【摘要】：目的睪丸的精子發(fā)生是一個(gè)復(fù)雜的細(xì)胞分裂分化過程,是一系列基因按時(shí)空順序表達(dá)與睪丸組織內(nèi)外環(huán)境共同作用的結(jié)果。許多在睪丸中特異或高度表達(dá)的基因參與這一過程。本研究在實(shí)驗(yàn)室前期工作的基礎(chǔ)上,選擇三個(gè)差異表達(dá)的基因TSG23,T279和TSC21進(jìn)行研究。通過對上述三種基因進(jìn)行生物信息學(xué),mRNA和蛋白質(zhì)的表達(dá)特性,及在正常和無精癥睪丸中的表達(dá)差異分析,探討它們在精子發(fā)生過程中的作用,為男性不育癥的診斷和治療提供新的思路和方法。 方法(1)應(yīng)用芯片技術(shù)篩選精子發(fā)生相關(guān)基因。(2)應(yīng)用生物信息學(xué)方法分析TSG23和T279基因和編碼蛋白的理化結(jié)構(gòu)。(3)應(yīng)用PCR方法檢測TSG23和T279基因在人和小鼠多組織中及在小鼠不同發(fā)育階段睪丸組織和不同小鼠生殖細(xì)胞系中的表達(dá)特征;比較它們在正常睪丸及無精癥睪丸中的表達(dá)差異。(4)制備TSG23、T279和TSC21基因的RNA探針,采用原位雜交的方法檢測睪丸組織中mRNA的表達(dá)。(5)構(gòu)建小鼠pEGFP-N1-T279重組質(zhì)粒載體,轉(zhuǎn)染細(xì)胞系,確定T279蛋白的亞細(xì)胞定位。(6)構(gòu)建人pET32a-T279和TSC21表達(dá)載體,轉(zhuǎn)化到感受態(tài)BL21細(xì)菌,誘導(dǎo)表達(dá)并純化T279和TSC21蛋白;免疫小鼠,制備抗人的TSC21多克隆抗體和T279單克隆抗體;應(yīng)用合成的人多肽抗原免疫小鼠制備TSG23多克隆抗體。(7) Western Blot檢測TSG23和T279蛋白和抗體的特異性。(8)免疫熒光方法檢測T279在人睪丸組織中的表達(dá)。(9)應(yīng)用免疫組化方法檢測TSG23、T279和TSC21蛋白在睪丸組織中的表達(dá)定位,并檢測它們在正常人睪丸組織和生精阻滯患者睪丸組織中的表達(dá)差異。 結(jié)果(1)表達(dá)譜芯片結(jié)果顯示TSG23和T279基因在成人睪丸中高表達(dá),在小鼠睪丸中于18日齡睪丸中開始表達(dá),且隨年齡逐漸升高。(2)人TSG23定位在20q13.12,cDNA全長752bp,編碼227個(gè)氨基酸。小鼠TSG23定位在2H3,cDNA全長755bp,編碼226個(gè)氨基酸。人T279定位于13q34,cDNA全長773 bp,編碼195個(gè)氨基酸。小鼠T279定位于8A2,cDNA長度761bp,編碼182個(gè)氨基酸。TSG23和T279皆為人—小鼠同源基因。(3) PCR結(jié)果顯示TSG23和T279為睪丸組織特異性表達(dá)。小鼠TSG23和T279僅在出生后15天的睪丸組織中開始表達(dá),且隨著年齡的增加逐漸增強(qiáng),TSG23在Sertoli細(xì)胞,精原細(xì)胞和精母細(xì)胞系中均沒有表達(dá),而T279僅在精母細(xì)胞系中表達(dá)。與正常睪丸組織比較,TSG23和T279基因在無精癥睪丸組織的表達(dá)減弱或消失。(4)成功制備TSG23、T279和TSC21的正義和反義探針。原位雜交顯示人和小鼠睪丸生精上皮的精母細(xì)胞和圓形精子細(xì)胞內(nèi)存在TSG23的強(qiáng)陽性雜交信號,并呈現(xiàn)階段特異性表達(dá)模式。小鼠T279mRNA主要定位于靠近管腔的精子細(xì)胞及精子中,精母細(xì)胞也有陽性信號,TSC21 mRNA定位于小鼠精母細(xì)胞和精子細(xì)胞的胞漿中。(5)熒光顯微鏡觀察T279融合蛋白定位在精母細(xì)胞的內(nèi)質(zhì)網(wǎng)中。(6)成功制備人TSG23、T279和TSC21的鼠源性抗體。(7) Western Blot結(jié)果顯示TSG23蛋白只在睪丸組織中識別出約23KD大小的特異性條帶,T279蛋白在睪丸組織中識別出約22KD大小的特異性條帶,而在其他組織中兩者均為檢測到條帶。(8)免疫熒光結(jié)果表明T279蛋白主要在正常人睪丸組織的精母細(xì)胞和圓形精子細(xì)胞中表達(dá)。(9)免疫組化結(jié)果顯示在正常睪丸中TSG23、T279和TSC21蛋白主要在精母細(xì)胞和圓形精子細(xì)胞中表達(dá),且TSG23主要表達(dá)于生精過程中的Ⅲ和Ⅴ期,而在Ⅰ和Ⅱ期中表達(dá)很低。在生精阻滯的無精癥患者中三個(gè)蛋白表達(dá)均減弱或消失。 結(jié)論(1) TSG23、T279和TSC21均為睪丸特異性高表達(dá)基因,且是人—小鼠同源性基因。在小鼠睪丸組織中的表達(dá)呈年齡依賴性升高。(2) TSG23、T279和TSC21在無精子癥患者睪丸組織的表達(dá)明顯減少或消失。(3) TSG23、T279和TSC21基因?qū)τ诓G丸發(fā)育和精子發(fā)生可能具有重要作用,其表達(dá)降低或消失可能是男性不育癥發(fā)生的重要原因之一。
[Abstract]:Objective Testicular spermatogenesis is a complex process of cell division and differentiation, which is the result of a series of genes expressed in time-space sequence and interaction with the internal and external environment of testicular tissue. The three genes TSG23, T279 and TSC21 were studied. Their bioinformatics, expression characteristics of mRNA and protein, and differential expression in normal and azoospermic testis were analyzed to explore their roles in spermatogenesis, and to provide new ideas and methods for the diagnosis and treatment of male infertility.
Methods (1) Screening of spermatogenesis related genes by microarray technique. (2) Analyzing the physical and chemical structure of TSG2 3 and T279 genes and coding proteins by bioinformatics method. (3) Detecting the expression of TSG2 3 and T279 genes in human and mouse multiple tissues, testicular tissues at different developmental stages and different mouse germ cell lines by PCR method. The expression of TSG23, T279 and TSC21 was detected by in situ hybridization. (5) The recombinant plasmid vector of mouse pEGFP-N1-T279 was constructed and transfected into cell lines to determine the subcellular localization of T279 protein. (6) Human pET32a-T279 was constructed. TSC21 and TSC21 expression vectors were transformed into competent BL21 bacteria to induce the expression and purification of T279 and TSC21 proteins; mice were immunized with polyclonal antibodies against human TSC21 and monoclonal antibodies against T279; mice were immunized with synthetic human polypeptide antigens to prepare TSG23 polyclonal antibodies. (7) Western Blot was used to detect the specificity of TSG23 and T279 proteins and antibodies. (8) Immunization. The expression of TSG23, T279 and TSC21 in human testicular tissue was detected by fluorescence staining. (9) The expression and localization of TSG23, T279 and TSC21 proteins in testicular tissue were detected by immunohistochemical method, and the expression differences of TSG23, T279 and TSC21 proteins in normal human testicular tissue and testicular tissue of patients with spermatogenesis block were detected.
Results (1) TSG23 and T279 genes were highly expressed in adult testis, and began to express in 18-day-old testis of mice, and gradually increased with age. (2) Human TSG23 was localized at 20q13.12, with a full length of 752 bp, encoding 227 amino acids. TSG2 3 and T279 were homologous to human-mouse genes. (3) PCR results showed that TSG2 3 and T279 were specifically expressed in testicular tissues. TSG2 3 and T279 were expressed only in testicular tissues 15 days after birth and began to express with the passage of the year. The expression of TSG23 and T279 in spermatocyte, spermatogonia and spermatocyte was decreased or disappeared compared with normal testicular tissues. (4) Sensitive and antisense probes for TSG23, T279 and TSC21 were successfully prepared. The results showed that there were strong positive hybridization signals of TSG23 in spermatocytes and round sperm cells of human and mouse testicular seminiferous epithelium, and the expression pattern was stage-specific. In cytoplasm. (5) The fusion protein T279 was localized in the endoplasmic reticulum of spermatocytes by fluorescence microscopy. (6) Mouse-derived antibodies against human TSG23, T279 and TSC21 were successfully prepared. (7) Western Blot results showed that the TSG23 protein only recognized a specific band of about 23 KD in testicular tissue, and the T279 protein recognized a specific band of about 22 KD in testicular tissue. Specific bands were detected in other tissues. (8) Immunofluorescence showed that T279 protein was mainly expressed in spermatocytes and round sperm cells of normal human testis. (9) Immunohistochemistry showed that TSG23, T279 and TSC21 proteins were mainly expressed in spermatocytes and round sperm cells of normal testis. TSG23 was mainly expressed in stages III and V of spermatogenesis, but very low in stages I and II.
Conclusion (1) TSG23, T279 and TSC21 are testicular-specific high-expression genes, and are homologous to human-mouse genes. The expression of TSG23, T279 and TSC21 in testicular tissues of mice increases in age-dependent manner. (2) The expression of TSG23, T279 and TSC21 in testicular tissues of azoospermia patients decreases or disappears significantly. (3) The expression of TSG23, T279 and TSC21 genes in testicular development and sperm disappears. It may play an important role in the occurrence of male infertility, and the decrease or disappearance of its expression may be one of the important reasons for the occurrence of male infertility.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R321

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