中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2013年
5期
263-268
,共6页
王志刚%吴展%徐水凌%崔戈%阮玲娟
王誌剛%吳展%徐水凌%崔戈%阮玲娟
왕지강%오전%서수릉%최과%원령연
弧菌,创伤%树突细胞%Toll样受体2%Toll样受体4%DNA降解,坏死
弧菌,創傷%樹突細胞%Toll樣受體2%Toll樣受體4%DNA降解,壞死
호균,창상%수돌세포%Toll양수체2%Toll양수체4%DNA강해,배사
Vibrio vulnificus%Dendritic cells%Toll-like receptor 2%Toll-like receptor 4%DNA degradation,necrotic
目的 探索创伤弧菌(Vv)感染树突状细胞(DC),经Toll样受体(TLR)2、4途径致DC急性坏死的实验研究.方法 建立Vv1.1758株与DC 2.4混合培养感染模型,用光学显微镜观察细胞感染率,电子显微镜观察Vv定位与细胞结构变化,实时定量反转录PCR测定TLR2、4mRNA表达量,ELISA法检测TNF-α表达量,DNA梯度电泳定性检测细胞凋亡,流式细胞术定量检测细胞凋亡及坏死率.率的比较采用x2检验,多组间数据比较采用方差分析.结果 混合培养0.5、1、2、4、6h时的细胞感染率分别为(7.8±0.8)％、(13.9±1.1)％、(34.6±4.9)％、(77.8±10.2)％和(95.8±13.1)％,混合培养2h后的感染率与培养0.5h相比差异有统计学意义(均P＜0.05).细菌定位于DC2.4细胞内侧,2h核染色质明显活跃,核内出现凋亡小体；4h胞质内出现空泡,染色质聚集,胞膜毁损严重；6h线粒体高度肿胀变形,细胞坏死.TLR2、4 mRNA表达于0.5h已达峰值.TNF-α在1h时开始增高(P＜0.05),2h达峰值.DNA梯度电泳2h呈冲刷状坏死,4～5 h出现720 bp与900 bp凋亡带.2、4、6h时细胞早期凋亡率分别为(3.1±3.8)％、(7.8±4.7)％和(12.7±8.2)％,显著高于对照组的(0.5±0.7)％(均P＜0.05).细胞坏死率分别为(16.7±12.5) ％、(41.6±25.9)％和(75.5±33.6)％,显著高于对照组的(2.3±0.8)％(均P＜0.05).结论 Vv感染DC可通过TLR2、4表达上调,TNF-α增加导致DNA降解,期间以细胞凋亡与坏死同时并存,但以坏死方式降解为主.
目的 探索創傷弧菌(Vv)感染樹突狀細胞(DC),經Toll樣受體(TLR)2、4途徑緻DC急性壞死的實驗研究.方法 建立Vv1.1758株與DC 2.4混閤培養感染模型,用光學顯微鏡觀察細胞感染率,電子顯微鏡觀察Vv定位與細胞結構變化,實時定量反轉錄PCR測定TLR2、4mRNA錶達量,ELISA法檢測TNF-α錶達量,DNA梯度電泳定性檢測細胞凋亡,流式細胞術定量檢測細胞凋亡及壞死率.率的比較採用x2檢驗,多組間數據比較採用方差分析.結果 混閤培養0.5、1、2、4、6h時的細胞感染率分彆為(7.8±0.8)％、(13.9±1.1)％、(34.6±4.9)％、(77.8±10.2)％和(95.8±13.1)％,混閤培養2h後的感染率與培養0.5h相比差異有統計學意義(均P＜0.05).細菌定位于DC2.4細胞內側,2h覈染色質明顯活躍,覈內齣現凋亡小體；4h胞質內齣現空泡,染色質聚集,胞膜燬損嚴重；6h線粒體高度腫脹變形,細胞壞死.TLR2、4 mRNA錶達于0.5h已達峰值.TNF-α在1h時開始增高(P＜0.05),2h達峰值.DNA梯度電泳2h呈遲刷狀壞死,4～5 h齣現720 bp與900 bp凋亡帶.2、4、6h時細胞早期凋亡率分彆為(3.1±3.8)％、(7.8±4.7)％和(12.7±8.2)％,顯著高于對照組的(0.5±0.7)％(均P＜0.05).細胞壞死率分彆為(16.7±12.5) ％、(41.6±25.9)％和(75.5±33.6)％,顯著高于對照組的(2.3±0.8)％(均P＜0.05).結論 Vv感染DC可通過TLR2、4錶達上調,TNF-α增加導緻DNA降解,期間以細胞凋亡與壞死同時併存,但以壞死方式降解為主.
목적 탐색창상호균(Vv)감염수돌상세포(DC),경Toll양수체(TLR)2、4도경치DC급성배사적실험연구.방법 건립Vv1.1758주여DC 2.4혼합배양감염모형,용광학현미경관찰세포감염솔,전자현미경관찰Vv정위여세포결구변화,실시정량반전록PCR측정TLR2、4mRNA표체량,ELISA법검측TNF-α표체량,DNA제도전영정성검측세포조망,류식세포술정량검측세포조망급배사솔.솔적비교채용x2검험,다조간수거비교채용방차분석.결과 혼합배양0.5、1、2、4、6h시적세포감염솔분별위(7.8±0.8)％、(13.9±1.1)％、(34.6±4.9)％、(77.8±10.2)％화(95.8±13.1)％,혼합배양2h후적감염솔여배양0.5h상비차이유통계학의의(균P＜0.05).세균정위우DC2.4세포내측,2h핵염색질명현활약,핵내출현조망소체；4h포질내출현공포,염색질취집,포막훼손엄중；6h선립체고도종창변형,세포배사.TLR2、4 mRNA표체우0.5h이체봉치.TNF-α재1h시개시증고(P＜0.05),2h체봉치.DNA제도전영2h정충쇄상배사,4～5 h출현720 bp여900 bp조망대.2、4、6h시세포조기조망솔분별위(3.1±3.8)％、(7.8±4.7)％화(12.7±8.2)％,현저고우대조조적(0.5±0.7)％(균P＜0.05).세포배사솔분별위(16.7±12.5) ％、(41.6±25.9)％화(75.5±33.6)％,현저고우대조조적(2.3±0.8)％(균P＜0.05).결론 Vv감염DC가통과TLR2、4표체상조,TNF-α증가도치DNA강해,기간이세포조망여배사동시병존,단이배사방식강해위주.
Objective To explore how Vibrio vulnificus (Vv) invades dendritic cells (DC) and induces acute necrosis of DC via toll-like receptor (TLR) 2 and 4 pathways.Methods Vv 1.1758 strain and DC 2.4 mixed culture model was established,observed the infection rates of DC with optical microscope,the location of Vv and structural changes of DC by transmission electron microscope.The expression levels of TLR2 and TLR4 mRNA were determined by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and tumor necrosis factor-α (TNFα) protein titers were measured by enzyme-linked immunosorbent assay (ELISA).DNA ladder qualitative test was used to detect cell apoptosis,while flow cytometry was used to quantify cell apoptosis and necrosis rates.Statistical analysis was done by chi-square test and one-way ANOVE.Results The infection rates of DC after 0.5,1,2,4 and 6 h of mixed culture were (7.8±0.8) ％,(13.9± 1.1) ％,(34.6±4.9) ％,(77.8± 10.2)％ and (95.8 ± 13.1)％,respectively.Vv was generally located in the internal cell membrane of DC 2.4.After 2 h co-culture,nuclear chromatins of DC became active and intranuclear apoptosis bodies appeared.After 4 h,cytoplasmic vacuoles appeared,chromatin gathered,and cell membranes were seriously damaged.After 6 h,mitochondria was highly swelled and distorted,and cell apoptosis and necrosis occurred.TLR2 and TLR4 mRNA levels reached peak values after co-culture for 0.5 h; TNF-α level began to increase at 1 h (P＜0.05) and reached peak values at 2 h.DNA Ladder electrophoresis presented scouring necrosis after 2 h culture and apoptotic bands appeared between 720 bp and 900 bp after 4 to 5 h culture.Early apoptosis rates of DC after 2,4 and 6 h culture were (3.1±3.8)％,(7.8±4.7)％ and (12.7±8.2)％,and necrosis rates of DC were (16.7±12.5)％,(41.6±25.9)％ and (75.5±33.6)％,higher than that of control group (all P＜0.05).Conclusions Vv infects DC and induce DNA degradation through up-regulated expression of TLR2 and TLR4 and increasing of TNF-α inflammatory mediators.During cell degradation,apoptosis and necrosis coexist,while necrosis is predominant.
