CAJ | 학술논문

目的：探索低温保存狗肾细胞（MDCK）对 B 型流感病毒分离和增殖的影响。方法复苏冻存的 P17代 MDCK 细胞，传至2～4代后，选择生长状态及形态良好的单层细胞，换新鲜细胞生长液，于4℃保存3 d、6 d、9 d 后，在相同培养条件下同时接种流感样病例咽拭子标本。上毒24 h 后每天观察细胞病变，采用实时荧光 PCR 相对定量法测定病毒的增殖率，血清学试验测定病毒血凝效价，同时以同一代次常规培养的 MD-CK 细胞单层为对照。结果（1）细胞病变：与对照相比较，4℃低温保存3、6 d 的细胞细胞病变无差别，而保存9 d 的细胞细胞病变快，维持时间短。（2）荧光定量：4℃低温保存3、6 d 的细胞对流感病毒的增殖（25．86×105～30．25×106、26．31×105～30．54×106）与常规分离（24．82×105～29．86×106）相比无差别，其增值率在105～106倍之间。而9 d 的细胞（19．72×104～28．34×105）则较对照要低，增值率在104～105倍之间。（3）HA 效价：4℃保存3 d 和6 d 的细胞分离病毒血凝效价大于116的毒株数与常规细胞（10．92±0.79）比较，差异无统计学意义（P ＞0．05），而保存9 d 的细胞分离病毒血凝效价大于116的毒株数较正常细胞低并有统计学意义（P ＜0．01）。结论4℃低温保存1周内 MDCK 细胞单层对 B 型流感病毒的分离和增值无影响。
목적：탐색저온보존구신세포（MDCK）대 B 형류감병독분리화증식적영향。방법복소동존적 P17대 MDCK 세포，전지2～4대후，선택생장상태급형태량호적단층세포，환신선세포생장액，우4℃보존3 d、6 d、9 d 후，재상동배양조건하동시접충류감양병례인식자표본。상독24 h 후매천관찰세포병변，채용실시형광 PCR 상대정량법측정병독적증식솔，혈청학시험측정병독혈응효개，동시이동일대차상규배양적 MD-CK 세포단층위대조。결과（1）세포병변：여대조상비교，4℃저온보존3、6 d 적세포세포병변무차별，이보존9 d 적세포세포병변쾌，유지시간단。（2）형광정량：4℃저온보존3、6 d 적세포대류감병독적증식（25．86×105～30．25×106、26．31×105～30．54×106）여상규분리（24．82×105～29．86×106）상비무차별，기증치솔재105～106배지간。이9 d 적세포（19．72×104～28．34×105）칙교대조요저，증치솔재104～105배지간。（3）HA 효개：4℃보존3 d 화6 d 적세포분리병독혈응효개대우116적독주수여상규세포（10．92±0.79）비교，차이무통계학의의（P ＞0．05），이보존9 d 적세포분리병독혈응효개대우116적독주수교정상세포저병유통계학의의（P ＜0．01）。결론4℃저온보존1주내 MDCK 세포단층대 B 형류감병독적분리화증치무영향。
Objective To explore the effect of cryopreserved canine kidney cells (MDCK)single -layer on the isolation and proliferation of influenza virus B.Methods Revived P17 MDCK cells were passage for 2 -4 gener-ations,and subsequently preserved in 4℃ refrigerator for 3,6 and 9 days,respectively.Under same experimental con-ditions,the 4℃ refigerater preserved cells were co -incubated with influenza -like illness(ILI)throat swab speci-mens.Cytopathic effect (CPE)was observed,and the proliferation of virus was determined using real -time PCR and the hemagglutinin titers were determined by serological test.Results (1)CPE:The CPE of the MDCK cells pre-served in 4℃ refrigerator for 3 or 6 days had no significant differences compared with that in the control group,while the cell preserved in 4℃ refrigerator for 9 days showed CPE fastly and maintained for a short time.(2)Real -time PCR:the proliferation of influenza virus B in the MDCK cells preserved with 4℃refrigerator for 3 or 6 days (25.86 × 105 -30.25 ×106 ,26.31 ×105 -30.54 ×106 )had on difference compared with that of the control group (24.82 × 105 -29.86 ×106 ),with the proliferation rate of 105 to 106 times,while the proliferation cell with 4℃ cryopreserved for 9 days(19.72 ×104 -28.34 ×105 )the proliferation in cells preserved for 9 days was sharply decreased,with pro-liferation rate of 104 to 105 times.(3)The HA titer:The virus strains with hemagglutination titer above or equal to 116 (P >0.05)isolated with MDCK cells preserved in 4℃ refrigerator 3 or 6 days were not significantly different from that of the control group (10.92 ±0.79).And the cells with 4℃ cryopreserved for 9 days were significantly dicreased (P <0.01).Conclusion No significant effects on the isolation and proliferation of influenza virus B using MDCK cell preserved in 4℃ frigerator for near one week were observed in the present study.