蜱传脑炎病毒跨过血脑屏障的体外实验

In vitro experiment for tick borne encephalitis virus transmitting across the blood brain barrier

摘要:

[目的]构建蜱传脑炎病毒（Tick-borne encephalitis virus，TBEV）跨血脑屏障研究的体外细胞模型，研究2种不同细胞的TBEV培养物在病毒跨过血脑屏障中的主要差异，从而为进一步TBEV跨血脑屏障的分子机制研究奠定基础。[方法]利用人脑微血管内皮细胞（Human brain microvascular endothelial cells，hCMEC/D3）构建体外血脑屏障的细胞模型。用BHK-21细胞中培养的蜱传脑炎病毒感染人脑微血管内皮细胞，检测TBEV在hCMEC/D3中的复制增殖情况；将TBEV加入体外血脑屏障模型的上层微孔中，用实时荧光定量PCR和噬斑测定的方法检测跨过血脑屏障的病毒量；将感染TBEV的人单核细胞加入血脑屏障模型的上层微孔中，观察渗漏进下层孔中的淋巴细胞，并用实时荧光定量PCR和噬斑测定的方法检测跨过血脑屏障的病毒量。利用伊文思蓝标记的白蛋白确定血脑屏障细胞的渗透率变化。[结果]实时荧光定量PCR和病毒滴度测定结果表明，TBEV不能在hCMEC/D3细胞中复制增殖，也不能直接跨过血脑屏障；然而，人单核细胞THP-1感染TBEV后，尽管单核细胞不能直接携带TBEV跨过血脑屏障，但THP-1中产生的病毒却能跨过血脑屏障模型进入下层孔中，并引起血脑屏障渗透率的增高。[结论]单核细胞有助于TBEV跨过血脑屏障。

Abstract:

[Objective] To understand the route and mechanism of tick borne encephalitis virus transmitting across the blood brain barrier, we established an in vitro blood brain barrier model, and studied the difference of the virus cultured from two different cell lines for transmitting across the blood brain barrier. [Methods] We used human brain microvascular endothelial cells (hCMEC/D3) to establish the vitro blood brain barrier model. First, we detected the infection and replication of tick borne encephalitis virus in hCMEC/D3 using real time PCR and plague assay. Then, we conducted the transmitting assay by adding tick borne encephalitis virus into the transwell inserts of the blood brain barrier model and detected virus titer leaking from the upper inserts into the bottom wells using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and plaque assay. Meanwhile, we added the THP-1 cells infected with tick borne encephalitis virus into the transwell inserts and observed the bottom wells under the optical microscope to confirm if the monocyte THP-1 could leak across the vito blood brain barrier. The virus titer leaked into bottom wells were detected by using qRT-PCR and plaque assay. The permeability change of blood brain barrier at different time post infection was tested by using Bovine Serum Albumin conjugated evens blue.[Results] We found that no replication of tick borne encephalitis virus in hCMEC/D3 cells, and tick borne encephalitis virus cultured in BHK-21 was unable to transmigrate across the blood brain barrier directly. However, the monocyte cell line THP-1 infected with tick borne encephalitis virus could cause its transmitting across the blood brain barrier and increase the permeability of the blood brain barrier, though THP-1 did not transmit across the vitro blood brain barrier directly.[Conclusion] Tick borne encephalitis virus can transmigrate across the blood brain barrier in assistance of monocyte THP-1 cells.