本文選題：LCN-2　切入點(diǎn)：睪丸　出處：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：LCN-2(lipocalin-2)名為脂質(zhì)運(yùn)載蛋白2,又名中性粒細(xì)胞白明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白,屬于轉(zhuǎn)運(yùn)蛋白中親脂性的分泌型糖蛋白,參與動(dòng)物機(jī)體多種生物功能。本研究主要采用原位雜交、免疫印跡、實(shí)時(shí)熒光定量PCR等方法,并利用發(fā)育性表達(dá)、隱睪、白消安處理等模型,分別檢測(cè)LCN-2基因和蛋白在CD-1小鼠睪丸中的表達(dá)與分布情況,探討LCN-2基因與雄性生殖的關(guān)系以及調(diào)節(jié)機(jī)制,為進(jìn)一步的研究LCN-2基因在小鼠生殖發(fā)育過程中的功能提供理論依據(jù)。發(fā)育性表達(dá)模型中發(fā)現(xiàn)LCN-2基因在小鼠1日齡、10日齡和20日齡中未見表達(dá),30日齡、40日齡、2月齡、4月齡、8月齡、12月齡中有表達(dá),原位雜交結(jié)果顯示在4月齡時(shí)表達(dá)量最高。隱睪模型中睪丸體積變小,HE染色曲細(xì)精管形態(tài)結(jié)構(gòu)損傷,生精上皮和曲細(xì)精管出現(xiàn)不同程度的退化,曲細(xì)精管中只有少量或基本沒有精原細(xì)胞,m RNA表達(dá)量明顯上升,其中大部分在睪丸間質(zhì)細(xì)胞中表達(dá)(P=0.0378)。WB結(jié)果顯示蛋白水平也是明顯增加(P=0.0005)。白消安模型中睪丸體積明顯變小,曲細(xì)精管結(jié)構(gòu)嚴(yán)重破壞,只剩下一層很薄的生精上皮細(xì)胞,管腔中幾乎沒有精子細(xì)胞。原位雜交結(jié)果顯示,m RNA表達(dá)量明顯上調(diào)(P=0.0232)。WB結(jié)果如同隱睪模型(P=0.0004)。實(shí)時(shí)熒光定量PCR結(jié)果顯示隱睪模型和白消安模型中實(shí)驗(yàn)組相比于對(duì)照組變化顯著,這也與原位雜交的結(jié)果相吻合。綜上所述,LCN-2基因在小鼠睪丸中有表達(dá),且在小鼠不同生長(zhǎng)發(fā)育階段的睪丸中表達(dá)量有所不同,在隱睪模型和白消安模型中LCN-2的表達(dá)量明顯上調(diào),說明無精子模型能夠誘導(dǎo)LCN-2的表達(dá),LCN-2基因可能參與精子的發(fā)生及成熟,睪丸中生殖細(xì)胞的分化和凋亡,有望給雄性不育癥、睪丸炎等生殖障礙病提供一個(gè)新的研究方向,為治療雄性不育癥患者提供理論依據(jù)。
[Abstract]:LCN-2lipocalin-2 (LCN-2lipocalin-2), also known as neutrophil gelatinase-associated lipid transport protein (LCN-2lipocalin-2), belongs to the lipocalin-2 transport protein, which is a kind of lipocalin-2 protein, and is involved in many kinds of biological functions. The expression and distribution of LCN-2 gene and protein in the testis of CD-1 mice were detected by Western blot and real-time fluorescence quantitative PCR. To explore the relationship between LCN-2 gene and male reproduction and its regulatory mechanism. In order to further study the function of LCN-2 gene in mouse reproductive development, it was found in the developmental expression model that LCN-2 gene was not expressed at the age of 30 days or 40 days old or 2 months old in 10 days old and 20 days old mice. It was expressed in the age of 4 months and 8 months and 12 months of age. The results of in situ hybridization showed that the highest expression level was observed at 4 months old. In the model of cryptorchidism, the testis became smaller and the seminiferous tubules were damaged by HE staining, and the seminiferous epithelium and seminiferous tubules degenerated to different degrees. The expression of m RNA in seminiferous tubule was significantly increased in only a few or little or no spermatogonial cells, most of which were expressed in Leydig cells of testis. The results showed that the protein level was also significantly increased by 0.0005. The testicular volume in Baoxuan model became smaller obviously. The structure of the seminiferous tubules was severely damaged, leaving only a very thin layer of spermatogenic epithelial cells. There were almost no sperm cells in the lumen. The results of in situ hybridization showed that the expression of RNA was significantly up-regulated as that of the cryptorchidism model. The results of real-time fluorescence quantitative PCR showed that the experimental group was significantly different from the control group in the cryptorchidism model and the Baixiaan model. These results were consistent with the results of in situ hybridization. In conclusion, the expression of LCN-2 gene was found in mouse testis, and the expression of LCN-2 gene was different in mouse testis at different stages of growth and development, and the expression of LCN-2 was up-regulated in cryptorchidism model and Baoxuan model. These results suggest that the azoospermia model can induce the expression of LCN-2 and LCN-2 gene in spermatogenesis and maturation. The differentiation and apoptosis of germ cells in testis may provide a new research direction for male infertility, orchitis and other reproductive disorders. To provide theoretical basis for the treatment of male infertility.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S814.1

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