中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2014年
9期
517-521
,共5页
邢雅玲%陈晓娟%闫飞%杜鹃%周勇%王学军%陈忠斌
邢雅玲%陳曉娟%閆飛%杜鵑%週勇%王學軍%陳忠斌
형아령%진효연%염비%두견%주용%왕학군%진충빈
3 p-siRNA%抗病毒天然免疫%肝炎病毒,乙型%水动力注射%小鼠
3 p-siRNA%抗病毒天然免疫%肝炎病毒,乙型%水動力註射%小鼠
3 p-siRNA%항병독천연면역%간염병독,을형%수동력주사%소서
3p-siRNA%Anti-viral innate immunity%Hepatitis B virus%Hydrodynamic injection%Mice
目的：观察 HBV-三磷酸（3p）-小干扰 RNA（siRNA）激活受试小鼠Ⅰ型干扰素抗病毒天然免疫反应以及抑制 HBV 基因组复制的双重作用。方法针对 HBV 基因组 S/P mRNA 设计并通过体外转录法制备带有5′-3p-修饰的 HBV-3p-siRNA；以未经修饰的 HBV-siRNA 和阴性对照（NC）-siRNA 作为对照；利用尾静脉水动力注射法建立 HBV 感染小鼠模型。将40只小鼠分为4组，每组10只，模型组：仅注射 pGL3．0-HBV 1．2拷贝质粒；阴性对照组：腹腔注射 NC-siRNA；HBV-siRNA 组：腹腔注射 HBV-siRNA；HBV-3p-siRNA 组：腹腔注射 HBV-3p-siRNA。ELISA 法检测小鼠血清中HBsAg 的表达和β干扰素的分泌量；荧光定量 PCR 检测小鼠血清 HBV DNA。采用 LSD 或 Dunnett T3统计方法进行 One way-ANOVA 统计学分析。结果小鼠血清中β干扰素水平模型组为（12．37±5．32）pg/mL，阴性对照组为（22．61±6．29）pg/mL，HBV-siRNA 组为（26．40±5．39）pg/mL，HBV-3p-siRNA 组为（68．37±21．00）pg/mL，HBV-siRNA 组和 HBV-3p-siRNA 组与模型组相比，差异有统计学意义（F 值分别为23．988、46．523，均 P ＜0．01）。小鼠血清中 HBsAg 水平模型组为（2864．86±907．11）ng/mL，阴性对照组为（2198．86±456．89）ng/mL，HBV-siRNA 组为（1049．71±396．28）ng/mL， HBV-3p-siRNA 组为（640．86±383．08）ng/mL，HBV-siRNA 组和 HBV-3p-siRNA 组与模型组相比，差异有统计学意义（F 值分别为23．537、39．144，P 值分别为0．025、0．010）。小鼠血清中 HBV DNA 的水平模型组为（2．54×104±1．46×104）拷贝/mL，阴性对照组为（2．22×104±2．62×103）拷贝/mL， HBV-siRNA组为（3．59×103±2．88×103）拷贝/mL，HBV-3p-siRNA 组为（2．65×103±1．46×103）拷贝/mL，HBV-siRNA 组和 HBV-3p-siRNA 组与模型组相比，差异均有统计学意义（F 值分别为15．013、16．741，均 P ＜0．05）。HBV-3p-siRNA、HBV-siRNA，以及 NC-siRNA 对 HBV 感染小鼠的体质量无明显影响。结论HBV-3p-siRNA可以通过 RNA 干扰机制特异性抑制 HBV 复制，提高机体天然免疫功能，是一种新型抗 HBV 核酸分子。
目的：觀察 HBV-三燐痠（3p）-小榦擾 RNA（siRNA）激活受試小鼠Ⅰ型榦擾素抗病毒天然免疫反應以及抑製 HBV 基因組複製的雙重作用。方法針對 HBV 基因組 S/P mRNA 設計併通過體外轉錄法製備帶有5′-3p-脩飾的 HBV-3p-siRNA；以未經脩飾的 HBV-siRNA 和陰性對照（NC）-siRNA 作為對照；利用尾靜脈水動力註射法建立 HBV 感染小鼠模型。將40隻小鼠分為4組，每組10隻，模型組：僅註射 pGL3．0-HBV 1．2拷貝質粒；陰性對照組：腹腔註射 NC-siRNA；HBV-siRNA 組：腹腔註射 HBV-siRNA；HBV-3p-siRNA 組：腹腔註射 HBV-3p-siRNA。ELISA 法檢測小鼠血清中HBsAg 的錶達和β榦擾素的分泌量；熒光定量 PCR 檢測小鼠血清 HBV DNA。採用 LSD 或 Dunnett T3統計方法進行 One way-ANOVA 統計學分析。結果小鼠血清中β榦擾素水平模型組為（12．37±5．32）pg/mL，陰性對照組為（22．61±6．29）pg/mL，HBV-siRNA 組為（26．40±5．39）pg/mL，HBV-3p-siRNA 組為（68．37±21．00）pg/mL，HBV-siRNA 組和 HBV-3p-siRNA 組與模型組相比，差異有統計學意義（F 值分彆為23．988、46．523，均 P ＜0．01）。小鼠血清中 HBsAg 水平模型組為（2864．86±907．11）ng/mL，陰性對照組為（2198．86±456．89）ng/mL，HBV-siRNA 組為（1049．71±396．28）ng/mL， HBV-3p-siRNA 組為（640．86±383．08）ng/mL，HBV-siRNA 組和 HBV-3p-siRNA 組與模型組相比，差異有統計學意義（F 值分彆為23．537、39．144，P 值分彆為0．025、0．010）。小鼠血清中 HBV DNA 的水平模型組為（2．54×104±1．46×104）拷貝/mL，陰性對照組為（2．22×104±2．62×103）拷貝/mL， HBV-siRNA組為（3．59×103±2．88×103）拷貝/mL，HBV-3p-siRNA 組為（2．65×103±1．46×103）拷貝/mL，HBV-siRNA 組和 HBV-3p-siRNA 組與模型組相比，差異均有統計學意義（F 值分彆為15．013、16．741，均 P ＜0．05）。HBV-3p-siRNA、HBV-siRNA，以及 NC-siRNA 對 HBV 感染小鼠的體質量無明顯影響。結論HBV-3p-siRNA可以通過 RNA 榦擾機製特異性抑製 HBV 複製，提高機體天然免疫功能，是一種新型抗 HBV 覈痠分子。
목적：관찰 HBV-삼린산（3p）-소간우 RNA（siRNA）격활수시소서Ⅰ형간우소항병독천연면역반응이급억제 HBV 기인조복제적쌍중작용。방법침대 HBV 기인조 S/P mRNA 설계병통과체외전록법제비대유5′-3p-수식적 HBV-3p-siRNA；이미경수식적 HBV-siRNA 화음성대조（NC）-siRNA 작위대조；이용미정맥수동력주사법건립 HBV 감염소서모형。장40지소서분위4조，매조10지，모형조：부주사 pGL3．0-HBV 1．2고패질립；음성대조조：복강주사 NC-siRNA；HBV-siRNA 조：복강주사 HBV-siRNA；HBV-3p-siRNA 조：복강주사 HBV-3p-siRNA。ELISA 법검측소서혈청중HBsAg 적표체화β간우소적분비량；형광정량 PCR 검측소서혈청 HBV DNA。채용 LSD 혹 Dunnett T3통계방법진행 One way-ANOVA 통계학분석。결과소서혈청중β간우소수평모형조위（12．37±5．32）pg/mL，음성대조조위（22．61±6．29）pg/mL，HBV-siRNA 조위（26．40±5．39）pg/mL，HBV-3p-siRNA 조위（68．37±21．00）pg/mL，HBV-siRNA 조화 HBV-3p-siRNA 조여모형조상비，차이유통계학의의（F 치분별위23．988、46．523，균 P ＜0．01）。소서혈청중 HBsAg 수평모형조위（2864．86±907．11）ng/mL，음성대조조위（2198．86±456．89）ng/mL，HBV-siRNA 조위（1049．71±396．28）ng/mL， HBV-3p-siRNA 조위（640．86±383．08）ng/mL，HBV-siRNA 조화 HBV-3p-siRNA 조여모형조상비，차이유통계학의의（F 치분별위23．537、39．144，P 치분별위0．025、0．010）。소서혈청중 HBV DNA 적수평모형조위（2．54×104±1．46×104）고패/mL，음성대조조위（2．22×104±2．62×103）고패/mL， HBV-siRNA조위（3．59×103±2．88×103）고패/mL，HBV-3p-siRNA 조위（2．65×103±1．46×103）고패/mL，HBV-siRNA 조화 HBV-3p-siRNA 조여모형조상비，차이균유통계학의의（F 치분별위15．013、16．741，균 P ＜0．05）。HBV-3p-siRNA、HBV-siRNA，이급 NC-siRNA 대 HBV 감염소서적체질량무명현영향。결론HBV-3p-siRNA가이통과 RNA 간우궤제특이성억제 HBV 복제，제고궤체천연면역공능，시일충신형항 HBV 핵산분자。
Objective To observe the activation of anti-viral innate immune response of type Ⅰinterferon and inhibition of hepatitis B virus (HBV)genome replication in mice by HBV-3p-siRNA. Methods HBV-3p-siRNA was designed by targeting specific sequence of HBV S/P mRNA and was generated by in vitro transcription.Negative control siRNA (NC-siRNA)and non-modified HBV-siRNA were used as control groups.Blood samples were collected from tail vein of mice and the model of HBV-infected mice were established by hydrodynamic injection.Forty mice were divided into 4 groups with 10 in each group.The model group was only injected with pGL3.0-HBV1 .2 copy plasmid.The negative control group received peritoneal injection of NC-siRNA.HBV-siRNA group received peritoneal injection of HBV-siRNA and HBV-3p-siRNA group received peritoneal injection of HBV-3p-siRNA.The interferon-β(IFN-β)and hepatitis B surface antigen (HBsAg)in serum were detected by enzyme linked immunosorbent assay (ELISA).The copies of HBV DNA were assessed by fluore scence quantitative polymerase chain reaction (PCR ).The statistical difference between groups was determined using One way-ANOVA analysis by LSD or Dunnett T3.Results Serum level of IFN-β was (12.37±5 .32)pg/mL in model group,(22.61 ±6.29 )pg/mL in negative control group,(26.40±5 .39)pg/mL in HBV-siRNA group and (68.37± 21 .00 ) pg/mL in HBV-3p-siRNA group.The secretions of IFN-β into serum were significantly enhanced by HBV-siRNA and HBV-3p-siRNA compared with model group (F =23.988 and 46.523,respectively,both P <0.01).Serum level of HBsAg was (2 864.86±907.11 )ng/mL in model group,(2 198.86±456.89 )ng/mL in negative control group,(1 049.71 ± 396.28 )ng/mL in HBV-siRNA group and (640.86±383.08)ng/mL in HBV-3p-siRNA group.The expressions of HBsAg were inhibited by HBV-3p-siRNA and HBV-siRNA compared with model group (F = 23.537 and 39.144, respectively;P =0.025 and 0.010,respectively).Serum level of HBV DNA was (2.54 ×104 ±1 .46 × 104 )copy/mL in model group,(2.22×104 ±2.62×103 )copy/mL in negative control group,(3.59×103 ±2.88×103 )copy/mL in HBV-siRNA group and (2.65 ×103 ±1 .46×103 )copy/mL in HBV-3p-siRNA group.Serum level of HBV DNA were inhibited by HBV-3p-siRNA and HBV-siRNA compared with model group (F =15 .013 and 16.741 ,respectively,both P <0.05 ).All of the indicated siRNA used in the experiments showed no apparent effects on the body mass index of the mice models.Conclusion HBV-3p-siRNA,which induces the production of IFN-β and inhibits HBV replication through gene silencing in vivo ,may be a powerful bifunctional antiviral molecule.
