴露時間達12小時以上可能會誘發(fā)SPIO-hNIS-EGFP-hUCMSCs程序性死亡。 3.MRI示蹤顯像獲得較高質(zhì)量的圖像，SPIO-hNIS-EGFP-hUCMSCs成功受到植入磁體產(chǎn)生磁場梯度的影響，由磁場引導干細胞在皮下區(qū)域向皮膚缺損部位快速遷移，此效應在外磁場作用后的第一個24小時最為明顯。SPECT顯像示標記干細胞在在0小時、24小時、48小時、7天的檢測與MRI顯像示蹤移植干細胞趨勢相符，但空間分辨率較MRI低。活體小動物熒光成像在0小時，24小時和48小時的外部磁場促進SPIO-hNIS-EGFP-hUCMSCs與MRI顯示一致的趨勢，但第7天的活體熒光成像細胞移植的示蹤呈陰性，考慮單位細胞濃度過小。普魯士藍與冰凍熒光切片顯示外磁場組具有較多的干細胞進入皮膚創(chuàng)面附近。 結論： 首次運用慢病毒轉(zhuǎn)染系統(tǒng)成功建立SPIO-hNIS-EGFP-hUCMSCs細胞系，并在生物活性、細胞干性、增殖活性、分化能力與原始hUCMSCs無顯著差別；外磁場暴露時間對干細胞生物特性造成較大影響，每天暴露時間6小時是最佳時間；首次將外磁場應用于皮下干細胞移植治療以增加干細胞的歸巢，MRI與基于hNIS報告基因SPECT顯像相結合，可以更為精確的示蹤干細胞，提高敏感度，減少假陽性。
[Abstract]:Objective:
Using nanotechnology of magnetic targeting and cell biology, the human Na / iodide transporter (hNIS) reporter gene, enhanced green fluorescent protein (EGFP) reporter gene and superparamagnetic iron oxide particle (SPIO) were used to mark human umbilical cord mesenchymal stem cells, and the single photon emission computed tomography (SPECT) of mesenchymal stem cells was established. Nuclear magnetic resonance imaging (MRI), living small animal fluorescence fitting imaging system, using multi-mode imaging mechanism to accurately distinguish the aggregation of local organ stem cells; while the magnetic field makes the stem cells to get the ability to target movement, MRI and SPECT99mTc imaging, the fluorescent pseudo synthetic images of living small animals are traced to explore the establishment of dry cells. The feasibility of cell targeting migration and tracer multimodal molecular imaging technology.
Method:
1. the establishment of a co labeled stem cell line: isolation and identification of human umbilical cord mesenchymal stem cells, selected EGFP reporter gene to indirectly respond to the expression of the target gene hNIS, PCR amplification of the target gene hNIS to pcDNA3.1, then EGFP connected to the downstream of hNIS, and then the entry vector through BP reaction homologous recombination to get the introductory clone, finally will be PCR and sequencing confirmed that the recombinant plasmid pCMV-NIS-EF1-GFP-PGK-puro was successfully constructed, and human umbilical cord mesenchymal stem cells were successfully constructed by enzyme digestion, PCR and sequencing. After obtaining the stable expression of hNIS and EGFP stem cell lines, the hNIS protein function was identified by 125I flow and 125I flow test. A proper concentration of SP was used. IO was incubated with cell lines to obtain hNIS and EGFP, SPIO co labeled stem cells, and to verify their activity and dry matter.
2. in vitro study of different external magnetic field exposure time: the control group was unmarked hUCMSCs, the experimental group was SPIO-hNIS-EGFP-hUCMSCs cells and the magnetic field was placed under the culture plate (200mT) for 3 days. The exposure time was divided into 1 hours group, 6 hour group, 12 hour group, 24 hour group. Trypan blue staining was used to detect cell vitality, M TT assay was used to test the cell proliferation ability, Transwell test was used to measure the migration ability of magnetic field intervention, the differentiation ability of fat induced osteogenesis induced differentiation, and the effect of Western blot on the expression of BCL-1, Bax, Caspase-3, Caspase-9 and so on, and to screen the appropriate exposure time to provide the basis for the study in vivo.
3. the target migration of stem cells and tracing in vivo under the action of 3. external magnetic field: a nude mouse model of full layer skin defect was established. The magnetic resonance imaging external magnetic field MRI, SPECT, and living small animals were traced at 0 hours, 24 hours, 48 hours and 7 days after the cell transplantation. The area reduction rate and the signal to noise ratio of the MRI map in situ stem cell mass were measured respectively. Prussian blue staining and frozen fluorescence staining were used to identify the number of stem cells in skin wounds. The healing time of skin wounds was compared between the two groups.
Result:
1. the human umbilical cord mesenchymal stem cells were successfully isolated and cultured, and their differentiation and proliferation ability and their dry character were identified. Under the third generation lentivirus packaging system, the true nuclear particles containing the target gene hNIS were constructed and the stable expression of hNIS-EGFP-hUCMSCs cell lines were obtained. The hNIS expression good.HNIS-EGFP-hUCMSCs experimental group cells were identified by Western blot The 125I active 125I flow test and the Exodus experiment proved that human umbilical cord mesenchymal stem cells have good function after hNIS expression. The human umbilical cord mesenchymal stem cell line marked by hNIS EGFP was named SPIO-hNIS-EGFP-hUCMSCs, and Prussian blue staining confirmed that SPIO entered the cell and the cell labeling rate was 98%.SPIO-hNIS-EGFP-hUCMSCs to 98%.SPIO-hNIS-EGFP-hUCMSCs. There is no significant difference in bioactivity, cell dry, proliferative activity and differentiation ability from the original hUCMSCs, which can be used as a therapeutic cell for animal models and prepare for the next SPECT, MRI, and living small animal fluorescence imaging to trace the stem cells.
Under the action of permanent magnet of 2. surface magnetic 200mT, the exposure time of magnetic field was 6 hours a day for three days. There was no difference between the blank control group and the cell activity, proliferation ability and differentiation ability. The exposure time of the magnetic field was over 12 hours and the cell biological activity and differentiation ability were completely inhibited by the.Transwell cell migration experiment to prove the time of magnetic field exposure. After 6 hours, the migration ability of SPIO-hNIS-EGFP-hUCMSCs cells does not increase with the exposure time. The exposure time of the external magnetic field does not exceed 6 hours / day and does not induce excessive expression of the apoptotic gene. The SPIO-hNIS-EGFP-hUCMSCs programmed death may be induced by the exposure time of the magnetic field for more than 12 hours.
3.MRI tracer imaging obtained high quality images. SPIO-hNIS-EGFP-hUCMSCs was successfully affected by magnetic gradient of implanted magnets. The magnetic field guided stem cells in the subcutaneous region to rapidly migrate to the skin defect. The effect of the first 24 hours after the external magnetic field was the most obvious.SPECT imaging labeled stem cells in 0 hours, 2 4 hours, 48 hours, 7 days and MRI imaging traced the trend of transplanted stem cells, but the spatial resolution was lower than that of MRI. The fluorescent imaging of living small animals in 0 hours, 24 hours and 48 hours promoted the consistent trend of SPIO-hNIS-EGFP-hUCMSCs and MRI, but the tracing of living fluorescent imaging cells for seventh days was negative. Prussian blue and frozen fluorescence sections showed that more stem cells entered the skin near the wound in the external magnetic field group.
Conclusion:
The SPIO-hNIS-EGFP-hUCMSCs cell line was successfully established by the lentivirus transfection system for the first time, and there was no significant difference in biological activity, cell dry, proliferation activity and differentiation ability with the original hUCMSCs; the exposure time of external magnetic field had a great influence on the biological characteristics of stem cells, and the time of 6 hours a day was the best time; the application of the external magnetic field for the first time was applied. In the treatment of subcutaneous stem cell transplantation to increase the homing of stem cells, the combination of MRI and hNIS based SPECT imaging can more accurately trace the stem cells, raise sensitivity and reduce false positive.
【學位授予單位】：暨南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R329;R814.42
本文編號：2164250
[Abstract]:Objective:
Using nanotechnology of magnetic targeting and cell biology, the human Na / iodide transporter (hNIS) reporter gene, enhanced green fluorescent protein (EGFP) reporter gene and superparamagnetic iron oxide particle (SPIO) were used to mark human umbilical cord mesenchymal stem cells, and the single photon emission computed tomography (SPECT) of mesenchymal stem cells was established. Nuclear magnetic resonance imaging (MRI), living small animal fluorescence fitting imaging system, using multi-mode imaging mechanism to accurately distinguish the aggregation of local organ stem cells; while the magnetic field makes the stem cells to get the ability to target movement, MRI and SPECT99mTc imaging, the fluorescent pseudo synthetic images of living small animals are traced to explore the establishment of dry cells. The feasibility of cell targeting migration and tracer multimodal molecular imaging technology.
Method:
1. the establishment of a co labeled stem cell line: isolation and identification of human umbilical cord mesenchymal stem cells, selected EGFP reporter gene to indirectly respond to the expression of the target gene hNIS, PCR amplification of the target gene hNIS to pcDNA3.1, then EGFP connected to the downstream of hNIS, and then the entry vector through BP reaction homologous recombination to get the introductory clone, finally will be PCR and sequencing confirmed that the recombinant plasmid pCMV-NIS-EF1-GFP-PGK-puro was successfully constructed, and human umbilical cord mesenchymal stem cells were successfully constructed by enzyme digestion, PCR and sequencing. After obtaining the stable expression of hNIS and EGFP stem cell lines, the hNIS protein function was identified by 125I flow and 125I flow test. A proper concentration of SP was used. IO was incubated with cell lines to obtain hNIS and EGFP, SPIO co labeled stem cells, and to verify their activity and dry matter.
2. in vitro study of different external magnetic field exposure time: the control group was unmarked hUCMSCs, the experimental group was SPIO-hNIS-EGFP-hUCMSCs cells and the magnetic field was placed under the culture plate (200mT) for 3 days. The exposure time was divided into 1 hours group, 6 hour group, 12 hour group, 24 hour group. Trypan blue staining was used to detect cell vitality, M TT assay was used to test the cell proliferation ability, Transwell test was used to measure the migration ability of magnetic field intervention, the differentiation ability of fat induced osteogenesis induced differentiation, and the effect of Western blot on the expression of BCL-1, Bax, Caspase-3, Caspase-9 and so on, and to screen the appropriate exposure time to provide the basis for the study in vivo.
3. the target migration of stem cells and tracing in vivo under the action of 3. external magnetic field: a nude mouse model of full layer skin defect was established. The magnetic resonance imaging external magnetic field MRI, SPECT, and living small animals were traced at 0 hours, 24 hours, 48 hours and 7 days after the cell transplantation. The area reduction rate and the signal to noise ratio of the MRI map in situ stem cell mass were measured respectively. Prussian blue staining and frozen fluorescence staining were used to identify the number of stem cells in skin wounds. The healing time of skin wounds was compared between the two groups.
Result:
1. the human umbilical cord mesenchymal stem cells were successfully isolated and cultured, and their differentiation and proliferation ability and their dry character were identified. Under the third generation lentivirus packaging system, the true nuclear particles containing the target gene hNIS were constructed and the stable expression of hNIS-EGFP-hUCMSCs cell lines were obtained. The hNIS expression good.HNIS-EGFP-hUCMSCs experimental group cells were identified by Western blot The 125I active 125I flow test and the Exodus experiment proved that human umbilical cord mesenchymal stem cells have good function after hNIS expression. The human umbilical cord mesenchymal stem cell line marked by hNIS EGFP was named SPIO-hNIS-EGFP-hUCMSCs, and Prussian blue staining confirmed that SPIO entered the cell and the cell labeling rate was 98%.SPIO-hNIS-EGFP-hUCMSCs to 98%.SPIO-hNIS-EGFP-hUCMSCs. There is no significant difference in bioactivity, cell dry, proliferative activity and differentiation ability from the original hUCMSCs, which can be used as a therapeutic cell for animal models and prepare for the next SPECT, MRI, and living small animal fluorescence imaging to trace the stem cells.
Under the action of permanent magnet of 2. surface magnetic 200mT, the exposure time of magnetic field was 6 hours a day for three days. There was no difference between the blank control group and the cell activity, proliferation ability and differentiation ability. The exposure time of the magnetic field was over 12 hours and the cell biological activity and differentiation ability were completely inhibited by the.Transwell cell migration experiment to prove the time of magnetic field exposure. After 6 hours, the migration ability of SPIO-hNIS-EGFP-hUCMSCs cells does not increase with the exposure time. The exposure time of the external magnetic field does not exceed 6 hours / day and does not induce excessive expression of the apoptotic gene. The SPIO-hNIS-EGFP-hUCMSCs programmed death may be induced by the exposure time of the magnetic field for more than 12 hours.
3.MRI tracer imaging obtained high quality images. SPIO-hNIS-EGFP-hUCMSCs was successfully affected by magnetic gradient of implanted magnets. The magnetic field guided stem cells in the subcutaneous region to rapidly migrate to the skin defect. The effect of the first 24 hours after the external magnetic field was the most obvious.SPECT imaging labeled stem cells in 0 hours, 2 4 hours, 48 hours, 7 days and MRI imaging traced the trend of transplanted stem cells, but the spatial resolution was lower than that of MRI. The fluorescent imaging of living small animals in 0 hours, 24 hours and 48 hours promoted the consistent trend of SPIO-hNIS-EGFP-hUCMSCs and MRI, but the tracing of living fluorescent imaging cells for seventh days was negative. Prussian blue and frozen fluorescence sections showed that more stem cells entered the skin near the wound in the external magnetic field group.
Conclusion:
The SPIO-hNIS-EGFP-hUCMSCs cell line was successfully established by the lentivirus transfection system for the first time, and there was no significant difference in biological activity, cell dry, proliferation activity and differentiation ability with the original hUCMSCs; the exposure time of external magnetic field had a great influence on the biological characteristics of stem cells, and the time of 6 hours a day was the best time; the application of the external magnetic field for the first time was applied. In the treatment of subcutaneous stem cell transplantation to increase the homing of stem cells, the combination of MRI and hNIS based SPECT imaging can more accurately trace the stem cells, raise sensitivity and reduce false positive.
【學位授予單位】：暨南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R329;R814.42
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