烧伤,电%磷脂酰肌醇3-激酶%蛋白激酶类%血清%单核细胞%内皮细胞%黏附作用
燒傷,電%燐脂酰肌醇3-激酶%蛋白激酶類%血清%單覈細胞%內皮細胞%黏附作用
소상,전%린지선기순3-격매%단백격매류%혈청%단핵세포%내피세포%점부작용
Burns,electric%Phosphatidylinositol 3-kinase%Protein kinases%Serum%Monocytes%Endothelial cells%Adhesion
目的 观察磷脂酰肌醇3激酶/蛋白激酶B(PI3 K/Akt)通路在电烧伤大鼠血清诱导的单核细胞中的变化,探讨其在单核细胞与血管内皮细胞黏附中的作用. 方法 取64只清洁级SD大鼠制作电烧伤模型,制备电烧伤大鼠血清;取24只大鼠不作处理,制备正常大鼠血清.(1)常规培养人单核细胞株THP-1细胞,按照随机数字表法将对数生长期细胞分为正常血清组(将细胞重新悬浮于含体积分数20％正常大鼠血清的RPMI 1640培养液中)和烧伤血清组(将细胞重新悬浮于含体积分数20％电烧伤大鼠血清的RPMI 1640培养液中),每组设6个复孔.正常血清组于培养24 h,烧伤血清组于培养3、6、24 h,观察THP-1细胞形态,双抗体夹心ELISA法测定2组细胞上清液中TNF-α含量.2组均于培养3、6、24 h收集细胞,蛋白质印迹法检测Akt活化状态.(2)另取THP-1细胞按照随机数字表法分为4组:正常血清组、烧伤血清组,2组各自培养方法同前;正常血清+阻断剂组、烧伤血清+阻断剂组,在正常血清组、烧伤血清组的培养液中加入渥曼青霉素100 nmol/L,余培养方法同前.每组设6个复孔.取培养3、6h的THP-1细胞,加入单层人血管内皮细胞株EA.hy926细胞,行单核-内皮细胞黏附检测.对数据行单因素方差分析、LSD-￡检验. 结果 (1)正常血清组培养24 h,THP-1细胞呈悬浮生长、形态一致.烧伤血清组培养3h,细胞膜完整,细胞形态不规则;培养6h细胞大小不一,细胞膜与细胞质膨胀;培养24 h细胞膜破裂,细胞死亡.正常血清组培养24 h和烧伤血清组培养3、6、24 h,THP-1细胞上清液中TNF-α含量分别为(38.5±1.4)、(75.1±1.5)、(91.5±1.8)、(117.0±1.4)pg/mL,总体比较差异明显(F=1 415.306,P＜0.01).烧伤血清组培养3、6、24 h THP-1细胞上清液中TNF-α含量均明显高于正常血清组培养24 h(￡值分别为29.614、42.852、63.485,P值均小于0.01).烧伤血清组培养3、6、24 h,THP-1细胞中磷酸化蛋白激酶B/Akt比值分别是正常血清组培养同时相点的2.66、3.69、1.17倍.(2)正常血清组、正常血清+阻断剂组、烧伤血清组、烧伤血清+阻断剂组培养3、6h,每100倍视野下黏附EA.hy926细胞的THP-1细胞数量分别为(231 ±45)、(280 ±47)、(703±169)、(335±85)个,(219±49)、(235±21)、(562±123)、(226±29)个,总体比较均差异明显(F值分别为25.630、18.975,P值均小于0.01).与正常血清组比较,烧伤血清组黏附EA.hy926细胞的THP-1细胞数在培养3、6h均明显增多(￡值分别为6.189、6.601,P值均小于0.01);烧伤血清+阻断剂组黏附EA.hy926细胞的THP-1细胞数在培养3、6h较烧伤血清组均明显减少(t值分别为6.821、6.465,P值均小于0.01). 结论 电烧伤大鼠血清可诱导单核细胞分泌TNF-α,促进单核-内皮细胞黏附.而阻断PI3K/Akt信号通路,可有效抑制单核-内皮细胞的黏附.
目的 觀察燐脂酰肌醇3激酶/蛋白激酶B(PI3 K/Akt)通路在電燒傷大鼠血清誘導的單覈細胞中的變化,探討其在單覈細胞與血管內皮細胞黏附中的作用. 方法 取64隻清潔級SD大鼠製作電燒傷模型,製備電燒傷大鼠血清;取24隻大鼠不作處理,製備正常大鼠血清.(1)常規培養人單覈細胞株THP-1細胞,按照隨機數字錶法將對數生長期細胞分為正常血清組(將細胞重新懸浮于含體積分數20％正常大鼠血清的RPMI 1640培養液中)和燒傷血清組(將細胞重新懸浮于含體積分數20％電燒傷大鼠血清的RPMI 1640培養液中),每組設6箇複孔.正常血清組于培養24 h,燒傷血清組于培養3、6、24 h,觀察THP-1細胞形態,雙抗體夾心ELISA法測定2組細胞上清液中TNF-α含量.2組均于培養3、6、24 h收集細胞,蛋白質印跡法檢測Akt活化狀態.(2)另取THP-1細胞按照隨機數字錶法分為4組:正常血清組、燒傷血清組,2組各自培養方法同前;正常血清+阻斷劑組、燒傷血清+阻斷劑組,在正常血清組、燒傷血清組的培養液中加入渥曼青黴素100 nmol/L,餘培養方法同前.每組設6箇複孔.取培養3、6h的THP-1細胞,加入單層人血管內皮細胞株EA.hy926細胞,行單覈-內皮細胞黏附檢測.對數據行單因素方差分析、LSD-￡檢驗. 結果 (1)正常血清組培養24 h,THP-1細胞呈懸浮生長、形態一緻.燒傷血清組培養3h,細胞膜完整,細胞形態不規則;培養6h細胞大小不一,細胞膜與細胞質膨脹;培養24 h細胞膜破裂,細胞死亡.正常血清組培養24 h和燒傷血清組培養3、6、24 h,THP-1細胞上清液中TNF-α含量分彆為(38.5±1.4)、(75.1±1.5)、(91.5±1.8)、(117.0±1.4)pg/mL,總體比較差異明顯(F=1 415.306,P＜0.01).燒傷血清組培養3、6、24 h THP-1細胞上清液中TNF-α含量均明顯高于正常血清組培養24 h(￡值分彆為29.614、42.852、63.485,P值均小于0.01).燒傷血清組培養3、6、24 h,THP-1細胞中燐痠化蛋白激酶B/Akt比值分彆是正常血清組培養同時相點的2.66、3.69、1.17倍.(2)正常血清組、正常血清+阻斷劑組、燒傷血清組、燒傷血清+阻斷劑組培養3、6h,每100倍視野下黏附EA.hy926細胞的THP-1細胞數量分彆為(231 ±45)、(280 ±47)、(703±169)、(335±85)箇,(219±49)、(235±21)、(562±123)、(226±29)箇,總體比較均差異明顯(F值分彆為25.630、18.975,P值均小于0.01).與正常血清組比較,燒傷血清組黏附EA.hy926細胞的THP-1細胞數在培養3、6h均明顯增多(￡值分彆為6.189、6.601,P值均小于0.01);燒傷血清+阻斷劑組黏附EA.hy926細胞的THP-1細胞數在培養3、6h較燒傷血清組均明顯減少(t值分彆為6.821、6.465,P值均小于0.01). 結論 電燒傷大鼠血清可誘導單覈細胞分泌TNF-α,促進單覈-內皮細胞黏附.而阻斷PI3K/Akt信號通路,可有效抑製單覈-內皮細胞的黏附.
목적 관찰린지선기순3격매/단백격매B(PI3 K/Akt)통로재전소상대서혈청유도적단핵세포중적변화,탐토기재단핵세포여혈관내피세포점부중적작용. 방법 취64지청길급SD대서제작전소상모형,제비전소상대서혈청;취24지대서불작처리,제비정상대서혈청.(1)상규배양인단핵세포주THP-1세포,안조수궤수자표법장대수생장기세포분위정상혈청조(장세포중신현부우함체적분수20％정상대서혈청적RPMI 1640배양액중)화소상혈청조(장세포중신현부우함체적분수20％전소상대서혈청적RPMI 1640배양액중),매조설6개복공.정상혈청조우배양24 h,소상혈청조우배양3、6、24 h,관찰THP-1세포형태,쌍항체협심ELISA법측정2조세포상청액중TNF-α함량.2조균우배양3、6、24 h수집세포,단백질인적법검측Akt활화상태.(2)령취THP-1세포안조수궤수자표법분위4조:정상혈청조、소상혈청조,2조각자배양방법동전;정상혈청+조단제조、소상혈청+조단제조,재정상혈청조、소상혈청조적배양액중가입악만청매소100 nmol/L,여배양방법동전.매조설6개복공.취배양3、6h적THP-1세포,가입단층인혈관내피세포주EA.hy926세포,행단핵-내피세포점부검측.대수거행단인소방차분석、LSD-￡검험. 결과 (1)정상혈청조배양24 h,THP-1세포정현부생장、형태일치.소상혈청조배양3h,세포막완정,세포형태불규칙;배양6h세포대소불일,세포막여세포질팽창;배양24 h세포막파렬,세포사망.정상혈청조배양24 h화소상혈청조배양3、6、24 h,THP-1세포상청액중TNF-α함량분별위(38.5±1.4)、(75.1±1.5)、(91.5±1.8)、(117.0±1.4)pg/mL,총체비교차이명현(F=1 415.306,P＜0.01).소상혈청조배양3、6、24 h THP-1세포상청액중TNF-α함량균명현고우정상혈청조배양24 h(￡치분별위29.614、42.852、63.485,P치균소우0.01).소상혈청조배양3、6、24 h,THP-1세포중린산화단백격매B/Akt비치분별시정상혈청조배양동시상점적2.66、3.69、1.17배.(2)정상혈청조、정상혈청+조단제조、소상혈청조、소상혈청+조단제조배양3、6h,매100배시야하점부EA.hy926세포적THP-1세포수량분별위(231 ±45)、(280 ±47)、(703±169)、(335±85)개,(219±49)、(235±21)、(562±123)、(226±29)개,총체비교균차이명현(F치분별위25.630、18.975,P치균소우0.01).여정상혈청조비교,소상혈청조점부EA.hy926세포적THP-1세포수재배양3、6h균명현증다(￡치분별위6.189、6.601,P치균소우0.01);소상혈청+조단제조점부EA.hy926세포적THP-1세포수재배양3、6h교소상혈청조균명현감소(t치분별위6.821、6.465,P치균소우0.01). 결론 전소상대서혈청가유도단핵세포분비TNF-α,촉진단핵-내피세포점부.이조단PI3K/Akt신호통로,가유효억제단핵-내피세포적점부.
Objective To observe the change in phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signal pathway in monocytes as induced by serum of rats with electrical burn,and to explore the effects of PI3K/Akt pathway on monocyte-endothelial cell adhesion.Methods Sixty-four SD rats of clean grade were inflicted with electrical burn for the collection of serum of rats with electrical burn ; another group of twenty-four SD rats were used to obtain normal serum without treatment.(1) Human monocyte line THP-1 was routinely cultured.The THP-1 cells in logarithmic phase were divided into normal serum group (resuspended in RPMI 1640 medium with 20％ normal rat serum) and burn serum group (resuspended with RPMI 1640 medium with 20％ serum of rats with electrical burn) according to the random number table,with 6 wells in each group.Morphology of THP-1 cells in normal serum group was observed at post culture hour (PCH) 24,and that in burn serum group at PCH 3,6,24.The contents of TNF-α in culture supernatant were determined by double-antibody sandwich ELISA at the corresponding time point in each group.The state of Akt activation was determined by Western blotting at PCH 3,6,24.(2) Another portion of THP-1 cells were divided into 4 groups according to the random number table,with 6 wells in each group.Cells in normal serum group and burn serum group were given with the same culture condition as above; cells in normal serum + inhibitor group and burn serum + inhibitor group were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 nmol/L wortmannin in the nutrient solution.At PCH 3 and 6,THP-1 cells were added into the well with a monolayer of endothelial cell line EA.hy926 to observe the monocyte-endothelial cell adhesion.Data were processed with one-way analysis of variance and LSD-t test.Results (1) In normal serum group,THP-1 cells showed growth in suspension,with uniform shape at PCH 24.In burn serum group,the cell shape became irregular though the membrane was complete at PCH 3 ; cellular size became irregular and cell membrane and cytoplasm were swollen at PCH 6; cell membrane was disrupted with death of cells at PCH 24.The contents of TNF-cα in culture supernatant in normal serum group at PCH 24 and in burn serum group at PCH 3,6,24 were respectively (38.5 ± 1.4),(75.1 ±1.5),(91.5 ± 1.8),(117.0 ±1.4) pg/mL (F =1 415.306,P ＜0.01).The contents of TNF-α in culture supernatant in burn serum group at PCH 3,6,24 were all significantly higher than the content of TNF-α in normal serum group at PCH 24 (with t values respectively 29.614,42.852,63.485,P values below 0.01).The ratio values of phosphoylated Akt to Akt in burn serum group at PCH 3,6,24 were respectively 2.66,3.69,1.17 times of those in normal serum group at the corresponding time point.(2) In normal serum group,normal serum + inhibitor group,burn serum group,and burn serum + inhibitor group at PCH 3 and 6,the numbers of THP-1 cells adherent to endothelial cells were respectively (231 ± 45),(280 ±47),(703±169),(335 ±85) per 100-time field; (219 ±49),(235 ±21),(562±123),(226 ±29) per 100-time field (with F values respectively 25.630 and 18.975,P values below 0.01).The number of THP-1 cells adhered to EA.hy926 cells was significantly more in burn serum group than in normal serum group at PCH 3 and 6 (with t values respectively 6.189 and 6.601,P values below 0.01).The number of THP-1 cells adherent to EA.hy926 cells was significantly fewer in burn serum + inhibitor group than in burn serum group at PCH 3 and 6 (with t values respectively 6.821 and 6.465,P values below 0.01).Conclusions The serum of rats suffering from electrical burn can induce the monocytes to secrete TNF-α,thus enhancing monocyte-endothelial cell adhesion,but it can be inhibited by blocking PI3K/Akt signal pathway.
