本文關(guān)鍵詞：染色體數(shù)目異常胚胎絨毛細胞著絲粒相關(guān)蛋白基因研究　出處：《重慶醫(yī)科大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 細胞培養(yǎng) 絨毛細胞 染色體數(shù)目分析 細胞轉(zhuǎn)染 RNA干擾 短發(fā)夾狀RNA hsMAD2 hBUB1 定量RT-PCR 染色體數(shù)目異常 hsMAD2基因 hBUB1基因 自然流產(chǎn)

【摘要】： 在胚胎發(fā)育的早期進行著旺盛的細胞有絲分裂,染色體正常分離到兩個子細胞是保證有絲分裂正常進行的重要基礎(chǔ),與之相關(guān)的蛋白質(zhì)種類至少應(yīng)包括:著絲粒相關(guān)蛋白(centromere associated proteins)(MAD,BUB等),著絲粒特異蛋白(centromere specific proteins)(CENPs等)和紡錘體微管相關(guān)蛋白(spindle microtube associated proteins)。在細胞分裂中染色體的正常分離受到有絲分裂紡錘體檢查點(mitotic spindle checkpoint)的調(diào)控,具有正常功能的紡錘體檢查點可保證姐妹染色單體正確地分離進入兩個子細胞。當(dāng)有絲分裂紡錘體未與染色單體附著時,檢查點處于激活狀態(tài),激活的檢查點會抑制后期啟動復(fù)合物(APC/C)的泛素連接酶活性,有絲分裂后期延遲。MAD2(mitosis arrest deficiency)和BUB1(budding uninhibited by benomyl)既是兩種著絲粒相關(guān)蛋白,也是組成紡錘體檢查點復(fù)合物的重要成分,因此又稱為檢查點蛋白(checkpoint proteins,CP),它們從酵母到人類都高度保守。這些蛋白確保只有在所有染色體著絲粒連接到紡錘體微管上時,細胞才能進入后期,染色體開始向細胞兩極移動,完成染色體的正常分離。哺乳動物細胞中,BUB1和MAD2蛋白在有絲分裂前期存在于著絲粒區(qū)域,中期它們將隨著紡錘體微管附著于著絲粒上而消失。Li和Ouyang認為這些CP蛋白在未與紡錘絲連接的著絲粒點上的濃度較高,在紡錘絲連接的著絲粒點上的濃度較低。鑒于hsMAD2和/或hBUB1可能具有重要的作用,我們對染色體數(shù)目異常胚胎絨毛組織hsMAD2和/或hBUB1基因表達進行了研究并發(fā)現(xiàn):hsMAD2和/或hBUB1基因蛋白水平的表達顯著降低。 RNA干擾(RAN interference,RNAi)技術(shù)是近年來廣泛應(yīng)用于哺乳動物細胞抑制基因表達的新技術(shù),主要用于基因功能學(xué)和基因治療學(xué)等方面的研究。本課題應(yīng)用RNAi技術(shù),探討染色體數(shù)目正常的絨毛細胞轉(zhuǎn)染靶向hsMAD2或hBUB1基因的RNAi重組體后,靶基因轉(zhuǎn)錄水平和蛋白表達水平的變化。我們的實驗數(shù)據(jù)顯示:shRNA-MAD2-1和shRNA-BUB1-2干擾序列能特異地、有效地抑制胚胎細胞中靶基因的表達,并發(fā)現(xiàn)染色體數(shù)目的異常細胞比例增加。在靶基因的臨床研究中,我們直接檢測到了染色體數(shù)目異常的自然流產(chǎn)的胚胎組織中靶蛋白表達的下降,得到了與RNAi干擾模型相同的實驗結(jié)果。 為此,我們推測在胚胎細胞中hsMAD2和/或hBUB1基因表達下調(diào)可能是導(dǎo)致胚胎細胞染色體數(shù)目的異常的原因之一。該研究為進一步探討染色體數(shù)目異常發(fā)生機理奠定了基礎(chǔ)。 第一部分 絨毛細胞的培養(yǎng)方法及轉(zhuǎn)染策略研究 目的: 1、流產(chǎn)胚胎絨毛組織原代細胞培養(yǎng),獲得染色體標(biāo)本制備方法; 2、流產(chǎn)胚胎絨毛組織原代培養(yǎng)、純化絨毛細胞并鑒定細胞種類; 3、了解絨毛細胞的生長特性和質(zhì)粒轉(zhuǎn)染條件。 方法: 1、制備流產(chǎn)組織絨毛細胞染色體標(biāo)本進行核型分析; 2、對流產(chǎn)胚胎進行細胞原代培養(yǎng)和純化。用抗-vimentin抗體和抗-cytokeratin抗體進行細胞種類鑒定; 3、采用MTT實驗繪制生長曲線,找到其對數(shù)生長期;用細胞貼壁率找出絨毛細胞生長所需的最適pH;同時采用轉(zhuǎn)染效率和細胞存活分數(shù)來評價絨毛細胞質(zhì)粒轉(zhuǎn)染的最佳脂質(zhì)體用量。 結(jié)果: 1、通過染色體分析篩選出40例染色體數(shù)目正常胚胎絨毛標(biāo)本; 2、人工流產(chǎn)胚胎絨毛組織的原代細胞培養(yǎng)、純化后絨毛細胞含量80%; 3、細胞傳代后12~36 h細胞處于對數(shù)生長期,48 h后進入平臺期;絨毛細胞生長最適的pH為6.8~7.0;綜合轉(zhuǎn)染效率和細胞存活分數(shù)找到最佳脂質(zhì)體用量。 結(jié)論:成功培養(yǎng)出胚胎絨毛細胞并初步了解其生長特性,同時對RNA干擾質(zhì)粒轉(zhuǎn)染所需的條件進行優(yōu)化,為后期的RNA干擾實驗作好準備。 第二部分 RNA干擾技術(shù)抑制內(nèi)源hsMAD2或hBUB1基因在絨毛細胞中的表達 目的: 1、構(gòu)建針對hsMAD2和hBUB1基因的短發(fā)夾狀RNA(shRNA)表達載體; 2、shRNA重組表達質(zhì)粒載體轉(zhuǎn)染染色體數(shù)目正常的絨毛細胞后,觀察靶基因表達的抑制,以及染色體數(shù)目異常的變化情況; 3、了解hsMAD2和hBUB1基因的表達抑制對絨毛細胞增殖的影響以及對細胞周期的調(diào)節(jié)。 方法: 1、設(shè)計、合成hsMAD2和hBUB1基因的shRNA序列,與載體pTZU6+1連接,構(gòu)建靶基因的shRNA重組表達質(zhì)粒; 2、shRNA重組質(zhì)粒轉(zhuǎn)染細胞48 h后,用免疫細胞化學(xué)和western blot觀察靶基因的蛋白水平,并用定量real-time PCR檢測mRNA水平; 3、有效干擾組胚胎絨毛細胞的染色體數(shù)目分析; 4、MTT試驗檢測干擾前后的細胞存活分數(shù); 5、用FCM評價兩基因?qū)ε咛ソq毛細胞周期的影響。 結(jié)果: 1、成功構(gòu)建針對靶基因的shRNA重組質(zhì)粒; 2、轉(zhuǎn)染有效干擾shRNA重組質(zhì)粒后,絨毛細胞靶基因的內(nèi)源性表達受到明顯抑制,其蛋白和mRNA水平都顯著下降; 3、有效干擾組細胞核型分析也發(fā)現(xiàn)多種類型的染色體數(shù)目異常; 4、MTT實驗發(fā)現(xiàn)有效干擾組細胞存活分數(shù)明顯下降,細胞增殖受到抑制;且經(jīng)FCM檢測知道有效干擾后使較多絨毛細胞停滯在分裂期(M期)。 結(jié)論:設(shè)計、合成的針對靶基因的shRNA重組質(zhì)粒在絨毛細胞中能有效抑制靶基因的表達,染色體數(shù)目異常絨毛細胞比率增加,說明MAD2和BUB1對染色體分離可能具有重要調(diào)控作用,成功構(gòu)建了MAD2和BUB1抑制后觀察染色體數(shù)目異常變化的體外細胞模型,為后期的臨床研究奠定了基礎(chǔ)。 第三部分 染色體數(shù)目異常的自然流產(chǎn)胚胎絨毛組織中hsMAD2和hBUB1基因表達的臨床研究 目的: 1、了解染色體數(shù)目異常的自然流產(chǎn)胚胎絨毛組織中是否存在MAD2和BUB1基因的蛋白和/(或)mRNA水平的異常,探索靶基因的表達在染色體數(shù)目異常的胚胎發(fā)生中可能具有的作用; 2、了解在自然流產(chǎn)染色體數(shù)目異常的胚胎組織中hsMAD2和hBUB1基因的蛋白編碼區(qū)域是否存在具有臨床意義的基因變異。 方法: 1、分別對早期自然流產(chǎn)的胚胎(12周)標(biāo)本絨毛細胞進行染色體數(shù)目進行分析;數(shù)目正常組為對照組,異常組為實驗組; 2、分別提取對照組和實驗組樣品絨毛組織中的總RNA和蛋白質(zhì); 3、用real-time RT-PCR檢測hsMAD2和hBUB1基因的mRNA量; 4、用western blot檢測每一蛋白樣品中hsMAD2和hBUB1水平; 5、實驗組中選擇8例蛋白表達量最低的標(biāo)本組織的cDNA,對兩靶基因的CDS區(qū)域用PCR分段擴增并測序,再用生物信息學(xué)方法將測序結(jié)果與genebank數(shù)據(jù)庫進行比對。 結(jié)果: 1、早期自然流產(chǎn)胚胎絨毛組織中篩選出25例染色體數(shù)目異常和31例染色體數(shù)目正常的組織標(biāo)本; 2、Real-time RT-PCR結(jié)果顯示,實驗組和對照組的hsMAD2和hBUB1基因的mRNA水平無顯著性差異; 3、Western Blot檢測結(jié)果顯示,hsMAD2和hBUB1蛋白在實驗組中的表達顯著低于對照組; 4、CDS測序結(jié)果與genebank數(shù)據(jù)庫比對,兩靶基因均無移位、缺失,僅發(fā)現(xiàn)3個同義點突變。 結(jié)論:在染色體數(shù)目異常的自然流產(chǎn)胚胎絨毛組織中hsMAD2和hBUB1表達顯著下降,提示其作為編碼CP的重要基因在染色體數(shù)目異常胚胎的發(fā)生中可能具有重要意義。
[Abstract]:In the early embryonic development of exuberant cell mitosis, normal chromosome separated into two daughter cells is an important basis to ensure the normal mitosis, related proteins should include at least: centromere associated protein (centromere associated proteins) (MAD, BUB), centromere specific proteins (centromere protein) (CENPs) and spindle microtubule associated protein (spindle microtube associated proteins). The normal separation of chromosomes during cell division by mitotic spindle checkpoint (mitotic spindle checkpoint) regulation, with the normal function of the spindle checkpoint can ensure the correct separation of sister chromatids into two daughter cells when there is. Mitotic spindle and chromatid attachment, the checkpoint is activated and the activation of the checkpoint inhibits late start complexes (APC/C) of the Ubiquitin ligase activity, mitotic anaphase delay.MAD2 (mitosis arrest deficiency) and BUB1 (budding uninhibited by benomyl) is the two important components of centromere associated protein, is composed of spindle checkpoint complexes, also known as checkpoint protein (checkpoint proteins, CP), they are highly conserved from yeast to humans. These proteins ensure that only in all chromosomes attached to spindle microtubules when cells can enter anaphase, the chromosomes began to move to opposite poles of the cell, the normal separation of chromosomes. Mammalian animal cells, BUB1 and MAD2 protein in the prophase of mitosis in the centromeric region, they will be with the mid spindle microtubule attachment to centromere and.Li and Ouyang argue that higher concentrations of disappearance of these CP proteins is not connected with the spindle of the centromere on the spindle connected to the centromere The concentration of hsMAD2 and / or hBUB1 may play an important role. We studied the gene expression of hsMAD2 and / or hBUB1 in the chorionic villi of chromosome number abnormality, and found that the expression level of hsMAD2 and / or hBUB1 protein decreased significantly.
RNA interference (RAN interference RNAi) technology is a new technology in recent years, widely used in mammalian cells to inhibit gene expression, mainly used for the research of gene function and gene therapy and so on. This paper discusses the application of RNAi technology, villus cells transfected with target normal chromosome number to the hsMAD2 or hBUB1 gene recombinant RNAi and the expression of target gene transcription and protein. Our data showed that shRNA-MAD2-1 and shRNA-BUB1-2 interference sequence can specifically and effectively inhibit the expression of target genes in embryonic cells, and abnormal chromosome number increased. The proportion of cells in the clinical research of target genes, we directly detected decreased expression of the target protein embryo abnormal chromosome number of spontaneous abortion, obtained the same results with the RNAi interference model.
Therefore, we speculate that the down regulation of hsMAD2 and / or hBUB1 gene expression in embryonic cells may be one of the reasons leading to abnormal chromosome numbers. This study laid a foundation for further exploring the mechanism of chromosome abnormality.
Part one
Study on the culture of villous cells and the strategy of transfection
Objective:
1, the primary cells of the aborted embryo were cultured, and the methods of preparing the chromosomes were obtained.
2, the aborted embryo was cultured in the villi tissue for primary culture, and the villus cells were purified and the species of cells were identified.
3, to understand the growth characteristics of chorionic cells and plasmid transfection conditions.
Method:
1, the karyotype of the villus cells in the abortion tissue was analyzed.
2, the primary culture and purification of the aborted embryos were carried out. The type of cell species was identified by anti -vimentin antibody and anti -cytokeratin antibody.
3, the growth curve was drawn by MTT experiment, and its logarithmic growth phase was found. The optimal pH for villus cell growth was found by cell adherence rate, and the transfection efficiency and cell survival fraction were used to evaluate the optimal dosage of plasmid transfection.
Result:
1, 40 normal embryo villi specimens were screened by chromosome analysis.
2, the primary cell culture of the aborted embryo chorionic villus was 80%, and the content of the villus cells after purification.
3, after cell passage, the 12~36 h cells were in logarithmic growth phase, and entered the plateau stage after 48 h. The most suitable pH for villi cell growth was 6.8~7.0, and the optimal transfection efficiency and cell survival fraction were found to find the best liposome dosage.
Conclusion: embryo chorionic cells were successfully cultured, and their growth characteristics were preliminarily understood. Meanwhile, the necessary conditions of RNA interference plasmid transfection were optimized, so as to prepare for the later RNA interference experiment.
The second part
RNA interference technique inhibits the expression of endogenous hsMAD2 or hBUB1 genes in chorionic cells
Objective:
1, a short hairpin RNA (shRNA) expression vector for hsMAD2 and hBUB1 genes was constructed.
2, shRNA recombinant plasmid vector was transfected into villous cells with normal chromosome number, and the inhibition of target gene expression and the change of chromosome number were observed.
3, we know the effect of inhibition of hsMAD2 and hBUB1 gene expression on the proliferation of chorionic villi and the regulation of cell cycle.
Method:
1, design, synthesize shRNA sequence of hsMAD2 and hBUB1 gene, connect with carrier pTZU6+1, construct shRNA recombinant expression plasmid of target gene.
2, after transfection of shRNA recombinant plasmid to 48 h cells, the protein level of the target gene was observed by immunocytochemistry and Western blot, and the level of mRNA was detected by quantitative real-time PCR.
3, the analysis of the number of chromosomes in the chorionic villus cells was effectively interfered.
4, MTT test was used to detect the cell survival fraction before and after interference.
5, the effect of two gene on the cell cycle of embryonic chorionic villi was evaluated by FCM.
Result:
1, the recombinant plasmid shRNA for target gene was successfully constructed.
2, after transfecting the recombinant plasmid with effective interference shRNA, the endogenous expression of the target gene of the villus cells was obviously inhibited, and the protein and mRNA level were significantly decreased.
3, the cell karyotype analysis of effective interference group also found the number of chromosomes of various types.
4, MTT experiments showed that the survival fraction of the effective interference group was significantly decreased, and the cell proliferation was inhibited. After FCM detection, more chorionic cells were arrested during the split phase (M phase).
Conclusion: design, synthesis of the target gene shRNA recombinant plasmid can inhibit target gene expression in villi cells, abnormal chromosome number of villus cells ratio increased, MAD2 and BUB1 on chromosome segregation may play an important role in regulation, successfully constructed an in vitro model of abnormal changes in chromosome number MAD2 and BUB1 inhibition was observed after. Lay a foundation for further clinical researches.
The third part
A clinical study on the expression of hsMAD2 and hBUB1 genes in the villous tissue of spontaneous abortion embryos with abnormal chromosome number
Objective:
1, we need to know whether there are abnormalities in protein and / or mRNA levels of MAD2 and BUB1 genes in natural chorionic villus tissues with abnormal chromosome numbers, and explore the possible role of target gene expression in the development of abnormal chromosome numbers.
2, to understand whether the protein coding region of hsMAD2 and hBUB1 genes in fetal tissues with abnormal chromosome number of spontaneous abortion has a genetic variation in clinical significance.
Method:
1, the number of chromosomes in the chorionic cells of early spontaneous abortion (12 weeks) was analyzed, and the normal group was the control group, and the abnormal group was the experimental group.
2, the total RNA and protein in the villi tissues of the control group and the experimental group were extracted respectively.
3, the mRNA amount of hsMAD2 and hBUB1 genes was detected by real-time RT-PCR.
4, the level of hsMAD2 and hBUB1 in each protein sample was detected by Western blot.
5, in the experimental group, 8 cases of cDNA with minimal protein expression were selected. The CDS region of the two target gene was amplified by PCR and sequenced, and the sequencing results were compared with genebank database by bioinformatics.
Result:
1, 25 cases of abnormal chromosome number and 31 normal chromosome number were selected from the villi tissue of early spontaneous abortion.
2, Real-time RT-PCR results showed that there was no significant difference in the mRNA level between the hsMAD2 and the hBUB1 genes in the experimental group and the control group.
3, the results of Western Blot detection showed that the expression of hsMAD2 and hBUB1 protein in the experimental group was significantly lower than that in the control group.
4, the results of CDS sequencing were compared with the genebank database, and the two target genes were not displaced and missing, and only 3 mutation of the synonymous point were found.
Conclusion: the expression of hsMAD2 and hBUB1 in natural chorionic villus tissues with chromosome number abnormalities is significantly decreased, suggesting that as an important gene encoding CP, it is of great significance in the occurrence of abnormal chromosome numbers.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R394

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