人单倍体圆形精子细胞识别方法的研究
发布时间：2018-10-11 07:01
【摘要】：目的资料显示，在男性不育中，约10%的患者为无精子症，却能产生正常的变态前的圆形精子细胞。第1例人ROSI技术获得的妊娠由Tesarik等于1995年报道，他们将从无精子症病人精液中获得的ROS注入卵子后出生了2位正常婴儿。虽然ROSI的成功为NOA患者带来了希望，但是ROSI的成功率很低，并且ROSI后胚胎的染色体非整倍性增加，准确的挑选出正常的单倍体精子细胞是该技术的一个巨大挑战。然而，鉴别活的单倍体精子细胞不是一个容易的过程，本实验为了建立稳定的精子细胞Puresperm非连续梯度离心和FISH（荧光原位杂交）检测技术，得到密度较高的单倍体圆形精子细胞，并对挑选出的精子细胞作倍性验证分析，初步了解单倍体圆形精子细胞的基本形态特征，为今后准确识别单倍体精子细胞提高准确率，为确切诊断NOA患者提供依据，为科学研究提供识别单倍体圆形精子细胞的方法。 方法睾丸活检标本来自安徽医科大学某医院无精子症患者睾丸活检组织，根据病理检查报告为非梗阻性无精子症，收集非梗阻性无精子症患者13例。然后对13例患者睾丸组织经过细胞涂片，利用FISH技术初步筛选出含有单倍体精子细胞的睾丸组织进行下一步实验，并在荧光显微镜下测量单倍体精子细胞的直径，并记录。实验进行前已告知患者取得其知情同意。将睾丸组织经过眼科小剪和TB针头撕碎后，经过酶消化、离心等处理，制备成单细胞悬液，再经过Puresperm非连续梯度离心，将含有单倍体精子细胞较多的分层单细胞悬液收集，最后在倒置显微镜下利用激光镜头（可以测量细胞直径）分别挑选出直径5μm、6μm，7μm，8μm精子细胞，每组细胞30枚，然后采用FISH（荧光原位杂交）技术进行杂交，检测每组单倍体精子细胞所占总数的比例。 结果①13例非梗阻性无精子症患者对其睾丸组织经过处理制备成单细胞悬液进行细胞涂片后，，经过FISH检测发现其中6例存在单倍体精子细胞，并初步在荧光倒置显微镜下测量细胞直径，X单倍体精子细胞平均直径在5.5μm，Y单倍体精子细胞平均直径在5.3μm。二倍体XX精子细胞平均直径为8.5μm，二倍体YY精子细胞平均直径为8μm。②Puresperm非连续梯度离心处理得到结果：70%分离层中圆形精子细胞的数量最多，而70%、70%与50%、50%层细胞较少。通过倒置显微镜下观察发现，在70%和100%交界面处各级生精细胞均有存在，但与未离心处理后单细胞悬液对比发现，离心后的细胞相对均匀，尽管未能排除其他的各级生精细胞。③单倍体圆形精子细胞的挑选在400×Hoffman倒置显微镜下，利用激光镜头测量细胞直径，选择出的圆形精子细胞基于细胞的外形、细胞直径、细胞核特征、顶体帽的出现、细胞质的特征，圆形精子细胞直径在5-7μm，细胞核直径在4-6μm，细胞核质比例较固定，细胞圆形，细胞表面光滑，细胞质较亮，细胞核较均匀，在圆形精子细胞较早阶段细胞核多位于细胞的中心位置，较后阶段，细胞核较多位于细胞的边缘。顶体泡/帽结构偶尔被观察到，与细胞核毗连，位于细胞的的边缘，顶体泡/帽较小，成灰色的点状结构，在倒置显微镜下观察与精子的顶体帽结构颜色像似。细胞质围绕细胞核类似薄的环状结构。圆形精子细胞的区分依靠细胞的大小，与圆形精子细胞区分较为困难的是次级精母细胞，因为它们的细胞形态学特征极其像似，通常次级精母细胞直径稍大一点，但是次级精母细胞通常也很少被观测到在射出的精液中和睾丸组织中，因为与存在两周以上的初级精母细胞相比较而言，他们存在的过程较短，不足24小时，（1976Burger），圆形精子细胞与其他直径较大的血细胞相比较容易区分。淋巴细胞直径在7-12μm，但是他们的细胞核较大，而且淋巴细胞核成圆形或者锯齿状结构，细胞质较窄不是连续的包围着细胞核。④使用双色FISH杂交，每组细胞均获得清晰的荧光信号，绿色代表X号染色体，橙色代表Y号染色体。在5-8μm范围内，直径越小的细胞，为单倍体精子细胞的可能性越大，挑选的准确率较高。 结论Puresperm非连续梯度离心和FISH技术对于评估非梗阻性无精子症患者睾丸组织内有没有单倍体圆形精子细胞是一种比较有用的一种方法，二者结合能够确定单倍体精子细胞的基本特征，能够指导临床医生准确的识别单倍体精子细胞，并可以起到质量控制的重要作用，同时FISH技术也可以检测圆形精子细胞染色体的组成。
[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,
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8
本文编号：2263287
[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,
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8
【参考文献】
相关期刊论文 前1条
1 黄毅,Ashok Agarwal;人精液中未成熟生殖细胞的检测及临床意义[J];中华男科学;2004年04期
本文编号：2263287
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