中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
5期
1094-1097
,共4页
陶海鹰%贺斌%卫爱林%刘世清
陶海鷹%賀斌%衛愛林%劉世清
도해응%하빈%위애림%류세청
羧甲基壳聚糖%雪旺细胞%增殖%细胞周期蛋白
羧甲基殼聚糖%雪旺細胞%增殖%細胞週期蛋白
최갑기각취당%설왕세포%증식%세포주기단백
Carboxymethylated chitosan%Schwann cells%Proliferation%Cyclin
目的 观察羧甲基壳聚糖(CMCS)对雪旺细胞(SCs)增殖及c-fos、c-jun及细胞周期素(Cyclin)E表达的影响.方法 取3～5d鼠龄无特定病原体(SPF)级SD大鼠20只,雌雄不限,体质量25～ 30 g,取双侧坐骨神经体外培养SCs,经S-100免疫荧光标记鉴定.取处于对数生长期的第2代SCs,应用噻唑蓝(MTT)比色法及5-溴脱氧尿嘧啶核苷(BrdU)染色法检测不同浓度CMCS对SCs增殖的影响.将SCs分为4组,加入CMCS调整终质量浓度为50 mg/L(B组)、100 mg/L(C组)、200 mg/L(D组),对照组(A组)加入等量磷酸盐缓冲液(PBS);逆转录-聚合酶链反应(RT-PCR)检测SCs内c-fos、c-jun及Cyclin E基因的表达;Western blot法检测c-fos、c-jun及Cyclin E蛋白表达的改变.结果 S-100免疫荧光标记鉴定结果显示细胞阳性率达90％以上.MTT结果表明各组SCs吸光度(A)值分别为:对照组为0.141 3 ±0.005 2,10 mg/L CMCS处理组为0.152 5±0.006 1,50 mg/L CMCS处理组为0.1648±0.0072,100 mg/L CMCS处理组为0.1726±0.0062,200 mg/L CMCS处理组为0.193 8±0.008 1,500 mg/L CMCS处理组为0.1947 ±0.0062,随着CMCS浓度的升高而增加.BrdU染色结果表明CMCS在50 ～ 200 mg/L浓度范围内可以促进SCs内DNA合成,且随着CMCS浓度的增加而增加,200 mg/L CMCS作用最明显,与对照组比较增加2～3倍.RT-PCR和Western blot检测均表明B、C、D组c-foc、c-jun及Cyclin E mRNA和蛋白表达均显著高于A组(P＜0.05);其中D组作用最明显.结论 CMCS可以促进SCs增殖并促进增殖周期相关基因c-fos、c-jun及Cyclin E表达.
目的 觀察羧甲基殼聚糖(CMCS)對雪旺細胞(SCs)增殖及c-fos、c-jun及細胞週期素(Cyclin)E錶達的影響.方法 取3～5d鼠齡無特定病原體(SPF)級SD大鼠20隻,雌雄不限,體質量25～ 30 g,取雙側坐骨神經體外培養SCs,經S-100免疫熒光標記鑒定.取處于對數生長期的第2代SCs,應用噻唑藍(MTT)比色法及5-溴脫氧尿嘧啶覈苷(BrdU)染色法檢測不同濃度CMCS對SCs增殖的影響.將SCs分為4組,加入CMCS調整終質量濃度為50 mg/L(B組)、100 mg/L(C組)、200 mg/L(D組),對照組(A組)加入等量燐痠鹽緩遲液(PBS);逆轉錄-聚閤酶鏈反應(RT-PCR)檢測SCs內c-fos、c-jun及Cyclin E基因的錶達;Western blot法檢測c-fos、c-jun及Cyclin E蛋白錶達的改變.結果 S-100免疫熒光標記鑒定結果顯示細胞暘性率達90％以上.MTT結果錶明各組SCs吸光度(A)值分彆為:對照組為0.141 3 ±0.005 2,10 mg/L CMCS處理組為0.152 5±0.006 1,50 mg/L CMCS處理組為0.1648±0.0072,100 mg/L CMCS處理組為0.1726±0.0062,200 mg/L CMCS處理組為0.193 8±0.008 1,500 mg/L CMCS處理組為0.1947 ±0.0062,隨著CMCS濃度的升高而增加.BrdU染色結果錶明CMCS在50 ～ 200 mg/L濃度範圍內可以促進SCs內DNA閤成,且隨著CMCS濃度的增加而增加,200 mg/L CMCS作用最明顯,與對照組比較增加2～3倍.RT-PCR和Western blot檢測均錶明B、C、D組c-foc、c-jun及Cyclin E mRNA和蛋白錶達均顯著高于A組(P＜0.05);其中D組作用最明顯.結論 CMCS可以促進SCs增殖併促進增殖週期相關基因c-fos、c-jun及Cyclin E錶達.
목적 관찰최갑기각취당(CMCS)대설왕세포(SCs)증식급c-fos、c-jun급세포주기소(Cyclin)E표체적영향.방법 취3～5d서령무특정병원체(SPF)급SD대서20지,자웅불한,체질량25～ 30 g,취쌍측좌골신경체외배양SCs,경S-100면역형광표기감정.취처우대수생장기적제2대SCs,응용새서람(MTT)비색법급5-추탈양뇨밀정핵감(BrdU)염색법검측불동농도CMCS대SCs증식적영향.장SCs분위4조,가입CMCS조정종질량농도위50 mg/L(B조)、100 mg/L(C조)、200 mg/L(D조),대조조(A조)가입등량린산염완충액(PBS);역전록-취합매련반응(RT-PCR)검측SCs내c-fos、c-jun급Cyclin E기인적표체;Western blot법검측c-fos、c-jun급Cyclin E단백표체적개변.결과 S-100면역형광표기감정결과현시세포양성솔체90％이상.MTT결과표명각조SCs흡광도(A)치분별위:대조조위0.141 3 ±0.005 2,10 mg/L CMCS처리조위0.152 5±0.006 1,50 mg/L CMCS처리조위0.1648±0.0072,100 mg/L CMCS처리조위0.1726±0.0062,200 mg/L CMCS처리조위0.193 8±0.008 1,500 mg/L CMCS처리조위0.1947 ±0.0062,수착CMCS농도적승고이증가.BrdU염색결과표명CMCS재50 ～ 200 mg/L농도범위내가이촉진SCs내DNA합성,차수착CMCS농도적증가이증가,200 mg/L CMCS작용최명현,여대조조비교증가2～3배.RT-PCR화Western blot검측균표명B、C、D조c-foc、c-jun급Cyclin E mRNA화단백표체균현저고우A조(P＜0.05);기중D조작용최명현.결론 CMCS가이촉진SCs증식병촉진증식주기상관기인c-fos、c-jun급Cyclin E표체.
Objective To investigate the effects of carboxymethylated chitosan (CMCS) on Schwann cells (SCs) proliferation and expression of c-fos,c-jun and Cyclin E.Methods Twenty 3-5 days old SPF SD rats were ures,weighing 25-30 g,take the sciatic nerves in vitro SCs,via S-100 immunofluorescence markers.In the logarithmic growth phase 2nd generation SCs,application methyl thiazol tet-razolium (MTT) and BrdU staining for different concentrations of CMCS SCs proliferation.The SCs are divided into four groups,adding a final concentration of CMCS adjustment 50 (B group),100 (C group),200 mg/L (D group) and control group (A) an equal volume phosphate buffer (PBS) ; reverse tran-scriptase PCR assay within SCs c-fos,c-jun and Cyclin E gene expression;Western blotting assay c-fos,c-jun and Cyclin E protein expression changes.Results S-100 immunofluorescence markers showing more than 90％ positive cells.MTT results showed that each group SCs absorbance (A) values were:control group (0.141 3 ±0.005 2),10 mg/L CMCS treatment group (0.152 5 ±0.006 1),50 mg/L CMCS treat-ment group (0.164 8 ±0.007 2),100 mg/L CMCS treatment group (0.172 6 ±0.006 2),200 mg/L CMCS treatment group (0.193 8 ±0.008 1),500 mg/L CMCS treatment group (0.194 7 ±0.006 2),with the concentration increased proliferation CMCS.CMCS BrdU staining results showed that in the concentration range of 50-200 mg/L can promote SCs DNA synthesis and increased with increasing concentrations of CMCS,200 mg/L CMCS most significant effect,compared with control group increased by 2 to 3 times.Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis showed,B,C,D group c-foc,c-jun and Cyclin E mRNA and protein expression were significantly higher in group A (P ＜0.05) ;among the most significant effect D group.Conclusion CMCS can promote the SCs proliferation and promotes cell cycle related gene c-fos,c-jun and Cyclin E expression.
