【摘要】：目的:構(gòu)建OATP1B1(Organic anion transporting polypeptide1B1)野生型和突變型過表達(dá)慢病毒載體基因平臺,并在此平臺上進(jìn)行OATP1B1388和521位點(diǎn)的突變對他莫昔芬及其代謝產(chǎn)物的攝取差異的比較研究。方法:從人肝臟組織提取總RNA并逆轉(zhuǎn)錄成c DNA,利用PCR技術(shù)釣取OATP1B1基因片段,同時(shí)對載體GV358進(jìn)行酶切,擴(kuò)增產(chǎn)物的兩末端序列與線性化克隆載體兩末端序列完全一致。以線性化載體和目的基因擴(kuò)增產(chǎn)物配制反應(yīng)體系,進(jìn)行重組反應(yīng),實(shí)現(xiàn)線性化載體和目的基因片段的體外環(huán)化。重組產(chǎn)物轉(zhuǎn)化到DH5α感受態(tài)細(xì)胞進(jìn)行擴(kuò)增,抽提、純化后進(jìn)行測序比對,測序比對正確的進(jìn)行定點(diǎn)突變得到OATP1B1388G(OATP1B1*1b)和521C(OATP1B1*5)的突變體質(zhì)粒。在脂質(zhì)體Lip2000介導(dǎo)下,把重組質(zhì)粒和慢病毒包裝質(zhì)粒p Helper1.0和p Helper2.0共同轉(zhuǎn)染293T細(xì)胞,包裝生產(chǎn)慢病毒,然后收獲病毒液,對病毒液進(jìn)行濃縮純化。符合實(shí)驗(yàn)要求且滴度合格的病毒液感染目的293T細(xì)胞,熒光定量PCR和蛋白免疫印跡法檢測293T細(xì)胞中OATP1B1野生型(OATP1B1*1a)及其突變型質(zhì)粒在基因和蛋白水平的表達(dá)情況。同時(shí),建立Z-他莫昔芬(Z-tamoxifen,縮寫為tamoxifen)及其活性代謝產(chǎn)物4-羥基-N-去甲基他莫昔芬(endoxifen)的高效液相串聯(lián)質(zhì)譜(HPLC-MS/MS)方法學(xué)。實(shí)驗(yàn)組分別為HEK293T細(xì)胞組、HEK293T細(xì)胞加藥組、陰性對照病毒-HEK293T組(HEK293T細(xì)胞轉(zhuǎn)染載體質(zhì)粒組)、OATP1B1*1a-HEK293T組、OATP1B1*1b-HEK293T組和OATP1B1*5-HEK293T組。六組細(xì)胞分別加入不同濃度梯度的tamoxifen和endoxifen作用24小時(shí)和48小時(shí)后用清水裂解細(xì)胞,運(yùn)用HPLC-MS/MS方法學(xué)比較OATP1B1基因多態(tài)性對tamoxifen和endoxifen的攝取差異。結(jié)果:1)加藥24小時(shí)檢測細(xì)胞裂解液中tamoxifen和endoxifen的含量,HEK293T細(xì)胞加藥組與陰性對照病毒-HEK293T加藥組,細(xì)胞裂解液中藥物含量對比,差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)。OATP1B1*1a-HEK293T組、OATP1B1*1b-HEK293T組和OATP1B1*5-HEK293T組,三組細(xì)胞裂解液中藥物含量明顯高于HEK293T細(xì)胞加藥組且差異具有統(tǒng)計(jì)學(xué)意義(P0.01),說明tamoxifen和endoxifen可以通過轉(zhuǎn)運(yùn)體OATP1B1攝取進(jìn)入細(xì)胞內(nèi)。2)加藥24小時(shí)細(xì)胞裂解液中tamoxifen和endoxifen的含量,OATP1B1*1b-HEK293T組細(xì)胞裂解液中藥物含量對比OATP1B1*1a-HEK293T組差異不具有統(tǒng)計(jì)學(xué)意義(P0.05);說明OATP1B1388位點(diǎn)堿基由A突變?yōu)镚時(shí),對轉(zhuǎn)運(yùn)體蛋白攝取能力沒有影響。OATP1B1*5-HEK293T組細(xì)胞裂解液中藥物含量對比OATP1B1*1a-HEK293T組含量偏低,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05);說明OATP1B1521位點(diǎn)堿基由T突變?yōu)镃時(shí)會抑制轉(zhuǎn)運(yùn)體蛋白的攝取轉(zhuǎn)運(yùn)功能。3)加藥48小時(shí)檢測細(xì)胞裂解液中tamoxifen和endoxifen的含量,HEK293T細(xì)胞加藥組與陰性對照病毒-HEK293T加藥組藥物含量對比,差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)。OATP1B1*1a-HEK293T組、OATP1B1*1b-HEK293T組和OATP1B1*5-HEK293T組,三組細(xì)胞裂解液中藥物含量明顯高于HEK293T細(xì)胞加藥組且差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。4)加藥48小時(shí),OATP1B1*1b-HEK293T組細(xì)胞裂解液中藥物含量對比OATP1B1*1a-HEK293T組差異不具有統(tǒng)計(jì)學(xué)意義(P0.05);OATP1B1*5-HEK293T組細(xì)胞裂解液中藥物濃度對比OATP1B1*1a-HEK293T組含量偏低,且差異具有統(tǒng)計(jì)學(xué)意義(P0.05);48小時(shí)各組細(xì)胞裂解液中藥物含量對比同一條件24小時(shí)細(xì)胞裂解液中藥物濃度略高,但是差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)說明24小時(shí)內(nèi)轉(zhuǎn)運(yùn)體OATP1B1對tamoxifen和endoxifen的攝取基本達(dá)到飽和。結(jié)論:1)過表達(dá)慢病毒質(zhì)粒細(xì)胞模型構(gòu)建成功,包括OATP1B1*1a-HEK29T、OATP1B1*1b-HEK293T和OATP1B1*5-HEK293T細(xì)胞模型,且感染效率達(dá)到80%以上,OATP1B1基因在m RNA、蛋白水平可以高度表達(dá)。2)tamoxifen及其主要代謝產(chǎn)物endoxifen可以通過OATP1B1攝取進(jìn)入細(xì)胞,OATP1B1521位點(diǎn)堿基T突變?yōu)镃時(shí),可以抑制轉(zhuǎn)運(yùn)體蛋白的攝取轉(zhuǎn)運(yùn)功能,導(dǎo)致細(xì)胞裂解液中藥物濃度對比野生組偏低且差異具有統(tǒng)計(jì)學(xué)意義(P0.05);OATP1B1388位點(diǎn)堿基由A突變?yōu)镚時(shí),細(xì)胞裂解液藥物的含量對比野生組差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)
[Abstract]:AIM: To construct a wild-type and mutant overexpression lentiviral gene platform of OATP1B1 (Organic anion transporting polypeptide 1B1) and to compare the uptake of tamoxifen and its metabolites by mutations at sites 1388 and 521. Methods: Total RNA was extracted from human liver tissues and reverse transcribed into C DNA. OATP1B1 gene fragments were harvested by PCR, and GV358 was digested by enzyme. The two terminal sequences of the amplified product were identical with those of the linearized cloning vector. The recombinant reaction system was prepared with the linearized vector and the target gene amplified product to realize in vitro cyclization of the linearized vector and the target gene fragment. The recombinant product was transformed into DH5a competent cells for amplification, extraction, purification and sequencing. The mutant plasmids of OATP1B1388G (OATP1B1*1b) and 521C (OATP1B1*5) were obtained by site-directed mutagenesis. The recombinant plasmids and lentiviral packaging plasmids P Helper 1.0 and P Helper 2.0 were co-transfected by liposome Lip2000. 293T cells were packaged to produce lentiviruses, and then harvested to concentrate and purify the virus solution. The expression of OATP1B1 wild type (OATP1B1 * 1a) and its mutant plasmids at gene and protein levels in 293T cells were detected by fluorescence quantitative PCR and Western blot. Meanwhile, a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method was established for the determination of Z-tamoxifen and its active metabolite 4-hydroxy-N-demethyltamoxifen. The experimental groups were HEK293T cell group, HEK293T cell dosage group, negative control virus-HEK293T cell transfection vector plasmid. Group A, OATP1B1*1a-HEK293T group, OATP1B1*1b-HEK293T group and OATP1B1*5-HEK293T group. Six groups of cells were treated with different concentration gradient of tamoxifen and endoxifen for 24 hours and 48 hours respectively. The uptake of tamoxifen and endoxifen was compared by HPLC-MS/MS. The contents of tamoxifen and endoxifen in cell lysate were detected by hourly method. There was no significant difference in the contents of tamoxifen and endoxifen between the HEK293T cell lysate group and the negative control virus-HEK293T cell lysate group (P 0.05). OATP1B1*1a-HEK293T group, OATP1B1*1b-HEK293T group and OATP1B1*5-HEK293T cell lysate group contained drugs. The amount of tamoxifen and endoxifen in the 24-hour cell lysate was significantly higher than that in the HEK293T group (P 0.01), indicating that tamoxifen and endoxifen could be uptaken into the cells by transporter OATP1B1. The content of tamoxifen and endoxifen in the 24-hour cell lysate was lower in the OATP1B1 * 1b-HEK293T group than in the OATP1B1 * 1a-HEK293T group. There was no significant difference between the two groups (P 0.05), indicating that there was no effect on transporter protein uptake when the base of OATP1B1388 changed from A to G. The drug content in cell lysate of OATP1B1*5-HEK293T group was lower than that of OATP1B1*1a-HEK293T group, and the difference was statistically significant (P 0.05); indicating that the base of OATP1B1521 mutated from T to C. It can inhibit the transporter protein uptake and transport function. 3) The content of tamoxifen and endoxifen in cell lysate was detected 48 hours after treatment. The content of tamoxifen and endoxifen in HEK293T cell treatment group was not significantly different from that in negative control virus-HEK293T treatment group (P 0.05). OATP1B1*1a-HEK293T group, OATP1B1*1b-HEK293T group and OATP1B1*5-HEK293T group were not significantly different (P 0.05). The drug content in cell lysate of OATP1B1*1b-HEK293T group was not significantly different from that of OATP1B1*1a-HEK293T group (P 0.05); the drug concentration in cell lysate of OATP1B1*5-HEK293T group was significantly higher than that of HEK293T group (P 0.01). Compared with OATP1B1*1a-HEK293T group, the content of drug in cell lysate was lower, and the difference was statistically significant (P 0.05); 48 hours after treatment, the drug concentration in cell lysate was slightly higher than that in 24 hours under the same condition, but the difference was not statistically significant (P 0.05). CONCLUSIONS: 1) Overexpression of lentivirus plasmid cell model was successfully constructed, including OATP1B1*1a-HEK29T, OATP1B1*1b-HEK293T and OATP1B1*5-HEK293T cell models, and the infection efficiency was above 80%. OATP1B1 gene was highly expressed in M RNA and protein level. 2) tamoxifen and its main metabolite endoxifen could be uptaken by OATP1B1. When OATP1B1521 base T mutated into C, the uptake and transport of transporter proteins were inhibited, resulting in a low and statistically significant difference in the concentration of drugs in cell lysate compared with the wild group (P 0.05); when OATP1B1388 base mutated from A to G, there was no uniform difference in the content of drugs in cell lysate compared with the wild group. Academic significance (P0.05)
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R96

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6 王龍強(qiáng);他莫昔芬對雌激素受體陰性乳腺癌細(xì)胞的殺傷能力的研究[D];華中科技大學(xué);2014年

7 陳多;選擇性雌激素受體調(diào)節(jié)劑對小鼠學(xué)習(xí)記憶功能的損傷作用及機(jī)理研究[D];沈陽藥科大學(xué);2001年

8 孫雪波;他莫昔芬在大鼠蛛網(wǎng)膜下腔出血后早期腦損傷中的神經(jīng)保護(hù)作用及機(jī)制研究[D];蘇州大學(xué);2013年

9 王帥;SHG-44人膠質(zhì)瘤細(xì)胞的離子通道特性及他莫昔芬抗膠質(zhì)瘤作用的機(jī)制研究[D];河北醫(yī)科大學(xué);2009年

10 周艷;乳腺癌ERβ表達(dá)的臨床意義及其與他莫昔芬治療耐藥相關(guān)意義的初步研究[D];第三軍醫(yī)大學(xué);2007年

相關(guān)碩士學(xué)位論文 前10條

1 楊墨琳;PAX2、AIB1在人乳腺癌細(xì)胞生長及他莫昔芬治療作用的實(shí)驗(yàn)研究[D];河北醫(yī)科大學(xué);2015年

2 王建偉;經(jīng)腹膜外術(shù)式加短期強(qiáng)的松和長期他莫昔芬治療特發(fā)性腹膜后纖維化[D];山東大學(xué);2016年

3 袁建梅;他莫昔芬與芳香化酶抑制劑對乳腺癌患者靜脈血栓發(fā)生影響的Meta分析[D];重慶醫(yī)科大學(xué);2016年

4 劉婕;疏肝寧心調(diào)陰陽方對絕經(jīng)前乳腺癌內(nèi)分泌治療療效影響[D];南京中醫(yī)藥大學(xué);2016年

5 陳緒;三黃煎劑抑制PI3K/AKT信號通路降低ROS促進(jìn)乳腺癌他莫昔芬耐藥細(xì)胞凋亡的實(shí)驗(yàn)研究[D];南京中醫(yī)藥大學(xué);2016年

6 謝周滔;他莫昔芬緩解包裹性腹膜硬化進(jìn)程中腹膜間皮—間質(zhì)轉(zhuǎn)分化過程的機(jī)制研究[D];浙江大學(xué);2016年

7 高春梅;OATP1B1基因多態(tài)性對他莫昔芬及其代謝產(chǎn)物的攝取研究[D];皖南醫(yī)學(xué)院;2016年

8 吳曉安;他莫昔芬通過GPR30介導(dǎo)CAF細(xì)胞外分泌CXCL16促進(jìn)乳腺癌MCF-7細(xì)胞遷移及侵襲[D];重慶醫(yī)科大學(xué);2016年

9 石統(tǒng)偉;利用基因表達(dá)的秩次關(guān)系預(yù)測他莫昔芬的治療效果[D];電子科技大學(xué);2014年

10 盛文;加味地黃湯治療他莫昔芬所致子宮內(nèi)膜增生的臨床研究[D];云南中醫(yī)學(xué)院;2014年

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