本文選題：TMC120 + 細(xì)胞凋亡��； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景： 盡管高效抗逆轉(zhuǎn)錄病毒治療(HAART)使人類免疫缺陷病毒(HIV)感染者的死亡率有所下降,艾滋病相關(guān)并發(fā)癥的發(fā)生率及死亡率也大幅降低,且艾滋病患者的平均壽命也顯著增加。但是惡性腫瘤始終是HIV感染者死亡的第二大常見原因。 最常見于艾滋病患者的腫瘤并發(fā)癥前三位分別是卡波西肉瘤,非霍奇金淋巴瘤,侵襲性宮頸癌,他們也稱為艾滋病特異性腫瘤。 與不斷下降的艾滋病相關(guān)并發(fā)癥的發(fā)生率與死亡率相比,艾滋病特異性腫瘤以及艾滋病非特異性腫瘤,如肺癌、肛管直腸癌等,卻是逐年增長的。 雖然對于艾滋病并發(fā)惡性腫瘤的患者,聯(lián)合應(yīng)用抗腫瘤化療和HAART已經(jīng)顯示出可行性和有效性,但是,很多抗腫瘤藥物與HAART合用可能導(dǎo)致藥物的蓄積和毒性增加、或者藥物的排泄增加和效力降低。不幸的是,揭示藥物相互作用,并可用于安全指導(dǎo)HAART和抗腫瘤化療聯(lián)合應(yīng)用的研究數(shù)據(jù)非常有限。對于抗腫瘤藥物與抗逆轉(zhuǎn)錄病毒藥物的聯(lián)合應(yīng)用,缺乏基于臨床試驗(yàn)的普遍可行的詳細(xì)指南。所以癌癥,始終是艾滋病患者生存的重大隱患。 因此,我們選取常用的各類抗HIV藥物作用于腫瘤細(xì)胞,篩查抗HIV藥物是否存在抗腫瘤細(xì)胞毒性,結(jié)果發(fā)現(xiàn),TMC120單獨(dú)應(yīng)用時(shí)對多種腫瘤細(xì)胞有顯著的細(xì)胞毒性。 TMC120(也叫達(dá)匹韋林),非核苷類逆轉(zhuǎn)錄酶抑制劑(NNRTI),目前主要研究作為陰道的殺微生物劑,以陰道環(huán)的形式在體外實(shí)驗(yàn)及間接體內(nèi)療法中均能有效地阻止HIV-1的感染。TMC120在抗HIV的臨床前研究中,其活性得以充分肯定,如果其兼具抗腫瘤活性,無疑給腫瘤患者,特別是艾滋病并發(fā)腫瘤的患者帶來福音。最重要的是,TMC120的應(yīng)用避免了抗HIV藥物與抗腫瘤藥物聯(lián)合應(yīng)用帶來的潛在的相互作用。 因此,TMC120有望用于預(yù)防及治療艾滋病并發(fā)腫瘤的患者,基于其潛在的抗腫瘤及抗逆轉(zhuǎn)錄病毒活性。 目的： 研究非核苷類抗HIV藥物TMC120的抗腫瘤活性,探討TMC120的抗腫瘤機(jī)制。并且,通過建立裸鼠宮頸癌移植瘤模型,進(jìn)一步研究TMC120體內(nèi)的抗腫瘤活性。 方法： 1.MTT法檢測細(xì)胞活力 多種抗HIV藥物及順鉑(20μmol/L)處理結(jié)腸癌HT-29細(xì)胞,多種抗HIV藥物及吉非替尼(40μmol/L)處理肺癌A549細(xì)胞,多種抗HIV藥物及紫杉醇(10μmol/L)處理宮頸癌HeLa細(xì)胞,48h后停止培養(yǎng),加入用培養(yǎng)基現(xiàn)配的MTT溶液,處理3h后,棄上清,加入DMSO振蕩混勻,于酶標(biāo)儀中測定吸光度值(OD570)。 多類腫瘤細(xì)胞,如肺癌細(xì)胞(A549、95D),肝癌細(xì)胞(HepG2),結(jié)腸癌細(xì)胞(colon26、 HT-29),食管癌細(xì)胞(EC109),乳腺癌細(xì)胞(MCF-7),子宮內(nèi)膜癌細(xì)胞(HEC-1),宮頸癌細(xì)胞(HeLa、 SiHa)與TMC120(0、1.56、3.13、6.25、12.5、25、50μmol/L)共同孵育48h后停止培養(yǎng),加入用培養(yǎng)基現(xiàn)配的MTT溶液,處理3h后,棄上清,加入DMSO振蕩混勻,于酶標(biāo)儀中測定吸光度值(OD570)。2.流式細(xì)胞儀檢測細(xì)胞凋亡情況 EC109細(xì)胞、HeLa細(xì)胞與0、6.25、12.5μmol/L的TMC120, SiHa細(xì)胞與0、、12.5、25μmol/L的TMC120共同孵育48h后停止培養(yǎng),用不含EDTA的胰蛋白酶消化細(xì)胞,離心后細(xì)胞用PBS洗2次,再用FITC Annexin V Apoptosis Detection Kit中的1×緩沖液100μL重懸細(xì)胞,加入FITC和PI各5μL,避光,室溫放置15min。每管細(xì)胞中再分別加入400μL的1×緩沖液,充分混勻后于1h內(nèi)上樣,用流式細(xì)胞儀檢測細(xì)胞凋亡情況。3.流式細(xì)胞儀測細(xì)胞周期分布 EC109, HeLa和SiHa細(xì)胞分別與0、6.25.12.5、25μmol/L的TMC120共同孵育24h后收集細(xì)胞,細(xì)胞用PBS洗2次,再用預(yù)冷的體積分?jǐn)?shù)為75%乙醇混懸細(xì)胞,置于4℃冰箱密封保存過夜。離心收集細(xì)胞,PBS洗1次,每管細(xì)胞加入500μL含PI(50μg/mL)及RNA酶(10μg/mL)的PBS溶液,細(xì)胞置于37℃避光孵育30rmin,后于1h內(nèi)上樣,用流式細(xì)胞儀檢測細(xì)胞周期的分布情況。 4. Western blotting測G2/M檢測點(diǎn)相關(guān)蛋白及M期標(biāo)志蛋 TMC120(12.5μmol/L)分別與HeLa和SiHa細(xì)胞共同孵育0、1、3、6、12、24、48h,紫杉醇(100nmol/L)與HeLa細(xì)胞共同孵育0、1、3、6、12、24、48h。裂解細(xì)胞,蛋白定量,Western Blotting檢測G2/M檢查點(diǎn)相關(guān)蛋白p21waf1/cip1、cyclin B1、 phospho-cdc2(Tyrl5),以及M期標(biāo)志蛋白phospho-Histone H3(Ser10)、phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198)的表達(dá)情況。 5.免疫熒光共聚焦檢測細(xì)胞微管形態(tài) HeLa和SiHa細(xì)胞分別用TMC120(12.5μmol/L)和紫杉醇(100nmol/L或1μmol/L)處理12h后,用4%多聚甲醛固定15min,再用100%甲醇通透,置于4℃孵育10min,吸棄甲醇,細(xì)胞用10%山羊血清室溫封閉1h,后用含有抗a/β-tubulin一抗的山羊血清于4℃孵育過夜。細(xì)胞用PBS洗2次,再用含有CY3熒光標(biāo)記的二抗室溫孵育2h。細(xì)胞用PBS洗2次,最后加入DAIP標(biāo)記細(xì)胞核,于共聚焦熒光顯微鏡下觀察細(xì)胞微管形態(tài),并拍照。 6.微管蛋白聚合實(shí)驗(yàn) 微管蛋白聚合檢測試劑盒測在TMC120或紫杉醇作用下,體外純微管蛋白的聚合情況。即酶標(biāo)儀溫度恒定37℃,每分鐘一次動態(tài)檢測OD340的值,以吸光度值表示微管蛋白聚合程度。 7.裸鼠移植瘤模型研究TMC120的體內(nèi)抗腫瘤活性 4到6周的雌性BALB/c nu/nu裸鼠用于實(shí)驗(yàn)研究。將SiHa細(xì)胞懸液皮下接種到裸鼠的左腋下,每只裸鼠接種約3×106個(gè)細(xì)胞。然后3次／周用游標(biāo)卡尺測量腫瘤的大小。腫瘤的大小按照以下公式計(jì)算：V(體積)=L×W2/2,其中L是腫瘤的長度,W是腫瘤的寬度。接種后10天,所有老鼠都長出明顯腫瘤。將裸鼠隨機(jī)分為5組,按以下方案給藥：(1)對照組；(2) TMC12012.5mg/kg，，每周給藥3次；(3)TMC12025mg/kg,每周給藥3次；(4) TMC12050mg/kg,每周給藥3次；(5)紫杉醇15mg/kg,每周給藥2次。5組裸鼠均采用腹腔注射給藥,給藥18天后處死裸鼠并取出腫瘤組織。 當(dāng)對照組腫瘤體積超過1000mm3后結(jié)束給藥,處死裸鼠取出腫瘤組織。腫瘤組織用4%的多聚甲醛固定,脫水后石蠟包埋；自動切片機(jī)將包埋組織切成5μm厚度的石蠟切片。石蠟切片脫蠟再水化組織,按TUNEL試劑盒的指示,將TUNEL反應(yīng)混合液加入樣品上,濕盒、暗室、37℃中加蓋孵育60min；加入轉(zhuǎn)化-POD于樣品上,濕盒、暗室、37℃中加蓋孵育30min；然后加入DAB底物,于室溫孵育10min；鏡檢前用蘇木素復(fù)染,中性樹脂封片保存。正置顯微鏡下拍照并計(jì)數(shù)凋亡細(xì)胞(即棕褐色的TUNEL陽性細(xì)胞)所占比例。 8.統(tǒng)計(jì)學(xué)分析 數(shù)據(jù)分析和作圖均是由GraphPad Prism5.0(數(shù)據(jù)分析和作圖軟件)提供,數(shù)據(jù)結(jié)果以x±s表示；多組間均數(shù)比較采用One way-ANOVA方法,各組分別與對照組比較采用Dunnett檢驗(yàn)以及各組間比較采用Tukey檢驗(yàn)。 結(jié)果： 1、MTT結(jié)果顯示ritonavir和TMC120單獨(dú)用藥時(shí)對結(jié)腸癌、肺癌、宮頸癌細(xì)胞都具有明顯的細(xì)胞毒性作用。 2、MTT結(jié)果顯示TMC120對腫瘤細(xì)胞具有濃度依耐性的抑制作用,且其對于各類腫瘤細(xì)胞的半數(shù)抑制率IC50值基本上在10μmol/L以內(nèi)。這表明TMC120對多種腫瘤細(xì)胞具有較強(qiáng)的細(xì)胞毒性作用。 3、流式測細(xì)胞凋亡的結(jié)果顯示,與對照組相比較,經(jīng)TMC120處理的腫瘤細(xì)胞,其FITC Annexin V陽性細(xì)胞比率顯著增加。結(jié)果表明,與抗腫瘤藥物順鉑類似,TMC120也能誘導(dǎo)腫瘤細(xì)胞的凋亡,通過介導(dǎo)凋亡而抑制腫瘤細(xì)胞的增殖。 4、通過流式細(xì)胞儀對細(xì)胞周期分布的檢測表明,經(jīng)TMC120處理的細(xì)胞,其G2/M期所占比率呈濃度依耐性的增多。即TMC120能夠促使腫瘤細(xì)胞阻滯在G2/M期,發(fā)揮類似于紫杉醇一樣的作用。 5、Western Blotting結(jié)果表明TMC120發(fā)揮類似紫杉醇的作用特征,即能夠削弱G2/M檢測點(diǎn)功能,使宮頸癌細(xì)胞阻滯在有絲分裂期。 6、Confocal的結(jié)果顯示,類似于紫杉醇,TMC120處理的宮頸癌細(xì)胞的微管伸展受限、短縮在核周圍,且有絲分裂期細(xì)胞出現(xiàn)異常的、單極的紡錘體結(jié)構(gòu)。結(jié)果表明,TMC120能夠引起微管的形態(tài)變化,破壞正常的有絲分裂期紡錘體結(jié)構(gòu),從而使腫瘤細(xì)胞難以維持正常的有絲分裂。 7、體外微管聚合實(shí)驗(yàn)顯示,TMC120呈現(xiàn)出劑量依耐性的促進(jìn)微管聚合的作用,表現(xiàn)出堪比于紫杉醇的微管穩(wěn)定效應(yīng)。結(jié)果證實(shí),TMC120能夠靶向微管,發(fā)揮穩(wěn)定微管的作用,從而破壞微管的動態(tài)平衡,干擾細(xì)胞的有絲分裂。 8、裸鼠移植瘤實(shí)驗(yàn)顯示,TMC120能夠明顯延遲腫瘤的生長,具有抗腫瘤體內(nèi)活性,且在各劑量的藥物處理中均沒有出現(xiàn)明顯的毒性反應(yīng)；并且,TMC120能夠介導(dǎo)體內(nèi)腫瘤組織的細(xì)胞凋亡,即通過介導(dǎo)腫瘤細(xì)胞的凋亡而抑制腫瘤組織的增殖。 結(jié)論： TMC120對多種腫瘤細(xì)胞均有較強(qiáng)的毒性作用,能夠介導(dǎo)腫瘤細(xì)胞的凋亡。類似于紫杉醇的作用特征,TMC120能夠削弱G2/M檢測點(diǎn)功能,使宮頸癌細(xì)胞阻滯在有絲分裂期；能夠促進(jìn)微管蛋白聚合,穩(wěn)定微管,改變微管的正常形態(tài),從而使腫瘤細(xì)胞難以進(jìn)行分裂增殖。裸鼠體內(nèi)實(shí)驗(yàn)證明,TMC120能夠延緩腫瘤的增長,并且介導(dǎo)裸鼠腫瘤細(xì)胞的凋亡。
[Abstract]:Background of Study :

Although high - efficiency antiretroviral therapy ( HAART ) has decreased the mortality rate of HIV - infected persons , the incidence and mortality of AIDS - related complications have also significantly decreased , and the average life expectancy of AIDS patients is also significantly increased . However , malignancies have always been the second most common cause of the death of HIV - infected persons .

The first three of the most common tumor complications in AIDS patients were Kaposi ' s sarcoma , non - Hodgkin ' s lymphoma , invasive cervical cancer , also known as AIDS - specific tumors .

Compared with the declining incidence and mortality of AIDS - related complications , AIDS - specific tumors and non - specific HIV / AIDS tumors , such as lung cancer , anal and rectal cancer , are growing year by year .

Although the combination of anti - tumor chemotherapy and HAART has shown the feasibility and effectiveness for patients with AIDS complicated with malignant tumors , the combination of many anti - tumor drugs with HAART may lead to increased accumulation and toxicity of drugs , or increased excretion and efficacy of drugs . Unfortunately , there is a very limited study data available for safety - directed combination of HAART and anti - retroviral drugs .

Therefore , we select the common anti - HIV drugs to act on tumor cells and screen anti - HIV drugs for anti - tumor cytotoxicity . As a result , it is found that the TMC120 has significant cytotoxicity to multiple tumor cells when used alone .

TMC120 , a non - nucleoside reverse transcriptase inhibitor ( NNRTI ) , is currently being used as a vaginal microbicide to effectively prevent the infection of HIV - 1 in both in vitro and indirect in vivo therapy . TMC120 , in preclinical studies against HIV , is sufficiently active to provide the gospel to patients with tumors , especially AIDS patients , in preclinical studies against HIV . Most importantly , the use of TMC120 avoids potential interactions between anti - HIV drugs and anti - tumor drugs .

Therefore , TMC120 is expected to be used in the prevention and treatment of AIDS patients with tumors , based on their potential anti - tumor and anti - retroviral activity .

Purpose :

To study the anti - tumor activity of non - nucleoside anti - HIV drug TMC120 and to investigate the anti - tumor mechanism of TMC120 .

Method :

1 . MTT assay to detect cell viability

A variety of anti - HIV drugs and cisplatin ( 20 渭mol / L ) were used to treat HT - 29 cells of colon cancer , multiple anti - HIV drugs and gemcitabine ( 40 渭mol / L ) for the treatment of human lung cancer A549 cells , multiple anti - HIV drugs and paclitaxel ( 10 渭mol / L ) for cervical cancer HeLa cells . After 48 hours culture was stopped , the MTT solution prepared with the culture medium was added , the supernatant was discarded , the DMSO oscillation was added , and the absorbance value was measured in the microplate reader ( OD570 ) .

Multiple tumor cells , such as lung cancer cells ( A549 , 95D ) , liver cancer cells ( HepG2 ) , colon cancer cells ( A549 , HT - 29 ) , esophageal cancer cells ( EC109 ) , breast cancer cells ( MCF - 7 ) , endometrial cancer cells ( HEC - 1 ) , cervical cancer cells ( HeLa , SiHa ) and TMC120 ( 0 , 1 . 56 , 3 . 13 , 6.25 , 12.5 , 25 , 50 渭mol / L ) were incubated for 3 h .

EC109 cells , HeLa cells and 0 , 6.25 , 12.5 渭mol / L TMC120 , SiHa cells and 0 , 12.5 , 25 渭mol / L TMC120 were incubated for 48 hours . Cells were digested with PBS without EDTA . After centrifugation , the cells were washed twice with PBS and incubated at room temperature for 15 min .

EC109 , HeLa and SiHa cells were incubated with 0 , 6.25 . 12.5 , 25 渭mol / L TMC120 for 24 h . The cells were collected by PBS . The cells were incubated overnight at 4.degree . C.

4 . Western blotting for G2 / M detection point - related protein and M - phase marker eggs

TMC120 ( 12.5 渭mol / L ) was incubated with HeLa and SiHa cells for 0 , 1 , 3 , 6 , 12 , 24 , 48 h , and paclitaxel ( 100 nmol / L ) was incubated with HeLa cells for 0 , 1 , 3 , 6 , 12 , 24 , 48 h . The expression of p21 waf1 / cip1 , cyclin B1 , bcl - cdc2 ( Tyrol 5 ) , and M - phase marker protein kinase - Histone H3 ( Ser10 ) , Ser - Aurora A ( Th28288 ) / Aurora B ( Thr 232 ) / Aurora C ( Thr 198 ) were detected by Western Blotting .

5 . Immunofluorescence confocal detection of cell microtubules morphology

HeLa and SiHa cells were treated with TMC120 ( 12.5 渭mol / L ) and paclitaxel ( 100 nmol / L or 1 渭mol / L ) for 15 min . After 12 h incubation with 100 % methanol , the cells were incubated overnight at 4.degree . C.

6 . Microtube protein polymerization experiment

In the presence of TMC120 or paclitaxel , the micro - tube protein polymerization detection kit was used to measure the polymerization of pure microtube protein in vitro , i.e . , the temperature of the microplate reader was constant 37 鈩

本文編號：1745435