本文選題：鎘 + 肝細(xì)胞�。� 參考：《南京師范大學(xué)》2006年碩士論文

【摘要】：本文通過原代培養(yǎng)新生乳鼠肝細(xì)胞模型，運(yùn)用細(xì)胞培養(yǎng)、熒光分光光度計(jì)、激光共聚焦顯微鏡和生化分析等生物學(xué)技術(shù)，系統(tǒng)研究了鎘負(fù)荷誘發(fā)肝細(xì)胞損傷以及肝細(xì)胞存活性、胞內(nèi)MDA含量、Ca~(2+)-Mg~(2+)-ATPase和Na~+-K~+-ATPase活性以及胞內(nèi)Ca_i~(2+)含量的變化，細(xì)胞培養(yǎng)上清液中LDH及AST的活性、白蛋白及Ca~(2+)含量變化，較為深入地探討了鎘導(dǎo)致的肝細(xì)胞胞內(nèi)Ca_i~(2+)穩(wěn)態(tài)失衡的可能機(jī)制；同時(shí)，還觀察了硒干預(yù)鎘誘發(fā)鼠肝細(xì)胞存活及其MDA含量和培養(yǎng)液中LDH活性的變化以及硒對(duì)鎘暴露肝細(xì)胞內(nèi)Ca~(2+)穩(wěn)態(tài)變化的影響。結(jié)果如下： 1 新生乳鼠肝細(xì)胞原代培養(yǎng)方法的建立 無(wú)菌分離新生乳鼠肝臟組織，采用膠原蛋白酶消化法分離肝細(xì)胞并使用無(wú)血清培養(yǎng)液HepatoZYME-SFM對(duì)其進(jìn)行體外培養(yǎng)，通過形態(tài)學(xué)及臺(tái)盼藍(lán)染色法鑒定，分離的肝細(xì)胞細(xì)胞完整，呈球形或橢球形，存活率在90％以上。說明該方法是比較理想的肝細(xì)胞培養(yǎng)法，為科學(xué)研究提供了細(xì)胞模型平臺(tái)。 2 鎘誘發(fā)肝細(xì)胞毒性及其胞內(nèi)游離Ca~(2+)變化研究 原代培養(yǎng)乳鼠肝細(xì)胞，分為三個(gè)鎘處理組和正常對(duì)照組，分別加入5、10、25μM CdCl_2染毒以及等量D-hank's溶液。結(jié)果顯示：實(shí)驗(yàn)后12h，CdCl_2導(dǎo)致肝細(xì)胞存活劑量依賴性下降；培養(yǎng)上清液LDH和AST的活力升高或顯著升高，，并且白蛋白含量大大降低。在CdCl_2暴露12h后，細(xì)胞內(nèi)MDA生成量增加處于較高水平，培養(yǎng)上清液Ca~(2+)含量顯著下降。進(jìn)一步觀察顯示，在CdCl_2暴露12h和24h后，肝細(xì)胞[Ca~(2+)]_i顯著升高：胞內(nèi)Ca~(2+)-Mg~(2+)-ATP酶及Na~+-K~+-ATP酶活力12h與對(duì)照組無(wú)明顯差異，至24h下降或明顯下降。提示：CdCl_2通過誘導(dǎo)肝細(xì)胞脂質(zhì)過氧化和損傷，引發(fā)細(xì)胞存活下降和功能障礙而導(dǎo)致強(qiáng)烈毒性；鎘暴露引發(fā)肝細(xì)胞[Ca~(2+)]_i異常增加可能是細(xì)胞損傷發(fā)展的重要機(jī)制，部分[Ca~(2+)]_i升高與胞外Ca~(2+)的進(jìn)入有密切關(guān)系。 3 鎘誘發(fā)肝細(xì)胞胞內(nèi)游離Ca~(2+)堆積的機(jī)制探討 原代培養(yǎng)乳鼠肝細(xì)胞，用Fluo-3／AM對(duì)生長(zhǎng)良好的原代培養(yǎng)肝細(xì)胞閉光孵育，然后清洗并重懸肝細(xì)胞于有鈣HBSS或無(wú)鈣HBSS中，同時(shí)設(shè)計(jì)加或不加2-APB抑制劑(100μM)的情況，用激光共聚焦顯微鏡觀察CdCl_2(10μM)急性暴露引起的胞內(nèi)鈣離子熒光強(qiáng)度變化。結(jié)果顯示：在僅有鈣的HBSS中，CdCl_2誘導(dǎo)肝細(xì)胞[Ca~(2+)]_i明顯上升，包括開始的緩慢上升期和隨后的持續(xù)升高期；在有鈣的HBSS+2-APB中，CdCl_2僅引起一個(gè)無(wú)開始上升期的緩緩升高；在無(wú)鈣的
[Abstract]:In this paper, the hepatocyte injury induced by cadmium loading and the viability of hepatocytes were systematically studied by using biological techniques such as cell culture, fluorescence spectrophotometer, laser confocal microscopy and biochemical analysis. The changes of intracellular MDA content, the activities of Ca ~ (2) -mg ~ (2) -ATPase and Na ~ -K ~ (-ATPase), the contents of Ca ~ (2), LDH and AST in supernatant of cell culture, and the changes of albumin and Ca ~ (2) contents were observed. The possible mechanism of cai ~ (2) homeostasis in hepatocytes induced by cadmium was discussed. The effects of selenium on cadmium induced hepatocyte survival and the changes of MDA content and LDH activity in culture medium were also observed. The effects of selenium on the changes of Ca ~ (2) homeostasis in hepatocytes exposed to cadmium were also observed. The results are as follows: 1 Establishment of primary culture method of neonatal rat hepatocytes Aseptic isolation of liver tissue from newborn rat Hepatocytes were isolated by collagenase digestion and cultured in vitro with serum-free medium HepatoZYME-SFM. The isolated hepatocytes were identified by morphology and trypan blue staining. The isolated hepatocytes were spherical or ellipsoid, and the survival rate was over 90%. It shows that this method is an ideal method for hepatocyte culture. 2 cadmium induced hepatocytotoxicity and its intracellular free Ca ~ (2) changes. Primary culture of neonatal rat hepatocytes, The rats were divided into three cadmium treatment groups and normal control group, which were treated with 5 ~ 10 ~ 10 渭 m CD _ Cl _ 2 and D-hankos solution respectively. The results showed that CDCl2 induced a dose-dependent decrease in the survival of hepatocytes, the activities of LDH and AST in the supernatant increased or increased significantly, and the albumin content decreased significantly. After exposure to CDCl2 for 12 h, the content of MDA increased at a high level, and the content of Ca2 in culture supernatant decreased significantly. Further observation showed that after CDCl2 exposure for 12 h and 24 h, [Ca2] tii in hepatocytes increased significantly: there was no significant difference in intracellular Ca ~ (2) -Mg- (2) -ATPase activity and Na ~ -K ~ -ATPase activity at 24 h after exposure to CDCl2, but decreased or decreased significantly at 24 h. The results suggest that the increase of [Ca ~ (2)] I in hepatocytes induced by cadmium exposure may be an important mechanism for the development of hepatocyte injury, which is caused by inducing lipid peroxidation and injury of hepatocytes, resulting in the decrease of cell survival and dysfunction of hepatocyte function, and the abnormal increase of [Ca ~ (2)] I in hepatocytes induced by cadmium exposure. The increase of partial [Ca ~ (2)] I is closely related to the entry of extracellular Ca ~ (2). 3 the mechanism of cadmium induced intracellular free Ca ~ (2) accumulation in hepatocytes Primary culture of neonatal rat hepatocytes, The cultured primary cultured hepatocytes were incubated with Fluo-3 / AM in closed light, then washed and suspended in the presence or absence of calcium HBSS, and the condition of adding or not adding 2-APB inhibitor (100 渭 M) was designed. The changes of intracellular calcium fluorescence intensity induced by CDCl2 (10 渭 M) acute exposure were observed by laser confocal microscopy. The results showed that CDCL _ 2 induced a marked increase of [Ca ~ (2)] _ I in the calcium-only HBSS, including the initial slow ascending period and the subsequent continuous rising period; in the calcium HBSS _ 2-APB, CDCL _ 2 only caused a slow rise in the non-initial ascendant phase; in the calcium free HBSS _ 2-APB, CDCL _ 2 induced a slow rise; in the calcium free HBSS _ 2-APB, CDCL _ 2 induced a slow rise in the liver cell [Ca ~ (2)].
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 袁燕;鎘對(duì)大鼠大腦皮質(zhì)神經(jīng)細(xì)胞毒性損傷的機(jī)制[D];揚(yáng)州大學(xué);2012年

相關(guān)碩士學(xué)位論文 前2條

1 張健;缺硒對(duì)雞腦組織損傷的研究[D];東北農(nóng)業(yè)大學(xué);2008年

2 孫婭;鈣離子在鎘致體外培養(yǎng)大鼠大腦皮質(zhì)神經(jīng)細(xì)胞凋亡中的作用[D];揚(yáng)州大學(xué);2011年

本文編號(hào)：2077033