多器官人源细胞系共培养模型的建立与初步应用

发布时间：2018-05-11 08:50

本文选题：人源细胞系 + 共培养　；参考：《第二军医大学》2014年博士论文

【摘要】：随着美国国家研究委员会（NRC)“21世纪的毒性测试：愿景与策略（Toxicitytesting in the21st century: a vision and a strategy)”这一具有里程碑意义研究报告的发表，预示着毒理学实验方法将发生革命性的改变。基于人体细胞的靶向性测试和毒性通路测试将取代以实验动物为中心的毒性评价方法成为当前毒理学的研究热点和发展趋势。美国EPA和NTP制定和实施了多项研究计划，不少国家的研究机构也开展了研究。当前的研究思路主要是从三个方面展开：一是不同人体细胞系毒性的比较，试图为靶向性测试提供依据；二是不同化学物毒性作用通路的研究；三是毒性通路测试和分析方法的研究。本课题主要针对靶向性测试问题进行研究。在靶向性测试研究方面，目前采用选择可能毒性靶器官的细胞，或采用多种细胞的比较研究等方法。其存在的问题：一是很多毒物未知其毒性靶器官；二是尽管可采用多种（器官）细胞的比较研究，但由于均是单一细胞培养条件下的分别测试，不是在同一条件下进行的，与体内情况不符，难以较快、较准确地确定靶器官细胞；三是很多毒物进入体内后需要经过肝脏和其它器官的代谢转化后才产生毒性或者消除毒性，现行的应用添加肝S9形成活化系统的方法不足以模拟体内的真实情况。此外体内细胞间通过微环境的相互作用状况在单一细胞培养中也不能实现。因此，现行方法难以较好解决靶向性测试问题。为此，本研究提出选择以人肝细胞系和常见靶器官人源器官细胞系，在同一条件下共培养，以构建一个不同器官人源细胞系共培养模型的研究思路。纳米物质与常规尺度物质相比，由于其特殊的理化性质，出现了很多新的生物学效应和特性，给毒理学安全性评价提出了新挑战。充分发现其与常规尺度物质相比有无新的或不同的毒效应、揭示其毒作用特征是解决纳米性物质安全性评价的关键。研究纳米物质的毒作用靶器官，比较其与常规尺度物质的异同，可为纳米性物质安全性评价问题的解决提供科学依据。本研究的目的是建立人源多器官细胞系体外共培养模型；探讨人源多器官细胞体外共培养方法作为毒性靶器官筛选的可能性；观察肝来源细胞系在该共培养体系下的代谢作用；将所建方法初步应用于纳米物质的靶向性研究。一、人源多器官细胞系体外共培养模型的建立选择常用于靶器官毒性研究的代表细胞系，即肺脏(A549)、肺脏（MRC-5）、肝脏(HepG-2)、神经系统(SH-SY5Y)、肾脏(HEK-293)和心脏(CCC-HEH-2)六种细胞；采用市售的48孔培养板经自制改造成共培养板，即以5孔为一组，在一组内的各孔壁上打洞，使一组内各孔之间的培养基能相互流通。将5种细胞分别种植于5孔中培养，比较单培养条件下和共培养条件下各细胞系生长情况。同时，比较了应用DMEM和美国典型培养物保藏中心（ATCC）推荐培养基对细胞的生长效果。结果：除MRC-5细胞在DMEM培养基中生长不佳外，其它细胞在DMEM培养基上均生长良好。因此，最终选定肺脏（A549)、肝脏(HepG-2)、神经系统(SH-SY5Y)、肾脏(HEK-293)和心脏(CCC-HEH-2)五种细胞系；以DMEM作为培养基。结果显示，各种细胞在共培养条件下生长良好，共培养细胞和单独培养细胞的生长曲线基本重合，表明所建的人源多器官细胞系共培养模型是成功的。二、人源多器官细胞系共培养模型在毒性靶器官筛选上的可行性研究选用已知毒性的四氯化碳、氯化镉、氟化钠、盐酸阿霉素、硫酸链霉素、羟基多巴胺、百草枯和白消安为受试物，应用MTT法检测受试物对各细胞24小时和/或72小时连续染毒的靶器官细胞毒性效应。结果：染毒24小时各细胞对各受试物毒性从大到小的顺序分别如下：1）四氯化碳：HepG-2 HEK-293 CCC-HEH-2 SH-SY5Y（基于IC50和IC10值）；2）氟化钠：HEK-293 CCC-HEH-2 HepG-2 A549 SH-SY5Y（基于IC50和IC10值）；3）羟基多巴胺：SH-SY5Y CCC-HEH-2 HEK-293 A549 HepG-2（基于IC50值），SH-SY5Y A549 HEK-293 CCC-HEH-2 HepG-2（基于IC10值）；4）硫酸链霉素：SH-SY5Y HEK-293 CCC-HEH-2 HepG-2 A549（基于IC50值），HEK-293SH-SY5Y CCC-HEH-2 HepG-2 A549（基于IC10值）；5）盐酸阿霉素：HepG-2HEK-293 SH-SY5Y CCC-HEH-2（基于IC50值），CCC-HEH-2 HEK-293HepG-2 SH-SY5Y A54（9基于IC10值）；6）百草枯：HepG-2 HEK-293 SH-SY5YA549 CCC-HEH-2（基于IC50值），HepG-2 HEK-293 A549 CCC-HEH-2 SH-SY5Y（基于IC10值）；7)白消安：HEK-293 HepG-2 CCC-HEH-2 SH-SY5Y A549（基于IC50和IC10值）；8）氯化镉：HEK-293 HepG-2 SH-SY5Y CCC-HEH-2A549（基于IC50值），HEK-293 CCC-HEH-2 HepG-2 SH-SY5YA549（基于IC10值）。72小时染毒各细胞对各受试物毒性从大到小的顺序分别如下：1）百草枯HEK-293 HepG-2 A549 SH-SY5Y CCC-HEH-2（基于IC50和IC10值）；2)白消安：HEK-293 HepG-2 SH-SY5Y A549 CCC-HEH-2（基于IC50和IC10值）。结果表明，除在体内以肺脏为靶器官毒性的百草枯和白消安未显示对肺脏来源的细胞系A549最敏感之外，其余化合物对其对应的靶细胞在IC50和/或IC10上显示良好毒性靶向性。但在本文人源细胞系共培养模型代谢能力的初步验证部分，发现共培养条件下，环磷酰胺（肺脏是其靶器官之一）对A549的IC10值为最小，提示肺脏是环磷酰胺的靶器官。三、人源细胞系共培养模型代谢能力的初步验证比较对乙酰氨基酚和环磷酰胺（两者均是通过代谢物表现活性）对细胞在与HepG-2共培养和单独培养条件下毒性差异性，环磷酰胺诱导HepG-2细胞体外微核试验，探讨HepG-2细胞的代谢功能。人源多器官细胞系共培养条件下，给予受试物对乙酰氨基酚和环磷酰胺（二者毒性主要由其代谢物表现），与细胞A549，HEK-293，SH-SY5Y和CCC-HEH-2单独培养给予相同浓度相应受试物相比，毒性有所增加，IC50和IC10值均低于其单独培养时的值。环磷酰胺可以诱导HepG-2细胞微核率升高。表明肝来源细胞HepG-2存在一定的代谢能力。四、人源多器官细胞系共培养模型在3种纳米物质毒性检测中的初步应用研究选择纳米氧化锌(30nm，50nm，，100nm，常规尺度)，纳米二氧化硅（15nm，30nm，50nm，100nm,常规尺度）和硫化镉量子点（荧光发射波长分别为380nm（晶体粒径为1.6nm），420nm（晶体粒径为1.6nm至7.3nm之间），480nm（晶体粒径为7.3nm），和常规尺度）三种纳米材料，应用IdMOC系统，通过MTT法检测各纳米尺度和其常规尺度材料对共培养体系下各细胞的毒性作用。结果：纳米氧化锌的靶细胞主要是HEK-293细胞，随尺度减小SH-SY5Y和HepG-2细胞对其敏感性增加，提示随着纳米尺度减小，神经和肝脏也可能是氧化锌的靶器官。纳米二氧化硅的靶细胞主要是SH-SY5Y细胞，随尺度减小，HepG-2细胞和HEK-293细胞对其敏感性增加，提示二氧化硅可能对神经系统毒性较大，在低尺度下对肝肾也有损伤。硫化镉量子点对HEK-293细胞毒性最大，其次为HepG-2和SH-SY5Y细胞。提示，硫化镉量子点的靶器官主要为肾脏，其次为肝脏和神经系统。表明三种受试纳米材料细胞毒性大于常规其常规尺度。纳米氧化锌和纳米二氧化硅，随尺度减小对各细胞的毒性增加，同时受影响的器官细胞数增加。荧光发射波长为420nm硫化镉量子点毒性大于荧光发射峰为380和480nm硫化镉量子点，以肾脏细胞最敏感。总结全文结论： 1.首次建立了基于主要毒作用靶器官的人源多器官细胞系共培养模型。 2.初步验证表明，人源多器官细胞系共培养方法可作为毒性靶器官筛选的模型。利用毒物对各细胞系IC50和IC10值比较，能较有效的辨别出毒物的靶器官毒性。IC50值和IC10值结合考虑，更能发现敏感细胞在低剂量下的毒性作用。本研究尚是初步的，所选用的毒性物质有限，对于所选用的细胞系、毒性评价的指标还有待于进一步验证和优化。 3. HepG-2细胞系保留了肝脏细胞一定的代谢活性，能够对试验中所选毒物前体（对乙酰氨基酚和环磷酰胺）进行代谢活化。 4.纳米尺寸的氧化锌、二氧化硅和硫化镉量子点对五种细胞的毒性都分别大于它们常规尺度的相应物质。对于纳米氧化锌和硫化镉量子点最敏感的靶器官可能是肾脏，纳米二氧化硅是神经系统。同时在本实验体系下随着纳米氧化锌和纳米二氧化硅尺度变小靶器官增加，毒性增强。发射峰为420nm的硫化镉量子点比另外两种硫化镉量子点毒性大，对应的靶器官也最多。人源多细胞系共培养模型或许能成为新的研究纳米毒性作用特征的方法。
[Abstract]:As the National Research Council ( NRC ) " The 21st century : a vision and a strategy " , the publication of a landmark study suggests that the toxicological test method will have a revolutionary change . A number of research programs have been developed and implemented by U.S . EPA and NTP . A number of research institutions have also conducted research .
Second , the study on the pathways of toxic action of different chemicals ;
The third is the study of toxicity pathway testing and analysis methods . The subject mainly focuses on the research of targeting test .

In the aspect of targeted test research , methods such as selecting cells that may be toxic target organs or comparative studies using a variety of cells are currently being used . The problem is that a lot of poisons are unknown to their toxic target organs ;
Second , although a variety of ( organ ) cells can be used for comparative studies , it is difficult to determine target organ cells faster and more accurately due to their respective tests under single cell culture conditions , not under the same condition ;
Therefore , the present method is difficult to solve the targeting test problem . Therefore , the present method has the advantages that the human liver cell line and the common target organ human source organ cell line are cultured under the same condition , so as to construct a research idea of the co - culture model of the human source cell line of different organs .

Compared with conventional scale substances , the nano - substance has many new biological effects and characteristics , and presents a new challenge for the evaluation of toxicological safety . It is found that it has no new or different toxic effects compared with conventional scale substances , and reveals that its toxic action is the key to solve the safety evaluation of nano substances .

The aim of this study was to establish a human multi - organ cell line in vitro co - culture model .
To explore the possibility of the in vitro co - culture method of human multi - organ cells as toxic target organs ;
To observe the metabolic function of hepatic cell line under the co - culture system ;
The proposed method was applied to the targeting study of nano - substance .

Establishment of co - culture model of human multi - organ cell line in vitro

Representative cell lines commonly used in target organ toxicity studies , i.e . lung ( A549 ) , lung ( MRC - 5 ) , liver ( HepG - 2 ) , nervous system ( SH - SYY ) , kidney ( HEK - 293 ) and heart ( CCC - HEH - 2 ) , were selected ;
The growth of each cell line under the condition of single culture and co - culture was compared with DMEM and American Type Culture Collection Center ( ATCC ) .

Results : Except MRC - 5 cells grew poorly in DMEM medium , other cells grew well on DMEM medium . Therefore , five cell lines of lung ( A549 ) , liver ( HepG - 2 ) , nervous system ( SH - SYY ) , kidney ( HEK - 293 ) and heart ( CCC - HEH - 2 ) were selected .
The results showed that the growth curves of cultured cells and cultured cells were substantially coincident with the growth curve of cultured cells and cultured cells under co - culture conditions , indicating that the co - culture model of human multi - organ cell lines was successful .

Study on the feasibility of co - culture model of human multi - organ cell line on the screening of toxic target organs

The cytotoxic effect of the test article on the target organ cytotoxic effect on 24 hours and / or 72 hours of each cell was determined by MTT assay .

Results : The order of toxicity of each cell to each test article was as follows : 1 ) carbon tetrachloride : HepG - 2 HEK - 293 CCC - HEH - 2 SH - SYY ( based on IC50 and IC10 ) ;
2 ) Sodium fluoride : HEK - 293 CCC - HEH - 2 HepG - 2 A549 SH - SYY ( based on IC50 and IC10 values ) ;
3 ) hydroxydopamine : SH - SYSYY CCC - HEH - 2 HEK - 293 A549 HepG - 2 ( based on IC50 value ) , SH - SYY A549 HEK - 293 CCC - HEH - 2 HepG - 2 ( based on IC10 value ) ;
4 ) streptomycin sulfate : SH - SY5HEK - 293 CCC - HEH - 2 HepG - 2 A549 ( based on IC50 value ) , HEK - 293SH - SYY CCC - HEH - 2 HepG - 2 A549 ( based on IC10 value ) ;
5 ) doxorubicin hydrochloride : HepG - 2HEK - 293 SH - SYY CCC - HEH - 2 ( based on IC50 value ) , CCC - HEH - 2 HEK - 293HepG - 2 SH - SYY A54 ( 9 based on IC10 value ) ;
6 ) : HepG - 2 HEK - 293 SH - SY5YA549 CCC - HEH - 2 ( based on IC50 value ) , HepG - 2 HEK - 293 , CCC - HEH - 2 SH - SYY ( based on IC10 value ) ;
7 ) Bai Xiaoan : HEK - 293 HepG - 2 CCC - HEH - 2 SH - SYY A549 ( based on IC50 and IC10 values ) ;
8 ) Cadmium chloride : HEK - 293 HepG - 2 SH - SYY CCC - HEH - 2A549 ( based on IC50 value ) , HEK - 293 CCC - HEH - 2 HepG - 2 SH - SY5YA549 ( based on IC10 value ) . The order of toxicity of each cell to each test substance from large to small in 72 hours was as follows : 1 ) 100 - grass - dried HEK - 293 HepG - 2 A549 SH - SYY CCC - HEH - 2 ( based on IC50 and IC10 values ) ;
2 ) Bai Xiaoan : HEK - 293 HepG - 2 SH - SYY A549 CCC - HEH - 2 ( based on IC50 and IC10 values ) .

The results showed that , in addition to the most sensitive lung - derived cell lines A549 , the remaining compounds showed good toxicity targeting on IC50 and / or IC10 . However , under the co - culture conditions , cyclophosphamide ( lung was one of its target organs ) was minimal to the IC10 value of A549 , suggesting that the lung was the target organ of cyclophosphamide .

III . Preliminary verification of metabolic capacity of co - culture model of human source cell line

In vitro micronucleus test of HepG - 2 cells was induced by cyclophosphamide and cyclophosphamide ( both by metabolite expression activity ) , and the metabolism function of HepG - 2 cells was investigated .

Under the co - culture conditions of human multi - organ cell lines , acetaminophen and cyclophosphamide were administered to the test article ( both toxicity was mainly represented by their metabolites ) , and the toxicity increased , the IC50 and IC10 values were lower than those of cell A549 , HEK - 293 , SH - SYY and CCC - HEH - 2 alone .

Study on the Preliminary Application of Co - culture Model of Human Multi - organ Cell Line in the Toxicity Test of Three Kinds of Nanomaterials

Nano - zinc oxide ( 30 nm , 50 nm , 100 nm , regular scale ) , nano - silica ( 15 nm , 30 nm , 50 nm , 100 nm , conventional scale ) and cadmium sulfide quantum dots ( fluorescence emission wavelength of 380 nm ( crystal grain size 1.6 nm ) , 420 nm ( crystal grain size of 1.6 nm to 7.3 nm ) , 480 nm ( crystal grain size of 7.3 nm ) and conventional scale ) were selected .

Results : The target cells of nano - zinc oxide were mainly HEK - 293 cells . The sensitivity of the cells was increased with the decrease of the size of S H - SYY and HepG - 2 cells .

The results showed that the cytotoxicity of three kinds of nano - materials was more than that of conventional nano - ZnO and nano - silica . The toxicity of nano - zinc oxide and nano - silica was increased with the decrease of the scale , while the number of the affected organ cells increased . The toxicity of the fluorescence emission was 420 nm , and the quantum dot toxicity was more than 380 and 480 nm .

Conclusion of full - text :

1 . A co - culture model of human multi - organ cell lines based on target organ of major toxic effect was established for the first time .

2 . Preliminary verification shows that the method of co - culture of human multi - organ cell lines can be used as a model for the screening of toxic target organs . The IC50 value and IC10 value of each cell line are compared with the IC50 value and IC10 value . The IC50 value and IC10 value are considered to be more effective . The toxicity of sensitive cells under low dose can be found more effectively . The present study is still preliminary , and the toxicity evaluation index is still to be further verified and optimized for the selected cell line .

3 . HepG - 2 cell line retains a certain metabolic activity of the liver cells and is capable of metabolic activation of the drug precursors selected in the assay ( acetaminophen and cyclophosphamide ) .

4 . The toxicity of nano - sized zinc oxide , silicon dioxide and cadmium sulfide quantum dots to five cells is larger than that of their conventional scales . The most sensitive target organs for the nano - zinc oxide and cadmium sulfide quantum dots may be the kidney , and the nano - silica is the nervous system . At the same time , the quantum dots of cadmium sulfide with the emission peak of 420 nm are more toxic than the other two cadmium sulfide quantum dots , and the corresponding target organs are also the most . The human multi - cell line co - culture model may be a new method to study the nano - toxicity .

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

【参考文献】