南通大学学报（医学版）
南通大學學報（醫學版）
남통대학학보（의학판）
JOURNAL OF NANTONG UNIVERSITY(MEDICAL SCIENCES)
2015年
3期
173-177
,共5页
冯杰%花娟%胡遵鲁%严莹莹%巫荣华%刘梅
馮傑%花娟%鬍遵魯%嚴瑩瑩%巫榮華%劉梅
풍걸%화연%호준로%엄형형%무영화%류매
Kinesin-12%Kinesin-5%星形胶质细胞%凋亡%细胞周期%大鼠
Kinesin-12%Kinesin-5%星形膠質細胞%凋亡%細胞週期%大鼠
Kinesin-12%Kinesin-5%성형효질세포%조망%세포주기%대서
Kinesin-12%Kinesin-5%astrocyte%apoptosis%cell cycle%rat
目的：比较驱动蛋白家族成员Kinesin-12和Kinesin-5对星形胶质细胞周期和凋亡的影响差异，探讨Kinesin-12在细胞增殖中的作用机制。方法：采用siRNA方法干扰Kinesin-12、Kinesin-5基因表达，通过实时定量PCR、Western Blot检测干扰表达效率；Hoechst33342染色观察细胞凋亡；经AnnexinⅤ/PI双染色检测细胞凋亡率；流式细胞术检测细胞周期变化；细胞免疫化学染色观察 Kinesin-12失功能后 MyosinⅡB 的分布改变。结果：Kinesin-12和Kinesin-5蛋白表达下调达65%以上；Hoechst33342染色和AnnexinⅤ/PI双染色法发现干扰Kinesin-12和Kinesin-5表达后细胞凋亡率显著增加(P＜0.05)；流式细胞术检测发现Kinesin-12 siRNA处理后细胞周期被阻滞在G1期，而Kinesin-5 siRNA处理后，细胞周期被阻滞在G2/M期。细胞免疫化学染色发现，抑制Kinesin-12表达后MyosinⅡB(即Myh 10)的分布发生改变。结论：抑制Kinesin-12或Kinesin-5表达，均可明显阻滞细胞分裂并诱导细胞凋亡，但作用机制与Kinesin-5不同，推测Kinesin-12可能与MyosinⅡB相互作用形成异源聚合体作用于微管微丝骨架重构。
目的：比較驅動蛋白傢族成員Kinesin-12和Kinesin-5對星形膠質細胞週期和凋亡的影響差異，探討Kinesin-12在細胞增殖中的作用機製。方法：採用siRNA方法榦擾Kinesin-12、Kinesin-5基因錶達，通過實時定量PCR、Western Blot檢測榦擾錶達效率；Hoechst33342染色觀察細胞凋亡；經AnnexinⅤ/PI雙染色檢測細胞凋亡率；流式細胞術檢測細胞週期變化；細胞免疫化學染色觀察 Kinesin-12失功能後 MyosinⅡB 的分佈改變。結果：Kinesin-12和Kinesin-5蛋白錶達下調達65%以上；Hoechst33342染色和AnnexinⅤ/PI雙染色法髮現榦擾Kinesin-12和Kinesin-5錶達後細胞凋亡率顯著增加(P＜0.05)；流式細胞術檢測髮現Kinesin-12 siRNA處理後細胞週期被阻滯在G1期，而Kinesin-5 siRNA處理後，細胞週期被阻滯在G2/M期。細胞免疫化學染色髮現，抑製Kinesin-12錶達後MyosinⅡB(即Myh 10)的分佈髮生改變。結論：抑製Kinesin-12或Kinesin-5錶達，均可明顯阻滯細胞分裂併誘導細胞凋亡，但作用機製與Kinesin-5不同，推測Kinesin-12可能與MyosinⅡB相互作用形成異源聚閤體作用于微管微絲骨架重構。
목적：비교구동단백가족성원Kinesin-12화Kinesin-5대성형효질세포주기화조망적영향차이，탐토Kinesin-12재세포증식중적작용궤제。방법：채용siRNA방법간우Kinesin-12、Kinesin-5기인표체，통과실시정량PCR、Western Blot검측간우표체효솔；Hoechst33342염색관찰세포조망；경AnnexinⅤ/PI쌍염색검측세포조망솔；류식세포술검측세포주기변화；세포면역화학염색관찰 Kinesin-12실공능후 MyosinⅡB 적분포개변。결과：Kinesin-12화Kinesin-5단백표체하조체65%이상；Hoechst33342염색화AnnexinⅤ/PI쌍염색법발현간우Kinesin-12화Kinesin-5표체후세포조망솔현저증가(P＜0.05)；류식세포술검측발현Kinesin-12 siRNA처리후세포주기피조체재G1기，이Kinesin-5 siRNA처리후，세포주기피조체재G2/M기。세포면역화학염색발현，억제Kinesin-12표체후MyosinⅡB(즉Myh 10)적분포발생개변。결론：억제Kinesin-12혹Kinesin-5표체，균가명현조체세포분렬병유도세포조망，단작용궤제여Kinesin-5불동，추측Kinesin-12가능여MyosinⅡB상호작용형성이원취합체작용우미관미사골가중구。
Objective:To investigate the mechanism of Kinesin-12 on cell proliferation, by comparing the effect of Kinesin-12, Kinesin-5 knockdown on cell cycle arrest and apoptosis in rat astrocytes. Methods: Kinesin-12, Kinesin-5 gene expression was silenced by siRNA transfection, and the gene expression level was detected by real-time quantitative PCR and Western Blot. Morphological changes were observed under the fluorescence microscope after being treated by Hoechst33342. Apoptotic cell and DNA contents were measured by flow cytometry after Kinesin-12, Kinesin-5 siRNA treatment for 4 days. Results: The protein level of Kinesin-12 and Kinesin-5 were decreased by over 65%. The results of Hoechst33342 staining showed the apoptotic cells significantly increased in Kinesin-12, Kinesin-5 knockdown groups compared with the control group (P<0.05). High apoptosis rate in the two genes knockdown cells was also found by AnnexinⅤ/PI double staining. The results of cell cycle analysis indicated depleting Kinesin-5 arrested more cells in G 2/M phase, however Kinesin-12 knockdown arrested cells in G1 phase. The distribution of Myosin ⅡB altered after Kinesin-12 depletion. Conclusion:Depletion of Kinesin-12 or Kinesin-5 leads to cell cycle arrest and promotes cell apoptosis, nevertheless, the mechanism of two motor proteins are different. We propose Kinesin-12 may play a role during cytokinesis by interacting with Myosin ⅡB, an actin dependent molecular motor protein.
