中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2014年
8期
598-605
,共8页
RNA干扰%OPTN%RGC5%亚细胞结构%细胞存活
RNA榦擾%OPTN%RGC5%亞細胞結構%細胞存活
RNA간우%OPTN%RGC5%아세포결구%세포존활
RNA interference%OPTN%RGC5%Ultra-structure%Cell survival
目的 观察大鼠OPTN基因的低表达对视网膜神经节细胞系(RGC5)亚细胞形态结构及存活的影响.方法 实验研究.设计并合成特异性针对Sprague-Dawley(SD)大鼠OPTN的3对RNA干扰片段(siRNA),体外培养SD大鼠星型胶质细胞,取对数生长期的细胞做实验,分别将3对siRNA(si-OPTN-001、si-OPTN-002、si-OPTN-003)通过脂质体Lipofectemine 2000转染入星型胶质细胞,24 ～ 48 h后采用实时PCR和Western Blot检测siRNA的干扰效果,筛选出抑制效果最佳的序列;以筛选出来的siRNA序列构建绿色荧光蛋白EGFP标记的si-OPTN,并通过Lipofectemine 2000包裹转染RGC5,将RGC5分为4组:空白对照组,绿色荧光蛋白真核细胞表达载体(pEGFP)阳性对照组,si-OPTN组,脂质体阴性对照组.转染24～48 h后用细胞器特异荧光染料标记细胞内不同细胞器结构,通过共聚焦荧光显微镜来观察表达的si-OPTN在RGC5内的分布、定位及其对细胞器形态结构的影响;通过形态学观察和Hoechst33342-PI荧光染色观察si-OPTN对RGC5存活的影响.实验中不同组间的总体比较采用单因素方差分析.结果 实时PCR及Western Blot的结果显示:转染si-OPTN-001、si-OPTN-002和si-OPTN-003 24 h后星型胶质细胞内OPTN的mRNA表达下降,分别为阴性对照组的(61.71±0.84)％、(48.13±0.92)％和(46.22±0.73)％,而转染si-OPTN-001、si-OPTN-002和si-OPTN-003 48 h后星型胶质细胞内OPTN的蛋白表达水平下降,分别为阴性对照组的(64.44±2.01)％、(57.78±1.97)％和(37.78±1.84)％,其中si-OPTN-003与阴性对照组相比降低显著;si-OPTN转染RGC5细胞24 h后的转染效率为(17.43±0.94)％;转染48 h后的转染效率为(20.13±1.24)％;表达的si-OPTN在细胞内基本上均匀分布于整个细胞,覆盖胞质和胞核,与阴性对照组细胞器染色情况比较,转染细胞内的肌丝蛋白、线粒体、溶酶体及高尔基体形态结构未见明显损伤性改变;Hochest33342/PI荧光染色结果显示:转染24 h,与空白对照组(0.74±0.34)％和阴性对照组(0.96±0.41)％比较,EGFP阳性对照组及si-OPTN组整组的RGC5细胞凋亡率均增加,差异有统计学意义(F=5.457,P＜0.05),但si-OPTN组转染质粒的细胞凋亡率明显低于阳性对照组,差异有统计学意义(F=6.541,P＜0.05).随转染时间延长,在转染48 h时,阳性对照组凋亡率较24 h时下降,si-OPTN组凋亡率稍升高但无统计学意义(F=3.212,P＞0.05),且仍低于阳性对照组.结论 相较于另2对si-RNA,si-OPTN-003更能有效地抑制OPTN的表达;si-OPTN转染RGC5后均匀分布于全细胞,对细胞内的亚细胞器结构未见明显损伤性改变;通过转染si-OPTN使OPTN表达降低可能对RGC5的存活有保护作用.
目的 觀察大鼠OPTN基因的低錶達對視網膜神經節細胞繫(RGC5)亞細胞形態結構及存活的影響.方法 實驗研究.設計併閤成特異性針對Sprague-Dawley(SD)大鼠OPTN的3對RNA榦擾片段(siRNA),體外培養SD大鼠星型膠質細胞,取對數生長期的細胞做實驗,分彆將3對siRNA(si-OPTN-001、si-OPTN-002、si-OPTN-003)通過脂質體Lipofectemine 2000轉染入星型膠質細胞,24 ～ 48 h後採用實時PCR和Western Blot檢測siRNA的榦擾效果,篩選齣抑製效果最佳的序列;以篩選齣來的siRNA序列構建綠色熒光蛋白EGFP標記的si-OPTN,併通過Lipofectemine 2000包裹轉染RGC5,將RGC5分為4組:空白對照組,綠色熒光蛋白真覈細胞錶達載體(pEGFP)暘性對照組,si-OPTN組,脂質體陰性對照組.轉染24～48 h後用細胞器特異熒光染料標記細胞內不同細胞器結構,通過共聚焦熒光顯微鏡來觀察錶達的si-OPTN在RGC5內的分佈、定位及其對細胞器形態結構的影響;通過形態學觀察和Hoechst33342-PI熒光染色觀察si-OPTN對RGC5存活的影響.實驗中不同組間的總體比較採用單因素方差分析.結果 實時PCR及Western Blot的結果顯示:轉染si-OPTN-001、si-OPTN-002和si-OPTN-003 24 h後星型膠質細胞內OPTN的mRNA錶達下降,分彆為陰性對照組的(61.71±0.84)％、(48.13±0.92)％和(46.22±0.73)％,而轉染si-OPTN-001、si-OPTN-002和si-OPTN-003 48 h後星型膠質細胞內OPTN的蛋白錶達水平下降,分彆為陰性對照組的(64.44±2.01)％、(57.78±1.97)％和(37.78±1.84)％,其中si-OPTN-003與陰性對照組相比降低顯著;si-OPTN轉染RGC5細胞24 h後的轉染效率為(17.43±0.94)％;轉染48 h後的轉染效率為(20.13±1.24)％;錶達的si-OPTN在細胞內基本上均勻分佈于整箇細胞,覆蓋胞質和胞覈,與陰性對照組細胞器染色情況比較,轉染細胞內的肌絲蛋白、線粒體、溶酶體及高爾基體形態結構未見明顯損傷性改變;Hochest33342/PI熒光染色結果顯示:轉染24 h,與空白對照組(0.74±0.34)％和陰性對照組(0.96±0.41)％比較,EGFP暘性對照組及si-OPTN組整組的RGC5細胞凋亡率均增加,差異有統計學意義(F=5.457,P＜0.05),但si-OPTN組轉染質粒的細胞凋亡率明顯低于暘性對照組,差異有統計學意義(F=6.541,P＜0.05).隨轉染時間延長,在轉染48 h時,暘性對照組凋亡率較24 h時下降,si-OPTN組凋亡率稍升高但無統計學意義(F=3.212,P＞0.05),且仍低于暘性對照組.結論 相較于另2對si-RNA,si-OPTN-003更能有效地抑製OPTN的錶達;si-OPTN轉染RGC5後均勻分佈于全細胞,對細胞內的亞細胞器結構未見明顯損傷性改變;通過轉染si-OPTN使OPTN錶達降低可能對RGC5的存活有保護作用.
목적 관찰대서OPTN기인적저표체대시망막신경절세포계(RGC5)아세포형태결구급존활적영향.방법 실험연구.설계병합성특이성침대Sprague-Dawley(SD)대서OPTN적3대RNA간우편단(siRNA),체외배양SD대서성형효질세포,취대수생장기적세포주실험,분별장3대siRNA(si-OPTN-001、si-OPTN-002、si-OPTN-003)통과지질체Lipofectemine 2000전염입성형효질세포,24 ～ 48 h후채용실시PCR화Western Blot검측siRNA적간우효과,사선출억제효과최가적서렬;이사선출래적siRNA서렬구건록색형광단백EGFP표기적si-OPTN,병통과Lipofectemine 2000포과전염RGC5,장RGC5분위4조:공백대조조,록색형광단백진핵세포표체재체(pEGFP)양성대조조,si-OPTN조,지질체음성대조조.전염24～48 h후용세포기특이형광염료표기세포내불동세포기결구,통과공취초형광현미경래관찰표체적si-OPTN재RGC5내적분포、정위급기대세포기형태결구적영향;통과형태학관찰화Hoechst33342-PI형광염색관찰si-OPTN대RGC5존활적영향.실험중불동조간적총체비교채용단인소방차분석.결과 실시PCR급Western Blot적결과현시:전염si-OPTN-001、si-OPTN-002화si-OPTN-003 24 h후성형효질세포내OPTN적mRNA표체하강,분별위음성대조조적(61.71±0.84)％、(48.13±0.92)％화(46.22±0.73)％,이전염si-OPTN-001、si-OPTN-002화si-OPTN-003 48 h후성형효질세포내OPTN적단백표체수평하강,분별위음성대조조적(64.44±2.01)％、(57.78±1.97)％화(37.78±1.84)％,기중si-OPTN-003여음성대조조상비강저현저;si-OPTN전염RGC5세포24 h후적전염효솔위(17.43±0.94)％;전염48 h후적전염효솔위(20.13±1.24)％;표체적si-OPTN재세포내기본상균균분포우정개세포,복개포질화포핵,여음성대조조세포기염색정황비교,전염세포내적기사단백、선립체、용매체급고이기체형태결구미견명현손상성개변;Hochest33342/PI형광염색결과현시:전염24 h,여공백대조조(0.74±0.34)％화음성대조조(0.96±0.41)％비교,EGFP양성대조조급si-OPTN조정조적RGC5세포조망솔균증가,차이유통계학의의(F=5.457,P＜0.05),단si-OPTN조전염질립적세포조망솔명현저우양성대조조,차이유통계학의의(F=6.541,P＜0.05).수전염시간연장,재전염48 h시,양성대조조조망솔교24 h시하강,si-OPTN조조망솔초승고단무통계학의의(F=3.212,P＞0.05),차잉저우양성대조조.결론 상교우령2대si-RNA,si-OPTN-003경능유효지억제OPTN적표체;si-OPTN전염RGC5후균균분포우전세포,대세포내적아세포기결구미견명현손상성개변;통과전염si-OPTN사OPTN표체강저가능대RGC5적존활유보호작용.
Objective To investigate the effects of decreased OPTN expression on the morphological alteration of sub-cellular structure and the survival of the rat retinal ganglion cell line RGC5.Methods Experimental study.OPTN specific siRNA was designed,synthesized and transfected into the astrocyte cell by Lipofectemine 2000.The mRNA and the protein of OPTN were assessed by real time-polymerase chain reaction (real time-PCR) and Western Blot.Eukaryotic expressing plasimid of OPTN specific siRNA (siOPTN) was constructed with the most effective RNA interference sequence and transfected into RGC5 by Lipofectemine 2000.The RGC5 was divided into 4 groups:blank control group,pEGFP positive group,siOPTN group,and liposome negative group.Specific fluorescent labeling dyes were used to mark the corresponding cell organelle.The distribution of optineurin and the morphology alteration of sub-cellular structure induced by the abnormal expression of optineurin in RGC5 were observed with confocal fluorescence microscopy.The effect of si-OPTN on cell survival was monitored by morphologic observation and PIHoechst33342 fluorescence staining of cells.One-way ANOVA was used between groups.Results After a 24 h transfection,the expression of OPTN mRNA was significantly inhibited by transfection of si-OPTN-001,si-OPTN-002 and si-OPTN-003 which was (61.71 ±0.84)％,(48.13 ±0.92)％ and (46.22 ±0.73)％ respectively comparing with negative control.Forty-eight hours after transfection,the protein expression of OPTN decreased to (64.44 ± 2.01) ％,(57.78 ± 1.97) ％ and (37.78 ± 1.84) ％ comparing with negative control.When cells were transfected after 24 h and 48 h,the transfecting efficiency of plasmids to RGC5 was (17.43 ± 0.94)％ and (20.13 ± 1.24)％ respectively.The distribution of si-OPTN was similar to EGFP.The green fluorescence distributed evenly in the cytoplasm and the nucleus.Compared with negative control,the morphology of filaments,mitochondria,lysosomes,and Golgi body had no significant alteration in siOPTN group.By using fluorescent double staining of Hoechst33342 and PI and comparing with blank control group (0.74 ±0.34) ％ and negative control group (0.96 ±0.41) ％,increased cell apoptosis was observed in positive control group and si-OPTN group obviously after a 24 h transfection (F =5.457,P ＜0.05),but the apoptosis rate decreased in si-OPTN group comparing with that of positive control group (F =6.541,P ＜ 0.05).As the transfecting time extended,the apoptosis rate of positive control group increased when tranfecteing cultures reached 48 h.The apoptosis rate in si-OPTN group increased slightly with no significance (F =3.212,P ＞0.05).Conclusions Si-OPTN-003 is the most effective siRNA in inhibiting the OPTN expression.The expression of si-OPTN distributed evenly in the whole cell with no obvious injuries in the transfected cell.The down regulation of optineurin induced by si-OPTN could be decreased RGC5 cell death.
