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

【摘要】：隨著美國國家研究委員會（NRC)“21世紀(jì)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(bào)告的發(fā)表，預(yù)示著毒理學(xué)實(shí)驗(yàn)方法將發(fā)生革命性的改變。基于人體細(xì)胞的靶向性測試和毒性通路測試將取代以實(shí)驗(yàn)動物為中心的毒性評價(jià)方法成為當(dāng)前毒理學(xué)的研究熱點(diǎn)和發(fā)展趨勢。美國EPA和NTP制定和實(shí)施了多項(xiàng)研究計(jì)劃，不少國家的研究機(jī)構(gòu)也開展了研究。當(dāng)前的研究思路主要是從三個(gè)方面展開：一是不同人體細(xì)胞系毒性的比較，試圖為靶向性測試提供依據(jù)；二是不同化學(xué)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(jìn)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(xì)胞，或采用多種細(xì)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(xì)胞的比較研究，但由于均是單一細(xì)胞培養(yǎng)條件下的分別測試，不是在同一條件下進(jìn)行的，與體內(nèi)情況不符，難以較快、較準(zhǔn)確地確定靶器官細(xì)胞；三是很多毒物進(jìn)入體內(nèi)后需要經(jīng)過肝臟和其它器官的代謝轉(zhuǎn)化后才產(chǎn)生毒性或者消除毒性，現(xiàn)行的應(yīng)用添加肝S9形成活化系統(tǒng)的方法不足以模擬體內(nèi)的真實(shí)情況。此外體內(nèi)細(xì)胞間通過微環(huán)境的相互作用狀況在單一細(xì)胞培養(yǎng)中也不能實(shí)現(xiàn)。因此，現(xiàn)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(xì)胞系和常見靶器官人源器官細(xì)胞系，在同一條件下共培養(yǎng)，以構(gòu)建一個(gè)不同器官人源細(xì)胞系共培養(yǎng)模型的研究思路。 納米物質(zhì)與常規(guī)尺度物質(zhì)相比，由于其特殊的理化性質(zhì)，出現(xiàn)了很多新的生物學(xué)效應(yīng)和特性，給毒理學(xué)安全性評價(jià)提出了新挑戰(zhàn)。充分發(fā)現(xiàn)其與常規(guī)尺度物質(zhì)相比有無新的或不同的毒效應(yīng)、揭示其毒作用特征是解決納米性物質(zhì)安全性評價(jià)的關(guān)鍵。研究納米物質(zhì)的毒作用靶器官，比較其與常規(guī)尺度物質(zhì)的異同，可為納米性物質(zhì)安全性評價(jià)問題的解決提供科學(xué)依據(jù)。 本研究的目的是建立人源多器官細(xì)胞系體外共培養(yǎng)模型；探討人源多器官細(xì)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(xì)胞系在該共培養(yǎng)體系下的代謝作用；將所建方法初步應(yīng)用于納米物質(zhì)的靶向性研究。 一、人源多器官細(xì)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(xì)胞系，即肺臟(A549)、肺臟（MRC-5）、肝臟(HepG-2)、神經(jīng)系統(tǒng)(SH-SY5Y)、腎臟(HEK-293)和心臟(CCC-HEH-2)六種細(xì)胞；采用市售的48孔培養(yǎng)板經(jīng)自制改造成共培養(yǎng)板，即以5孔為一組，在一組內(nèi)的各孔壁上打洞，使一組內(nèi)各孔之間的培養(yǎng)基能相互流通。將5種細(xì)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(xì)胞系生長情況。同時(shí)，比較了應(yīng)用DMEM和美國典型培養(yǎng)物保藏中心（ATCC）推薦培養(yǎng)基對細(xì)胞的生長效果。 結(jié)果：除MRC-5細(xì)胞在DMEM培養(yǎng)基中生長不佳外，其它細(xì)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(jīng)系統(tǒng)(SH-SY5Y)、腎臟(HEK-293)和心臟(CCC-HEH-2)五種細(xì)胞系；以DMEM作為培養(yǎng)基。結(jié)果顯示，各種細(xì)胞在共培養(yǎng)條件下生長良好，共培養(yǎng)細(xì)胞和單獨(dú)培養(yǎng)細(xì)胞的生長曲線基本重合，表明所建的人源多器官細(xì)胞系共培養(yǎng)模型是成功的。 二、人源多器官細(xì)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(yīng)用MTT法檢測受試物對各細(xì)胞24小時(shí)和/或72小時(shí)連續(xù)染毒的靶器官細(xì)胞毒性效應(yīng)。 結(jié)果：染毒24小時(shí)各細(xì)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(shí)染毒各細(xì)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(jié)果表明，除在體內(nèi)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(xì)胞系A(chǔ)549最敏感之外，其余化合物對其對應(yīng)的靶細(xì)胞在IC50和/或IC10上顯示良好毒性靶向性。但在本文人源細(xì)胞系共培養(yǎng)模型代謝能力的初步驗(yàn)證部分，發(fā)現(xiàn)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(xì)胞系共培養(yǎng)模型代謝能力的初步驗(yàn)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(xiàn)活性）對細(xì)胞在與HepG-2共培養(yǎng)和單獨(dú)培養(yǎng)條件下毒性差異性，環(huán)磷酰胺誘導(dǎo)HepG-2細(xì)胞體外微核試驗(yàn)，探討HepG-2細(xì)胞的代謝功能。 人源多器官細(xì)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(xiàn)），與細(xì)胞A549，HEK-293，SH-SY5Y和CCC-HEH-2單獨(dú)培養(yǎng)給予相同濃度相應(yīng)受試物相比，毒性有所增加，IC50和IC10值均低于其單獨(dú)培養(yǎng)時(shí)的值。環(huán)磷酰胺可以誘導(dǎo)HepG-2細(xì)胞微核率升高。表明肝來源細(xì)胞HepG-2存在一定的代謝能力。 四、人源多器官細(xì)胞系共培養(yǎng)模型在3種納米物質(zhì)毒性檢測中的初步應(yīng)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(diǎn)（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(yīng)用IdMOC系統(tǒng)，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(xì)胞的毒性作用。 結(jié)果：納米氧化鋅的靶細(xì)胞主要是HEK-293細(xì)胞，隨尺度減小SH-SY5Y和HepG-2細(xì)胞對其敏感性增加，提示隨著納米尺度減小，神經(jīng)和肝臟也可能是氧化鋅的靶器官。納米二氧化硅的靶細(xì)胞主要是SH-SY5Y細(xì)胞，隨尺度減小，HepG-2細(xì)胞和HEK-293細(xì)胞對其敏感性增加，提示二氧化硅可能對神經(jīng)系統(tǒng)毒性較大，在低尺度下對肝腎也有損傷。硫化鎘量子點(diǎn)對HEK-293細(xì)胞毒性最大，其次為HepG-2和SH-SY5Y細(xì)胞。提示，硫化鎘量子點(diǎn)的靶器官主要為腎臟，其次為肝臟和神經(jīng)系統(tǒng)。 表明三種受試納米材料細(xì)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(xì)胞的毒性增加，同時(shí)受影響的器官細(xì)胞數(shù)增加。熒光發(fā)射波長為420nm硫化鎘量子點(diǎn)毒性大于熒光發(fā)射峰為380和480nm硫化鎘量子點(diǎn)，以腎臟細(xì)胞最敏感。 總結(jié)全文結(jié)論： 1.首次建立了基于主要毒作用靶器官的人源多器官細(xì)胞系共培養(yǎng)模型。 2.初步驗(yàn)證表明，人源多器官細(xì)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(xì)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(jié)合考慮，更能發(fā)現(xiàn)敏感細(xì)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(zhì)有限，對于所選用的細(xì)胞系、毒性評價(jià)的指標(biāo)還有待于進(jìn)一步驗(yàn)證和優(yōu)化。 3. HepG-2細(xì)胞系保留了肝臟細(xì)胞一定的代謝活性，能夠?qū)υ囼?yàn)中所選毒物前體（對乙酰氨基酚和環(huán)磷酰胺）進(jìn)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(diǎn)對五種細(xì)胞的毒性都分別大于它們常規(guī)尺度的相應(yīng)物質(zhì)。對于納米氧化鋅和硫化鎘量子點(diǎn)最敏感的靶器官可能是腎臟，納米二氧化硅是神經(jīng)系統(tǒng)。同時(shí)在本實(shí)驗(yàn)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(qiáng)。發(fā)射峰為420nm的硫化鎘量子點(diǎn)比另外兩種硫化鎘量子點(diǎn)毒性大，對應(yīng)的靶器官也最多。人源多細(xì)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(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R114

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