【摘要】：第一章視紫紅質(zhì)T17M突變體的亞細(xì)胞定位 目的：研究視紫紅質(zhì)T17M突變體的亞細(xì)胞定位及意義。 方法：構(gòu)建pCDNA-3.1-T17M rhodopsin-myc質(zhì)粒和pCDNA-3.1-WT rhodopsin-myc質(zhì)粒,使用EcoR Ⅰ和BamH Ⅰ雙酶切法進(jìn)行鑒定,基因測(cè)序。將構(gòu)建成功的質(zhì)粒轉(zhuǎn)染到HEK293細(xì)胞,采用Western blot檢測(cè)視紫紅質(zhì)T17M突變體和野生型的蛋白表達(dá)差異。免疫熒光顯微鏡下觀察視紫紅質(zhì)T17M突變體和野生型的亞細(xì)胞定位。 結(jié)果：經(jīng)過(guò)PCR擴(kuò)增和雙酶切鑒定,獲得約1000bp大小的條帶,基因測(cè)序顯示第50位堿基C突變成T,成功構(gòu)建pCDNA-3.1-T17M rhodopsin-myc質(zhì)粒和pCDNA-3.1-WT rhodopsin-myc質(zhì)粒。質(zhì)粒轉(zhuǎn)染后,Western blot檢測(cè)到約40KD的條帶,轉(zhuǎn)染可高效表達(dá)視紫紅質(zhì)T17M突變體和野生型蛋白。熒光顯微鏡檢查發(fā)現(xiàn),視紫紅質(zhì)T17突變體聚集在內(nèi)質(zhì)網(wǎng),與高爾基體不存在共定位；而野生型主要位于細(xì)胞膜。 結(jié)論：視紫紅質(zhì)T17M突變體異常定位于內(nèi)質(zhì)網(wǎng),與高爾基體不存在共定位。視紫紅質(zhì)野生型主要位于細(xì)胞膜。 第二章視紫紅質(zhì)T17M突變體的降解途徑 目的：研究視紫紅質(zhì)T17M突變體的降解途徑及意義。 方法：使用MTT法檢測(cè)視紫紅質(zhì)T17M突變體和野生型的降解速度。Western blot檢測(cè)溶酶體抑制劑CQ和蛋白酶體抑制劑MG132對(duì)視紫紅質(zhì)T17M突變體和野生型降解的影響。免疫沉淀檢測(cè)視紫紅質(zhì)泛素化水平。Western blot檢測(cè)p97/VCP-QQ(?)Erasin siRNA對(duì)視紫紅質(zhì)T17M突變體和野生型的半衰期的影響。 結(jié)果：蛋白合成抑制劑CHX處理HEK293細(xì)胞和ARPE-19細(xì)胞6h后,CHX處理前視紫紅質(zhì)蛋白標(biāo)化為1, HEK293細(xì)胞的視紫紅質(zhì)T17M突變體和野生型蛋白相對(duì)值分別為0.219±0.032和0.635±0.072(P0.01), ARPE-19細(xì)胞的視紫紅質(zhì)T17M突變體和野生型蛋白相對(duì)值分別為0.302±0.041和0.531±0.052(P0.01)。溶酶體抑制劑CQ處理HEK293細(xì)胞12h后,視紫紅質(zhì)T17M突變體由1增加到1.023±0.265,視紫紅質(zhì)野生型蛋白相對(duì)值由1增加到1.433±0.159(P0.05)。蛋白酶體抑制劑MG132處L理HEK293細(xì)胞6h后,視紫紅質(zhì)T17M突變體由1增加到7.213±2.108(P0.01),視紫紅質(zhì)野生型蛋白相對(duì)值由1增加到2.011±0.221(P0.05)。免疫沉淀后,泛素化視紫紅質(zhì)T17M突變體由1增加到2.200±0.361(P0.01),野生型蛋白相對(duì)值由1增加到1.160±0.162。在ARPE-19細(xì)胞中過(guò)表達(dá)p97/VCP-QQ后,對(duì)照組和p97/VCP-QQ組的視紫紅質(zhì)T17M突變體蛋白相對(duì)值分別為0.159±0.052和0.558±0.095(P0.01)。Erasin siRNA處理后,視紫紅質(zhì)T17M突變體的對(duì)照組Erasin siRNA組的蛋白相對(duì)值分別為0.230±0.059和0.602±0.064(P0.01),而視紫紅質(zhì)野生型降解速度沒(méi)有明顯變化。 結(jié)論：與視紫紅質(zhì)野生型相比,T17M突變體降解加速。視紫紅質(zhì)T17M突變體只能通過(guò)蛋白酶體系統(tǒng)降解,而野生型可以通過(guò)自噬溶酶體系統(tǒng)和蛋白酶體系統(tǒng)降解。視紫紅質(zhì)T17M突變體的降解與泛素化的ERAD相關(guān)。過(guò)表達(dá)p97/VCP-QQ和Erasin siRNA干擾可以抑制視紫紅質(zhì)T17M突變體通過(guò)ERAD途徑降解。 第三章視紫紅質(zhì)T17M突變誘導(dǎo)細(xì)胞死亡的機(jī)制 目的：研究視紫紅質(zhì)T17M突變誘導(dǎo)細(xì)胞死亡的機(jī)制。 方法：建立pEGFP-CL1-ARPE-19細(xì)胞系,采用Western blot方法檢測(cè)其蛋白酶體活性。過(guò)表達(dá)視紫紅質(zhì)蛋白,誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),采用Western blot方法檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白BIP、GRP94、CHOP、 peIF-2a、eIF-2a、active ATF-6a的表達(dá)差異,并用PBA處理細(xì)胞,觀察PBA對(duì)以上內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白表達(dá)的影響。Tunicamycin處理ARPE-19細(xì)胞,流式細(xì)胞儀檢測(cè)細(xì)胞死亡數(shù)目。過(guò)表達(dá)視紫紅質(zhì)T17M突變體,采用流式細(xì)胞儀檢測(cè)細(xì)胞內(nèi)ROS水平,并使用ROS清除劑NAC和BHA處理,觀察細(xì)胞死亡變化情況。 結(jié)果：pEGFP-CL1-ARPE-19細(xì)胞系可穩(wěn)定表達(dá)uGFP,視紫紅質(zhì)T17M突變、野生型的uGFP表達(dá)量沒(méi)有顯著變化。T17M突變可使細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激蛋白BIP、GRP94、CHOP、peIF-2a、eIF-2a、active ATF-6a表達(dá)上調(diào),與視紫紅質(zhì)野生型相比,BIP、CHOP、GRP94、 peIF-2a/eIF-2a、active ATF-6a表達(dá)量分別增加2.439±0.363倍(P0.01)、2.433±0.802倍(P0.01)、1.600±0.212倍(P0.05)、1.567±0.153(P0.05)、2.167±0.306倍(P0.01)。與視紫紅質(zhì)野生型相比,CHOP表達(dá)量在使用PBA處理前后分別增加了2.600±0.854倍和1.467±0.306倍(P0.05),GRP94表達(dá)量處理前后增加1.921±0.557倍和1.203±0.239倍(P0.05),peIF-2a/eIF-2a為1.733±0.154倍和1.167±0.252倍(P0.05),active ATF-6a為2.564±0.406倍和1.349±0.529倍(P0.05)。而使用內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)劑‘Tunicamycin前后,空載體、視紫紅質(zhì)野生型和T17M突變體的ARPE-19細(xì)胞死亡率分別為4.156%±0.501%和4.814%±0.531%、3.879%±0.413%和5.712%±0.574%、7.021%±0.612%和16.213%±3.419%,視紫紅質(zhì)T17M突變體過(guò)表達(dá)的細(xì)胞死亡率較空載體和野生型明顯增加(P0.05)。與空載體組相比,視紫紅質(zhì)野生型和視紫紅質(zhì)T17M突變體ROS的相對(duì)比分別為1.136±0.055,1.935±0.088(P0.01)。使用ROS清除劑NAC和BHA后,空載體組、DMSO組、NAC組和BHA組的死亡率分別為3.716%±0.523%、7.322%±1.924%、4.857%±1.369%(相比DMSO組,P0.05)和4.271%±0.988%(相比DMSO組,P0.01)。 結(jié)論：視紫紅質(zhì)T17M突變不影響蛋白酶體活性。T17M突變能誘導(dǎo)細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激,使內(nèi)質(zhì)網(wǎng)應(yīng)激蛋白BIP、GRP94、CHOP、peIF-2a、 eIF-2a、active ATF-6a表達(dá)上調(diào)�；瘜W(xué)分子伴侶PBA能緩解T17M誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激。視紫紅質(zhì)T17M突變?cè)黾蛹?xì)胞對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)劑Tunicamycin的敏感性。突變的T17M視紫紅質(zhì)增加細(xì)胞內(nèi)ROS的水平,ROS清除劑NAC和BHA能抑制視紫紅質(zhì)T17M突變導(dǎo)致的細(xì)胞死
[Abstract]:Chapter 1 subcellular localization of rhodopsin T17M mutant
Objective: To study the subcellular localization and significance of rhodopsin T17M mutant.
Methods: pCDNA-3.1-T17M rhodopsin-myc plasmids and pCDNA-3.1-WT rhodopsin-myc plasmids were constructed and identified by EcoR I and BamH I double enzyme digestion and gene sequencing. The constructed plasmids were transfected into HEK293 cells, and Western blot was used to detect the differences in the expression of rhodopsin T17M mutants and wild type proteins. Immunofluorescence microscopy was used. Subcellular localization of rhodopsin T17M mutant and wild type was observed.
Results: after PCR amplification and double enzyme digestion, the 1000bp size bands were obtained. Gene sequencing showed that fiftieth base C was transformed into T, and pCDNA-3.1-T17M rhodopsin-myc plasmids and pCDNA-3.1-WT rhodopsin-myc plasmids were successfully constructed. After plasmid transfection, Western blot detected approximately 40KD bands. The transfection could efficiently express rhodopsin T17M mutation The fluorescence microscope showed that the T17 mutant of rhodopsin was clustered in the endoplasmic reticulum, and there was no co location with the Golgi body, while the wild type was mainly in the cell membrane.
Conclusion: the rhodopsin T17M mutant is located in the endoplasmic reticulum and has no co localization with Golgi apparatus. The rhodopsin type is mainly located in the cell membrane.
The second chapter of degradation pathway of rhodopsin T17M mutant
Objective: To study the degradation pathway and significance of rhodopsin T17M mutant.
Methods: MTT method was used to detect the T17M mutant of rhodopsin and the degradation rate of wild type.Western blot to detect the effect of lysosome inhibitor CQ and proteasome inhibitor MG132 on the degradation of rhodopsin T17M mutants and wild type. Immunoprecipitating detection of.Western blot detection p97/VCP-QQ (?) Erasin siRNA against rhodopsin The half-life of T17M mutant and wild type of purplish red.
Results: after the protein synthesis inhibitor CHX treated the HEK293 cells and ARPE-19 cells 6h, the CHX treated rhodopsin was labeled as 1, the relative values of the rhodopsin T17M mutants and the wild type proteins were 0.219 + 0.032 and 0.635 + 0.072 (P0.01) respectively, and the relative values of the rhodopsin T17M mutants of ARPE-19 cells and the relative values of the wild type proteins in ARPE-19 cells were divided. Don't be 0.302 + 0.041 and 0.531 + 0.052 (P0.01). After the lysosome inhibitor CQ treated HEK293 cell 12h, the T17M mutant of the rhodopsin increased from 1 to 1.023 + 0.265, and the relative value of the rhodopsin wild type protein increased from 1 to 1.433 + 0.159 (P0.05). After the proteasome inhibitor MG132 was L rational HEK293 cell 6h, the T17M mutant of the rhodopsin increased from 1 to 7.21. 3 + 2.108 (P0.01), the relative value of rhodopsin wild type protein increased from 1 to 2.011 + 0.221 (P0.05). After immunoprecipitation, the ubiquitination rhodopsin T17M mutant was increased from 1 to 2.200 + 0.361 (P0.01), and the relative value of wild type protein increased from 1 to 1.160 + 0.162. in ARPE-19 cells, and the control group and p97/VCP-QQ group of rhodopsin The relative values of the qualitative T17M mutant proteins were 0.159 + 0.052 and 0.558 + 0.095 (P0.01).Erasin siRNA respectively. The relative values of the Erasin siRNA group of the control group of the rhodopsin T17M mutant were 0.230 + 0.059 and 0.602 + 0.064 (P0.01), respectively, while the degradation rate of the rhodopsin wild type was not significantly changed.
Conclusion: compared with the wild type of rhodopsin, the degradation of T17M mutant is accelerated. The T17M mutant of rhodopsin can be degraded only through the proteasome system, while the wild type can be degraded by autophagosome system and proteasome system. The degradation of the T17M mutant of the rhodopsin is related to the ERAD of the ubiquitination. P97/VCP-QQ and Erasin siRNA are overexpressed by over expression of the mutant of the rhodopsin. Disturbance can inhibit the degradation of rhodopsin T17M mutant via ERAD pathway.
The third chapter is about the mechanism of cell death induced by T17M mutation of rhodopsin.
Objective: To study the mechanism of cell death induced by T17M mutation of rhodopsin.
Methods: the pEGFP-CL1-ARPE-19 cell line was established and the activity of proteasome was detected by Western blot. Overexpression of rhodopsin, induced endoplasmic reticulum stress reaction, and Western blot method was used to detect the expression difference of endoplasmic reticulum stress related protein BIP, GRP94, CHOP, peIF-2a, eIF-2a, active ATF-6a. The expression of stress related protein in the endoplasmic reticulum was affected by.Tunicamycin treatment of ARPE-19 cells, the number of cell deaths was detected by flow cytometry. The T17M mutant of rhodopsin was overexpressed. The level of intracellular ROS was detected by flow cytometry, and the ROS scavenger NAC and BHA were used to observe the change of cell death.
Results: pEGFP-CL1-ARPE-19 cell lines can express uGFP, T17M mutation of rhodopsin, and there is no significant change in the expression of uGFP in the wild type,.T17M mutation can make the cell endoplasmic reticulum stress protein BIP, GRP94, CHOP, peIF-2a, eIF-2a, active ATF-6a, compared with the wild type of rhodopsin The amount increased by 2.439 + 0.363 times (P0.01), 2.433 + 0.802 times (P0.01), 1.600 + 0.212 times (P0.05), 1.567 + 0.153 (P0.05), 2.167 + 0.306 times (P0.01). Compared with the wild type of rhodopsin, the expression of CHOP increased by 2.600 + 0.854 times and 1.467 + 1.600 times (P0.05) before and after PBA treatment, and the GRP94 expression was increased before and after the treatment. It was 1.203 + 0.239 times (P0.05), peIF-2a/eIF-2a was 1.733 + 0.154 times and 1.167 + 0.252 times (P0.05), active ATF-6a was 2.564 + 0.406 times and 1.349 + 0.529 times (P0.05). And the ARPE-19 cell death rate was 4.156% + 0.501% and 4.8 by using endoplasmic reticulum stress inducer before and after Tunicamycin. 14% + 0.531%, 3.879% + 0.413% and 5.712% + 0.574%, 7.021% + 0.612% and 16.213% + 3.419%, the overexpressed cell mortality of the rhodopsin T17M mutant was significantly higher than that of the empty vector and the wild type (P0.05). Compared with the no-load group, the relative ratio of the rhodopsin wild type and the rhodopsin T17M mutant ROS was respectively (P0.01) + 0.088 (P0.01). After the use of ROS scavenger NAC and BHA, the mortality of the unloaded body group, the DMSO group, the NAC group and the BHA group were 3.716% + 0.523%, 7.322% + 1.924%, 4.857% + 1.369% (compared to the DMSO group, P0.05) and 4.271% + 0.988% (compared to the DMSO group, P0.01).
Conclusion: T17M mutation of rhodopsin does not affect the.T17M mutation of proteasome activity can induce endoplasmic reticulum stress and up-regulated expression of BIP, GRP94, CHOP, peIF-2a, eIF-2a, active ATF-6a expression of endoplasmic reticulum stress protein. Chemical chaperone PBA can alleviate the T17M induced endoplasmic reticulum stress. Rhodopsin T17M mutation increases the induction of endoplasmic reticulum stress induced by the rhodopsin T17M mutation The sensitivity of the agent Tunicamycin. Mutant T17M rhodopsin increased the level of ROS in cells. ROS scavenger NAC and BHA could inhibit cell death caused by T17M mutation of rhodopsin.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R774.13

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