化学功能团对骨肉瘤细胞生物学行为的影响
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摘要
研究背景
细胞内外环境等多种因素综合影响细胞的粘附、迁移、增殖、凋亡和坏死等生物学行为,因此细胞在支架材料上面的表现形式肯定会受到材料物理性质、表面化学性质、表面形貌和化学成分等多种因素的影响。近来在材料的化学改性方面的研究日益增多,材料的影响因素较多,得出的结论也很多样化,而运用自组装膜技术进行化学功能团修饰材料后的基础研究,可保证单一表面化学特性对细胞生物学行为的影响,避免多种综合因素的交叉影响,得出的结论会更可靠。
自组装膜技术发展较早,技术成熟,国内外很多行业都有应用。在国外,将这种技术运用到医学方面的基础研究开展早且较多,而在国内则相对较少且迟。目前已经开展了对破骨细胞、人成纤维细胞、间充质干细胞、神经干细胞等正常组织细胞的基础研究,近来国内部分学者开始重视这些化学功能团对肿瘤细胞的作用研究,目前已经发现部分化学功能团有抑制乳腺癌细胞、肝癌细胞粘附、增殖的作用。通过这些研究还发现,不同的化学功能团对细胞的不同生物学特性的影响不一样,且对不同细胞类型的影响也不一样,有的功能团具有明显的抑制肿瘤细胞生长的作用,反之也有化学功能团促进肿瘤细胞生长。
骨肉瘤是一种好发于青少年的恶性肿瘤,治疗棘手,预后差,尚没有非常有效的治愈办法。鉴于此,在国内外尚没有关于化学功能团对骨肉瘤细胞生物学功能影响方面的报道前提下,我们开始着手进行化学功能团对骨肉瘤细胞生物学功能方面影响的研究。
本课题采用传统的硅-金作为基质,选择甲基(-CH3)、氨基(-NH2)、羧基(-COOH)和羟基(-OH)四种化学功能团进行自组装膜研究。分别研究这四种化学功能团对骨肉瘤细胞的粘附、增殖及凋亡等三种重要的生物学行为的影响,明确哪些化学功能团对骨肉瘤细胞的哪些行为会产生抑制,对哪些行为会起促进作用,也就是说是否具有抑制肿瘤作用。
目的
本课题是一种运用自组装膜技术,观察化学功能团对骨肉瘤细胞生物学行为影响的探索性研究,研究的目的主要从以下几个方面进行明确。
明确白组装膜表面不同化学功能团的接触角等物理特性不同。
明确不同化学功能团对骨肉瘤细胞粘附的影响
明确不同化学功能团对骨肉瘤细胞增殖的影响
明确不同化学功能团对骨肉瘤细胞凋亡的影响
研究方法
本课题是运用自组装膜技术,观察化学功能团对骨肉瘤细胞生物学行为影响的探索性研究,因此从镀金硅片的制备及表面化学基团修饰、骨肉瘤细胞培养、细胞在化学功能团表面的粘附、增殖、凋亡等方面进行观察研究,具体方法如下：
1.镀金硅片的制备及其表面化学基团修饰。
采用自组装膜技术在镀金硅片基板上分别接枝甲基、氨基、羧基、羟基等四种不同化学官能团,然后对材料进行了接触角方面的测量。由于合作单位良好的接枝技术的保证,本实验没有重复对材料表面进行X射线光电子能谱(XPS,X-ray photoelectron spectroscopy)和原子力显微镜(AFM)技术表征。
2.骨肉瘤细胞的培养。
将冻存的U2-OS型骨肉瘤细胞株,放入37℃水浴箱进行复苏,移入25cm培养瓶内,放入37℃含5%C02恒温培养箱内过夜,更换10%胎牛血清RPMI1640培养基继续培养,每日观察,随时换液培养,待生长至细胞融合70%-80%时用0.25%胰酶消化液消化后传代培养,取对数生长期细胞做后续实验。
3.骨肉瘤细胞在材料表面的粘附观察。
每孔一枚接枝后金片,每组3孔,每孔加浓度为1×104/m1细胞悬液250μl,入恒温箱培养,分别选择3小时,6小时和9小时进行光镜观察,选择粘附6小时进行细胞形态学的扫描电镜观察。
4.骨肉瘤细胞在材料表面的增殖情况观察。
每孔一枚接枝后金片,每组3孔,每孔加浓度为1x104/ml细胞悬液2501μl,入恒温箱培养,采用MTT法计算2天、4天和6天时的增殖情况,了解不同化学功能团的细胞毒性情况；并结合活／死细胞荧光染色标记和测定培养24小时后细胞外液中的乳酸脱氢酶含量观察骨肉瘤细胞24小时后的细胞活力情况。
5.骨肉瘤细胞在材料表面的凋亡情况观察。
每孔一枚接枝后金片,每组3孔,每孔加浓度为5x104/ml细胞液0.5ml,入恒温箱培养,采用流式细胞仪计算24小时的细胞凋亡情况。
结果
1.自组装膜
经过镀金、切割处理后,去除表面划痕严重及标准不符合的金片,得到标准统一无划痕的镀金硅片,这些金片的大小为1.2cm×0.8cm；再将这些镀金硅片经分别含甲基、氨基、羧基和羟基四种不同的硫醇溶液浸泡后,成功接种四种不同化学功能团,得到表面结构均匀的单分子自组装膜。为了进一步对这些接种有不同化学功能团的自组装单分子膜表面结构进行表征,我们对其进行有关接触角的测量。所测甲基、氨基、羧基和羟基接触角分别为105。±9.1。、59.7°±3.80、18.3°±3.90和9.5。±1.20.。统计学分析进一步证实不同化学基团修饰表面的接触角差异具有显著性(Welch值=1488.41,P=0.000)。以上结果也表明甲基具有较强的疏水性,羟基具有最强的亲水性,羧基中等程度亲水性,氨基的亲水性较小。
2.骨肉瘤细胞的培养
在细胞传代培养的实验初期,共有3次6瓶细胞受到污染,培养基颜色明显浑浊,原来贴壁的骨肉瘤细胞成片脱落坏死。经及时查找总结,结果均是继发细菌污染,具体是分装胎牛血清时污染血清一次,PBS污染一次,传代操作过程污染细胞一次。污染后广泛查阅文献,分析原因,向实验室老师请教相关细胞培养技术及关键步骤并及时整改,结果在整个后续实验过程中均未再次出现细胞污染问题。经过及时换液、传代后的细胞均贴壁良好,细胞分裂增殖力旺盛,有利于后续试验的开展。
3.骨肉瘤细胞粘附情况
本实验中,我们利用光学倒置显微镜和扫描电镜观察U-20S在四种化学功能团修饰金片表面粘附情况。相差显微镜下,培养3小时后,各组粘附于金片表面的细胞基本都成小圆形,少许呈小椭圆形,甲基表面粘附的细胞不紧密,呈要脱落状。6小时后细胞形态开始多样化,在氨基、羧基、羟基及对照组金片表面均可见细胞体积增大,可见大椭圆形、梭形及多边形细胞,而在甲基表面细胞仍呈小圆形状。而9小时后,光镜下各组细胞形态变化差异更加明显,有更多梭形、不规则形细胞开始出现,而甲基表面的细胞仍多数呈圆形或椭圆形,梭形、不规则形细胞较少。
镜下见甲基表面的细胞数增加最不明显,氨基和羧基表面的细胞数明显增加,而羟基和对照组细胞数增长较少,进一步统计学结果不拒绝球形假设(W=0.813,P=0.394),且检测时间点与组间有交互作用(F=89.315,P=0.000),统计学上不同时间点各组间细胞数具有显著性差异(F=122.008,P=0.000)。3小时后粘附的细胞数以氨基表面稍多,甲基、羟基和对照组表面较少,统计学上氨基组与甲基、羟基和对照组相比有显著性差异(P<0.05),与羧基组差异无显著性(P=0.786),甲基、羟基和对照组之间差异无显著性(P>0.05)。6小时后甲基表面细胞数增加不明显,其他各组表面细胞数均有不同程度增加,氨基组增加最明显,与其他各组相比统计学差异具有显著性(P<0.05),而甲基与对照组表面细胞数统计学差异无显著性(P=0.094)。9小时后氨基、羧基和羟基组细胞数均明显增多,其中氨基组增加最明显,甲基组增加不明显,统计学上各组之间细胞数差异均具有显著性(P<0.05),各组金片表面粘附细胞数均数总体趋势为-CH3<<-OH<-COOH≈-NH2。
扫描电镜下更进一步观察各组功能团表面粘附细胞的形态学变化,U-20S细胞与各组金片混合培养6小时后,细胞在羟基和羧基组金片表面多呈现出椭圆形和多边形,细胞体积较大,胞浆丰富,可见较多伪足伸出,部分较大伪足相互融合。而在氨基功能团表面的细胞多数为长梭形,甲基表面的细胞仍呈小圆形,细胞体积较小。
4.骨肉瘤细胞增殖情况
4.1MTT结果
在细胞接种浓度相同的条件下,随着培养时间的推移,所有化学官能团表面的骨肉瘤细胞都有不同程度的增殖情况出现,不过甲基表面骨肉瘤细胞增殖的OD值较低,羟基组OD值较高,而在2d和4d时对照组和羧基组的增殖情况相似(P值分别为0.203,0.700)。不同时间点甲基表面细胞增殖情况同其他4组相比均有显著性差异(P=0.000),进一步多重比较显示羟基表面细胞增殖在2d和4d时与其他各组均有统计学差异(P<0.05),尤其是在培养时间达到6天后其增殖均数更加明显,但与氨基、羧基组相比无统计学差异(P值分别为0.057,0.948)；氨基和羧基表面的骨肉瘤细胞均明显增殖,但相互比较结果在2天和6天时无统计学差异(2d和6d时的P值分别为0.752和0.051),培养4天时差异具有显著性(P=0.006)。以上结果似乎显示氨基、羧基和羟基均有促进骨肉瘤细胞增殖的可能。反过来说,上述结果提示甲基似乎有抑制骨肉瘤细胞增殖的可能。整体均数上看,不同化学官能团表面骨肉瘤细胞的增殖趋势为：羧基>羟基≥氨基>>甲基。该趋势说明甲基官能团抑制了骨肉瘤细胞的增殖。
4.2不同化学功能团表面粘附细胞的活力情况
本实验中,我们选择培养24小时作为检测细胞活力试验的时间点,用活／死细胞检测盒进行检测,荧光显微镜下观察。其中活细胞标记为绿色,死细胞标记为红色。结果可以看出甲基表面的细胞体积都很小,荧光染色显示为死细胞。相反的是,在氨基、羧基和羟基表面,大量活细胞存在,并呈现出较好的生长状态。氨基表面细胞多数呈长梭形,羧基和羟基表面的骨肉瘤细胞多为椭圆形或多边形,生长状况与扫描电镜下所见基本相符。
整体上,不同化学功能团表面粘附细胞的生存率趋势为：氨基>羟基≥羧基>>甲基。以上结果也充分证明氨基功能团对骨肉瘤细胞的毒性小,而甲基显示有较高的骨肉瘤细胞毒性,是抑制骨肉瘤细胞增殖的有力证据。
4.3细胞内乳酸脱氢酶释放情况
我们从细胞增殖、细胞活力检测结果分析,认为甲基致细胞死亡的原因可能系引起骨肉瘤细胞膜的损伤,进而致细胞内物质的流出。因此本组实验通过测定细胞质内乳酸脱氢酶的浓度情况来判断甲基功能团致细胞死亡的假设。基于细胞活力试验结果,我们选择培养24小时作为检测的时间点,实验结果显示甲基功能团可以致大量乳酸脱氢酶释放,而氨基功能团组的乳酸脱氢酶活性最低,也低于对照组。统计分析结果方差齐性(F=1.716,P=0.223),各组间比较具有显著性差异(F=111.549,P=0.000),进一步相互比较得出甲基与各组均有显著性差异(P=0.000),羟基与各组也有统计学差异(P<0.05),氨基、羧基和对照组之间无统计学差异(P>0.05)。从均数上看,乳酸脱氢酶活性趋势：甲基>羟基≥羧基>氨基。以上结果与MTT法一致,也充分显示细胞内乳酸脱氢酶的释放程度与化学功能团的细胞毒性一致。
5.骨肉瘤细胞凋亡情况
本试验中将Annexin V与7-AAD一起使用,用来检测不同时期的凋亡细胞,结果发现甲基功能团表面骨肉瘤细胞的凋亡率为7.6%,坏死率为12.5%,而氨基功能团表面的骨肉瘤细胞凋亡率仅约4.3%,但坏死率却达到12.6%,羟基组的凋亡率最低,仅2.1%,坏死率为12.3%,羧基组的凋亡率约为4.8%,坏死率为11.9%。统计学分析方差齐性(凋亡组F=1.075,P=0.413,坏死组F=0.162,P=0.919),各组间凋亡率统计学差异有显著性(F=396.20,P=0.000),进一步分析各组凋亡率之间均有显著性差异(p=0.000)；各组间坏死率比较也具有差异性(F=7.29,P=0.01)。进一步分析显示甲基组与羧基组差异具有显著性(p=0.007),氨基组和羧基、羟基组比较具有统计学差异(P<0.05)。各组化学功能团诱导骨肉瘤细胞凋亡的总体趋势为甲基>羧基>氨基≥羟基。以上结果显示各组功能团均有一定程度诱导骨肉瘤细胞发生凋亡的作用,但总体作用不显著。综合分析得出：氨基具有较甲基更佳的生物相容性,而甲基可以诱导骨肉瘤细胞发生早期凋亡及坏死。也就是说,甲基具有更强的抗骨肉瘤细胞活性。
结论
1.自组装膜技术成熟,接枝后可以得到表面均匀结构单一的立体结构,接触角测量结果说明甲基具有明显疏水性,羟基的亲水性最高,而羧基和氨基的亲水性稍弱。亲水性大小顺序为：羟基>羧基>氨基>甲基
2.骨肉瘤细胞的传代培养属于贴壁细胞培养,技术成熟,但在整个培养过程中仍有可能出现细菌、真菌、支原体等污染情况的发生,致实验失败。需要做到早期发现,认真查找污染源并持续改进,方能保证后续实验的有效开展。
3.研究证明了生物材料表面化学特性对骨肉瘤细胞的生长会产生影响,不同的化学功能团会有不同的作用。氨基和羧基功能团虽然能够促进骨肉瘤细胞的凋亡,但也有较强的促粘附、增殖作用；羟基功能团不仅诱导骨肉瘤细胞的凋亡率低,而且还促进其粘附、增殖；而甲基功能团具有一致的抑制骨肉瘤细胞粘附、增殖和促进骨肉瘤细胞凋亡坏死的发生,也就是说具有明显的抑制骨肉瘤细胞生长的能力,具有较强的抗癌作用。
Background
A variety of factors including the internal and external environments of cell,may have effect on the biological behavior of cell adhesion, migration, proliferation, apoptosis and necrosis.Therefore, the form of cell on the scaffold would certainly be influenced by many factors like the physical properties of materials, surface chemistry, surface morphology and chemical composition, etc. Recently, more and more researches about the chemical modification of materials increased, and the conclusion is various because of the interference of many material features.The basic research of chemical functional groups,using self-assembled monolayers(SAMs) technology,can ensure the impact of the material surface chemistry on cells only by the single chemical character of the materials, avoiding a variety of intersect factors influence, and the conclusion probably be more reliable.
Self-assembled monolayers technology developed earlier, and is a mature technology,have been widely used in many industries all over the world. The SAMs technology has been applied to much more medical basic research earlier in other countries,but which is relatively less and later in our country.Up to now,SAMs has been used to the basic research of some normal tissue cells, like the osteoclasts, human fibroblasts, messenchymal stem cells and neural stem cells. Recently, some domestic scholars began to pay much more attention to a few tumor cells.According to their report, some chemical functional groups have been found to have effect of inhibiting the adhesion and proliferation of breast cancer and hepatoma cells. These studies also found that different chemical functional groups have not the identical effects on the different biological properties of the same cell, and also on the different cell types. That is to say, some chemical functional groups can inhibit the growth of tumor cells, and some other chemical functional groups probably promote tumor cell growth on the contrary.
As a kind of malignant bone tumor, osteosarcoma often occurs in young people, and has unsatisfactory treatment, and has the poor prognosis. And so far, there is no very effective way to cure this disease. Due to the fact that there is no report to explain the relationship between the chemical functional groups and the biological behaviors of the osteosarcoma cells, we are working on the research.
In our study, the traditional silicon-gold was chosen as the SAMs substrate, and methyl(-CH3), amino(-NH2), carboxyl(-COOH) and hydroxyl(-OH) functional groups were selected as the influence factors.The main purpose is to find the exact effect of these chemical functional groups on these important biological behaviors of osteosarcoma cell,such as the adhesion, proliferation and apoptosis.That is to say, Learn to know what kind of chemical functional groups can give rise to what kind of behavior change of osteosarcoma cells,and learn to know whether the chemical functional groups can inhibit the tumor growth or not.
Objective
This topic is an exploratory study of observing the effect of chemical functional groups on the biological behaviors of osteosarcoma cells with the self-assembled monolayers technology.The main purpose will be expained explicitly from the following aspects:
To clear the different physical characteristics of the self-assembled monolayers surface by measuring the contact angle of materials.
To clear the impact of different chemical functional groups on osteosarcoma cell adhesion.
To clear the impact of different chemical functional groups on osteosarcoma cell proliferation.
To clear the impact of different chemical functional groups on osteosarcoma cell apoptosis.
Methods
This topic is an exploratory study of observing the effect of chemical functional groups on the biological behavior of osteosarcoma cells with the self-assembled monolayers technology.Therefore, the preparation of silicon-gold substrate, and its modification of chemical functional groups, and osteosarcoma cell culture, and the observation of the cells adhesion,proliferation and apoptosis on the surface of SAMs will be included in our experiment.Specific methods are shown as follows:
1. Preparation of gold-silicon substrate and self-assembled with different chemical group.
Using self-assembled monolayers technology, the methyl, amino, carboxyl and hydroxyl functional groups were self-assembled on the Au layers respectively. We characterized these surfaces to determine the equivalent surface densities of chemical functional groups by measuring their contact angles. Due to good self-assembled monolayers technique of co-operation unit,the further examination for the morphology of these groups surface by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) was missed in our experiment.
2. Osteosarcoma cell culture.
Firstly, the frozen U2-OS osteosarcoma cell lines were put into37℃water bath for resuscitation, and then shifted into25cm culture bottles. Finally the culture bottles were placed at37℃in5%CO2and95%air.24hours later, the cell culture and daily observation go on after replacing10%FBS RPMI1640medium.Culture medium was changed at any time.After cells growth to be70%to80%confluence, followed with0.25%trypsin digestion, and then passaged culture.The logarithmic growth phase cells are the best choice to do follow-up experiments.
3.Adhesion of osteosarcoma cells on the surface of the SAMs.
Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. U2-OS cells were seeded on these substrates with a concentration1×104cells/ml at37℃with5%CO2.After3h,6h and9h culture, cell adhesion analyses were done under inverted microscopy. And further adhesion analysis of the cell cultured for6h was done by scanning electron microscopy (SEM).
4.Proliferation of osteosarcoma cells on the surface of the SAMs.
Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. Cells were seeded on these substrates with a concentration1×104cells/ml at37℃with5%CO2. The ability of cell proliferation cultured in2d,4d and6d was checked by the MTT assay (n=3donors) in our experiments. The use of Live/Dead cell staining is to observe osteosarcoma cell viability after24hours culture on the gold substrates.The activity of LDH was measured spectrophotometrically after culturing with gold substrates in24-well plates for24hours, so as to further understand the cytotoxicity of the chemical functional groups.
4. Apoptosis of osteosarcoma cells on the surface of the SAMs.
Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. Cells were seeded on these substrates with a concentration5x104cells/ml at37℃with5%CO2,and each hole with the medium500ul.The cell apoptosis was analyzed by the flow cytometry (Millipore, USA) after24hours culture on the gold substrates.
Result
1.Self-assembled monolayers
After gold spraying and cutting process, the gold pieces with severe surface scratches and unqualified standard were removed.Then we can get the standard uniform gold-silicon plates without any scratches, all of these gold pieces size is1.2cm×0.8cm.Once these gold-silicon plates were immersed into different thiol solution containing methyl, amino, carboxy and hydroxy for12h, successful self-assembled process happened with the four different chemical functional groups, and an uniform surface of self-assembled monolayers was obtained. To further characterize these different surface of SAMs, we measured their contact angles. Among these test surfaces, the-CH3surface had the highest contact angle of 105°±9.1°, which was the most hydrophobic surface in these test.the-OH surface was the most hydrophilic with a contact angle of9.5°±1.2°. The surfaces of-COOH had the moderate hydrophilic with a contact angle of18.3°±3.9°.The hydrophilic of-NH2surface was less because of its contact angle of-59.7°±3.8°. Statistical analysis results showwed the difference between these chemical groups is significant(Welch=1488.41,p=0.000).
2.Osteosarcoma cell culture.
In the early experiments, a total of3times,6bottles of cells were contaminated by some bacterials, and the color of the medium change to muddy significantly, and original adherent osteosarcoma cell became necrosis falling into pieces. After the contamination of the cell culture, we tried to find the pollution factors timely.On summary,the first contamination happened during packing fetal bovine serum, and the second was PBS pollution, and the last happened during the period of cell passaged culture. At last,with the help of the extensive literature review related to the cell culture, and with the help of lab teachers for the relevant cell culture technology and key steps,cell pollution problems were not repeated in subsequent experiments throughout the process. After the timely exchange of medium, the passaged cells show good adhesion and proliferation,which is favourable for the follow-up tests.
3. Osteosarcoma cell adhesion
In the experiment,the U2-OS cells adhesion on different chemical functional groups was checked by the phase contrast microscope and SEM. Under phase contrast microscope, after3hours culture, almost all of the U2-OS cells adhered onto the surface of the gold sustrates began to present a small round shape, and little cells became small oval. Especially,the cells on the-CH3surface seemed to be very loose.6hours later, the U2-OS cells adhered onto the surface of the gold sustrates began to present more and more morphology changes.The cells volume on-NH2,-COOH,--OH and the bare gold substrate (the control group)surface increased, and large oval, polygonal, spindle cells were seen on these surface,but the cells on-CH3surface were still small round. After9hours incubation, the cell morphology difference existed significantly. The cell on-CH3surface still remained round or oval, but more spindle and irregularly shaped cells began to appear on the other three surfaces.
Under phase contrast microscope,the cell number increasing on the-CH3surface was least,and the cell number increasing on the-OH and control group surface with time changes was not so obvious as on the-NH2and-COOH surfaces,the statistical analysis result is not against the Sphericity Assumed(W=:0.813,P=0.394),and the time had cross effect on the group(F=89.315,P=0.000),and the difference between groups at each time point was significant(F=122.008,P=0.000). After3hours culture, the cell number on-NH2surface was similar as-COOH surface(P=0.786), but was higher than the other three groups (P<0.05).In addition,the cell number on-CH3,-OH and Control group surface was also similar(P>0.05).6hours later, no significant increase in cell number on-CH3surface, on the contrary, the number of cells on-NH2surface increased significantly,the difference between-NH2and the others was significant(P<0.05),but the difference between-CH3and the control group was not significant(P=0.094). After9hours incubation, for-NH2,-OH,-COOH groups, the number of cells raised accordingly.,In particular, the number on-NH2surface increased significantly, but it was no obvious on-CH3surface,and there were significant differences among these groups(P<0.05). In addition,the adhesion number of U-2OS followed the trend:-CH3<<-OH<-COOH≈-NH2.
In order to further prove the different morphology changes, SEM indicated that U-2OS cells cultured on OH and COOH functional groups after6h incubation exhibitted polygonal and oval morphology,those on NH2showed a spindle shape, while cells cultured on-CH3group were smaller and in a spherical shape,.In addition, these cells on OH and COOH surfaces had bigger volume and abundant cytoplasm, and more pseudopodia extended to a larger area.But the cell on-CH3surface remained round and small volume yet.
4.Osteosarcoma cell proliferation
4.1MTT results
The U-2OS cells on different surfaces were uniformly seeded at the beginning of experiment. From day2to day6, there was a significant cell proliferation increasing on each group surface. However, the cells on-CH3surface were at a low O.D. level compared with-OH functional group, and the cell proliferation on-COOH and Control group surfaces was similar at day2(P=0.203)and at day4(P=0.700).But the cell proliferation difference between on-CH3and the other group surface at any time was also significant(P=0.000).The cell proliferation difference between on-OH surface and on the other group surfaces at day2and day4were also significant(P<0.05), especially the number increasing was more obvious at day6,but the difference was not significant compared with-NH2and -COOH surface (P=0.057and0.948respectively). The proliferation on-NH2and-COOH surface were all obvious,the difference was not significant at day2(P=0.752) and day6(P=0.051),but the difference was significant at day4(P=0.006).It appeared that-COOH、-OH and-NH2groups had a promoting effect on U-2OS proliferation in a longer culture period. On the contrary, the-CH3group had a negative effect on U-2OS proliferation. Furthermore, the proliferation capacity of U-2OS on different surfaces followed the trend:-COOH>-OH>-NH2>>-CH3.
4.2cell vitality on different chemical functional groups surface
In this experiment, we selected24hours incubation as a time point to test the cell viability with Live/Dead cell staining kit, and the marked result was recorded under fluorescent microscope. Wherein the living cells labeled in green, dead cells marked in red. We found that there were little cells on CH3surface, which consisted most of dead cells. In contrast, on-COOH,-NH2and-OH surfaces, there were much more viable cells in a much larger contact area. The U-2OS cells cultured on OH and COOH functional groups exhibitted polygonal and oval morphology,those on NH2showed a spindle shape, which were consistent with the SEM results. The survival rates of cells on different surfaces followed the trend:-NH2>-OH>-COOH>>-CH3. These results supported the idea that the-NH2functional group diminished cell toxicity whereas-CH3exhibited cell toxicity.The result is a strong evidence that-CH3group has the effect of inhibiting osteosarcoma cell proliferation.
4.3LDH analysis
From the result of MTT and Live/Dead cell staining,we can have a hypothesis that the cells on-CH3surface died from cell membrane damaged and the outflow of intracellular substances. To test this assumption, we measured the release of the cytoplasmic enzyme LDH by adherent cells on chemical groups modified substrates. Based on the results of the cell viability test, we chose a24h incubation time as the test time point.The result proved that the largest number of methyl lactate dehydrogenase release on-CH3group, and the lowest lactate dehydrogenase activity on-NH2functional group, and the lower one on the control group. Statistical analysis result showed homogeneity of variance(F=1.716,P=0.223),and the difference between groups was significant (F=111.549,P:=0.000).Further analysis showed the difference between-CH3and the others was also significant(P=0.000),and the difference between-OH and the others was also significant(P<0.05),but the lactate dehydrogenase activity was similar among-NH2、-COOH and Control groups(P>0.05). Assays of LDH activities were observed in the followed trend:-CH3>-OH≥-COOH>-NH2, which were agreed with the MTT results. It showed the relationship between cell toxicity and the release of LDH enzymes.
5.Osteosarcoma cell apoptosis
U-2OS cells on different chemical groups were applied for apoptosis analysis using Annexin V-PE and7-AAD. The combination of Annexin V-PE and7-AAD is useful for apoptosis cells analysis whether in the early stage of apoptosis or not. As is shown, the-CH3group caused about7.6%apoptosis and12.5%necrosis, whereas the-NH2group caused approximately4.3%apoptosis and12.6%necrosis. The apoptosis rate on the-OH surface was only2.1%,which was the lowest,but its necrosis rate was12.3%. The-COOH group caused approximately4.8%apoptosis and11.9%necrosis. Statistical analysis result showed homogeneity of variance whether apoptosis rate(F=1.075,P=0.413) or necrosis rate(F=0.162,P=0.919),and the difference between groups was significant (F=396.20,P=0.000). Further analysis showed the difference of apoptosis rate between groups was significant(P=0.000),and the difference of necrosis rate between groups was also significant(F=7.29,P=0.01).The difference of apotosis rate between-CH3and-COOH was significant(P=0.007),and the difference of apotosis rate between-NH2and-COOH or-OH was also significant(P<0.05).The apoptosis rate was in the followed trend:-CH3>-COOH>-NH2>-OH.
These results indicated that all of cells on different chemical functional groups surface had a certain degree of apoptosis, but the overall effect was not significant.From all of results aboved, we may have a conclusion that NH2surface had the best biocompatibility than-CH3surface, and-CH3can induce apoptosis and necrosis of osteosarcoma cells earlier. That is to say,-CH3group has a stronger anti-osteosarcoma cell activity.
Conclusion
1. Surface of self-assembled mono layers presents us an equivalent surface densities of chemical functional groups, and a three-dimensional structure. The results of contact angle measurement prove-CH3surface be a most hydrophobic group, and-OH surface be the most hydrophilic group, and-NH2,-COOH be the lower hydrophilic groups.The hydrophilic ability followed the trend:-OH>-COOH>-NH2>-CH3.
2. Subculture of osteosarcoma cells belongs to adherent cell culture technology,which is a mature cell culture technology. But there may be contamination in the whole cell culture process, causing the experiment failed. It is important to find pollution factors timely and seriously, and to improve our cell culture technology continuously, which ensure the follow-up experiments effectively.
3.Our work provides evidence that chemical properties of the biological material surface have an impact on the growth of osteosarcoma cells, and different chemical functional groups have not the same effects on the cells.Although-NH2and-COOH promoted cell apoptosis,but sustained visible cell adhesion and promoting cell growth. Not only-OH surfaces promoted cell proliferation,but also exhibited the weakest ability of inducing cell apoptosis. In contrast,-CH3surfaces showed anti-cancer effect from inhibiting cell growth, poor cell adhesion, high apoptosis and necrosis.
细胞内外环境等多种因素综合影响细胞的粘附、迁移、增殖、凋亡和坏死等生物学行为,因此细胞在支架材料上面的表现形式肯定会受到材料物理性质、表面化学性质、表面形貌和化学成分等多种因素的影响。近来在材料的化学改性方面的研究日益增多,材料的影响因素较多,得出的结论也很多样化,而运用自组装膜技术进行化学功能团修饰材料后的基础研究,可保证单一表面化学特性对细胞生物学行为的影响,避免多种综合因素的交叉影响,得出的结论会更可靠。
自组装膜技术发展较早,技术成熟,国内外很多行业都有应用。在国外,将这种技术运用到医学方面的基础研究开展早且较多,而在国内则相对较少且迟。目前已经开展了对破骨细胞、人成纤维细胞、间充质干细胞、神经干细胞等正常组织细胞的基础研究,近来国内部分学者开始重视这些化学功能团对肿瘤细胞的作用研究,目前已经发现部分化学功能团有抑制乳腺癌细胞、肝癌细胞粘附、增殖的作用。通过这些研究还发现,不同的化学功能团对细胞的不同生物学特性的影响不一样,且对不同细胞类型的影响也不一样,有的功能团具有明显的抑制肿瘤细胞生长的作用,反之也有化学功能团促进肿瘤细胞生长。
骨肉瘤是一种好发于青少年的恶性肿瘤,治疗棘手,预后差,尚没有非常有效的治愈办法。鉴于此,在国内外尚没有关于化学功能团对骨肉瘤细胞生物学功能影响方面的报道前提下,我们开始着手进行化学功能团对骨肉瘤细胞生物学功能方面影响的研究。
本课题采用传统的硅-金作为基质,选择甲基(-CH3)、氨基(-NH2)、羧基(-COOH)和羟基(-OH)四种化学功能团进行自组装膜研究。分别研究这四种化学功能团对骨肉瘤细胞的粘附、增殖及凋亡等三种重要的生物学行为的影响,明确哪些化学功能团对骨肉瘤细胞的哪些行为会产生抑制,对哪些行为会起促进作用,也就是说是否具有抑制肿瘤作用。
目的
本课题是一种运用自组装膜技术,观察化学功能团对骨肉瘤细胞生物学行为影响的探索性研究,研究的目的主要从以下几个方面进行明确。
明确白组装膜表面不同化学功能团的接触角等物理特性不同。
明确不同化学功能团对骨肉瘤细胞粘附的影响
明确不同化学功能团对骨肉瘤细胞增殖的影响
明确不同化学功能团对骨肉瘤细胞凋亡的影响
研究方法
本课题是运用自组装膜技术,观察化学功能团对骨肉瘤细胞生物学行为影响的探索性研究,因此从镀金硅片的制备及表面化学基团修饰、骨肉瘤细胞培养、细胞在化学功能团表面的粘附、增殖、凋亡等方面进行观察研究,具体方法如下：
1.镀金硅片的制备及其表面化学基团修饰。
采用自组装膜技术在镀金硅片基板上分别接枝甲基、氨基、羧基、羟基等四种不同化学官能团,然后对材料进行了接触角方面的测量。由于合作单位良好的接枝技术的保证,本实验没有重复对材料表面进行X射线光电子能谱(XPS,X-ray photoelectron spectroscopy)和原子力显微镜(AFM)技术表征。
2.骨肉瘤细胞的培养。
将冻存的U2-OS型骨肉瘤细胞株,放入37℃水浴箱进行复苏,移入25cm培养瓶内,放入37℃含5%C02恒温培养箱内过夜,更换10%胎牛血清RPMI1640培养基继续培养,每日观察,随时换液培养,待生长至细胞融合70%-80%时用0.25%胰酶消化液消化后传代培养,取对数生长期细胞做后续实验。
3.骨肉瘤细胞在材料表面的粘附观察。
每孔一枚接枝后金片,每组3孔,每孔加浓度为1×104/m1细胞悬液250μl,入恒温箱培养,分别选择3小时,6小时和9小时进行光镜观察,选择粘附6小时进行细胞形态学的扫描电镜观察。
4.骨肉瘤细胞在材料表面的增殖情况观察。
每孔一枚接枝后金片,每组3孔,每孔加浓度为1x104/ml细胞悬液2501μl,入恒温箱培养,采用MTT法计算2天、4天和6天时的增殖情况,了解不同化学功能团的细胞毒性情况；并结合活／死细胞荧光染色标记和测定培养24小时后细胞外液中的乳酸脱氢酶含量观察骨肉瘤细胞24小时后的细胞活力情况。
5.骨肉瘤细胞在材料表面的凋亡情况观察。
每孔一枚接枝后金片,每组3孔,每孔加浓度为5x104/ml细胞液0.5ml,入恒温箱培养,采用流式细胞仪计算24小时的细胞凋亡情况。
结果
1.自组装膜
经过镀金、切割处理后,去除表面划痕严重及标准不符合的金片,得到标准统一无划痕的镀金硅片,这些金片的大小为1.2cm×0.8cm；再将这些镀金硅片经分别含甲基、氨基、羧基和羟基四种不同的硫醇溶液浸泡后,成功接种四种不同化学功能团,得到表面结构均匀的单分子自组装膜。为了进一步对这些接种有不同化学功能团的自组装单分子膜表面结构进行表征,我们对其进行有关接触角的测量。所测甲基、氨基、羧基和羟基接触角分别为105。±9.1。、59.7°±3.80、18.3°±3.90和9.5。±1.20.。统计学分析进一步证实不同化学基团修饰表面的接触角差异具有显著性(Welch值=1488.41,P=0.000)。以上结果也表明甲基具有较强的疏水性,羟基具有最强的亲水性,羧基中等程度亲水性,氨基的亲水性较小。
2.骨肉瘤细胞的培养
在细胞传代培养的实验初期,共有3次6瓶细胞受到污染,培养基颜色明显浑浊,原来贴壁的骨肉瘤细胞成片脱落坏死。经及时查找总结,结果均是继发细菌污染,具体是分装胎牛血清时污染血清一次,PBS污染一次,传代操作过程污染细胞一次。污染后广泛查阅文献,分析原因,向实验室老师请教相关细胞培养技术及关键步骤并及时整改,结果在整个后续实验过程中均未再次出现细胞污染问题。经过及时换液、传代后的细胞均贴壁良好,细胞分裂增殖力旺盛,有利于后续试验的开展。
3.骨肉瘤细胞粘附情况
本实验中,我们利用光学倒置显微镜和扫描电镜观察U-20S在四种化学功能团修饰金片表面粘附情况。相差显微镜下,培养3小时后,各组粘附于金片表面的细胞基本都成小圆形,少许呈小椭圆形,甲基表面粘附的细胞不紧密,呈要脱落状。6小时后细胞形态开始多样化,在氨基、羧基、羟基及对照组金片表面均可见细胞体积增大,可见大椭圆形、梭形及多边形细胞,而在甲基表面细胞仍呈小圆形状。而9小时后,光镜下各组细胞形态变化差异更加明显,有更多梭形、不规则形细胞开始出现,而甲基表面的细胞仍多数呈圆形或椭圆形,梭形、不规则形细胞较少。
镜下见甲基表面的细胞数增加最不明显,氨基和羧基表面的细胞数明显增加,而羟基和对照组细胞数增长较少,进一步统计学结果不拒绝球形假设(W=0.813,P=0.394),且检测时间点与组间有交互作用(F=89.315,P=0.000),统计学上不同时间点各组间细胞数具有显著性差异(F=122.008,P=0.000)。3小时后粘附的细胞数以氨基表面稍多,甲基、羟基和对照组表面较少,统计学上氨基组与甲基、羟基和对照组相比有显著性差异(P<0.05),与羧基组差异无显著性(P=0.786),甲基、羟基和对照组之间差异无显著性(P>0.05)。6小时后甲基表面细胞数增加不明显,其他各组表面细胞数均有不同程度增加,氨基组增加最明显,与其他各组相比统计学差异具有显著性(P<0.05),而甲基与对照组表面细胞数统计学差异无显著性(P=0.094)。9小时后氨基、羧基和羟基组细胞数均明显增多,其中氨基组增加最明显,甲基组增加不明显,统计学上各组之间细胞数差异均具有显著性(P<0.05),各组金片表面粘附细胞数均数总体趋势为-CH3<<-OH<-COOH≈-NH2。
扫描电镜下更进一步观察各组功能团表面粘附细胞的形态学变化,U-20S细胞与各组金片混合培养6小时后,细胞在羟基和羧基组金片表面多呈现出椭圆形和多边形,细胞体积较大,胞浆丰富,可见较多伪足伸出,部分较大伪足相互融合。而在氨基功能团表面的细胞多数为长梭形,甲基表面的细胞仍呈小圆形,细胞体积较小。
4.骨肉瘤细胞增殖情况
4.1MTT结果
在细胞接种浓度相同的条件下,随着培养时间的推移,所有化学官能团表面的骨肉瘤细胞都有不同程度的增殖情况出现,不过甲基表面骨肉瘤细胞增殖的OD值较低,羟基组OD值较高,而在2d和4d时对照组和羧基组的增殖情况相似(P值分别为0.203,0.700)。不同时间点甲基表面细胞增殖情况同其他4组相比均有显著性差异(P=0.000),进一步多重比较显示羟基表面细胞增殖在2d和4d时与其他各组均有统计学差异(P<0.05),尤其是在培养时间达到6天后其增殖均数更加明显,但与氨基、羧基组相比无统计学差异(P值分别为0.057,0.948)；氨基和羧基表面的骨肉瘤细胞均明显增殖,但相互比较结果在2天和6天时无统计学差异(2d和6d时的P值分别为0.752和0.051),培养4天时差异具有显著性(P=0.006)。以上结果似乎显示氨基、羧基和羟基均有促进骨肉瘤细胞增殖的可能。反过来说,上述结果提示甲基似乎有抑制骨肉瘤细胞增殖的可能。整体均数上看,不同化学官能团表面骨肉瘤细胞的增殖趋势为：羧基>羟基≥氨基>>甲基。该趋势说明甲基官能团抑制了骨肉瘤细胞的增殖。
4.2不同化学功能团表面粘附细胞的活力情况
本实验中,我们选择培养24小时作为检测细胞活力试验的时间点,用活／死细胞检测盒进行检测,荧光显微镜下观察。其中活细胞标记为绿色,死细胞标记为红色。结果可以看出甲基表面的细胞体积都很小,荧光染色显示为死细胞。相反的是,在氨基、羧基和羟基表面,大量活细胞存在,并呈现出较好的生长状态。氨基表面细胞多数呈长梭形,羧基和羟基表面的骨肉瘤细胞多为椭圆形或多边形,生长状况与扫描电镜下所见基本相符。
整体上,不同化学功能团表面粘附细胞的生存率趋势为：氨基>羟基≥羧基>>甲基。以上结果也充分证明氨基功能团对骨肉瘤细胞的毒性小,而甲基显示有较高的骨肉瘤细胞毒性,是抑制骨肉瘤细胞增殖的有力证据。
4.3细胞内乳酸脱氢酶释放情况
我们从细胞增殖、细胞活力检测结果分析,认为甲基致细胞死亡的原因可能系引起骨肉瘤细胞膜的损伤,进而致细胞内物质的流出。因此本组实验通过测定细胞质内乳酸脱氢酶的浓度情况来判断甲基功能团致细胞死亡的假设。基于细胞活力试验结果,我们选择培养24小时作为检测的时间点,实验结果显示甲基功能团可以致大量乳酸脱氢酶释放,而氨基功能团组的乳酸脱氢酶活性最低,也低于对照组。统计分析结果方差齐性(F=1.716,P=0.223),各组间比较具有显著性差异(F=111.549,P=0.000),进一步相互比较得出甲基与各组均有显著性差异(P=0.000),羟基与各组也有统计学差异(P<0.05),氨基、羧基和对照组之间无统计学差异(P>0.05)。从均数上看,乳酸脱氢酶活性趋势：甲基>羟基≥羧基>氨基。以上结果与MTT法一致,也充分显示细胞内乳酸脱氢酶的释放程度与化学功能团的细胞毒性一致。
5.骨肉瘤细胞凋亡情况
本试验中将Annexin V与7-AAD一起使用,用来检测不同时期的凋亡细胞,结果发现甲基功能团表面骨肉瘤细胞的凋亡率为7.6%,坏死率为12.5%,而氨基功能团表面的骨肉瘤细胞凋亡率仅约4.3%,但坏死率却达到12.6%,羟基组的凋亡率最低,仅2.1%,坏死率为12.3%,羧基组的凋亡率约为4.8%,坏死率为11.9%。统计学分析方差齐性(凋亡组F=1.075,P=0.413,坏死组F=0.162,P=0.919),各组间凋亡率统计学差异有显著性(F=396.20,P=0.000),进一步分析各组凋亡率之间均有显著性差异(p=0.000)；各组间坏死率比较也具有差异性(F=7.29,P=0.01)。进一步分析显示甲基组与羧基组差异具有显著性(p=0.007),氨基组和羧基、羟基组比较具有统计学差异(P<0.05)。各组化学功能团诱导骨肉瘤细胞凋亡的总体趋势为甲基>羧基>氨基≥羟基。以上结果显示各组功能团均有一定程度诱导骨肉瘤细胞发生凋亡的作用,但总体作用不显著。综合分析得出：氨基具有较甲基更佳的生物相容性,而甲基可以诱导骨肉瘤细胞发生早期凋亡及坏死。也就是说,甲基具有更强的抗骨肉瘤细胞活性。
结论
1.自组装膜技术成熟,接枝后可以得到表面均匀结构单一的立体结构,接触角测量结果说明甲基具有明显疏水性,羟基的亲水性最高,而羧基和氨基的亲水性稍弱。亲水性大小顺序为：羟基>羧基>氨基>甲基
2.骨肉瘤细胞的传代培养属于贴壁细胞培养,技术成熟,但在整个培养过程中仍有可能出现细菌、真菌、支原体等污染情况的发生,致实验失败。需要做到早期发现,认真查找污染源并持续改进,方能保证后续实验的有效开展。
3.研究证明了生物材料表面化学特性对骨肉瘤细胞的生长会产生影响,不同的化学功能团会有不同的作用。氨基和羧基功能团虽然能够促进骨肉瘤细胞的凋亡,但也有较强的促粘附、增殖作用；羟基功能团不仅诱导骨肉瘤细胞的凋亡率低,而且还促进其粘附、增殖；而甲基功能团具有一致的抑制骨肉瘤细胞粘附、增殖和促进骨肉瘤细胞凋亡坏死的发生,也就是说具有明显的抑制骨肉瘤细胞生长的能力,具有较强的抗癌作用。
Background
A variety of factors including the internal and external environments of cell,may have effect on the biological behavior of cell adhesion, migration, proliferation, apoptosis and necrosis.Therefore, the form of cell on the scaffold would certainly be influenced by many factors like the physical properties of materials, surface chemistry, surface morphology and chemical composition, etc. Recently, more and more researches about the chemical modification of materials increased, and the conclusion is various because of the interference of many material features.The basic research of chemical functional groups,using self-assembled monolayers(SAMs) technology,can ensure the impact of the material surface chemistry on cells only by the single chemical character of the materials, avoiding a variety of intersect factors influence, and the conclusion probably be more reliable.
Self-assembled monolayers technology developed earlier, and is a mature technology,have been widely used in many industries all over the world. The SAMs technology has been applied to much more medical basic research earlier in other countries,but which is relatively less and later in our country.Up to now,SAMs has been used to the basic research of some normal tissue cells, like the osteoclasts, human fibroblasts, messenchymal stem cells and neural stem cells. Recently, some domestic scholars began to pay much more attention to a few tumor cells.According to their report, some chemical functional groups have been found to have effect of inhibiting the adhesion and proliferation of breast cancer and hepatoma cells. These studies also found that different chemical functional groups have not the identical effects on the different biological properties of the same cell, and also on the different cell types. That is to say, some chemical functional groups can inhibit the growth of tumor cells, and some other chemical functional groups probably promote tumor cell growth on the contrary.
As a kind of malignant bone tumor, osteosarcoma often occurs in young people, and has unsatisfactory treatment, and has the poor prognosis. And so far, there is no very effective way to cure this disease. Due to the fact that there is no report to explain the relationship between the chemical functional groups and the biological behaviors of the osteosarcoma cells, we are working on the research.
In our study, the traditional silicon-gold was chosen as the SAMs substrate, and methyl(-CH3), amino(-NH2), carboxyl(-COOH) and hydroxyl(-OH) functional groups were selected as the influence factors.The main purpose is to find the exact effect of these chemical functional groups on these important biological behaviors of osteosarcoma cell,such as the adhesion, proliferation and apoptosis.That is to say, Learn to know what kind of chemical functional groups can give rise to what kind of behavior change of osteosarcoma cells,and learn to know whether the chemical functional groups can inhibit the tumor growth or not.
Objective
This topic is an exploratory study of observing the effect of chemical functional groups on the biological behaviors of osteosarcoma cells with the self-assembled monolayers technology.The main purpose will be expained explicitly from the following aspects:
To clear the different physical characteristics of the self-assembled monolayers surface by measuring the contact angle of materials.
To clear the impact of different chemical functional groups on osteosarcoma cell adhesion.
To clear the impact of different chemical functional groups on osteosarcoma cell proliferation.
To clear the impact of different chemical functional groups on osteosarcoma cell apoptosis.
Methods
This topic is an exploratory study of observing the effect of chemical functional groups on the biological behavior of osteosarcoma cells with the self-assembled monolayers technology.Therefore, the preparation of silicon-gold substrate, and its modification of chemical functional groups, and osteosarcoma cell culture, and the observation of the cells adhesion,proliferation and apoptosis on the surface of SAMs will be included in our experiment.Specific methods are shown as follows:
1. Preparation of gold-silicon substrate and self-assembled with different chemical group.
Using self-assembled monolayers technology, the methyl, amino, carboxyl and hydroxyl functional groups were self-assembled on the Au layers respectively. We characterized these surfaces to determine the equivalent surface densities of chemical functional groups by measuring their contact angles. Due to good self-assembled monolayers technique of co-operation unit,the further examination for the morphology of these groups surface by atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) was missed in our experiment.
2. Osteosarcoma cell culture.
Firstly, the frozen U2-OS osteosarcoma cell lines were put into37℃water bath for resuscitation, and then shifted into25cm culture bottles. Finally the culture bottles were placed at37℃in5%CO2and95%air.24hours later, the cell culture and daily observation go on after replacing10%FBS RPMI1640medium.Culture medium was changed at any time.After cells growth to be70%to80%confluence, followed with0.25%trypsin digestion, and then passaged culture.The logarithmic growth phase cells are the best choice to do follow-up experiments.
3.Adhesion of osteosarcoma cells on the surface of the SAMs.
Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. U2-OS cells were seeded on these substrates with a concentration1×104cells/ml at37℃with5%CO2.After3h,6h and9h culture, cell adhesion analyses were done under inverted microscopy. And further adhesion analysis of the cell cultured for6h was done by scanning electron microscopy (SEM).
4.Proliferation of osteosarcoma cells on the surface of the SAMs.
Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. Cells were seeded on these substrates with a concentration1×104cells/ml at37℃with5%CO2. The ability of cell proliferation cultured in2d,4d and6d was checked by the MTT assay (n=3donors) in our experiments. The use of Live/Dead cell staining is to observe osteosarcoma cell viability after24hours culture on the gold substrates.The activity of LDH was measured spectrophotometrically after culturing with gold substrates in24-well plates for24hours, so as to further understand the cytotoxicity of the chemical functional groups.
4. Apoptosis of osteosarcoma cells on the surface of the SAMs.
Each hole of24-well plates with a substrate assembled by these chemical functional groups, and each group with three holes. Cells were seeded on these substrates with a concentration5x104cells/ml at37℃with5%CO2,and each hole with the medium500ul.The cell apoptosis was analyzed by the flow cytometry (Millipore, USA) after24hours culture on the gold substrates.
Result
1.Self-assembled monolayers
After gold spraying and cutting process, the gold pieces with severe surface scratches and unqualified standard were removed.Then we can get the standard uniform gold-silicon plates without any scratches, all of these gold pieces size is1.2cm×0.8cm.Once these gold-silicon plates were immersed into different thiol solution containing methyl, amino, carboxy and hydroxy for12h, successful self-assembled process happened with the four different chemical functional groups, and an uniform surface of self-assembled monolayers was obtained. To further characterize these different surface of SAMs, we measured their contact angles. Among these test surfaces, the-CH3surface had the highest contact angle of 105°±9.1°, which was the most hydrophobic surface in these test.the-OH surface was the most hydrophilic with a contact angle of9.5°±1.2°. The surfaces of-COOH had the moderate hydrophilic with a contact angle of18.3°±3.9°.The hydrophilic of-NH2surface was less because of its contact angle of-59.7°±3.8°. Statistical analysis results showwed the difference between these chemical groups is significant(Welch=1488.41,p=0.000).
2.Osteosarcoma cell culture.
In the early experiments, a total of3times,6bottles of cells were contaminated by some bacterials, and the color of the medium change to muddy significantly, and original adherent osteosarcoma cell became necrosis falling into pieces. After the contamination of the cell culture, we tried to find the pollution factors timely.On summary,the first contamination happened during packing fetal bovine serum, and the second was PBS pollution, and the last happened during the period of cell passaged culture. At last,with the help of the extensive literature review related to the cell culture, and with the help of lab teachers for the relevant cell culture technology and key steps,cell pollution problems were not repeated in subsequent experiments throughout the process. After the timely exchange of medium, the passaged cells show good adhesion and proliferation,which is favourable for the follow-up tests.
3. Osteosarcoma cell adhesion
In the experiment,the U2-OS cells adhesion on different chemical functional groups was checked by the phase contrast microscope and SEM. Under phase contrast microscope, after3hours culture, almost all of the U2-OS cells adhered onto the surface of the gold sustrates began to present a small round shape, and little cells became small oval. Especially,the cells on the-CH3surface seemed to be very loose.6hours later, the U2-OS cells adhered onto the surface of the gold sustrates began to present more and more morphology changes.The cells volume on-NH2,-COOH,--OH and the bare gold substrate (the control group)surface increased, and large oval, polygonal, spindle cells were seen on these surface,but the cells on-CH3surface were still small round. After9hours incubation, the cell morphology difference existed significantly. The cell on-CH3surface still remained round or oval, but more spindle and irregularly shaped cells began to appear on the other three surfaces.
Under phase contrast microscope,the cell number increasing on the-CH3surface was least,and the cell number increasing on the-OH and control group surface with time changes was not so obvious as on the-NH2and-COOH surfaces,the statistical analysis result is not against the Sphericity Assumed(W=:0.813,P=0.394),and the time had cross effect on the group(F=89.315,P=0.000),and the difference between groups at each time point was significant(F=122.008,P=0.000). After3hours culture, the cell number on-NH2surface was similar as-COOH surface(P=0.786), but was higher than the other three groups (P<0.05).In addition,the cell number on-CH3,-OH and Control group surface was also similar(P>0.05).6hours later, no significant increase in cell number on-CH3surface, on the contrary, the number of cells on-NH2surface increased significantly,the difference between-NH2and the others was significant(P<0.05),but the difference between-CH3and the control group was not significant(P=0.094). After9hours incubation, for-NH2,-OH,-COOH groups, the number of cells raised accordingly.,In particular, the number on-NH2surface increased significantly, but it was no obvious on-CH3surface,and there were significant differences among these groups(P<0.05). In addition,the adhesion number of U-2OS followed the trend:-CH3<<-OH<-COOH≈-NH2.
In order to further prove the different morphology changes, SEM indicated that U-2OS cells cultured on OH and COOH functional groups after6h incubation exhibitted polygonal and oval morphology,those on NH2showed a spindle shape, while cells cultured on-CH3group were smaller and in a spherical shape,.In addition, these cells on OH and COOH surfaces had bigger volume and abundant cytoplasm, and more pseudopodia extended to a larger area.But the cell on-CH3surface remained round and small volume yet.
4.Osteosarcoma cell proliferation
4.1MTT results
The U-2OS cells on different surfaces were uniformly seeded at the beginning of experiment. From day2to day6, there was a significant cell proliferation increasing on each group surface. However, the cells on-CH3surface were at a low O.D. level compared with-OH functional group, and the cell proliferation on-COOH and Control group surfaces was similar at day2(P=0.203)and at day4(P=0.700).But the cell proliferation difference between on-CH3and the other group surface at any time was also significant(P=0.000).The cell proliferation difference between on-OH surface and on the other group surfaces at day2and day4were also significant(P<0.05), especially the number increasing was more obvious at day6,but the difference was not significant compared with-NH2and -COOH surface (P=0.057and0.948respectively). The proliferation on-NH2and-COOH surface were all obvious,the difference was not significant at day2(P=0.752) and day6(P=0.051),but the difference was significant at day4(P=0.006).It appeared that-COOH、-OH and-NH2groups had a promoting effect on U-2OS proliferation in a longer culture period. On the contrary, the-CH3group had a negative effect on U-2OS proliferation. Furthermore, the proliferation capacity of U-2OS on different surfaces followed the trend:-COOH>-OH>-NH2>>-CH3.
4.2cell vitality on different chemical functional groups surface
In this experiment, we selected24hours incubation as a time point to test the cell viability with Live/Dead cell staining kit, and the marked result was recorded under fluorescent microscope. Wherein the living cells labeled in green, dead cells marked in red. We found that there were little cells on CH3surface, which consisted most of dead cells. In contrast, on-COOH,-NH2and-OH surfaces, there were much more viable cells in a much larger contact area. The U-2OS cells cultured on OH and COOH functional groups exhibitted polygonal and oval morphology,those on NH2showed a spindle shape, which were consistent with the SEM results. The survival rates of cells on different surfaces followed the trend:-NH2>-OH>-COOH>>-CH3. These results supported the idea that the-NH2functional group diminished cell toxicity whereas-CH3exhibited cell toxicity.The result is a strong evidence that-CH3group has the effect of inhibiting osteosarcoma cell proliferation.
4.3LDH analysis
From the result of MTT and Live/Dead cell staining,we can have a hypothesis that the cells on-CH3surface died from cell membrane damaged and the outflow of intracellular substances. To test this assumption, we measured the release of the cytoplasmic enzyme LDH by adherent cells on chemical groups modified substrates. Based on the results of the cell viability test, we chose a24h incubation time as the test time point.The result proved that the largest number of methyl lactate dehydrogenase release on-CH3group, and the lowest lactate dehydrogenase activity on-NH2functional group, and the lower one on the control group. Statistical analysis result showed homogeneity of variance(F=1.716,P=0.223),and the difference between groups was significant (F=111.549,P:=0.000).Further analysis showed the difference between-CH3and the others was also significant(P=0.000),and the difference between-OH and the others was also significant(P<0.05),but the lactate dehydrogenase activity was similar among-NH2、-COOH and Control groups(P>0.05). Assays of LDH activities were observed in the followed trend:-CH3>-OH≥-COOH>-NH2, which were agreed with the MTT results. It showed the relationship between cell toxicity and the release of LDH enzymes.
5.Osteosarcoma cell apoptosis
U-2OS cells on different chemical groups were applied for apoptosis analysis using Annexin V-PE and7-AAD. The combination of Annexin V-PE and7-AAD is useful for apoptosis cells analysis whether in the early stage of apoptosis or not. As is shown, the-CH3group caused about7.6%apoptosis and12.5%necrosis, whereas the-NH2group caused approximately4.3%apoptosis and12.6%necrosis. The apoptosis rate on the-OH surface was only2.1%,which was the lowest,but its necrosis rate was12.3%. The-COOH group caused approximately4.8%apoptosis and11.9%necrosis. Statistical analysis result showed homogeneity of variance whether apoptosis rate(F=1.075,P=0.413) or necrosis rate(F=0.162,P=0.919),and the difference between groups was significant (F=396.20,P=0.000). Further analysis showed the difference of apoptosis rate between groups was significant(P=0.000),and the difference of necrosis rate between groups was also significant(F=7.29,P=0.01).The difference of apotosis rate between-CH3and-COOH was significant(P=0.007),and the difference of apotosis rate between-NH2and-COOH or-OH was also significant(P<0.05).The apoptosis rate was in the followed trend:-CH3>-COOH>-NH2>-OH.
These results indicated that all of cells on different chemical functional groups surface had a certain degree of apoptosis, but the overall effect was not significant.From all of results aboved, we may have a conclusion that NH2surface had the best biocompatibility than-CH3surface, and-CH3can induce apoptosis and necrosis of osteosarcoma cells earlier. That is to say,-CH3group has a stronger anti-osteosarcoma cell activity.
Conclusion
1. Surface of self-assembled mono layers presents us an equivalent surface densities of chemical functional groups, and a three-dimensional structure. The results of contact angle measurement prove-CH3surface be a most hydrophobic group, and-OH surface be the most hydrophilic group, and-NH2,-COOH be the lower hydrophilic groups.The hydrophilic ability followed the trend:-OH>-COOH>-NH2>-CH3.
2. Subculture of osteosarcoma cells belongs to adherent cell culture technology,which is a mature cell culture technology. But there may be contamination in the whole cell culture process, causing the experiment failed. It is important to find pollution factors timely and seriously, and to improve our cell culture technology continuously, which ensure the follow-up experiments effectively.
3.Our work provides evidence that chemical properties of the biological material surface have an impact on the growth of osteosarcoma cells, and different chemical functional groups have not the same effects on the cells.Although-NH2and-COOH promoted cell apoptosis,but sustained visible cell adhesion and promoting cell growth. Not only-OH surfaces promoted cell proliferation,but also exhibited the weakest ability of inducing cell apoptosis. In contrast,-CH3surfaces showed anti-cancer effect from inhibiting cell growth, poor cell adhesion, high apoptosis and necrosis.
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