背景 诱导细胞间隙连接蛋白(Cx)基因在瘤细胞中过表达是全反式维甲酸(ATRA)抑制肿瘤细胞生长的重要机制.我们先前的研究已证实,ATRA抑制视网膜母细胞瘤(RB)细胞增生、分化作用的机制与诱导Cx43过表达有关,但药物作用位点及其对在体肿瘤组织生长的影响尚未明确. 目的研究ATRA对RB细胞中Cx43基因及其蛋白表达的影响,探讨其作用机制. 方法 用无水乙醇将ATRA溶解并配制成浓度为1×10-2 mol/L的溶液,使用前再用细胞培养液配制成不同浓度ATRA液.用含体积分数10％热灭活胎牛血清(FBS)的RPMI-1640培养液常规培养人RB细胞株HXO-RB44,将1×10-5、1×10-6和1×10-2mol/L的ATRA分别加至培养基中,分别于添加后2、4、6d采集细胞,分别采用Western blot法和逆转录PCR法检测细胞中Cx43蛋白及mRNA的表达变化.选取15只BALB/c裸鼠,将RB细胞悬液1μl(含5×105个细胞)进行裸鼠右眼前房注射,建立裸鼠眼前房RB模型.将造模成功的11只裸鼠随机分为空白对照组、阴性对照组和1×10-5 mol/L ATRA组,阴性对照组和1×10-5 mol/L ATRA组裸鼠分别行0.5％无水乙醇的生理盐水或1×10-5 mol/L ATRA前房注射,每3天1次,共注射3周,裂隙灯显微镜下观察各组裸鼠肿瘤生长情况;实验结束时摘除实验眼,用苏木精-伊红染色法进行组织病理学检查. 结果 Western blot法检测结果显示,随ATRA作用时间的延长,各组Cx43蛋白表达量(ACx43/AGAPDH)均逐渐增加,总体比较差异有统计学意义(F时间=71.31,P=0.00;F分组=7.66,P=0.00);药物作用后2 d 1×10-5 mol/L ATRA组、作用后4 d 1×10-6 mol/L ATRA组、作用后6 d 1×10-7 mol/L ATRA组Cx43蛋白表达量均明显高于空白对照组,差异均有统计学意义(￡=3.34,P＜0.01;t=2.33,P＜0.05;t=3.12,P＜0.01).逆转录PCR检测结果显示,随ATRA作用时间的延长,各组Cx43 mRNA表达量(ACx43mRNA/Aβ-actin)均逐渐升高,差异均有统计学意义(F时间=90.90,P=0.00;F分组=6.86,P=0.00),药物作用后2 d 1×10-5 mol/L ATRA组、作用后4 d 1×10-6mol/L ATRA组和1×10-7mol/LATRA组Cx43 mRNA表达量均明显高于空白对照组,差异均有统计学意义(t=3.57,P＜0.01;t=6.31,P＜0.01;t=2.22,P＜0.05).RB细胞悬液前房注射后6～9d,11眼成功建立裸鼠RB肿瘤模型,造模后20 d,空白对照组裸鼠肿瘤长满前房,眼球突出;而同时间点1×10-5 mol/L ATRA组肿瘤生长仅占前房的1/2.组织病理学检查发现,空白对照组裸鼠肿瘤细胞充满前房、后房和玻璃体腔,而1×10-5 mol/L ATRA组裸鼠肿瘤主要在前房生长,后房及玻璃体腔内未见瘤细胞. 结论 ATRA在转录水平诱导RB细胞中Cx43表达的上调,从而抑制和限制裸鼠前房RB肿瘤的生长.
揹景 誘導細胞間隙連接蛋白(Cx)基因在瘤細胞中過錶達是全反式維甲痠(ATRA)抑製腫瘤細胞生長的重要機製.我們先前的研究已證實,ATRA抑製視網膜母細胞瘤(RB)細胞增生、分化作用的機製與誘導Cx43過錶達有關,但藥物作用位點及其對在體腫瘤組織生長的影響尚未明確. 目的研究ATRA對RB細胞中Cx43基因及其蛋白錶達的影響,探討其作用機製. 方法 用無水乙醇將ATRA溶解併配製成濃度為1×10-2 mol/L的溶液,使用前再用細胞培養液配製成不同濃度ATRA液.用含體積分數10％熱滅活胎牛血清(FBS)的RPMI-1640培養液常規培養人RB細胞株HXO-RB44,將1×10-5、1×10-6和1×10-2mol/L的ATRA分彆加至培養基中,分彆于添加後2、4、6d採集細胞,分彆採用Western blot法和逆轉錄PCR法檢測細胞中Cx43蛋白及mRNA的錶達變化.選取15隻BALB/c裸鼠,將RB細胞懸液1μl(含5×105箇細胞)進行裸鼠右眼前房註射,建立裸鼠眼前房RB模型.將造模成功的11隻裸鼠隨機分為空白對照組、陰性對照組和1×10-5 mol/L ATRA組,陰性對照組和1×10-5 mol/L ATRA組裸鼠分彆行0.5％無水乙醇的生理鹽水或1×10-5 mol/L ATRA前房註射,每3天1次,共註射3週,裂隙燈顯微鏡下觀察各組裸鼠腫瘤生長情況;實驗結束時摘除實驗眼,用囌木精-伊紅染色法進行組織病理學檢查. 結果 Western blot法檢測結果顯示,隨ATRA作用時間的延長,各組Cx43蛋白錶達量(ACx43/AGAPDH)均逐漸增加,總體比較差異有統計學意義(F時間=71.31,P=0.00;F分組=7.66,P=0.00);藥物作用後2 d 1×10-5 mol/L ATRA組、作用後4 d 1×10-6 mol/L ATRA組、作用後6 d 1×10-7 mol/L ATRA組Cx43蛋白錶達量均明顯高于空白對照組,差異均有統計學意義(￡=3.34,P＜0.01;t=2.33,P＜0.05;t=3.12,P＜0.01).逆轉錄PCR檢測結果顯示,隨ATRA作用時間的延長,各組Cx43 mRNA錶達量(ACx43mRNA/Aβ-actin)均逐漸升高,差異均有統計學意義(F時間=90.90,P=0.00;F分組=6.86,P=0.00),藥物作用後2 d 1×10-5 mol/L ATRA組、作用後4 d 1×10-6mol/L ATRA組和1×10-7mol/LATRA組Cx43 mRNA錶達量均明顯高于空白對照組,差異均有統計學意義(t=3.57,P＜0.01;t=6.31,P＜0.01;t=2.22,P＜0.05).RB細胞懸液前房註射後6～9d,11眼成功建立裸鼠RB腫瘤模型,造模後20 d,空白對照組裸鼠腫瘤長滿前房,眼毬突齣;而同時間點1×10-5 mol/L ATRA組腫瘤生長僅佔前房的1/2.組織病理學檢查髮現,空白對照組裸鼠腫瘤細胞充滿前房、後房和玻璃體腔,而1×10-5 mol/L ATRA組裸鼠腫瘤主要在前房生長,後房及玻璃體腔內未見瘤細胞. 結論 ATRA在轉錄水平誘導RB細胞中Cx43錶達的上調,從而抑製和限製裸鼠前房RB腫瘤的生長.
배경 유도세포간극련접단백(Cx)기인재류세포중과표체시전반식유갑산(ATRA)억제종류세포생장적중요궤제.아문선전적연구이증실,ATRA억제시망막모세포류(RB)세포증생、분화작용적궤제여유도Cx43과표체유관,단약물작용위점급기대재체종류조직생장적영향상미명학. 목적연구ATRA대RB세포중Cx43기인급기단백표체적영향,탐토기작용궤제. 방법 용무수을순장ATRA용해병배제성농도위1×10-2 mol/L적용액,사용전재용세포배양액배제성불동농도ATRA액.용함체적분수10％열멸활태우혈청(FBS)적RPMI-1640배양액상규배양인RB세포주HXO-RB44,장1×10-5、1×10-6화1×10-2mol/L적ATRA분별가지배양기중,분별우첨가후2、4、6d채집세포,분별채용Western blot법화역전록PCR법검측세포중Cx43단백급mRNA적표체변화.선취15지BALB/c라서,장RB세포현액1μl(함5×105개세포)진행라서우안전방주사,건립라서안전방RB모형.장조모성공적11지라서수궤분위공백대조조、음성대조조화1×10-5 mol/L ATRA조,음성대조조화1×10-5 mol/L ATRA조라서분별행0.5％무수을순적생리염수혹1×10-5 mol/L ATRA전방주사,매3천1차,공주사3주,렬극등현미경하관찰각조라서종류생장정황;실험결속시적제실험안,용소목정-이홍염색법진행조직병이학검사. 결과 Western blot법검측결과현시,수ATRA작용시간적연장,각조Cx43단백표체량(ACx43/AGAPDH)균축점증가,총체비교차이유통계학의의(F시간=71.31,P=0.00;F분조=7.66,P=0.00);약물작용후2 d 1×10-5 mol/L ATRA조、작용후4 d 1×10-6 mol/L ATRA조、작용후6 d 1×10-7 mol/L ATRA조Cx43단백표체량균명현고우공백대조조,차이균유통계학의의(￡=3.34,P＜0.01;t=2.33,P＜0.05;t=3.12,P＜0.01).역전록PCR검측결과현시,수ATRA작용시간적연장,각조Cx43 mRNA표체량(ACx43mRNA/Aβ-actin)균축점승고,차이균유통계학의의(F시간=90.90,P=0.00;F분조=6.86,P=0.00),약물작용후2 d 1×10-5 mol/L ATRA조、작용후4 d 1×10-6mol/L ATRA조화1×10-7mol/LATRA조Cx43 mRNA표체량균명현고우공백대조조,차이균유통계학의의(t=3.57,P＜0.01;t=6.31,P＜0.01;t=2.22,P＜0.05).RB세포현액전방주사후6～9d,11안성공건립라서RB종류모형,조모후20 d,공백대조조라서종류장만전방,안구돌출;이동시간점1×10-5 mol/L ATRA조종류생장부점전방적1/2.조직병이학검사발현,공백대조조라서종류세포충만전방、후방화파리체강,이1×10-5 mol/L ATRA조라서종류주요재전방생장,후방급파리체강내미견류세포. 결론 ATRA재전록수평유도RB세포중Cx43표체적상조,종이억제화한제라서전방RB종류적생장.
Background One of the important machanisms of all trans retinoic acid (ATRA) is to regulate the expression of connexin (Cx) gene.ATRA inhibits the proliferation and differentiation of retinoblastoma (RB) cells,which is related to Cx43.However,the control site of ATRA and its effect on RB tumor in vivo have not been identified.Objective This study was to investigate the effect of ATRA on Cx43 expression in RB cells and its approach mechanisms.Methods ATRA solution of 1 × 10 2 mol/L was prepared with ethanol and formulated into 1×10 5,1×10-6and 1 × 10 7 mol/L of solution with culture medium further.Human RB cell line (HXO-RB44) was cultured and treated with different concentrations of ATRA for 2,4 and 6 days,respectively.The expressions of Cx43 protein and mRNA in RB cells were detected by Western blot and reverse transcription PCR (RT-PCR),respectively.RB models were established by injecting HXO-RB44 cell suspension into anterior chamber in the right eyes of 15 athymic mice.Eleven successful models were divided into the blank control group,negative control group and 1 × 10-5 mol/L ATRA group,and 0.5％ normal saline solution with athymic or 1 ×10-5 mol/L ATRA solution was injected into the anterior chamber in the negative control group and 1 × 10-5 mol/L ATRA group in the 3-day interval for 3 weeks.The model eyes were examined under the slit lamp microscope.The eyeballs were extracted at the end of the experiment for hematoxylin and eosin staining.Results Western blot assay showed that the absorbance values of Cx43 protein (ACx43/AGAPDH) were increased gradually as time lapse of ATRA treatment among the groups (Ftime =71.31,P =0.00; Fgroup =7.66,P =0.00).The expressions of Cx43 protein were significantly higher in the 1 × 10 5 mol/L ATRA group after 2 days,1 × 10-6 mol/L ATRA group after 4 days,1 × 10-7 mol/L ATRA group after 6 days than those in the blank control group at various time points (t =3.34,P＜0.01 ;t =2.33,P＜0.05;t =3.12,P＜ 0.01).RT-PCR showed that the absorbance values of Cx43 mRNA (ACx43mRXA/Aβ-actin) were significantly enhanced as the prolong of treatment time of ATRT among the groups (Ftime =90.90,P =0.00 ; Fgroup =6.86,P =0.00).The expressions of Cx43 mRNA were significantly higher in the 1 × 10-5 mol/L ATRA group after 2 days,1 × 10 6 mol/L ATRA group and 1 ×10-7 mol/L ATRA group after 4 days than those in the blank control group at various time points (t=3.57,P＜0.01 ;t=6.31,P＜0.01 ;t=2.22,P＜0.05).RB models were successfully created in 11 eyes on the 6-9 days following the intrachamber injection of RB cell suspension.The RB cells were filled with chamber in the blank control group 20 days after injection,and RB only occupied half of the anterior chamber in the 1 × 10-5 mol/L ATRA group.Histopathological examination exhibited that the RB cells were seen in the anterior and posterior chamber as well as vitreous in the blank control group,however,the cells were only found in the anterior chamber in the 1 × 10 5 mol/L ATRA group.Conclusions ATRA can inhibit the growth of RB in vitro and in vivo by inducing the expression of Cx43 gene in transcription process.
