GAPDH基因5’非編碼區(qū)突變及過(guò)表達(dá)突變?cè)谂两鹕≈械淖饔醚芯?/H1>
發(fā)布時(shí)間：2019-07-02 11:15
【摘要】：第一部分帕金森病基因多態(tài)性研究 第一節(jié)GAPDH基因多態(tài)性與PD關(guān)聯(lián)分析目的：采用病例對(duì)照研究,對(duì)PD患者與正常對(duì)照組中GAPDH基因進(jìn)行遺傳學(xué)分析,并探討GAPDH基因與PD發(fā)病風(fēng)險(xiǎn)及相關(guān)性。 方法：收集265例原發(fā)性PD患者和性別、年齡匹配的269例正常人外周血,采用外周血DNA提取試劑盒,提取基因組DNA。采用SNaPshot的方法檢測(cè)目的基因并分析結(jié)果。 結(jié)果：所測(cè)GAPDH基因中其5’非編碼區(qū)點(diǎn)rs1136666基因多態(tài)性,與PD發(fā)病相關(guān)。其中G為保護(hù)堿基,且此點(diǎn)男性病例對(duì)照研究統(tǒng)計(jì)學(xué)有明顯差異性,而女性研究無(wú)統(tǒng)計(jì)學(xué)意義。此點(diǎn)在早發(fā)組病例對(duì)照研究中等位基因頻率研究有統(tǒng)計(jì)學(xué)意義,但基因型研究無(wú)統(tǒng)計(jì)學(xué)意義。在晚發(fā)組等位基因頻率研究及基因型研究均有統(tǒng)計(jì)學(xué)意義。 結(jié)論：GAPDH基因5’非編碼區(qū)點(diǎn)rs1136666多態(tài)性與PD發(fā)病相關(guān),且其相關(guān)性與性別、年齡有關(guān)。男性、老齡是該基因與PD相關(guān)發(fā)病的危險(xiǎn)因素。 第二節(jié)NCAPD2、GAPDH家族、PKP2P1、PPM1H、CD9、ETV6基因多態(tài)性與PD相關(guān)性分析 目的：采用病例對(duì)照研究,對(duì)PD患者與正常對(duì)照組中基因組其他入組基因進(jìn)行遺傳學(xué)分析,并探討這些基因與PD發(fā)病風(fēng)險(xiǎn)及相關(guān)性。 方法：收集265例原發(fā)性PD患者和性別、年齡匹配的269例正常人外周血,采用外周血DNA提取試劑盒,提取基因組DNA。采用SNaPshot的方法檢測(cè)目的基因并分析結(jié)果。 結(jié)果：GAPDH基因上游NCAPD2基因中rs7311174、rs2072374位點(diǎn)基因多態(tài)性在PD病例對(duì)照研究中有統(tǒng)計(jì)學(xué)意義,而rs740850位點(diǎn)基因多態(tài)性在PD病例對(duì)照研究中無(wú)統(tǒng)計(jì)學(xué)意義。GAPDH家族位點(diǎn)中rs2029721、s4806173、rs12984928基因多態(tài)性在PD病例對(duì)照研究中沒(méi)有統(tǒng)計(jì)學(xué)意義。rs10492243在此次研究中與PD有強(qiáng)烈的關(guān)聯(lián)性。rs2008134、rs342169、rs740839、rs732868在此次病例對(duì)照研究中沒(méi)有統(tǒng)計(jì)學(xué)意義。 結(jié)論：NCAPD2基因與PD發(fā)病相關(guān)。GAPDH家族中位點(diǎn)所納入此次研究的三個(gè)點(diǎn)未發(fā)現(xiàn)與PD相關(guān)聯(lián)。 第二部分GAPDH基因突變與PD關(guān)系研究 第一節(jié)魚(yú)藤酮處理SH-SY5Y細(xì)胞的氧化水平研究 目的：觀察魚(yú)藤酮干預(yù)SH-SY5Y細(xì)胞后細(xì)胞內(nèi)氧化水平的變化。 方法：進(jìn)行SH-SY5Y細(xì)胞培養(yǎng)。將細(xì)胞按濃度梯度分組,MTT法摸索最佳干預(yù)濃度。光鏡下觀察各濃度梯度細(xì)胞形態(tài)學(xué)變化。使用DCFH-DA處理細(xì)胞后流式細(xì)胞儀觀察細(xì)胞內(nèi)氧化水平變化；采用SOD Assay kit檢測(cè)細(xì)胞內(nèi)SOD活性水平；采用TBARS Assay kit檢測(cè)細(xì)胞內(nèi)脂質(zhì)過(guò)氧化水平。 結(jié)果：MTT檢測(cè)細(xì)胞活力摸索出細(xì)胞最適干預(yù)濃度為500nnM,且細(xì)胞活力與干預(yù)濃度呈濃度依賴(lài)性。光鏡下觀察細(xì)胞發(fā)現(xiàn)經(jīng)不同濃度處理的細(xì)胞,200nM少量細(xì)胞出現(xiàn)形態(tài)學(xué)改變,隨著濃度的改變,細(xì)胞形態(tài)學(xué)及細(xì)胞數(shù)量減少的更為明顯,2μM的魚(yú)藤酮加入培養(yǎng)基后細(xì)胞幾乎停止生長(zhǎng),突起消失變圓。使用DCFH-DA處理500nnM魚(yú)藤酮處理后的細(xì)胞發(fā)現(xiàn)細(xì)胞內(nèi)ROS水平升高；SOD Assay kit檢測(cè)細(xì)胞內(nèi)SOD活性水平降低；采用TBARS Assay kit檢測(cè)細(xì)胞內(nèi)脂質(zhì)過(guò)氧化增強(qiáng)。 結(jié)論：魚(yú)藤酮干預(yù)細(xì)胞后能使細(xì)胞內(nèi)氧化水平升高,SOD水平降低,脂質(zhì)過(guò)氧化增強(qiáng)。 第二節(jié)GAPDH基因5,非編碼區(qū)突變對(duì)細(xì)胞功能的影響 目的：構(gòu)建rs1136666點(diǎn)突變載體,探討GAPDH基因突變與PD的關(guān)系。 方法：進(jìn)行SH-SY5Y細(xì)胞培養(yǎng),克隆點(diǎn)rs1136666點(diǎn)所在片段。根據(jù)分組采用魚(yú)滕湒或載體單個(gè)或同時(shí)進(jìn)行干預(yù)細(xì)胞。摸索載體轉(zhuǎn)染細(xì)胞的合適濃度及轉(zhuǎn)染時(shí)間以及檢驗(yàn)時(shí)間。使用實(shí)時(shí)熒光定量PCR觀察各組中GAPDH基因表達(dá)情況。使用western blot觀察各組細(xì)胞中GAPDH表達(dá)的情況及觀察各組中凋亡的情況。使用SOD Assay kit檢測(cè)細(xì)胞內(nèi)SOD活性水平；采用TBARS Assay kit檢測(cè)細(xì)胞內(nèi)脂質(zhì)過(guò)氧化水平。使用流式細(xì)胞儀對(duì)各組細(xì)胞中神經(jīng)元凋亡進(jìn)行檢測(cè),比較各組細(xì)胞經(jīng)干預(yù)后細(xì)胞凋亡的情況；使用DCFH-DA處理細(xì)胞后在流式細(xì)胞儀中檢測(cè),對(duì)各組細(xì)胞中神經(jīng)元中氧化應(yīng)激水平作出比較。 結(jié)果：實(shí)時(shí)熒光定量PCR示GAPDH基因5’非編碼區(qū)突變引起細(xì)胞內(nèi)mRNA表達(dá)降低;western blot示GAPDH基因5’非編碼區(qū)突變可引起細(xì)胞內(nèi)凋亡蛋白表達(dá)增高,抗凋亡蛋白表達(dá)降低,GAPDH表達(dá)增高。而GAPDH突變轉(zhuǎn)染與rot聯(lián)用可增強(qiáng)凋亡作用。流式細(xì)胞儀中觀察到GAPDH突變轉(zhuǎn)染后的細(xì)胞凋亡增加,加用魚(yú)藤酮后,凋亡作用更強(qiáng)。DCFH-DA處理后的細(xì)胞可見(jiàn)GAPDH突變轉(zhuǎn)染的細(xì)胞中ROS水平增高,魚(yú)藤酮可增強(qiáng)這一作用。GAPDH基因5’非編碼區(qū)突變轉(zhuǎn)染后的細(xì)胞內(nèi)SOD水平下降,GAPDH基因5’非編碼區(qū)突變轉(zhuǎn)染后細(xì)胞內(nèi)脂質(zhì)過(guò)氧化水平增高,而魚(yú)藤酮使SOD水平下降及脂質(zhì)過(guò)氧化增高得更明顯。 結(jié)論：GAPDH基因5’非編碼區(qū)突變,可引起mRNA表達(dá)異常,蛋白折疊障礙,異常GAPDH聚集,最終引起細(xì)胞死亡,加入魚(yú)藤酮后,細(xì)胞內(nèi)凋亡各項(xiàng)指標(biāo)發(fā)生改變。 第三節(jié)GAPDH過(guò)表達(dá)對(duì)細(xì)胞功能的影響 目的：構(gòu)建GAPDH過(guò)表達(dá)載體,探討GAPDH基因過(guò)表達(dá)與PD的關(guān)系。GAPDH基因過(guò)表達(dá)與GAPDH突變共同在PD中的關(guān)系研究。 方法：進(jìn)行SH-SY5Y細(xì)胞培養(yǎng),構(gòu)建GAPDH過(guò)表達(dá)載體。根據(jù)分組采用魚(yú)滕湒或載體單個(gè)或同時(shí)進(jìn)行干預(yù)細(xì)胞。摸索載體轉(zhuǎn)染細(xì)胞的合適濃度及轉(zhuǎn)染時(shí)間以及檢驗(yàn)時(shí)間。使用DCFH-DA處理細(xì)胞后在流式細(xì)胞儀中檢測(cè),對(duì)各組細(xì)胞中神經(jīng)元中氧化應(yīng)激水平作出比較。用SOD Assay kit檢測(cè)細(xì)胞內(nèi)SOD活性水平；采用TBARS Assay kit檢測(cè)細(xì)胞內(nèi)脂質(zhì)過(guò)氧化水平。使用western blot觀察各組細(xì)胞中GAPDH表達(dá)的情況及觀察各組中凋亡情況。使用流式細(xì)胞儀對(duì)各組細(xì)胞中神經(jīng)元凋亡進(jìn)行檢測(cè),比較各組細(xì)胞經(jīng)干預(yù)后細(xì)胞凋亡的情況。使用非變性PAGE膠印跡GAPDH基因5’非編碼區(qū)突變及GAPDH過(guò)表達(dá)以及魚(yú)藤酮處理細(xì)胞后細(xì)胞內(nèi)GAPDH多聚體表達(dá)情況。 結(jié)果：western blot示過(guò)表達(dá)載體轉(zhuǎn)染的細(xì)胞內(nèi)GAPDH表達(dá)增高,流式細(xì)胞儀觀察細(xì)胞凋亡增多。DCFH-DA示過(guò)表達(dá)載體轉(zhuǎn)染后的細(xì)胞中ROS水平增高。而當(dāng)GAPDH突變與過(guò)表達(dá)同時(shí)存在時(shí)這一作用被增強(qiáng)。非變性PAGE膠印跡示GAPDH基因5’非編碼區(qū)突變及GAPDH過(guò)表達(dá)以及魚(yú)藤酮處理細(xì)胞后細(xì)胞內(nèi)異常GAPDH表達(dá)增多。 結(jié)論：GAPDH過(guò)表達(dá)時(shí)細(xì)胞內(nèi)氧化水平增高,促進(jìn)細(xì)胞凋亡,當(dāng)過(guò)表達(dá)與突變同時(shí)存在時(shí),細(xì)胞凋亡作用更強(qiáng)。
[Abstract]:Study on the polymorphism of the first part of parkinson's disease Section 1. GAPDH gene polymorphism and PD association analysis: a case-control study was used to make a genetic analysis of the GAPDH gene in the PD patients and the normal control group, and to explore the risk of GAPDH gene and PD and the correlation. Methods:265 patients with primary PD and 269 normal subjects with sex and age were collected, and the peripheral blood DNA extraction kit was used to extract the genome. DNA. The target gene was detected by the method of SNaPshot and divided Results: The gene polymorphism of rs1136666 in the 5 'non-coding region of GAPDH gene and P D. The G is the protective base, and there is a significant difference in the male case-control study of this point, and there is no female study. Statistical significance. This point was statistically significant in the study of the frequency of the allele in the early-onset case-control study, but the genotypic study did not Statistical significance. The frequency and genotype of the allele in the late-onset group were all Conclusion: The polymorphism of rs1136666 in the 5 'non-coding region of GAPDH gene is related to the occurrence of PD, and its correlation is related to that of PD. Sex, age-related. Male, old is the gene related to PD Risk factors for morbidity. Section II NCAPD2, GAPDH family, PKP2P1, PPM1H, CD9, ETV6 gene Objective: To study the genetic analysis of the other enrolled genes in the genome of PD patients and the normal control group by case-control study, and to explore these genes. Methods:265 patients with primary PD and 269 normal subjects with sex and age were collected, and the peripheral blood was extracted from peripheral blood. taking the kit, extracting the genomic DNA, using the SNaPshot, Results: The genetic polymorphism of rs7311174 and rs2072374 in the NCAPD2 gene in the upstream of the GAPDH gene was of statistical significance in the control study of PD. There was no statistical significance in the control of PD cases. rs2029721, s4806173, rs12984928 in the GAPDH family site were polymorphic at P There was no statistical significance in the D case control study. rs10492243 was There is a strong association with PD in this study. rs2008134, rs342169, rs740839, rs732868 are here There was no statistical significance in the secondary case control study. On the relationship between the NCAPD2 gene and the PD, the site of the GAPDH family is included. The three points of this study were not found to be associated with PD. The first part of the study on the relationship between the gene mutation of the second part of GAPDH and Study on the level of oxidation of SH-SY5Y cells treated with rotenone The intracellular oxygen of SH-SY5Y cells after the intervention of ketone Methods: SH-SY5Y cell culture was performed. culturing; grouping the cells according to a concentration gradient, and the MTT method The changes of cell morphology were observed under light microscope. The level of intracellular oxidation was observed by flow cytometry using DCFH-DA, and the level of SOD activity in cells was detected by using the SOD Assay kit. Detection of the level of lipid peroxidation in the cells by the TBARS Assay kit. The pre-concentration was 500 nnM, and the cell viability and the concentration of the intervention were in a concentration-dependent manner. The results showed that the level of ROS in the cells was increased by using DCFH-DA, and the level of SOD activity in the cells was decreased. The increase of lipid peroxidation in the cells was detected by the TBARS Asay kit. After the cell, the level of internal oxidation of the cell is increased, the level of SOD is reduced, and the lipid peroxidation is reduced. The effect of the mutation of the second GAPDH gene 5 and the non-coding region on the function of the cell: the structure The mutation vector of rs1136666 was constructed to study the relationship between GAPDH gene mutation and PD. Method: SH-SY5Y cell culture, clone point rs1 The segment where the 136666 point is located. The fish-tented or carrier sheet is used according to the grouping. One or the same time for the intervention of the cells. The appropriate concentration of the carrier-transfected cells and the time of transfection A real-time fluorescence quantitative PCR was used to observe the expression of GAPDH gene in each group. The expression of GAPDH in each group was observed by ern-blot and the apoptosis in each group was observed. The level of SOD activity was detected. The level of lipid peroxidation in the cells was detected by the TBARS Assay kit. The apoptosis of the cells in each group was detected by flow cytometry. Results: The 5 'non-coding region mutation of GAPDH gene was reduced by real-time fluorescence quantitative PCR, and 5' non-coding of GAPDH gene was shown by western blot. The mutation of the region can cause the expression of the apoptosis protein in the cell to be increased, and the anti-apoptotic protein table The increase of GAPDH expression and the increase of GAPDH expression and the combination of GAPDH mutation and rot can enhance the effect of apoptosis. The cell apoptosis increased after the GAPDH mutation was observed in the cytometer, and the apoptosis was stronger after the addition of the rotenone. The DCFH-DA treatment The level of ROS in the cells transfected with the GAPDH mutation was increased in the cells of the GAPDH gene. The level of SOD in the cells of the 5 'non-coding region of the GAPDH gene was decreased, and the non-coding region of the GAPDH gene was mutated. Conclusion: The 5 'non-coding region of GAPDH gene can cause abnormal expression of mRNA and protein folding. Abnormal GAPDH aggregation, resulting in cell death, After the addition of the rotenone, the apoptosis indexes of the cells were changed. The purpose of cellular function: to construct a GAPDH overexpression vector and to explore the GAP The relationship between the overexpression of DH gene and PD and the overexpression of GAPDH gene and GAPDH A study of the relationship between mutations in PD. Method: SH- SY5Y cell culture to construct a GAPDH overexpression vector. The appropriate concentration of the transfected cells and the time of transfection and the time of the test were found in the group. The level of oxidative stress in the neurons of each group was compared by using DCFH-DA to process the cells, and the levels of oxidative stress in the cells of each group were compared. Assay of the level of SOD activity in the cells by the ay kit; the detection of intracellular lipid with the TBARS Assay kit The expression of GAPDH in each group was observed by western blot and the expression of GAPDH in each group was observed by western blot. Apoptosis was detected by flow cytometry. The apoptosis of the cells in each group was compared by flow cytometry. The non-modified PAGE gel was used to print the GAPD. H gene 5 'non-coding region mutation and GAPDH overexpression and the expression of GAPDH multimer in cells after the treatment of cells with rotenone. : Western blot showed the intracellular GAPD in the cells transfected with the expression vector H-expression, flow cytometry to observe the increase of apoptosis, DCFH-D A showed an increase in the level of ROS in the cells transfected with the expression vector. This action was enhanced when the GAPDH mutation was present at the same time as the overexpression. APDH gene 5 'non-coding region mutation and GAPDH overexpression and abnormal GAPDH expression in the cells after the treatment of the cells with the rotenone.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R742.5
【共引文獻(xiàn)】
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8 陳春暖;王濤;;3-磷酸甘油醛脫氫酶與帕金森病[J];中國(guó)臨床神經(jīng)科學(xué);2011年05期
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10 簡(jiǎn)崇書(shū);侯婷婷;殷榮康;程和平;王顯花;;線粒體穩(wěn)定高鈣信號(hào)誘發(fā)超氧炫[J];中國(guó)科學(xué):生命科學(xué);2013年10期
相關(guān)博士學(xué)位論文 前10條
1 許文嶸;毛發(fā)周期的調(diào)控網(wǎng)絡(luò)與雄激素性脫發(fā)基因治療的基礎(chǔ)研究[D];南京醫(yī)科大學(xué);2010年
2 鄒菁;維持體外擴(kuò)增臍帶血干細(xì)胞干性：下調(diào)異常升高的ROS和p38MAPKα信號(hào)[D];華中科技大學(xué);2011年
3 盧秉國(guó);龍眼子葉胚發(fā)育相關(guān)的若干基因克隆及序列分析[D];福建農(nóng)林大學(xué);2009年
4 黃金莎;GAPDH過(guò)表達(dá)及異常聚集在帕金森病發(fā)病機(jī)制中作用的研究[D];華中科技大學(xué);2009年
5 周亮;胸腺素α1的克隆表達(dá)及其作用機(jī)理研究[D];浙江大學(xué);2008年
6 劉慧君;過(guò)氧化氫、Ca~（2+）對(duì)骨骼肌線粒體移動(dòng)和動(dòng)態(tài)變化的調(diào)節(jié)[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年
7 張李峰;紅芪和黃芪的免疫調(diào)節(jié)作用及抗免疫老化機(jī)制比較研究[D];蘭州大學(xué);2012年
8 李梅;TIGAR通過(guò)增加NADPH在腦缺血再灌注損傷中發(fā)揮神經(jīng)保護(hù)作用[D];蘇州大學(xué);2013年
9 王虹;真核生物AU-rich元件結(jié)合蛋白HuR和GAPDH3的結(jié)構(gòu)功能研究[D];中國(guó)科學(xué)技術(shù)大學(xué);2013年
10 張付;甲基苯丙胺對(duì)PC12細(xì)胞的毒性損傷、蛋白質(zhì)硝基化、L-257抗損傷等作用的機(jī)制研究[D];南方醫(yī)科大學(xué);2012年
相關(guān)碩士學(xué)位論文 前10條
1 何影影;一種檢測(cè)細(xì)胞內(nèi)RNA轉(zhuǎn)錄的熒光探針前體的合成研究[D];南京理工大學(xué);2012年
2 胡穎;喹唑啉化合物庫(kù)內(nèi)小分子促小鼠胚胎干細(xì)胞心肌分化活性篩選及相關(guān)分子事件研究[D];浙江大學(xué);2010年
3 徐莉;纈沙坦對(duì)擴(kuò)張型心肌病大鼠心肌線粒體結(jié)構(gòu)及功能的影響[D];江蘇大學(xué);2010年
4 齊卡;鈣轉(zhuǎn)運(yùn)異常與高尿酸導(dǎo)致的內(nèi)皮細(xì)胞損傷的相關(guān)關(guān)系及其分子機(jī)制研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2010年
5 許藝惠;補(bǔ)腎復(fù)方對(duì)MES23.5神經(jīng)細(xì)胞氧化損傷后抗氧化酶的影響[D];福建中醫(yī)藥大學(xué);2013年
6 劉輝;腦脊液GAPDH抗體表達(dá)與多發(fā)性硬化的相關(guān)性研究[D];河北醫(yī)科大學(xué);2013年
7 李艷麗;PI3K/Akt信號(hào)通路在頭部淺低溫減輕大鼠全腦缺血再灌注損傷中的作用[D];河北醫(yī)科大學(xué);2013年
8 李明亮;內(nèi)質(zhì)網(wǎng)應(yīng)激聯(lián)合線粒體在高血糖加重腦缺血再灌注損傷中作用的體外實(shí)驗(yàn)研究[D];遵義醫(yī)學(xué)院;2013年
9 宋慶亮;何首烏飲調(diào)節(jié)衰老大鼠睪丸Leydig細(xì)胞分泌睪酮的機(jī)制[D];河北大學(xué);2013年
10 謝云鵬;β1,4半乳糖基轉(zhuǎn)移酶I與激素、TGF-β1在胚胎著床調(diào)控中的作用[D];大連醫(yī)科大學(xué);2012年
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本文編號(hào)：2508887
本文鏈接：http://sikaile.net/yixuelunwen/shenjingyixue/2508887.html
[Abstract]:Study on the polymorphism of the first part of parkinson's disease Section 1. GAPDH gene polymorphism and PD association analysis: a case-control study was used to make a genetic analysis of the GAPDH gene in the PD patients and the normal control group, and to explore the risk of GAPDH gene and PD and the correlation. Methods:265 patients with primary PD and 269 normal subjects with sex and age were collected, and the peripheral blood DNA extraction kit was used to extract the genome. DNA. The target gene was detected by the method of SNaPshot and divided Results: The gene polymorphism of rs1136666 in the 5 'non-coding region of GAPDH gene and P D. The G is the protective base, and there is a significant difference in the male case-control study of this point, and there is no female study. Statistical significance. This point was statistically significant in the study of the frequency of the allele in the early-onset case-control study, but the genotypic study did not Statistical significance. The frequency and genotype of the allele in the late-onset group were all Conclusion: The polymorphism of rs1136666 in the 5 'non-coding region of GAPDH gene is related to the occurrence of PD, and its correlation is related to that of PD. Sex, age-related. Male, old is the gene related to PD Risk factors for morbidity. Section II NCAPD2, GAPDH family, PKP2P1, PPM1H, CD9, ETV6 gene Objective: To study the genetic analysis of the other enrolled genes in the genome of PD patients and the normal control group by case-control study, and to explore these genes. Methods:265 patients with primary PD and 269 normal subjects with sex and age were collected, and the peripheral blood was extracted from peripheral blood. taking the kit, extracting the genomic DNA, using the SNaPshot, Results: The genetic polymorphism of rs7311174 and rs2072374 in the NCAPD2 gene in the upstream of the GAPDH gene was of statistical significance in the control study of PD. There was no statistical significance in the control of PD cases. rs2029721, s4806173, rs12984928 in the GAPDH family site were polymorphic at P There was no statistical significance in the D case control study. rs10492243 was There is a strong association with PD in this study. rs2008134, rs342169, rs740839, rs732868 are here There was no statistical significance in the secondary case control study. On the relationship between the NCAPD2 gene and the PD, the site of the GAPDH family is included. The three points of this study were not found to be associated with PD. The first part of the study on the relationship between the gene mutation of the second part of GAPDH and Study on the level of oxidation of SH-SY5Y cells treated with rotenone The intracellular oxygen of SH-SY5Y cells after the intervention of ketone Methods: SH-SY5Y cell culture was performed. culturing; grouping the cells according to a concentration gradient, and the MTT method The changes of cell morphology were observed under light microscope. The level of intracellular oxidation was observed by flow cytometry using DCFH-DA, and the level of SOD activity in cells was detected by using the SOD Assay kit. Detection of the level of lipid peroxidation in the cells by the TBARS Assay kit. The pre-concentration was 500 nnM, and the cell viability and the concentration of the intervention were in a concentration-dependent manner. The results showed that the level of ROS in the cells was increased by using DCFH-DA, and the level of SOD activity in the cells was decreased. The increase of lipid peroxidation in the cells was detected by the TBARS Asay kit. After the cell, the level of internal oxidation of the cell is increased, the level of SOD is reduced, and the lipid peroxidation is reduced. The effect of the mutation of the second GAPDH gene 5 and the non-coding region on the function of the cell: the structure The mutation vector of rs1136666 was constructed to study the relationship between GAPDH gene mutation and PD. Method: SH-SY5Y cell culture, clone point rs1 The segment where the 136666 point is located. The fish-tented or carrier sheet is used according to the grouping. One or the same time for the intervention of the cells. The appropriate concentration of the carrier-transfected cells and the time of transfection A real-time fluorescence quantitative PCR was used to observe the expression of GAPDH gene in each group. The expression of GAPDH in each group was observed by ern-blot and the apoptosis in each group was observed. The level of SOD activity was detected. The level of lipid peroxidation in the cells was detected by the TBARS Assay kit. The apoptosis of the cells in each group was detected by flow cytometry. Results: The 5 'non-coding region mutation of GAPDH gene was reduced by real-time fluorescence quantitative PCR, and 5' non-coding of GAPDH gene was shown by western blot. The mutation of the region can cause the expression of the apoptosis protein in the cell to be increased, and the anti-apoptotic protein table The increase of GAPDH expression and the increase of GAPDH expression and the combination of GAPDH mutation and rot can enhance the effect of apoptosis. The cell apoptosis increased after the GAPDH mutation was observed in the cytometer, and the apoptosis was stronger after the addition of the rotenone. The DCFH-DA treatment The level of ROS in the cells transfected with the GAPDH mutation was increased in the cells of the GAPDH gene. The level of SOD in the cells of the 5 'non-coding region of the GAPDH gene was decreased, and the non-coding region of the GAPDH gene was mutated. Conclusion: The 5 'non-coding region of GAPDH gene can cause abnormal expression of mRNA and protein folding. Abnormal GAPDH aggregation, resulting in cell death, After the addition of the rotenone, the apoptosis indexes of the cells were changed. The purpose of cellular function: to construct a GAPDH overexpression vector and to explore the GAP The relationship between the overexpression of DH gene and PD and the overexpression of GAPDH gene and GAPDH A study of the relationship between mutations in PD. Method: SH- SY5Y cell culture to construct a GAPDH overexpression vector. The appropriate concentration of the transfected cells and the time of transfection and the time of the test were found in the group. The level of oxidative stress in the neurons of each group was compared by using DCFH-DA to process the cells, and the levels of oxidative stress in the cells of each group were compared. Assay of the level of SOD activity in the cells by the ay kit; the detection of intracellular lipid with the TBARS Assay kit The expression of GAPDH in each group was observed by western blot and the expression of GAPDH in each group was observed by western blot. Apoptosis was detected by flow cytometry. The apoptosis of the cells in each group was compared by flow cytometry. The non-modified PAGE gel was used to print the GAPD. H gene 5 'non-coding region mutation and GAPDH overexpression and the expression of GAPDH multimer in cells after the treatment of cells with rotenone. : Western blot showed the intracellular GAPD in the cells transfected with the expression vector H-expression, flow cytometry to observe the increase of apoptosis, DCFH-D A showed an increase in the level of ROS in the cells transfected with the expression vector. This action was enhanced when the GAPDH mutation was present at the same time as the overexpression. APDH gene 5 'non-coding region mutation and GAPDH overexpression and abnormal GAPDH expression in the cells after the treatment of the cells with the rotenone.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R742.5
【共引文獻(xiàn)】
相關(guān)期刊論文 前10條
1 趙靜;程楠;王訓(xùn);;金屬元素銅與神經(jīng)變性疾病的研究進(jìn)展[J];安徽醫(yī)學(xué);2009年11期
2 盧秉國(guó);何煒毅;申艷紅;蔣際謀;陳曉靜;陳偉;呂柳新;;龍眼子葉胚3-磷酸甘油醛脫氫酶基因的cDNA克隆及序列分析[J];福建農(nóng)林大學(xué)學(xué)報(bào)(自然科學(xué)版);2009年05期
3 曹云霞;高明;;精子發(fā)生障礙的遺傳學(xué)研究進(jìn)展[J];國(guó)際生殖健康/計(jì)劃生育雜志;2014年03期
4 YANG Ming;PAN Chenyuan;BO Jun;WANG Kejian;;Regulation of metallothionein gene expression in response to benzo[a]pyrene exposure and bacterial challenge in marine cultured black porgy(Acanthopagrus schlegelii)[J];Chinese Journal of Geochemistry;2014年04期
5 Jin-Ying Zhang;Fan Zhang;Chao-Qun Hong;Armando E.Giuliano;Xiao-Jiang Cui;Guang-Ji Zhou;Guo-Jun Zhang;Yu-Kun Cui;;Critical protein GAPDH and its regulatory mechanisms in cancer cells[J];Cancer Biology & Medicine;2015年01期
6 馮軍;鄭彩霞;;植物響應(yīng)寒冷脅迫的CBF基因表達(dá)調(diào)控[J];吉林農(nóng)業(yè);2011年04期
7 陸征宇;趙虹;汪濤;陳潔;金環(huán);李文婷;張琦君;;補(bǔ)腎養(yǎng)肝熄風(fēng)方藥對(duì)帕金森病小鼠紋狀體抗氧化酶和細(xì)胞凋亡的影響[J];江蘇中醫(yī)藥;2011年03期
8 陳春暖;王濤;;3-磷酸甘油醛脫氫酶與帕金森病[J];中國(guó)臨床神經(jīng)科學(xué);2011年05期
9 李強(qiáng);趙延欣;劉學(xué)源;;SIAH蛋白研究進(jìn)展[J];中國(guó)臨床神經(jīng)科學(xué);2013年02期
10 簡(jiǎn)崇書(shū);侯婷婷;殷榮康;程和平;王顯花;;線粒體穩(wěn)定高鈣信號(hào)誘發(fā)超氧炫[J];中國(guó)科學(xué):生命科學(xué);2013年10期
相關(guān)博士學(xué)位論文 前10條
1 許文嶸;毛發(fā)周期的調(diào)控網(wǎng)絡(luò)與雄激素性脫發(fā)基因治療的基礎(chǔ)研究[D];南京醫(yī)科大學(xué);2010年
2 鄒菁;維持體外擴(kuò)增臍帶血干細(xì)胞干性：下調(diào)異常升高的ROS和p38MAPKα信號(hào)[D];華中科技大學(xué);2011年
3 盧秉國(guó);龍眼子葉胚發(fā)育相關(guān)的若干基因克隆及序列分析[D];福建農(nóng)林大學(xué);2009年
4 黃金莎;GAPDH過(guò)表達(dá)及異常聚集在帕金森病發(fā)病機(jī)制中作用的研究[D];華中科技大學(xué);2009年
5 周亮;胸腺素α1的克隆表達(dá)及其作用機(jī)理研究[D];浙江大學(xué);2008年
6 劉慧君;過(guò)氧化氫、Ca~（2+）對(duì)骨骼肌線粒體移動(dòng)和動(dòng)態(tài)變化的調(diào)節(jié)[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年
7 張李峰;紅芪和黃芪的免疫調(diào)節(jié)作用及抗免疫老化機(jī)制比較研究[D];蘭州大學(xué);2012年
8 李梅;TIGAR通過(guò)增加NADPH在腦缺血再灌注損傷中發(fā)揮神經(jīng)保護(hù)作用[D];蘇州大學(xué);2013年
9 王虹;真核生物AU-rich元件結(jié)合蛋白HuR和GAPDH3的結(jié)構(gòu)功能研究[D];中國(guó)科學(xué)技術(shù)大學(xué);2013年
10 張付;甲基苯丙胺對(duì)PC12細(xì)胞的毒性損傷、蛋白質(zhì)硝基化、L-257抗損傷等作用的機(jī)制研究[D];南方醫(yī)科大學(xué);2012年
相關(guān)碩士學(xué)位論文 前10條
1 何影影;一種檢測(cè)細(xì)胞內(nèi)RNA轉(zhuǎn)錄的熒光探針前體的合成研究[D];南京理工大學(xué);2012年
2 胡穎;喹唑啉化合物庫(kù)內(nèi)小分子促小鼠胚胎干細(xì)胞心肌分化活性篩選及相關(guān)分子事件研究[D];浙江大學(xué);2010年
3 徐莉;纈沙坦對(duì)擴(kuò)張型心肌病大鼠心肌線粒體結(jié)構(gòu)及功能的影響[D];江蘇大學(xué);2010年
4 齊卡;鈣轉(zhuǎn)運(yùn)異常與高尿酸導(dǎo)致的內(nèi)皮細(xì)胞損傷的相關(guān)關(guān)系及其分子機(jī)制研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2010年
5 許藝惠;補(bǔ)腎復(fù)方對(duì)MES23.5神經(jīng)細(xì)胞氧化損傷后抗氧化酶的影響[D];福建中醫(yī)藥大學(xué);2013年
6 劉輝;腦脊液GAPDH抗體表達(dá)與多發(fā)性硬化的相關(guān)性研究[D];河北醫(yī)科大學(xué);2013年
7 李艷麗;PI3K/Akt信號(hào)通路在頭部淺低溫減輕大鼠全腦缺血再灌注損傷中的作用[D];河北醫(yī)科大學(xué);2013年
8 李明亮;內(nèi)質(zhì)網(wǎng)應(yīng)激聯(lián)合線粒體在高血糖加重腦缺血再灌注損傷中作用的體外實(shí)驗(yàn)研究[D];遵義醫(yī)學(xué)院;2013年
9 宋慶亮;何首烏飲調(diào)節(jié)衰老大鼠睪丸Leydig細(xì)胞分泌睪酮的機(jī)制[D];河北大學(xué);2013年
10 謝云鵬;β1,4半乳糖基轉(zhuǎn)移酶I與激素、TGF-β1在胚胎著床調(diào)控中的作用[D];大連醫(yī)科大學(xué);2012年
，本文編號(hào)：2508887
本文鏈接：http://sikaile.net/yixuelunwen/shenjingyixue/2508887.html
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