發(fā)布時間：2018-05-11 08:50

  本文選題：人源細胞系 + 共培養(yǎng)　； 參考：《第二軍醫(yī)大學》2014年博士論文

【摘要】：隨著美國國家研究委員會（NRC)“21世紀的毒性測試：愿景與策略（Toxicitytesting in the21st century: a vision and a strategy)”這一具有里程碑意義研究報告的發(fā)表，預示著毒理學實驗方法將發(fā)生革命性的改變。基于人體細胞的靶向性測試和毒性通路測試將取代以實驗動物為中心的毒性評價方法成為當前毒理學的研究熱點和發(fā)展趨勢。美國EPA和NTP制定和實施了多項研究計劃，不少國家的研究機構也開展了研究。當前的研究思路主要是從三個方面展開：一是不同人體細胞系毒性的比較，試圖為靶向性測試提供依據；二是不同化學物毒性作用通路的研究；三是毒性通路測試和分析方法的研究。本課題主要針對靶向性測試問題進行研究。 在靶向性測試研究方面，目前采用選擇可能毒性靶器官的細胞，或采用多種細胞的比較研究等方法。其存在的問題：一是很多毒物未知其毒性靶器官；二是盡管可采用多種（器官）細胞的比較研究，但由于均是單一細胞培養(yǎng)條件下的分別測試，不是在同一條件下進行的，與體內情況不符，難以較快、較準確地確定靶器官細胞；三是很多毒物進入體內后需要經過肝臟和其它器官的代謝轉化后才產生毒性或者消除毒性，現行的應用添加肝S9形成活化系統的方法不足以模擬體內的真實情況。此外體內細胞間通過微環(huán)境的相互作用狀況在單一細胞培養(yǎng)中也不能實現。因此，現行方法難以較好解決靶向性測試問題。為此，本研究提出選擇以人肝細胞系和常見靶器官人源器官細胞系，在同一條件下共培養(yǎng)，以構建一個不同器官人源細胞系共培養(yǎng)模型的研究思路。 納米物質與常規(guī)尺度物質相比，由于其特殊的理化性質，出現了很多新的生物學效應和特性，給毒理學安全性評價提出了新挑戰(zhàn)。充分發(fā)現其與常規(guī)尺度物質相比有無新的或不同的毒效應、揭示其毒作用特征是解決納米性物質安全性評價的關鍵。研究納米物質的毒作用靶器官，比較其與常規(guī)尺度物質的異同，可為納米性物質安全性評價問題的解決提供科學依據。 本研究的目的是建立人源多器官細胞系體外共培養(yǎng)模型；探討人源多器官細胞體外共培養(yǎng)方法作為毒性靶器官篩選的可能性；觀察肝來源細胞系在該共培養(yǎng)體系下的代謝作用；將所建方法初步應用于納米物質的靶向性研究。 一、人源多器官細胞系體外共培養(yǎng)模型的建立 選擇常用于靶器官毒性研究的代表細胞系，即肺臟(A549)、肺臟（MRC-5）、肝臟(HepG-2)、神經系統(SH-SY5Y)、腎臟(HEK-293)和心臟(CCC-HEH-2)六種細胞；采用市售的48孔培養(yǎng)板經自制改造成共培養(yǎng)板，即以5孔為一組，在一組內的各孔壁上打洞，使一組內各孔之間的培養(yǎng)基能相互流通。將5種細胞分別種植于5孔中培養(yǎng)，比較單培養(yǎng)條件下和共培養(yǎng)條件下各細胞系生長情況。同時，比較了應用DMEM和美國典型培養(yǎng)物保藏中心（ATCC）推薦培養(yǎng)基對細胞的生長效果。 結果：除MRC-5細胞在DMEM培養(yǎng)基中生長不佳外，其它細胞在DMEM培養(yǎng)基上均生長良好。因此，最終選定肺臟（A549)、肝臟(HepG-2)、神經系統(SH-SY5Y)、腎臟(HEK-293)和心臟(CCC-HEH-2)五種細胞系；以DMEM作為培養(yǎng)基。結果顯示，各種細胞在共培養(yǎng)條件下生長良好，共培養(yǎng)細胞和單獨培養(yǎng)細胞的生長曲線基本重合，表明所建的人源多器官細胞系共培養(yǎng)模型是成功的。 二、人源多器官細胞系共培養(yǎng)模型在毒性靶器官篩選上的可行性研究 選用已知毒性的四氯化碳、氯化鎘、氟化鈉、鹽酸阿霉素、硫酸鏈霉素、羥基多巴胺、百草枯和白消安為受試物，應用MTT法檢測受試物對各細胞24小時和/或72小時連續(xù)染毒的靶器官細胞毒性效應。 結果：染毒24小時各細胞對各受試物毒性從大到小的順序分別如下：1）四氯化碳：HepG-2 HEK-293 CCC-HEH-2 SH-SY5Y（基于IC50和IC10值）；2）氟化鈉：HEK-293 CCC-HEH-2 HepG-2 A549 SH-SY5Y（基于IC50和IC10值）；3）羥基多巴胺：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）硫酸鏈霉素：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）鹽酸阿霉素：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）氯化鎘：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小時染毒各細胞對各受試物毒性從大到小的順序分別如下：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）。 結果表明，除在體內以肺臟為靶器官毒性的百草枯和白消安未顯示對肺臟來源的細胞系A549最敏感之外，其余化合物對其對應的靶細胞在IC50和/或IC10上顯示良好毒性靶向性。但在本文人源細胞系共培養(yǎng)模型代謝能力的初步驗證部分，發(fā)現共培養(yǎng)條件下，環(huán)磷酰胺（肺臟是其靶器官之一）對A549的IC10值為最小，提示肺臟是環(huán)磷酰胺的靶器官。 三、人源細胞系共培養(yǎng)模型代謝能力的初步驗證 比較對乙酰氨基酚和環(huán)磷酰胺（兩者均是通過代謝物表現活性）對細胞在與HepG-2共培養(yǎng)和單獨培養(yǎng)條件下毒性差異性，環(huán)磷酰胺誘導HepG-2細胞體外微核試驗，探討HepG-2細胞的代謝功能。 人源多器官細胞系共培養(yǎng)條件下，給予受試物對乙酰氨基酚和環(huán)磷酰胺（二者毒性主要由其代謝物表現），與細胞A549，HEK-293，SH-SY5Y和CCC-HEH-2單獨培養(yǎng)給予相同濃度相應受試物相比，毒性有所增加，IC50和IC10值均低于其單獨培養(yǎng)時的值。環(huán)磷酰胺可以誘導HepG-2細胞微核率升高。表明肝來源細胞HepG-2存在一定的代謝能力。 四、人源多器官細胞系共培養(yǎng)模型在3種納米物質毒性檢測中的初步應用研究 選擇納米氧化鋅(30nm，50nm，，100nm，常規(guī)尺度)，納米二氧化硅（15nm，30nm，50nm，100nm,常規(guī)尺度）和硫化鎘量子點（熒光發(fā)射波長分別為380nm（晶體粒徑為1.6nm），420nm（晶體粒徑為1.6nm至7.3nm之間），480nm（晶體粒徑為7.3nm），和常規(guī)尺度）三種納米材料，應用IdMOC系統，通過MTT法檢測各納米尺度和其常規(guī)尺度材料對共培養(yǎng)體系下各細胞的毒性作用。 結果：納米氧化鋅的靶細胞主要是HEK-293細胞，隨尺度減小SH-SY5Y和HepG-2細胞對其敏感性增加，提示隨著納米尺度減小，神經和肝臟也可能是氧化鋅的靶器官。納米二氧化硅的靶細胞主要是SH-SY5Y細胞，隨尺度減小，HepG-2細胞和HEK-293細胞對其敏感性增加，提示二氧化硅可能對神經系統毒性較大，在低尺度下對肝腎也有損傷。硫化鎘量子點對HEK-293細胞毒性最大，其次為HepG-2和SH-SY5Y細胞。提示，硫化鎘量子點的靶器官主要為腎臟，其次為肝臟和神經系統。 表明三種受試納米材料細胞毒性大于常規(guī)其常規(guī)尺度。納米氧化鋅和納米二氧化硅，隨尺度減小對各細胞的毒性增加，同時受影響的器官細胞數增加。熒光發(fā)射波長為420nm硫化鎘量子點毒性大于熒光發(fā)射峰為380和480nm硫化鎘量子點，以腎臟細胞最敏感。 總結全文結論： 1.首次建立了基于主要毒作用靶器官的人源多器官細胞系共培養(yǎng)模型。 2.初步驗證表明，人源多器官細胞系共培養(yǎng)方法可作為毒性靶器官篩選的模型。利用毒物對各細胞系IC50和IC10值比較，能較有效的辨別出毒物的靶器官毒性。IC50值和IC10值結合考慮，更能發(fā)現敏感細胞在低劑量下的毒性作用。本研究尚是初步的，所選用的毒性物質有限，對于所選用的細胞系、毒性評價的指標還有待于進一步驗證和優(yōu)化。 3. HepG-2細胞系保留了肝臟細胞一定的代謝活性，能夠對試驗中所選毒物前體（對乙酰氨基酚和環(huán)磷酰胺）進行代謝活化。 4.納米尺寸的氧化鋅、二氧化硅和硫化鎘量子點對五種細胞的毒性都分別大于它們常規(guī)尺度的相應物質。對于納米氧化鋅和硫化鎘量子點最敏感的靶器官可能是腎臟，納米二氧化硅是神經系統。同時在本實驗體系下隨著納米氧化鋅和納米二氧化硅尺度變小靶器官增加，毒性增強。發(fā)射峰為420nm的硫化鎘量子點比另外兩種硫化鎘量子點毒性大，對應的靶器官也最多。人源多細胞系共培養(yǎng)模型或許能成為新的研究納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 .

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R114

【參考文獻】

相關期刊論文 前10條

1 楊鳳霞;劉其麗;畢磊;;納米氧化鋅的應用綜述[J];安徽化工;2006年01期

2 孫景萍;王素華;高艷榮;;納米級和微米級二氧化硅粉塵對大鼠肺損傷的研究[J];包頭醫(yī)學院學報;2011年04期

3 馬華智;施暢;石富江;孫艷麗;于永生;吳純啟;王全軍;丁日高;;多遺傳學終點的遺傳毒性試驗組合的建立[J];癌變.畸變.突變;2013年06期

4 王久亮,劉寬,秦秀娟,邵光杰;納米氧化鋅的應用研究展望[J];哈爾濱工業(yè)大學學報;2004年02期

5 施暢,廖明陽;環(huán)磷酰胺與異環(huán)磷酰胺在體內的代謝特點分析[J];解放軍藥學學報;2001年02期

6 胡爭艷;孫珍;張軼;吳仁安;鄒漢法;;納米二氧化硅影響人肺癌細胞的定量糖蛋白質組學研究[J];化學學報;2012年19期

7 蔣龍川;牛雙蛟;尹荔松;何捷嫻;;納米SiO_2的制備及應用研究進展[J];化工新型材料;2013年07期

8 蔣利軍;符新;李光;董經緯;;納米二氧化硅復合材料的應用研究進展[J];熱帶農業(yè)科學;2007年06期

9 段佳;陳祥貴;帥培強;饒夙;劉振平;;HepG2和L5178Y細胞彗星試驗檢測甲磺酸甲酯和環(huán)磷酰胺遺傳毒性的比較研究[J];西華大學學報(自然科學版);2011年01期

10 余強;李坤剛;文興;李君;王玉秋;;納米硫化鎘量子點細胞毒性作用機制[J];生態(tài)毒理學報;2009年04期

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