【作者】 于雪；

【导师】 金景姬；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 在细胞生物学中,用于检测细胞活力及细胞增殖的方法主要有3 H-TdR掺入法和MTT比色法:3 H-TdR掺入法是一种直接通过测定分裂的细胞数来评价细胞增殖能力的方法;MTT法是一种间接的方法,即通过检测细胞活力(cell viability)来检测样品中健康细胞的数目,进而评价细胞的增殖能力。目前比较常用的是MTT比色法。MTT法(MTT中文名称为3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐),其检测原理是活细胞线粒体中的琥珀酸脱氢酶能将培养基中的MTT还原,产生不溶于水的紫色结晶——甲瓒颗粒,而死细胞不能产生琥珀酸脱氢酶,通过用酶联免疫检测仪在492nm波长处测定其光吸收值,建立甲瓒生成量与活细胞数量的关系,从而获得细胞存活率的一种测量方法【1】。研究表明,在一定细胞浓度范围内,光吸收值与活细胞数成正比【2】。该方法已广泛用于细胞毒性实验、生物相容性评价、生物活性因子的检测以及抗肿瘤药物的筛选等等。MTS是一种新型的四唑类化合物,原理与MTT相同,但其产生的甲瓚是水溶性的,因而实验操作中减少了MTT中用DMSO溶解甲瓚的步骤,比MTT法高效。另外,MTS还原产物颜色较深,吸光度值变异范围大,灵敏度更高,结果更准确。MTS法能更快捷准确地测定细胞的增殖反应,而且能够避免由于人为操作、实验环境等因素造成的实验误差,大大地提高了实验结果的准确性和重现性【3】。本文的研究分为两部分,首先利用MTT法和细胞成像技术研究了无机纳米材料——中空介孔纳米硅球(fHMSN)的生物相容性,并以抗肿瘤药物阿霉素(DOX)作为药物模型,研究了fHMSN担载药物DOX进入癌细胞后的药物释放作用。此外,还应用MTS方法和细胞成像技术研究了可溶性小分子有效碳(AC)溶液对体外培养的癌细胞及人胎盘间充质干细胞(PMSC细胞)增殖的影响。第一部分的结果表明,中空介孔纳米硅球fHMSN在浓度≤50ug/ml时,细胞存活率>90%,有较低的细胞毒性,可以作为抗癌药物载体用于临床应用;fHMSN在担载抗癌药物阿霉素进入癌细胞后,能够缓慢释放药物,避免了高剂量药物产生的毒副作用;同时利用细胞成像技术,可以观察到随着孵育时间的延长,fHMSN能够被细胞通过胞吞进入到细胞中,其担载药物阿霉素后也能够被逐渐胞吞进入细胞,并主要存在于细胞的胞质区。第二部分实验用MTS方法初步证实了可溶性小分子有效碳(AC)的抗肿瘤作用并不是通过直接作用于肿瘤细胞来完成的。实验同时还发现这种可溶性小分子有效碳AC虽然不会对肿瘤细胞有明显的抑制作用,却可以促进人正常细胞的增殖,因而选取了在体外常规培养条件下生长速度比较缓慢的人胎盘间充质干细胞(PMSC)为对象进行研究,在培养基中添加不同稀释度的AC后,与未添加组相比,实验组的胎盘间充质干细胞生长状态良好,并且活细胞数目远多于对照组的细胞数。这一发现有望解决常规体外培养条件下的间充质干细胞增殖速度缓慢的问题,为间充质干细胞在体外高效培养提供了可能的解决方法。更多还原

【Abstract】 In cell biology,the main methods for detecting cell activity and proliferation are the 3 H-TdR incorporation and MTT colorimetry method.3 H-TdR incorporation method is a method that directly through dividing cell numbers to evaluate cell proliferation.MTT method is an indirect method,namely through the detection of cell viability according to the number of healthy cells in test samples,and then evaluate the ability of cell proliferation.MTT is a frequently-used method for detecting cell survival and growth.The principle for MTT assay is that Succinate dehydrogenase in mitochondria of living cell can make the exogenous MTT be reduced to water insoluble purple crystal deposition called formazan in cells,however,dead cells can not produce the succinate dehydrogenase.By enzyme-linked immunosorbent assay to detect the absorbance in 492 nm wavelength,we can construct a relationship between the number of formazan formation and the living cell number,so as to obtain the cell viability.Researches have showed that in a certain concentration range of cells,the absorbance is proportional to the number of living cells.This method has been widely used in detecting the activity of some bioactive factors,mass screening the anti-tumor drugs,and the cell toxicity test.MTS is a new type of four azole compounds.The principle of MTS is the same as MTT,but its formazan is water-soluble,so the experimental operation don’t need to dissolve formazan by DMSO like the MTT method,so it is much more efficient compared with the MTT method.In addition,the color of MTS products is deeper,the absorbance variation range is larger,and it has higher sensitivity and more accurate results.MTS method could more quickly and accurately determine cell proliferation,and also avoid errors caused by manual operation or test environment,it greatly improves the accuracy of the test results.This paper is divided into two parts,firstly,we use the MTT method and cell imaging technology to study the bio-compatibility of inorganic nanomaterials-fHMSN,then we use the anticancer drug doxorubicin(DOX)as a drug model,to study how can the fHMSN take the drug into cells and release the drug gradually.In addition,we also use the MTS method and cell imaging technology to research the influence of small molecular organic carbon solution(AC)on proliferation of in-vitro cultured cancer cells and human placenta mesenchymal stem cells(PMSC).The first part of the results show that the hollow mesoporous silica nanospheres fHMSN at a concentration of less than 50ug/ml,the cell viability is >90%,so the fHMSN has low toxicity,and can be used as a carrier of anticancer drugs in clinic.After fHMSN loading the anticancer drug doxorubicin into cancer cells,it can gradually release the drug,to avoid toxic side effects of high doses of the drug.Then using cell imaging technology,it can be observed that with the incubation time goes on,fHMSN can be absorbed into the cells by endocytosis,the drug loaded by fHMSN can also be gradually endocytosed into cells,and mainly resists in the cytoplasm of cells.In the second part of the experiment,we use MTS method to confirm that the anti-tumor effect of small molecule available carbon(AC)is not through direct effection on tumor cells.The experiments also found that although the small molecule available carbon(AC)has no significant inhibitory effect on tumor cells,it can promote normal cell proliferation in vitro,so we select human mesenchymal stem cells whose growth rate was very slow when cultured in-vitro under normal culture condition,after add different dilutions of AC into the medium,the experimental group of mesenchymal stem cell’s growth is good,and the growth rate has accelerated noticeably,compared with the control group.This finding is expected to solve the slow proliferation rate of mesenchymal stem cells(MSCs)when cultured in vitro,it can provide a possible solution for the efficient in vitro culture of mesenchymal stem cells(MSCs).更多还原