无葡聚糖包被的超顺磁性纳米颗粒对大鼠骨髓间充质干细胞的细胞毒性及增殖活性的影响

发布时间：2018-07-05 04:06

本文选题：无葡聚糖包被的 + 超顺磁性纳米颗粒　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景：近年来,随着对干细胞研究认识的深入及分子生物学和细胞生物工程技术的发展,骨髓间充质干细胞(BMSCs)在再生医学及临床中应用愈加广泛。BMSCs分化来源于中胚层,存在于全身结缔组织和器官间质中,在骨髓中含量最高,具有强大的自我更新和增殖能力及多向分化潜能。该细胞因具有易获取、易分离、易培养,且免疫排斥反应低,易在宿主体内长期存活、易于外源基因转染等优点,已被广泛应用于当今医学研究中,尤其是BMSCs移植已成为骨科疾病研究中的热点。超顺磁性氧化铁纳米颗粒(SPIO)是一种新型磁共振细胞内对比剂,因其同时具有高热稳定性、低毒性、超顺磁性和纳米特性,并可包被不同生物大分子,已经越来越广泛地的应用于生物科学的研究中,包括靶向给药、肿瘤磁过热疗法、生物传感器以及特异靶点的浓度示踪等。SPIO是目前常用的干细胞标记物,在医学领域应用广泛,SPIO应用的基本原则是其生物安全性及生物相容性,所以其毒性问题不容忽视,而这些特性是由其尺寸和表征所决定的。铁为人体正常代谢需要的元素,但过量聚积则容易引起毒性。研究显示SPIO标记后的细胞传至第10代仍生长良好,SPIO具有生物可降解性,在活体内被红细胞溶酶体转化为体内铁(Fe2+或Fe3+),最终进入正常血浆铁池,参与血红蛋白或其他代谢过程,对细胞或组织器官无明显毒副作用。此外,SPIO已被证实在用于药物载体及肿瘤靶向治疗中具有良好的生物相容性及安全性。 SPIO的表面活性剂种类繁多,常见的有多聚赖氨酸、硫酸鱼精蛋白、繁枝体等,而葡聚糖作为表面活性剂的优点有：①葡聚糖分子带正电荷,能与带负电荷的Fe304产生静电吸附；②葡聚糖是一种完全由α-D-吡喃葡萄糖单体组成的多糖,其侧链由(1,4)和(1,6)连接的葡聚糖残基构成,每5个葡聚糖残基组成的重复单元有1个分支,位于主链葡聚糖残基的6-0位上,可与Fe产生共价键结合,能保持SPIO磁流体的稳定性；③葡聚糖结构中还包含有大量的“-OH"基团,能与一些生物大分子的氨基形成Schiff键,产生共价键紧密连接,为SPIO作为载体,将短链氨基酸、多肽、小分子蛋白质等靶向转运进入细胞内创造条件。但是传统的葡聚糖包被的SPIO,如AMI225(feridex)、AMI2227(ferumoxtran)、AMI2121(LUMIREM)和SHU555A(resovist)等用于干细胞标记亦存在缺陷：①均需先与鱼精蛋白或多聚赖氨酸(PLL)等转染剂混合,使SPIO表面由负电荷变为正电荷,才能高效率转染标记干细胞,操作不便,甚至影响结果的稳定性；②在体降解时间短,目前文献报道最长的示踪时间为12周,难以满足软骨修复示踪的时间要求。近年来越来越多的研究运用SPIO标记BMSCs,其纳米颗粒大多都有葡聚糖包被,颗粒直径较大。而目前国内外尚无无葡聚糖包被的SPIO的相关实验研究。目的：应用CCK-8法检测细胞增殖活性,通过检测细胞上清液中乳酸脱氢酶活性及细胞内过氧化物歧化酶活性来检测SPIO的细胞毒性,来探究无葡聚糖包被的SPIO对大鼠骨髓间充质干细胞(BMSCs)的细胞毒性、增殖等生物学活性的影响,为后续研究打下基础。方法： 1、BMSCs的分离、培养和鉴定：取2-4周龄,体重90-120g的Wistar大鼠1只,麻醉,双下肢手术部位消毒后,无菌分离出两侧股骨和胫骨。剪去股骨及胫骨两端,暴露骨髓腔,用注射器吸取培养基反复冲洗出股骨和胫骨中的骨髓组织,反复吹打均匀分散后,移入10ml离心管,1000r/min离心10min,细胞沉淀中加入体积分数为10%的胎牛血清和1%青、链霉素的DMEM/F12培养基,按照1×109/L的密度接种在25cm2培养瓶中,放入CO2培养箱中孵育。48h后去除未贴壁的细胞,并更换新鲜培养液,以后每3d换液1次。当细胞达90%以上融合后进行消化传代,扩增培养得到第三代BMSCs。收集5×105个细胞用流式细胞仪检测BMSCs表面标志CD29,CD90以鉴定干细胞。 2、无葡聚糖包被的SPIO标记BMSCs及阳性率检测：将无葡聚糖包被的SPIO用0.22μmm无菌过滤器过滤除菌后加入到新鲜DMEM/F12细胞培养基(含10%胎牛血清)中,振荡混匀60min。加入培养液将SPIO的最终浓度调整为0,25,50,75,100μg/ml。取第3代BMSCs,0.25%胰酶消化收集洗涤细胞2次,吹打成单细胞悬液,以每孔5×104/ml接种两块24孔培养板中,每孔加入培养基1m1,置于培养箱(37℃、体积分数5%CO2)中培养24小时。孵育24h小时后吸弃培养液加入1ml含SPIO培养液,阴性对照组加入不含SPIO的培养液,空白对照组中无BMSCs也不加入SPIO。24小时后行普鲁士蓝染色(吸出培养液,4%多聚甲醛固定30min,用蒸馏水洗3遍,将预先配置好的2%盐酸水溶液和2%亚铁氰化钾水溶液等量混合,加入培养板中染色过夜。用蒸馏水洗3遍,梯度酒精脱水),光镜下观察细胞染色情况并拍照。 3、无葡聚糖包被的SPIO标记的BMSCs的增殖活性检测：连续10天将相同代数的SPIO标记的细胞制为单细胞悬液,取9滴细胞悬液移入小试管内,加入1滴0.4%的台盼蓝染液,混匀,染色3min,随后在光镜下观察并进行细胞计数,死亡细胞被染为蓝色,活细胞不着色,计数200个细胞,并计算活细胞百分率(活细胞百分率=活细胞数／细胞总数×100%)。将5种不同浓度无葡聚糖包被的SPIO标记的BMSCs制成细胞悬液,调整细胞浓度至2×104/ml,外加无细胞的空白对照组,按每孔100μl接种于2块96孔板中,每组每天有3个复孔。培养24小时后第一天测量组每孔加入10μlCCK-8溶液,将培养板放入细胞培养箱中继续培养2h,酶标仪450nm波长读板,获得标本的吸光度值(D值)。连续培养6d,每天的相同时间重复以上操作。根据公式：细胞抑制率=(D实验组-D对照组)/(D实验组-D空白组)x100计算各浓度SPIO对细胞的抑制率,并进行统计学分析。 4、BMSCs细胞内生化指标的测定不同浓度的无葡聚糖包被的SPIO、标记BMSCs培养24小时后,收集上清液,按照乳酸脱氢酶(LDH)试剂盒使用说明进行检测5组细胞上清液中LDH活性；采用超氧化物歧化酶(SOD)测试盒,用黄嘌呤氧化酶法测定细胞内SOD的活性,根据试剂盒的操作指南,制备细胞裂解液测定SOD的活性。 5、统计学分析计量资料用均数±标准差表示,采用SPSS13.0统计软件进行数据分析。多组间定量数据采用单因素方差分析,组间两两比较采用LSD检验,检验水平a=0.05,P0.05为差异有统计学意义。结果：1、大鼠骨髓间充质干细胞的鉴定。我们与中国科学院苏州纳米技术与纳米仿生研究所合作成功研制出生物性能稳定的、尺寸可控的、单分散的SPIO,表面经修饰后,使其可在生理细胞培养液中保持稳定,颗粒直径约12nm。该颗粒有很强的饱和磁矩,在低温下(6K)有磁滞,但在室温下表现超顺磁性。采用全骨髓培养法培养法获得第四代的大鼠BMSCs,细胞形态为长梭形及多角形,呈漩涡状排列。流式细胞检测结果表明该细胞高表达CD29(99.44%)及CD90(96.40%)。标记细胞行普鲁士蓝染色后,可见细胞内SPIO颗粒被染为蓝色,散在分布于细胞核周围。试验中观察到,50μg/ml及以上浓度的SPIO培养基孵育的细胞,SPIO的标记率基本可达100%。25μg/ml组SPIO培养基孵育的细胞仍有少量未被染色,说明其未与SPIO充分结合。 2、CCK-8法测定不同浓度无葡聚糖包被的SPIO标记的BMSCs的增殖活性。在将无葡聚糖包被的SPIO标记的BMSCs用于体内实验之前,需寻找到一个合适的浓度,在这个浓度下,BMSCs既能获得最大的标记率,又不会影响其增殖及分化。为此,我们首先连续10天用台盼蓝染色计算活细胞百分率。结果显示,SPIO标记的BMSCs连续10天的活细胞百分率均在99%以上,说明SPIO对BMSCs的生存无明显影响。然后,我们将25μg/ml、50μg/ml、75μg/ml、100μg/ml等4种不同浓度SPIO标记的BMSCs以及无SPIO标记的阴性对照组细胞制成细胞悬液,分别接种于96孔板中,采用CCK-8法进行细胞增殖检测,酶联免疫检测仪测定各孔的吸光度值,然后根据公式计算各种浓度SPIO对细胞生长的抑制率,将其绘制成柱状图。结果显示：随着SPIO浓度的增高,样本吸光度值逐渐减小,SPIO对细胞的抑制率逐渐增大,25μg/ml组SPIO对细胞的抑制率最小,50μg/ml组SPIO虽然对细胞生长的抑制率较25μg/ml组为高,但二者在统计学上无明显差别(P=0.076)；25μg/ml组对细胞的标记率尚无法达到100%,而50gg/ml组可对细胞100%标记。 3、无葡聚糖包被的SPIO对BMSCs氧化损伤的检测 3.1、LDH活力检测结果。使用SPIO组中大鼠BMSCs的LDH释放率高于阴性对照组(P0.001),各组之间LDH释放率随无葡聚糖包被的SPIO颗粒浓度升高而增加,但25μg/ml和50μg/ml组之间的差异无统计学意义(P=0.110)。 3.2、SOD活性检测结果。随标记的SPIO的浓度的增加,细胞内SOD的活性逐渐降低,细胞内SOD的活性与SPIO存在剂量效应,但25μg/ml和50μg/ml组之间的差异无统计学意义(P=0.126)。结论： 1、随着SPIO浓度的增高,SPIO对细胞的抑制率及毒性逐渐增大,25μg/ml组SPIO对细胞的影响最小,50μg/ml组SPIO虽然对细胞生长的影响较25μg/ml组为高,但二者在统计学上无明显差别(P0.05)。 2、普鲁士蓝染色证实,50μg/ml及以上浓度的无葡聚糖包被的SPIO均可100%标记细胞,但25μg/ml组SPIO尚无法达到完全标记。 3、50μg/ml为SPIO的理想标记浓度,此浓度下BMSCs标记率高,且SPIO的细胞毒性及其对BMSCs的增殖活性影响较小。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R687

【参考文献】