西安交通大学学报（医学版）
西安交通大學學報（醫學版）
서안교통대학학보（의학판）
JOURNAL OF XI'AN JIAOTONG UNIVERSITY(MEDICAL SCIENCES)
2014年
3期
300-305
,共6页
庹清章%董江涛%田玺择%刘云霞%董伟杰%刘丹霞%李微%吴芳%章乐%张万江
庹清章%董江濤%田璽擇%劉雲霞%董偉傑%劉丹霞%李微%吳芳%章樂%張萬江
탁청장%동강도%전새택%류운하%동위걸%류단하%리미%오방%장악%장만강
结核分枝杆菌%Hsp16.3%肺泡巨噬细胞%凋亡%Caspase-3%Bcl-2
結覈分枝桿菌%Hsp16.3%肺泡巨噬細胞%凋亡%Caspase-3%Bcl-2
결핵분지간균%Hsp16.3%폐포거서세포%조망%Caspase-3%Bcl-2
Mycobacterium tuberculosis%Hsp16.3%alveolar macrophage%apoptosis%Caspase-3%Bcl-2
目的：探讨结核分枝杆菌(MTB)小分子热休克蛋白 Hsp16.3表达与感染小鼠肺泡巨噬细胞凋亡的关系。方法分别将 MTB 国际标准强毒株 H37Rv 株 Hsp16.3基因缺失突变菌株(△H37Rv)、结核杆菌国际标准强毒株H37Rv菌株(H37Rv)以及无菌生理盐水溶液(空白对照)通过小鼠尾静脉注入小鼠体内以建立小鼠的感染模型,并在感染后的1、3、5、7、9、11、13、15 d分离及鉴定小鼠肺泡巨噬细胞,用激光共聚焦显微镜技术检测及鉴定 MTB感染小鼠肺泡巨噬细胞；流式细胞术检测不同时间点感染小鼠肺泡巨噬细胞的凋亡率；Western blot 技术检测小鼠肺泡巨噬细胞内半胱天冬酶-3(Caspase-3)和B细胞淋巴瘤/白血病-2(Bcl-2)蛋白的表达。结果△H37Rv 菌株感染组和 H37Rv菌株感染组凋亡率均高于空白对照组,△H37Rv组的巨噬细胞凋亡率在感染1~7 d内逐渐升高,至感染7 d时达到高峰,随后呈下降趋势,与 H37Rv组相比,1~7 d及13~15 d,△H37Rv组的巨噬细胞凋亡率高于 H37Rv组,差异有统计学意义(P<0.05)；H37Rv组和△H37Rv 组的巨噬细胞 Caspase-3和 Bcl-2蛋白表达水平均高于空白对照组；1~7 d内,H37Rv感染组和△H37Rv感染组的巨噬细胞Caspase-3表达水平逐渐升高,至第7天达到高峰,且△H37Rv 感染组在第13天时出现第二次高峰,但Caspase-3表达水平均高于 H37Rv 感染组,差异有统计学意义(P<0.05)；在感染的早期(1~7 d),△H37Rv感染组Bcl-2的表达水平无明显变化(P<0.05),但在9 d后逐渐升高；H37Rv感染组巨噬细胞内Bcl-2的表达水平1~7 d内无明显变化(P<0.05),7 d 后逐渐升高,但△H37Rv 感染组 Bcl-2的表达水平始终低于 H37Rv组且7 d后表现更为显著。结论在 MTB感染的早期和晚期,MTB小分子热休克蛋白 Hsp16.3的表达能够有效抑制小鼠肺泡巨噬细胞的凋亡,而这种抑制作用可能是通过抑制凋亡蛋白酶 Caspase-3的表达,同时促进 Bcl-2蛋白的表达来实现的。
目的：探討結覈分枝桿菌(MTB)小分子熱休剋蛋白 Hsp16.3錶達與感染小鼠肺泡巨噬細胞凋亡的關繫。方法分彆將 MTB 國際標準彊毒株 H37Rv 株 Hsp16.3基因缺失突變菌株(△H37Rv)、結覈桿菌國際標準彊毒株H37Rv菌株(H37Rv)以及無菌生理鹽水溶液(空白對照)通過小鼠尾靜脈註入小鼠體內以建立小鼠的感染模型,併在感染後的1、3、5、7、9、11、13、15 d分離及鑒定小鼠肺泡巨噬細胞,用激光共聚焦顯微鏡技術檢測及鑒定 MTB感染小鼠肺泡巨噬細胞；流式細胞術檢測不同時間點感染小鼠肺泡巨噬細胞的凋亡率；Western blot 技術檢測小鼠肺泡巨噬細胞內半胱天鼕酶-3(Caspase-3)和B細胞淋巴瘤/白血病-2(Bcl-2)蛋白的錶達。結果△H37Rv 菌株感染組和 H37Rv菌株感染組凋亡率均高于空白對照組,△H37Rv組的巨噬細胞凋亡率在感染1~7 d內逐漸升高,至感染7 d時達到高峰,隨後呈下降趨勢,與 H37Rv組相比,1~7 d及13~15 d,△H37Rv組的巨噬細胞凋亡率高于 H37Rv組,差異有統計學意義(P<0.05)；H37Rv組和△H37Rv 組的巨噬細胞 Caspase-3和 Bcl-2蛋白錶達水平均高于空白對照組；1~7 d內,H37Rv感染組和△H37Rv感染組的巨噬細胞Caspase-3錶達水平逐漸升高,至第7天達到高峰,且△H37Rv 感染組在第13天時齣現第二次高峰,但Caspase-3錶達水平均高于 H37Rv 感染組,差異有統計學意義(P<0.05)；在感染的早期(1~7 d),△H37Rv感染組Bcl-2的錶達水平無明顯變化(P<0.05),但在9 d後逐漸升高；H37Rv感染組巨噬細胞內Bcl-2的錶達水平1~7 d內無明顯變化(P<0.05),7 d 後逐漸升高,但△H37Rv 感染組 Bcl-2的錶達水平始終低于 H37Rv組且7 d後錶現更為顯著。結論在 MTB感染的早期和晚期,MTB小分子熱休剋蛋白 Hsp16.3的錶達能夠有效抑製小鼠肺泡巨噬細胞的凋亡,而這種抑製作用可能是通過抑製凋亡蛋白酶 Caspase-3的錶達,同時促進 Bcl-2蛋白的錶達來實現的。
목적：탐토결핵분지간균(MTB)소분자열휴극단백 Hsp16.3표체여감염소서폐포거서세포조망적관계。방법분별장 MTB 국제표준강독주 H37Rv 주 Hsp16.3기인결실돌변균주(△H37Rv)、결핵간균국제표준강독주H37Rv균주(H37Rv)이급무균생리염수용액(공백대조)통과소서미정맥주입소서체내이건립소서적감염모형,병재감염후적1、3、5、7、9、11、13、15 d분리급감정소서폐포거서세포,용격광공취초현미경기술검측급감정 MTB감염소서폐포거서세포；류식세포술검측불동시간점감염소서폐포거서세포적조망솔；Western blot 기술검측소서폐포거서세포내반광천동매-3(Caspase-3)화B세포림파류/백혈병-2(Bcl-2)단백적표체。결과△H37Rv 균주감염조화 H37Rv균주감염조조망솔균고우공백대조조,△H37Rv조적거서세포조망솔재감염1~7 d내축점승고,지감염7 d시체도고봉,수후정하강추세,여 H37Rv조상비,1~7 d급13~15 d,△H37Rv조적거서세포조망솔고우 H37Rv조,차이유통계학의의(P<0.05)；H37Rv조화△H37Rv 조적거서세포 Caspase-3화 Bcl-2단백표체수평균고우공백대조조；1~7 d내,H37Rv감염조화△H37Rv감염조적거서세포Caspase-3표체수평축점승고,지제7천체도고봉,차△H37Rv 감염조재제13천시출현제이차고봉,단Caspase-3표체수평균고우 H37Rv 감염조,차이유통계학의의(P<0.05)；재감염적조기(1~7 d),△H37Rv감염조Bcl-2적표체수평무명현변화(P<0.05),단재9 d후축점승고；H37Rv감염조거서세포내Bcl-2적표체수평1~7 d내무명현변화(P<0.05),7 d 후축점승고,단△H37Rv 감염조 Bcl-2적표체수평시종저우 H37Rv조차7 d후표현경위현저。결론재 MTB감염적조기화만기,MTB소분자열휴극단백 Hsp16.3적표체능구유효억제소서폐포거서세포적조망,이저충억제작용가능시통과억제조망단백매 Caspase-3적표체,동시촉진 Bcl-2단백적표체래실현적。
Objective To study the relationship between the expression of Mycobacterium tuberculosis small heat shock protein Hsp16.3 and the apoptosis of infected mouse alveolar macrophages.Methods The laboratory mice were infected with bacterial suspension of the international standard virulent strain of Mycobacterium tuberculosis H37Rv strains (H37Rv),Hsp16.3 gene deletion mutants of the international standard virulent of Mycobacterium tuberculosis H37Rv strains(△H37Rv),or sterile saline solution (normal control)by the tail vein. After successful replication of mouse infection model in each group,we cleaved the alveolus of each group of mice and collected lavage fluid to obtain alveolar macrophages of the infected mice at days 1 ,3 ,5 ,7 ,9 ,1 1 ,1 3 and 1 5 .Then the infection status of macrophages was observed with confocal laser scanning microscopy;flow cytometry was used to detect the apoptosis rate of alveolar macrophages of the infected mice;Caspase-3 and Bcl-2 expressions were examined by Western blot.Results The apoptosis rate of Hsp16.3 gene was higher in deletion strain (△H37Rv)group and H37Rv strains (H37Rv)group than in control group.The apoptosis rate of alveolar macrophages in △ H37Rv group gradually increased,peaked at day 7 ,and then gradually decreased.It was significantly higher in H3 7 Rv group than in H3 7 Rv strain group from day 1 to 7 and from day 1 3 to 1 5 (P<0 .0 5 ).Caspase-3 and Bcl-2 protein expressions in the macrophages of△H37Rv group and H37Rv group were higher than those of control group.Caspase-3 expression in the microphages of △H3 7 Rv group and H3 7 Rv group gradually increased from day 1 to 7 and peaked at day 7;it peaked again at day 13 in H37Rv group.However,Caspase-3 expression remained significantly higher in△H37Rv group than in H3 7 Rv group (P<0 .0 5 ).Bcl-2 expression in △H3 7 Rv group did not change much at the early stage of infection (P<0 .0 5 ),but gradually increased after day 9 .Bcl-2 expression in H3 7 Rv group did did not change much from day 1 to 7 (P<0.05),but gradually increased after day 7.However,it remained lower in△H37Rv group than in H37Rv group,especially after 7 days(P<0.05).Conclusion Mycobacterium tuberculosis small heat shock protein Hsp16.3 can inhibit the apoptosis of macrophages during the early and late stages of infection,and this inhibition may be achieved by inhibiting the expression of apoptotic protease Caspase-3 and promoting the expression of Bcl-2 protein.
