本文選題：乙肝　切入點(diǎn)：原代肝細(xì)胞　出處：《北京協(xié)和醫(yī)學(xué)院》2013年博士論文　論文類型：學(xué)位論文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)的感染是目前世界范圍內(nèi)最為嚴(yán)重的公共衛(wèi)生問題之一。根據(jù)世界衛(wèi)生組織統(tǒng)計,截至目前為止,每年由于急性或慢性的HBV感染造成的死亡人數(shù)達(dá)到60萬左右。 在現(xiàn)有的HBV體外感染研究中,原代樹烝肝實(shí)質(zhì)細(xì)胞(primary tupaia hepatocytes, PTH)是一種被成功使用的模型。盡管如此,該系統(tǒng)仍然存在著相當(dāng)?shù)木窒扌浴ｓw內(nèi)外培養(yǎng)環(huán)境的差異使得從肝臟組織中分離出來的原代肝細(xì)胞僅能部分維持原有的HBV易感性,而且隨著體外培養(yǎng)時間的增加往往會迅速喪失。此外,實(shí)驗(yàn)動物的個體差別和分離技術(shù)的復(fù)雜性等因素也會增加不同批次分離的原代細(xì)胞的差異。這些問題導(dǎo)致了PTH作為HBV研究模型顯得不夠高效和不夠穩(wěn)定,降低了檢測結(jié)果的信噪比,加大了分析難度,使得一些真正影響感染過程的重要因素在實(shí)際研究過程中變得難以被發(fā)現(xiàn)。另一方面,短暫的易感性維持時間也限制了許多研究方法的應(yīng)用。 我們采用樹烝的肝星狀細(xì)胞(hepatic stellate cells,HSC)作為支持細(xì)胞,建立了一種新的PTH共培養(yǎng)模型。我們發(fā)現(xiàn),HBV在這種共培養(yǎng)系統(tǒng)上,感染得到了顯著增強(qiáng)。除了HSC之外,使用樹烝皮膚來源的原代成纖維細(xì)胞(primary tupaia fibroblast, PTF)建立的共培養(yǎng)系統(tǒng)也能對HBV的感染起到類似的促進(jìn)效果。與之相一致的是,共培養(yǎng)系統(tǒng)對肝細(xì)胞的部分生理功能,包括細(xì)胞色素酶P450(CYP450)超家族成員的活性等方面也起到了較為明顯的增強(qiáng)效果。這樣的共培養(yǎng)系統(tǒng)很好地解決了原有模型存在的低效和不穩(wěn)定的問題,有利于利用該模型揭示更多感染過程中的關(guān)鍵因素。 除了促進(jìn)感染之外,我們還發(fā)現(xiàn),共培養(yǎng)系統(tǒng)能夠顯著地延長體外培養(yǎng)的原代肝實(shí)質(zhì)細(xì)胞對HBV易感性的維持時問。這一優(yōu)勢拓寬了PTH作為HBV感染模型在實(shí)際研究中的應(yīng)用。 我們進(jìn)一步研究了共培養(yǎng)系統(tǒng)影響肝實(shí)質(zhì)細(xì)胞的相關(guān)機(jī)制,發(fā)現(xiàn)支持細(xì)胞對肝細(xì)胞的影響需要通過細(xì)胞間的直接接觸來實(shí)現(xiàn),并且兩種細(xì)胞接觸的時間點(diǎn)也會對最終促進(jìn)感染的程度產(chǎn)生影響。 我們通過表達(dá)水平、分布以及功能等多方面的檢測,發(fā)現(xiàn)新鑒定出的HBV受體,鈉離子-�；悄懰峁厕D(zhuǎn)運(yùn)蛋白(NTCP)在共培養(yǎng)系統(tǒng)中的肝細(xì)胞上表達(dá)沒有發(fā)生明顯的變化。另一方面,我們發(fā)現(xiàn)細(xì)胞內(nèi)與HBV復(fù)制相關(guān)的一些肝富集轉(zhuǎn)錄因子(liver enriched transcription factor, LETF)的表達(dá)量出現(xiàn)了上調(diào)。研究發(fā)現(xiàn)其中的兩種,HNF4A和HLF,在通過RNA干擾下調(diào)其表達(dá)水平后,HBV的感染效率也出現(xiàn)了明顯的下降。這些結(jié)果顯示,共培養(yǎng)系統(tǒng)中HBV的感染很有可能是在病毒的復(fù)制與生物合成等環(huán)節(jié)受到了增強(qiáng)。 我們對共培養(yǎng)系統(tǒng)中的肝細(xì)胞作了進(jìn)一步研究,發(fā)現(xiàn)兩種與細(xì)胞生理狀態(tài)密切相關(guān)的標(biāo)記蛋白,MRP2(multidrug resistance protein2)和ZO-1(zonula occludens protein1)在表達(dá)和分布上呈現(xiàn)出與體內(nèi)肝臟組織中的肝細(xì)胞類似的特征,而這些特征是單培養(yǎng)的肝細(xì)胞所不具備的。這說明共培養(yǎng)系統(tǒng)中的肝細(xì)胞在生理特征上更加接近于體內(nèi)微環(huán)境中的細(xì)胞。 因此,我們建立的原代肝細(xì)胞的共培養(yǎng)系統(tǒng),能夠作為一種體外研究HBV感染更加高效的模型,并且能夠模擬更為接近體內(nèi)生理狀態(tài)下發(fā)生的HBV感染過程。這對于我們進(jìn)一步揭示HBV感染和致病的相關(guān)機(jī)制有著十分重要的意義。
[Abstract]:The infection of hepatitis B virus (HBV) is one of the most serious public health problems in the world. According to WHO statistics, so far, the number of deaths caused by acute or chronic HBV infection has reached 600 thousand so far.
In the study of HBV infection in vitro. The primary hepatocytes (primary tree, Tupaia hepatocytes, PTH) is a successful model used. However, this system still has considerable limitations. In vivo and in vitro culture environment makes the difference of primary hepatocytes isolated from the liver tissues can only maintain the original HBV susceptibility, and it increases with time in vitro will be lost. In addition, individual differences and separation technology of experimental animal and other factors will also increase the complexity of different batches of isolated primary cells. The difference in these problems led to PTH HBV as the research model is not efficient and stable enough the test results, reduce the signal-to-noise ratio, increase the difficulty of analysis, some important factors that really affect the infection process in the course of practical research has become difficult to be found. On the other hand, short The duration of susceptibility also restricts the application of many research methods.
We use the hepatic stellate cells (hepatic, the stellate cells tree, HSC) as support cells, a new PTH co culture model. We found that HBV co cultured in this system, the infection has been significantly enhanced. In addition to HSC, primary fibroblasts, skin source (using a tree primary Tupaia fibroblast, PTF) to establish a co culture system can promote a similar effect on HBV infection. In line with this, co culture system on the part of the physiological function of liver cells, including cytochrome P450 (CYP450) superfamily member activity also plays an enhancement effect more obvious. This co culture system is a good solution to the original model is inefficient and unstable, is conducive to the use of the model reveal more key factors in the process of infection.
In addition to promoting infection, we also found that co culture system can significantly prolong the maintenance of HBV susceptibility in primary cultured hepatocytes in vitro. This advantage widens the application of PTH as a HBV infection model in practical research.
We further studied the mechanism of co culture system affecting liver parenchymal cells, and found that the influence of supporting cells on hepatocytes needs to be achieved through direct contact between cells. And the time of two cell contacts will also affect the degree of infection ultimately.
We through the expression of multiple testing of distribution and function, found the newly identified HBV receptor, Na + - taurocholate co transporter (NTCP) in liver cells expression have no obvious changes in co culture. On the other hand, we found that cells with HBV replication related liver enrichment of transcription factors (liver enriched transcription factor, LETF) expression was upregulated. The study found that two of them, HNF4A and HLF, through RNA interference inhibit the expression, the infection efficiency of HBV also decreased significantly. These results suggest that the most likely in viral replication and biosynthesis link has been enhanced in co culture system of HBV infection.
We made further research on the system of the liver cells were co cultured, found two kinds of state and closely related to the physiological cell marker protein, MRP2 (multidrug resistance protein2) and ZO-1 (zonula occludens protein1) on the expression and distribution characteristics showed in vivo and in the liver of liver cells were similar, and these features are single cultured liver cells do not have. This means that the system of hepatocytes on the physiological characteristics of more close to the in vivo microenvironment in cell culture.
Therefore, we established the primary hepatocyte coculture system, can be used as a more efficient model of HBV infection in vitro, and can simulate the process is more close to the physiological state of the infection occurred under HBV. This is for us to further reveal the HBV infection and pathogenesis of the relevant mechanism has a very important significance.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R512.62

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 周鳳娟,胡振林,戴建新,施柯,陳蕊雯,林懿,孫樹漢;乙肝DNA疫苗pVAX-PS對樹，

本文編號：1563061