【摘要】：目的資料顯示，在男性不育中，約10%的患者為無精子癥，卻能產(chǎn)生正常的變態(tài)前的圓形精子細胞。第1例人ROSI技術(shù)獲得的妊娠由Tesarik等于1995年報道，他們將從無精子癥病人精液中獲得的ROS注入卵子后出生了2位正常嬰兒。雖然ROSI的成功為NOA患者帶來了希望，但是ROSI的成功率很低，并且ROSI后胚胎的染色體非整倍性增加，準確的挑選出正常的單倍體精子細胞是該技術(shù)的一個巨大挑戰(zhàn)。然而，鑒別活的單倍體精子細胞不是一個容易的過程，本實驗為了建立穩(wěn)定的精子細胞Puresperm非連續(xù)梯度離心和FISH（熒光原位雜交）檢測技術(shù)，得到密度較高的單倍體圓形精子細胞，并對挑選出的精子細胞作倍性驗證分析，初步了解單倍體圓形精子細胞的基本形態(tài)特征，為今后準確識別單倍體精子細胞提高準確率，為確切診斷NOA患者提供依據(jù)，為科學研究提供識別單倍體圓形精子細胞的方法。 方法睪丸活檢標本來自安徽醫(yī)科大學某醫(yī)院無精子癥患者睪丸活檢組織，根據(jù)病理檢查報告為非梗阻性無精子癥，收集非梗阻性無精子癥患者13例。然后對13例患者睪丸組織經(jīng)過細胞涂片，利用FISH技術(shù)初步篩選出含有單倍體精子細胞的睪丸組織進行下一步實驗，并在熒光顯微鏡下測量單倍體精子細胞的直徑，并記錄。實驗進行前已告知患者取得其知情同意。將睪丸組織經(jīng)過眼科小剪和TB針頭撕碎后，經(jīng)過酶消化、離心等處理，制備成單細胞懸液，再經(jīng)過Puresperm非連續(xù)梯度離心，將含有單倍體精子細胞較多的分層單細胞懸液收集，最后在倒置顯微鏡下利用激光鏡頭（可以測量細胞直徑）分別挑選出直徑5μm、6μm，7μm，8μm精子細胞，每組細胞30枚，然后采用FISH（熒光原位雜交）技術(shù)進行雜交，檢測每組單倍體精子細胞所占總數(shù)的比例。 結(jié)果①13例非梗阻性無精子癥患者對其睪丸組織經(jīng)過處理制備成單細胞懸液進行細胞涂片后，，經(jīng)過FISH檢測發(fā)現(xiàn)其中6例存在單倍體精子細胞，并初步在熒光倒置顯微鏡下測量細胞直徑，X單倍體精子細胞平均直徑在5.5μm，Y單倍體精子細胞平均直徑在5.3μm。二倍體XX精子細胞平均直徑為8.5μm，二倍體YY精子細胞平均直徑為8μm。②Puresperm非連續(xù)梯度離心處理得到結(jié)果：70%分離層中圓形精子細胞的數(shù)量最多，而70%、70%與50%、50%層細胞較少。通過倒置顯微鏡下觀察發(fā)現(xiàn)，在70%和100%交界面處各級生精細胞均有存在，但與未離心處理后單細胞懸液對比發(fā)現(xiàn)，離心后的細胞相對均勻，盡管未能排除其他的各級生精細胞。③單倍體圓形精子細胞的挑選在400×Hoffman倒置顯微鏡下，利用激光鏡頭測量細胞直徑，選擇出的圓形精子細胞基于細胞的外形、細胞直徑、細胞核特征、頂體帽的出現(xiàn)、細胞質(zhì)的特征，圓形精子細胞直徑在5-7μm，細胞核直徑在4-6μm，細胞核質(zhì)比例較固定，細胞圓形，細胞表面光滑，細胞質(zhì)較亮，細胞核較均勻，在圓形精子細胞較早階段細胞核多位于細胞的中心位置，較后階段，細胞核較多位于細胞的邊緣。頂體泡/帽結(jié)構(gòu)偶爾被觀察到，與細胞核毗連，位于細胞的的邊緣，頂體泡/帽較小，成灰色的點狀結(jié)構(gòu)，在倒置顯微鏡下觀察與精子的頂體帽結(jié)構(gòu)顏色像似。細胞質(zhì)圍繞細胞核類似薄的環(huán)狀結(jié)構(gòu)。圓形精子細胞的區(qū)分依靠細胞的大小，與圓形精子細胞區(qū)分較為困難的是次級精母細胞，因為它們的細胞形態(tài)學特征極其像似，通常次級精母細胞直徑稍大一點，但是次級精母細胞通常也很少被觀測到在射出的精液中和睪丸組織中，因為與存在兩周以上的初級精母細胞相比較而言，他們存在的過程較短，不足24小時，（1976Burger），圓形精子細胞與其他直徑較大的血細胞相比較容易區(qū)分。淋巴細胞直徑在7-12μm，但是他們的細胞核較大，而且淋巴細胞核成圓形或者鋸齒狀結(jié)構(gòu)，細胞質(zhì)較窄不是連續(xù)的包圍著細胞核。④使用雙色FISH雜交，每組細胞均獲得清晰的熒光信號，綠色代表X號染色體，橙色代表Y號染色體。在5-8μm范圍內(nèi)，直徑越小的細胞，為單倍體精子細胞的可能性越大，挑選的準確率較高。 結(jié)論Puresperm非連續(xù)梯度離心和FISH技術(shù)對于評估非梗阻性無精子癥患者睪丸組織內(nèi)有沒有單倍體圓形精子細胞是一種比較有用的一種方法，二者結(jié)合能夠確定單倍體精子細胞的基本特征，能夠指導(dǎo)臨床醫(yī)生準確的識別單倍體精子細胞，并可以起到質(zhì)量控制的重要作用，同時FISH技術(shù)也可以檢測圓形精子細胞染色體的組成。
[Abstract]:Objective To show that about 10% of male infertility is azoospermia, but it can produce normal pre-morbid round sperm cells. The pregnancy, obtained in the first human ROSI technique, was reported by Tesarik in 1995, and they were born with 2 normal babies following the injection of ROS from the sperm of azoospermia patients. Although the success of ROSI brings hope to NOA patients, the success rate of ROSI is very low, and the chromosome non-ploidy of ROSI is increased, and the accurate selection of normal haploid spermatids is a great challenge to the technology. However, the identification of viable haploid spermatids is not an easy process, and in order to establish stable sperm cell Purespm non-continuous gradient centrifugation and FISH (fluorescence in situ hybridization) detection techniques, haploid round sperm cells having a higher density are obtained, and carrying out ploidy verification analysis on the selected sperm cells, preliminarily knowing the basic morphological characteristics of the haploid round sperm cells, improving the accuracy rate of the haploid sperm cells in the future, providing a basis for the accurate diagnosis of NOA patients, The invention provides a method for identifying haploid round sperm cells for scientific research. Methods Testicle biopsy specimens from patients with azoospermia in a hospital in Anhui Medical University were collected from testicular biopsy specimens, and non-obstructive azoospermia patients were collected according to the pathological examination report. 3 cases of testicular tissue of 13 patients were subjected to cell smear, the testis tissue containing haploid spermatids was initially screened by FISH technique, and the diameter of haploid spermatids was measured under fluorescence microscope. Record. The patient was informed of his informed consent before the experiment The method comprises the following steps: after the testicular tissue is torn apart by an ophthalmic mini-scissors and a TB needle, performing enzymolysis, centrifugation and the like to prepare a single-cell suspension liquid, and then carrying out non-continuous gradient centrifugation through Puresperm, and then carrying out multi-layer single-cell suspension liquid containing the haploid spermatids. Collected, the diameter of 5. m, 6. m, 7. m, 8. m sperm cells, 30 cells per group, and then FISH (fluorescence in situ hybridization) technique were selected using a laser lens (which can measure cell diameter) under an inverted microscope The total number of haploid sperm cells in each group. Results In 13 non-obstructive azoospermia patients treated with single cell suspension for cell smear, 6 cases of haploid spermatids were detected by FISH. The average diameter of X haploid spermatids was 5. 5. m and the average diameter of Y haploid spermatids was 5. 3. m 8. 5. m u.M, The average diameter of diploid YY sperm cells was 8. m u.m. The results showed that the number of round sperm cells in 70% separation layer was the largest, while 70%, 70% and 50%, 50%. At the interface of 70% and 100%, spermatogenic cells were present at the interface of 70% and 100%. However, after centrifugation, the cells were relatively uniform, although other cells could not be excluded. The cell diameter, cell diameter, cell nucleus characteristics, the appearance of the top cap and the appearance of the top cap were determined by the selection of the haploid round spermatids of the order of 400g Hoffman's inverted microscope. The cytoplasm features that the diameter of the circular sperm cells is 5-7. m u.m, the nucleus diameter is 4-6. m u.m, the nucleus mass ratio is relatively fixed, the cell circle is smooth, the surface of the cells is smooth, the cytoplasm is brighter, the nuclei are more uniform, the nuclei at the early stage of the round sperm cells are located at the central position of the cells, in that lat stage, the nuclei are more localized in the nucleus, The edge of the cell. The top blister/ cap structure is occasionally observed, contiguous with the nucleus, located at the edge of the cell, the top blister/ cap is small, is a gray dot-shaped structure, and the top cap junction of the sperm is observed under an inverted microscope It's like. The cytoplasm is thin around the nucleus. The circular structure of the round sperm cells depends on the size of the cells, and it is more difficult to distinguish the secondary spermatocytes from the round sperm cells because their cell morphological characteristics are extremely image-like, usually secondary spermatocytes. A slightly larger diameter, but secondary spermatocytes are often rarely observed in the ejected semen and in testicular tissue, as compared to the primary spermatocytes in the presence of more than two weeks, they have a relatively short process, less than 24 hours, (1976Bu), rger), round sperm cells compared to other large blood cells It is easier to differentiate. Lymphocyte diameters are 7-12. m u.m, but their nuclei are large, and the lymphocyte nuclei are rounded or serrated, and the cytoplasm is narrower than a continuous bag Two-color FISH hybridization is used for each group of cells to obtain a clear fluorescence signal, green represents the X chromosome, and the orange representative Y chromosome. In the range of 5-8. m u.m, the smaller the diameter of the cells, the larger the possibility of haploid spermatids, the larger the choice. Conclusion Purethrom non-continuous gradient centrifugation and FISH technique are a useful method for evaluating the existence of haploid round spermatids in testicle tissue of non-obstructive azoospermia patients. the basic characteristics of the sub-cells can guide the clinician to accurately identify the haploid sperm cells and can play an important role in quality control,
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.8

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1 黃毅,Ashok Agarwal;人精液中未成熟生殖細胞的檢測及臨床意義[J];中華男科學;2004年04期

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