本文選題：無葡聚糖包被的 + 超順磁性納米顆粒　； 參考：《南方醫(yī)科大學》2014年碩士論文

【摘要】：研究背景： 近年來,隨著對干細胞研究認識的深入及分子生物學和細胞生物工程技術(shù)的發(fā)展,骨髓間充質(zhì)干細胞(BMSCs)在再生醫(yī)學及臨床中應(yīng)用愈加廣泛。BMSCs分化來源于中胚層,存在于全身結(jié)締組織和器官間質(zhì)中,在骨髓中含量最高,具有強大的自我更新和增殖能力及多向分化潛能。該細胞因具有易獲取、易分離、易培養(yǎng),且免疫排斥反應(yīng)低,易在宿主體內(nèi)長期存活、易于外源基因轉(zhuǎn)染等優(yōu)點,已被廣泛應(yīng)用于當今醫(yī)學研究中,尤其是BMSCs移植已成為骨科疾病研究中的熱點。 超順磁性氧化鐵納米顆粒(SPIO)是一種新型磁共振細胞內(nèi)對比劑,因其同時具有高熱穩(wěn)定性、低毒性、超順磁性和納米特性,并可包被不同生物大分子,已經(jīng)越來越廣泛地的應(yīng)用于生物科學的研究中,包括靶向給藥、腫瘤磁過熱療法、生物傳感器以及特異靶點的濃度示蹤等。SPIO是目前常用的干細胞標記物,在醫(yī)學領(lǐng)域應(yīng)用廣泛,SPIO應(yīng)用的基本原則是其生物安全性及生物相容性,所以其毒性問題不容忽視,而這些特性是由其尺寸和表征所決定的。鐵為人體正常代謝需要的元素,但過量聚積則容易引起毒性。研究顯示SPIO標記后的細胞傳至第10代仍生長良好,SPIO具有生物可降解性,在活體內(nèi)被紅細胞溶酶體轉(zhuǎn)化為體內(nèi)鐵(Fe2+或Fe3+),最終進入正常血漿鐵池,參與血紅蛋白或其他代謝過程,對細胞或組織器官無明顯毒副作用。此外,SPIO已被證實在用于藥物載體及腫瘤靶向治療中具有良好的生物相容性及安全性。 SPIO的表面活性劑種類繁多,常見的有多聚賴氨酸、硫酸魚精蛋白、繁枝體等,而葡聚糖作為表面活性劑的優(yōu)點有：①葡聚糖分子帶正電荷,能與帶負電荷的Fe304產(chǎn)生靜電吸附；②葡聚糖是一種完全由α-D-吡喃葡萄糖單體組成的多糖,其側(cè)鏈由(1,4)和(1,6)連接的葡聚糖殘基構(gòu)成,每5個葡聚糖殘基組成的重復(fù)單元有1個分支,位于主鏈葡聚糖殘基的6-0位上,可與Fe產(chǎn)生共價鍵結(jié)合,能保持SPIO磁流體的穩(wěn)定性；③葡聚糖結(jié)構(gòu)中還包含有大量的“-OH"基團,能與一些生物大分子的氨基形成Schiff鍵,產(chǎn)生共價鍵緊密連接,為SPIO作為載體,將短鏈氨基酸、多肽、小分子蛋白質(zhì)等靶向轉(zhuǎn)運進入細胞內(nèi)創(chuàng)造條件。但是傳統(tǒng)的葡聚糖包被的SPIO,如AMI225(feridex)、AMI2227(ferumoxtran)、AMI2121(LUMIREM)和SHU555A(resovist)等用于干細胞標記亦存在缺陷：①均需先與魚精蛋白或多聚賴氨酸(PLL)等轉(zhuǎn)染劑混合,使SPIO表面由負電荷變?yōu)檎姾?才能高效率轉(zhuǎn)染標記干細胞,操作不便,甚至影響結(jié)果的穩(wěn)定性；②在體降解時間短,目前文獻報道最長的示蹤時間為12周,難以滿足軟骨修復(fù)示蹤的時間要求。近年來越來越多的研究運用SPIO標記BMSCs,其納米顆粒大多都有葡聚糖包被,顆粒直徑較大。而目前國內(nèi)外尚無無葡聚糖包被的SPIO的相關(guān)實驗研究。 目的： 應(yīng)用CCK-8法檢測細胞增殖活性,通過檢測細胞上清液中乳酸脫氫酶活性及細胞內(nèi)過氧化物歧化酶活性來檢測SPIO的細胞毒性,來探究無葡聚糖包被的SPIO對大鼠骨髓間充質(zhì)干細胞(BMSCs)的細胞毒性、增殖等生物學活性的影響,為后續(xù)研究打下基礎(chǔ)。 方法： 1、BMSCs的分離、培養(yǎng)和鑒定： 取2-4周齡,體重90-120g的Wistar大鼠1只,麻醉,雙下肢手術(shù)部位消毒后,無菌分離出兩側(cè)股骨和脛骨。剪去股骨及脛骨兩端,暴露骨髓腔,用注射器吸取培養(yǎng)基反復(fù)沖洗出股骨和脛骨中的骨髓組織,反復(fù)吹打均勻分散后,移入10ml離心管,1000r/min離心10min,細胞沉淀中加入體積分數(shù)為10%的胎牛血清和1%青、鏈霉素的DMEM/F12培養(yǎng)基,按照1×109/L的密度接種在25cm2培養(yǎng)瓶中,放入CO2培養(yǎng)箱中孵育。48h后去除未貼壁的細胞,并更換新鮮培養(yǎng)液,以后每3d換液1次。當細胞達90%以上融合后進行消化傳代,擴增培養(yǎng)得到第三代BMSCs。收集5×105個細胞用流式細胞儀檢測BMSCs表面標志CD29,CD90以鑒定干細胞。 2、無葡聚糖包被的SPIO標記BMSCs及陽性率檢測： 將無葡聚糖包被的SPIO用0.22μmm無菌過濾器過濾除菌后加入到新鮮DMEM/F12細胞培養(yǎng)基(含10%胎牛血清)中,振蕩混勻60min。加入培養(yǎng)液將SPIO的最終濃度調(diào)整為0,25,50,75,100μg/ml。取第3代BMSCs,0.25%胰酶消化收集洗滌細胞2次,吹打成單細胞懸液,以每孔5×104/ml接種兩塊24孔培養(yǎng)板中,每孔加入培養(yǎng)基1m1,置于培養(yǎng)箱(37℃、體積分數(shù)5%CO2)中培養(yǎng)24小時。孵育24h小時后吸棄培養(yǎng)液加入1ml含SPIO培養(yǎng)液,陰性對照組加入不含SPIO的培養(yǎng)液,空白對照組中無BMSCs也不加入SPIO。24小時后行普魯士藍染色(吸出培養(yǎng)液,4%多聚甲醛固定30min,用蒸餾水洗3遍,將預(yù)先配置好的2%鹽酸水溶液和2%亞鐵氰化鉀水溶液等量混合,加入培養(yǎng)板中染色過夜。用蒸餾水洗3遍,梯度酒精脫水),光鏡下觀察細胞染色情況并拍照。 3、無葡聚糖包被的SPIO標記的BMSCs的增殖活性檢測： 連續(xù)10天將相同代數(shù)的SPIO標記的細胞制為單細胞懸液,取9滴細胞懸液移入小試管內(nèi),加入1滴0.4%的臺盼藍染液,混勻,染色3min,隨后在光鏡下觀察并進行細胞計數(shù),死亡細胞被染為藍色,活細胞不著色,計數(shù)200個細胞,并計算活細胞百分率(活細胞百分率=活細胞數(shù)／細胞總數(shù)×100%)。 將5種不同濃度無葡聚糖包被的SPIO標記的BMSCs制成細胞懸液,調(diào)整細胞濃度至2×104/ml,外加無細胞的空白對照組,按每孔100μl接種于2塊96孔板中,每組每天有3個復(fù)孔。培養(yǎng)24小時后第一天測量組每孔加入10μlCCK-8溶液,將培養(yǎng)板放入細胞培養(yǎng)箱中繼續(xù)培養(yǎng)2h,酶標儀450nm波長讀板,獲得標本的吸光度值(D值)。連續(xù)培養(yǎng)6d,每天的相同時間重復(fù)以上操作。根據(jù)公式：細胞抑制率=(D實驗組-D對照組)/(D實驗組-D空白組)x100計算各濃度SPIO對細胞的抑制率,并進行統(tǒng)計學分析。 4、BMSCs細胞內(nèi)生化指標的測定不同濃度的無葡聚糖包被的SPIO、標記BMSCs培養(yǎng)24小時后,收集上清液,按照乳酸脫氫酶(LDH)試劑盒使用說明進行檢測5組細胞上清液中LDH活性；采用超氧化物歧化酶(SOD)測試盒,用黃嘌呤氧化酶法測定細胞內(nèi)SOD的活性,根據(jù)試劑盒的操作指南,制備細胞裂解液測定SOD的活性。 5、統(tǒng)計學分析計量資料用均數(shù)±標準差表示,采用SPSS13.0統(tǒng)計軟件進行數(shù)據(jù)分析。多組間定量數(shù)據(jù)采用單因素方差分析,組間兩兩比較采用LSD檢驗,檢驗水平a=0.05,P0.05為差異有統(tǒng)計學意義。結(jié)果：1、大鼠骨髓間充質(zhì)干細胞的鑒定。我們與中國科學院蘇州納米技術(shù)與納米仿生研究所合作成功研制出生物性能穩(wěn)定的、尺寸可控的、單分散的SPIO,表面經(jīng)修飾后,使其可在生理細胞培養(yǎng)液中保持穩(wěn)定,顆粒直徑約12nm。該顆粒有很強的飽和磁矩,在低溫下(6K)有磁滯,但在室溫下表現(xiàn)超順磁性。采用全骨髓培養(yǎng)法培養(yǎng)法獲得第四代的大鼠BMSCs,細胞形態(tài)為長梭形及多角形,呈漩渦狀排列。流式細胞檢測結(jié)果表明該細胞高表達CD29(99.44%)及CD90(96.40%)。標記細胞行普魯士藍染色后,可見細胞內(nèi)SPIO顆粒被染為藍色,散在分布于細胞核周圍。試驗中觀察到,50μg/ml及以上濃度的SPIO培養(yǎng)基孵育的細胞,SPIO的標記率基本可達100%。25μg/ml組SPIO培養(yǎng)基孵育的細胞仍有少量未被染色,說明其未與SPIO充分結(jié)合。 2、CCK-8法測定不同濃度無葡聚糖包被的SPIO標記的BMSCs的增殖活性。在將無葡聚糖包被的SPIO標記的BMSCs用于體內(nèi)實驗之前,需尋找到一個合適的濃度,在這個濃度下,BMSCs既能獲得最大的標記率,又不會影響其增殖及分化。為此,我們首先連續(xù)10天用臺盼藍染色計算活細胞百分率。結(jié)果顯示,SPIO標記的BMSCs連續(xù)10天的活細胞百分率均在99%以上,說明SPIO對BMSCs的生存無明顯影響。然后,我們將25μg/ml、50μg/ml、75μg/ml、100μg/ml等4種不同濃度SPIO標記的BMSCs以及無SPIO標記的陰性對照組細胞制成細胞懸液,分別接種于96孔板中,采用CCK-8法進行細胞增殖檢測,酶聯(lián)免疫檢測儀測定各孔的吸光度值,然后根據(jù)公式計算各種濃度SPIO對細胞生長的抑制率,將其繪制成柱狀圖。結(jié)果顯示：隨著SPIO濃度的增高,樣本吸光度值逐漸減小,SPIO對細胞的抑制率逐漸增大,25μg/ml組SPIO對細胞的抑制率最小,50μg/ml組SPIO雖然對細胞生長的抑制率較25μg/ml組為高,但二者在統(tǒng)計學上無明顯差別(P=0.076)；25μg/ml組對細胞的標記率尚無法達到100%,而50gg/ml組可對細胞100%標記。 3、無葡聚糖包被的SPIO對BMSCs氧化損傷的檢測 3.1、LDH活力檢測結(jié)果。使用SPIO組中大鼠BMSCs的LDH釋放率高于陰性對照組(P0.001),各組之間LDH釋放率隨無葡聚糖包被的SPIO顆粒濃度升高而增加,但25μg/ml和50μg/ml組之間的差異無統(tǒng)計學意義(P=0.110)。 3.2、SOD活性檢測結(jié)果。隨標記的SPIO的濃度的增加,細胞內(nèi)SOD的活性逐漸降低,細胞內(nèi)SOD的活性與SPIO存在劑量效應(yīng),但25μg/ml和50μg/ml組之間的差異無統(tǒng)計學意義(P=0.126)。 結(jié)論： 1、隨著SPIO濃度的增高,SPIO對細胞的抑制率及毒性逐漸增大,25μg/ml組SPIO對細胞的影響最小,50μg/ml組SPIO雖然對細胞生長的影響較25μg/ml組為高,但二者在統(tǒng)計學上無明顯差別(P0.05)。 2、普魯士藍染色證實,50μg/ml及以上濃度的無葡聚糖包被的SPIO均可100%標記細胞,但25μg/ml組SPIO尚無法達到完全標記。 3、50μg/ml為SPIO的理想標記濃度,此濃度下BMSCs標記率高,且SPIO的細胞毒性及其對BMSCs的增殖活性影響較小。
[Abstract]:Research background:
In recent years, with the deep understanding of stem cell research and the development of molecular biology and cell bioengineering technology, bone marrow mesenchymal stem cells (BMSCs) have been widely used in regenerative medicine and clinical application of.BMSCs differentiation from mesoderm, which exist in the connective tissue and interstitial tissue of the body, with the highest content in the bone marrow and strong in the bone marrow. It has been widely used in modern medical research, especially the BMSCs transplantation has become a hot spot in the research of disease in Department of orthopedics.
Superparamagnetic iron oxide nanoparticles (SPIO) is a new type of magnetic resonance intracellular contrast agent. Because of its high thermal stability, low toxicity, superparamagnetic and nano properties, and can be coated with different biological macromolecules, it has become more and more widely used in the research of biological science, including targeted drug delivery, tumor Magnetic superheat therapy, biological transmission. .SPIO is a commonly used marker of stem cells and is widely used in the field of medicine. The basic principle of SPIO application is its biological safety and biocompatibility, so its toxicity can not be ignored, and these characteristics are determined by its size and indication. Iron is the element of normal metabolism of human body. In the tenth generation, the SPIO labeled cells still grow well, and the SPIO has biodegradability. In vivo, the erythrocyte lysosomes are converted into the body iron (Fe2+ or Fe3+), eventually entering the normal plasma iron pool, participating in the hemoglobin or other metabolic processes, and there are no cells or tissue organs. In addition, SPIO has been proved to have good biocompatibility and safety in drug delivery and tumor targeting therapy.
There are a wide variety of surfactants in SPIO, such as polylysine, protamine sulfate and brandri, and dextran as a surface active agent. (1) the dextran molecule has positive charge and can produce electrostatic adsorption with Fe304 with negative charge; and dextran is a kind of polysaccharide composed of alpha -D- glucosglucose monomers. The chain is composed of the glucan residue linked by (1,4) and (1,6). The repeating unit of every 5 glucan residues has 1 branches, which is located in 6-0 bits of the main chain dextran residue. It can be combined with the covalent bond of the Fe to maintain the stability of the SPIO magnetic fluid; and the structure of glucan contains a large number of "-OH" groups and can be used with some biological macromolecules. The amino group forms Schiff bonds and produces covalent bonds tightly connected to create conditions for transporting short chain amino acids, peptides and small molecular proteins into cells to create conditions for SPIO as a carrier, but the traditional SPIO, such as AMI225 (Feridex), AMI2227 (ferumoxtran), AMI2121 (LUMIREM) and SHU555A (resovist), is used for stem cell labeling. There are also defects: first, it is necessary to mix the transfection agent such as protamine or polylysine (PLL) to make the SPIO surface from negative charge into positive charge, so that the transfection of labeled stem cells can be efficiently transfected, it is inconvenient to operate, and even affects the stability of the result; 2. The time of degradation in body is short and the longest tracing time in the previous literature is 12 weeks, it is difficult to satisfy the soft. The time requirements for bone repair tracer. In recent years, more and more studies have been made on the use of SPIO labeled BMSCs. Most of the nanoparticles have glucan envelope and the diameter of the particles is larger. At present, there is no related experimental research on SPIO without deglucan inclusion at home and abroad.
Objective:
The cell proliferation activity was detected by CCK-8, and the cytotoxicity of SPIO was detected by detecting the activity of lactate dehydrogenase in the supernatant and the activity of the peroxidase dismutase in cell. The effect of SPIO on the cytotoxicity and proliferation of rat bone marrow mesenchymal stem cells (BMSCs) was investigated. Lay the foundation.
Method:
1, the isolation, culture and identification of BMSCs:
After 2-4 weeks of age, 1 rats of body weight 90-120g were taken. After anaesthesia, the two lower extremities were sterilized. The two sides of the femur and tibia were isolated. The two ends of the femur and tibia were removed and the bone marrow cavity was exposed. The bone marrow tissues in the femur and tibia were rinsed out repeatedly with a syringe. After repeated blow, the 10ml centrifuge tube was transferred to 1000r/min. 1000r/min In the centrifuge 10min, the fetal bovine serum and 1% green and streptomycin medium were added to the cell precipitation, and the DMEM/F12 medium of streptomycin was inoculated in the 25cm2 culture bottle according to the density of 1 x 109/L. The cells were incubated in the CO2 incubator and incubated for.48h to remove the non adherent cells, and the fresh medium was replaced for 1 times per 3D. When the cells reached more than 90% fusion, the cells were fused more than 90%. After digestion and passage, third generations of BMSCs. were collected and cultured, and 5 * 105 cells were collected. BMSCs surface markers CD29 and CD90 were detected by flow cytometry to identify stem cells.
2, no dextran coated SPIO marker BMSCs and positive rate were detected:
After removing bacteria free SPIO with 0.22 u mm aseptic filter and adding fresh DMEM/F12 cell culture medium (containing 10% fetal bovine serum), the final concentration of SPIO was adjusted to 0,25,50,75100 mu g/ml. to take third generation BMSCs, 0.25% trypsin elimination and 2 washing cells, and blow into single cell suspension. Inoculated two block 24 hole culture plates with 5 x 104/ml per pore, each hole was added to culture medium 1m1 and cultured in culture box (37 C, volume fraction 5%CO2) for 24 hours. After incubating for 24h hours, the culture solution was added to 1ml containing SPIO culture solution, negative control group was added without SPIO, and no BMSCs in blank control group was not added to SPIO.24 hours after prusu. Blue staining (absorption of culture liquid, 4% polyformaldehyde fixed 30min, 3 times washed with distilled water, the pre configured 2% hydrochloric acid solution and 2% potassium ferrocyanide solution in the same amount, added to the culture plate for the night. 3 times with distilled water, gradient alcohol dehydration), light microscopy observation of cell dyeing conditions and photographed.
3, the proliferation activity of SPIO labeled BMSCs without dextran coated was detected.
The SPIO labeled cells of the same algebra were made into single cell suspension for 10 days, and 9 drops of cell suspension were transferred into the small test tube, 1 drops of 0.4% drops of trypan blue dye were added, mixed and stained for 3min. Then the cells were observed under the light microscope and counted. The dead cells were stained blue, the living cells were not coloured, 200 cells were counted, and the percentage of living cells was calculated. The percentage of living cells = the number of living cells / cell count * 100%.
5 different concentrations of SPIO labeled BMSCs without glucan envelope were made into cell suspension, and the cell concentration was adjusted to 2 x 104/ml, and the cells were inoculated in the blank control group with no cell. 2 96 hole plates were inoculated at 100 mu per pore. Each group had 3 compound pores a day. After 24 hours, the first day of the measurement group added 10 mu lCCK-8 solution to the cell culture. In the incubator, 2h was continued and the 450nm wavelength of the enzyme labeled instrument was read. The absorbance value (D value) of the specimen was obtained. 6D was continuously cultured and repeated operations were repeated at the same time every day. According to the formula, the inhibitory rate of the cells was (-D control group of D experimental group) / (D experimental group -D blank group) and X100 calculated the inhibition rate of each SPIO to the cell, and the statistical analysis was carried out.
4, the intracellular biochemical indexes of BMSCs cells were measured with different concentrations of SPIO without dextran envelope, and 24 hours after labeling BMSCs, the supernatant was collected and the LDH activity in the 5 groups of cell supernatants was detected in accordance with the use of the lactate dehydrogenase (LDH) kit. Using the superoxide dismutase (SOD) test box, the intracellular SO was determined by xanthine oxidase method. The activity of D was determined. According to the guide of the kit, cell lysate was prepared to determine the activity of SOD.
5, the statistical analysis of measurement data was expressed with mean standard deviation, and SPSS13.0 statistical software was used to analyze the data. The quantitative data of multiple groups were analyzed by single factor variance, 22 of the groups were compared with LSD test, and the level of a=0.05 was tested. The difference was statistically significant. Results: 1, the identification of bone marrow mesenchymal stem cells in rats. The National Academy of Sciences, Suzhou nanotechnology and nano biomimetic Research Institute, has successfully developed a stable, size controlled, monodisperse SPIO. After the surface modification, the surface can remain stable in the physiological cell culture, the particle size is about 12nm. and the particle has a strong saturation magnetic moment and the magnetic hysteresis at low temperature (6K), but at room temperature. Superparamagnetic. The BMSCs of fourth generations of rat was obtained by full bone marrow culture. The cell morphology was long spindle shaped and polygonal and swirled. The results of flow cytometry showed that the cells expressed CD29 (99.44%) and CD90 (96.40%). After the staining of Prussian blue, the SPIO particles in the cells were stained blue and scattered in the distribution. It was observed around the nucleus. In the experiment, the cells incubated in the SPIO medium with 50 g/ml and above were incubated in the SPIO medium. The labeling rate of SPIO was basically up to the cells incubated in the SPIO medium of group g/ml, and the cells still had little staining, indicating that the cells were not fully combined with SPIO.
2, CCK-8 method was used to determine the proliferation activity of the SPIO labeled BMSCs with different concentrations of dextran coated SPIO. A suitable concentration should be found before the BMSCs labeled by SPIO without dextran inclusion in vivo. At this concentration, the maximum labeling rate can be obtained, and it will not affect its proliferation and differentiation. The percentage of living cells was calculated by trypan blue staining for 10 days. The results showed that the percentage of living cells of the SPIO labeled BMSCs for 10 days was more than 99%, indicating that SPIO had no obvious effect on the survival of BMSCs. Then, we had 4 BMSCs and negative control groups with different concentrations of SPIO marked with different concentrations, including 25 mu g/ml, 50 mu g/ml, 75 mu g/ml, 100 u g/ml and so on. Cell suspension was made into cell suspension and inoculated in 96 orifice plates respectively. Cell proliferation was detected by CCK-8 method. The absorbance value of each hole was measured by enzyme immunoassay instrument. Then the inhibitory rate of various concentrations of SPIO on cell growth was calculated according to the formula. It was drawn into a columnar chart. The results showed that the sample absorbency was gradually increased with the increase of SPIO concentration. The inhibition rate of SPIO to cells increased gradually. The inhibition rate of SPIO to cells in the group of 25 mu g/ml was the smallest. The inhibition rate of SPIO in the group of 50 mu g/ml was higher than that of the 25 micron g/ml group, but there was no significant difference between the two groups (P=0.076), and the labeling rate of 25 mu g/ml group was still unable to reach 100%, and the 50gg/ml group could label the cells in 100%.
3, the detection of BMSCs oxidative damage by dextran coated SPIO.
3.1, LDH activity detection results. The LDH release rate of BMSCs in the SPIO group was higher than that of the negative control group (P0.001). The LDH release rate increased with the increase of SPIO particle concentration without dextran inclusion, but there was no significant difference between the 25 and 50 micron groups (P= 0.110).
3.2, SOD activity detection results. With the increase of the concentration of labeled SPIO, the activity of SOD in the cells gradually decreased, and the activity of SOD in the cells had a dose effect with SPIO, but there was no significant difference between the 25 and 50 g/ml groups (P=0.126).
Conclusion:
1, with the increase of SPIO concentration, the inhibition rate and toxicity of SPIO increased gradually. The effect of SPIO on cells in the group of 25 mu g/ml was the smallest. The effect of SPIO on cell growth in 50 mu g/ml group was higher than that of the 25 g/ml group, but there was no significant difference between the two groups (P0.05).
2, Prussian blue staining showed that SPIO without dextran coated at 50 g/ml and 100% could be labeled with 100% markers, but SPIO in 25 uh group could not be completely labeled.
3,50 g/ml was the ideal marker concentration of SPIO. The BMSCs labeling rate was high under this concentration, and the cytotoxicity of SPIO had little effect on BMSCs proliferation activity.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R687

【參考文獻】

相關(guān)期刊論文 前3條

1 唐大宗;夏黎明;;對比劑的發(fā)展史[J];放射學實踐;2007年06期

2 梁文英;莫潤陽;鄭敏;;SPIO磁性標記細胞技術(shù)研究進展[J];青海師范大學學報(自然科學版);2010年01期

3 陳長青;陳晨;鄧永文;方加勝;王小宜;;大鼠脂肪干細胞磁性標記及MRI成像研究[J];中華神經(jīng)外科疾病研究雜志;2008年01期

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