CAJ | 학술논문

目的：利用水凝胶（ hydrogel ）模拟人血管内皮细胞基底膜弹性硬度，以登革病毒2型（DENV-2）NGC株感染接种于水凝胶为基底的原代人脐静脉内皮细胞（human umbilical vein endotheli-al cells，HUVECs），观察HUVECs在不同弹性硬度材质上培养产生IL-29的情况。方法分离培养原代HUVECs，将细胞接种于水凝胶，以MOI（multiplicity of infection）＝10的DENV-2感染原代HUVECs，流式细胞术检测48 h细胞凋亡率及病毒感染率，并送检mRNA基因表达谱芯片，通过实时荧光定量PCR及双抗体夹心ELISA法检测IL-29的表达情况。结果 DENV-2感染原代HUVECs 48 h，培养于水凝胶上的细胞的病毒感染率较低，细胞凋亡率与未感染组比较，差异无统计学意义（P＞0．05），基因芯片检测发现IL-29的产生与普通塑料培养瓶接种的脐静脉内皮细胞受到DENV-2感染后存在差异，实时荧光定量PCR和双抗体夹心ELISA法检测IL-29的表达，差异均存在统计学意义（P＜0．05）。结论水凝胶模拟人血管内皮细胞基底膜硬度，用DENV-2感染接种于水凝胶上脐静脉内皮细胞，基因芯片检测发现IL-29的产生，与体外正常培养条件下受到DENV-2感染后存在差异，水凝胶的建立可能为DENV发病机制的研究提供一个新的模型。
목적：이용수응효（ hydrogel ）모의인혈관내피세포기저막탄성경도，이등혁병독2형（DENV-2）NGC주감염접충우수응효위기저적원대인제정맥내피세포（human umbilical vein endotheli-al cells，HUVECs），관찰HUVECs재불동탄성경도재질상배양산생IL-29적정황。방법분리배양원대HUVECs，장세포접충우수응효，이MOI（multiplicity of infection）＝10적DENV-2감염원대HUVECs，류식세포술검측48 h세포조망솔급병독감염솔，병송검mRNA기인표체보심편，통과실시형광정량PCR급쌍항체협심ELISA법검측IL-29적표체정황。결과 DENV-2감염원대HUVECs 48 h，배양우수응효상적세포적병독감염솔교저，세포조망솔여미감염조비교，차이무통계학의의（P＞0．05），기인심편검측발현IL-29적산생여보통소료배양병접충적제정맥내피세포수도DENV-2감염후존재차이，실시형광정량PCR화쌍항체협심ELISA법검측IL-29적표체，차이균존재통계학의의（P＜0．05）。결론수응효모의인혈관내피세포기저막경도，용DENV-2감염접충우수응효상제정맥내피세포，기인심편검측발현IL-29적산생，여체외정상배양조건하수도DENV-2감염후존재차이，수응효적건립가능위DENV발병궤제적연구제공일개신적모형。
Objective To analyze the effects of dengue virus 2 ( DENV-2 ) infection on the ex-pression of IL-29 in primary human umbilical vein endothelial cells ( HUVECs) cultured on hydrogel sub-strates .Methods Primary HUVECs were isolated and cultured on hydrogel substrates .DENV-2 stains were used to infect the primary HUVECs at a multiplicity of infection( MOI) of 10.Flow cytometry analysis was performed to detect the apoptosis and infection rate of HUVECs after 48 hours of culturing .The gene chip profiling was performed to analyze mRNA expression .The expression of IL-29 at mRNA and protein levels were measured by real-time fluorescent quantitative PCR analysis and double antibody sandwich ELISA as -say, respectively.Results Compared with 96.36%of baby hamster kidney (BHK) cells that were infected with DENV-2 stains, only 4.71%primary HUVECs cultured on hydrogel substrates were infected .The pri-mary HUVECs cultured on hydrogel substrates with or without DENV-2 infection showed no significant differ-ences with the rates of cell apoptosis and infection (P>0.05).A significant difference was observed with the expression of IL-29 at mRNA level between primary HUVECs cultured on hydrogel substrates and the cells cultured in plastic bottles (P<0.05).The results of the real-time quantitative PCR analysis and ELISA as-say showed that IL-29 was highly expressed in DENV-2 infected primary HUVECs cultured on hydrogel sub-strates as compared with those in control groups (P<0.05).Conclusion The expression of IL-29 was de-tected in DENV-2 infected primary HUVECs cultured on hydrogel substrates , which was significantly differ-ent from that in DENV-2 infected primary HUVECs cultured in plastic bottles .The successful establishment of hydrogel substrates as the model of vascular basement membranes might provide a new way for the investi -gation of the pathogenesis of DENV infection .