论文标题:小鼠睾丸及不育患者精液中精子细胞的分离与鉴定
Isolation and Identification of Spermatids from Mouse Testis and Semen of Male Infertile Patients
论文作者 朱培元
论文导师 黄宇烽,论文学位 硕士,论文专业 免疫学
论文单位 南京医科大学,点击次数 120,论文页数 63页File Size2237k
2002-04-01论文网 http://www.lw23.com/lunwen_18605177/ 精子细胞;非连续;Percoll梯度离心;小鼠;男性不育;精液;免疫细胞化学;流式细胞术;荧光原位杂交
Spermatid;Discontinuous Percoll gradient centrifugation; Mouse; Male infertile;Semen;Immunocytochemistry; Flow cytometry;Fluorescence in situ hybridization
1995年，人类首次利用圆形精子细胞和长形精子细胞体外授精获得成功后，预示着精子细胞显微注射授精可用于治疗临床无法治愈的男性不育患者。目前，完全不能产生精子的非梗阻性无精子症(non-obstmctive azoospermia,NOA)患者可采用卵胞质内圆形精子细胞注射(round spermatid injection,ROSI)或长形精子细胞注射(elongated spermatid inection,ELSI)治疗，精子细胞可从精液或从睾丸活检组织中获取。甚至已经有采用冻融睾丸组织中精子细胞行ROSI后获得持续妊娠的报告。 但精子细胞的受精率和妊娠率令人失望，限制这项技术成功和阻碍其临床应用的许多问题仍然没有解决。除精子细胞的核蛋白不完全成熟及卵子激活不足等因素外，如何正确选择存活精子细胞是个难题，如圆形精子细胞与精母细胞、单核细胞和多形核白细胞等其他圆形细胞的区分就比较困难。另一方面，已经报道了多种不同的方法可从睾丸中分离精子细胞。最常用的是机械方法，即利用细针将活检获得的小块睾丸组织撕开，释放出精子细胞；也可利用酶将睾丸组织消化成细胞悬液，其中包含所有类型的生精细胞。然后在显微镜下从中手工选择精子细胞供立即穿刺使用，但不可能获得大量纯化的精子细胞，而反复活检获取精子细胞将进一步损伤患者已有缺陷的睾丸精曲小管(seminiferous tubules)。因此，有必要建立一种比较简单的、可获取大量纯化精子细胞的方法，不但可满足立即使用所需，也可进行冷冻保存用于以后的治疗周期。 在第一部分试验中，首先利用连续3次组合酶消化成年小鼠睾丸制备睾丸细胞悬液，然后经6层非连续Percoll梯度离心法(15％、22％、30％、40％、50％和60％)分离，通过形态学和流式细胞术鉴定 南京医科大学硕士学位论文 各个Percoll组分中精子细胞的含量，并以伊红Y排斥试验测定细胞 的存活率。结果组合酶消化后获得的睾丸细胞悬液中，97％以上的细 胞仍然存活。经k。u梯度离。c后形成的6个组分中，22％Percu 组分主要含精子细胞（平均占86．7oho．05）85．5％以上细胞存活； 6 30OPercoll组分中细胞密度最高po0．05），含有各级来成熟生精 细胞，存活细胞超过92％。因此采用组合酶消化结合非连续Percoll 梯度离。G法，可有效地从小鼠睾丸中分离到较多较纯的存活精子细 胞。 将获得的小鼠精子细胞进行卵胞质内注射，共穿刺32个小鼠卵 子，15个行 ROSI和 17个行 ELSI。结果在培养的 3 d中，ROSI的 卵子没有发生卵裂，＆ ELSI的卵子中有 3个服至 2细胞期O7．6O）。 在第H部分试验中，利用这种非连续 Percoll梯度离。c法，对 15 例各种类型不育患者的精液细胞进行分离，并利用瑞－姬染色法、流 式细胞术、免疫细胞化学和荧光原位杂交oISffi等方法，从细胞形态 特征、DNA倍体、细胞表面标i己与分化抗原，以及原位杂交信号的 数目和位置结合细胞核特有的形态等方面加以鉴定。结果效果更好， 可分离出足够数量的精子细胞，还可将精子和白细胞与禾成熟生精 细胞分离。其中 22％Percoll组分中单倍体（In）精子细胞平均占四．85 t 5．18）％，显著高于其他组分（＜0．005X 平均密度为（l．010 i 0．786） xlo’／ml；而精子和白细胞王要位于 60％的 Percoll组分中。
The first reports on successful clinical use of round and elongated spermatids for assisted reproduction in 1995 suggest the benefits that the technique of spermatid conception can bring to some patients suffering from otherwise untreatable types of male factor infertility. Men with non-obstructive azoospermia(NOA) can now be treated by using intra-oocyte round spermatid injection(ROSI) or elongated spermatid injection(ELSI). Spermatids can be retrieved from semen or from testis biopsy specimens. An ongoing clinical pregnancy has even been reported following ROSI using frozen-thawed testicular spermatids.However, the rates of fertilization and pregnancy with spermatids have been disappointing. Many problems limiting success rates and binding a wide application of this technique still remain unresolved. Except the incomplete maturation of spermatid nuclear and oocyte activation, idendification of a live spermatid is a pivotal procedure. It is difficult to distinguish round spermatids from other round cells such as spermatocytes, monocytes, polymorphonuclear leukocytes and so on. On the other hand, various methods of isolation of spermatids from testis are available. Most commonly used is the mechanical method in which a small piece of testicular tissue is torn apart using needles and the spermatids are released. While enzymatic digestion of human testicular tissue can result in a mixture of cell types including a whole range of spermatogenic cell types. Although with these methods, spermatids can be isolated manually under the microscope for immediate use, it is notpossible to obtain large populations of purified spermatids. But repeated biopsies for retrieving spermatids can further compromise the already deficient seminiferous tubules of patients. Therefore there is a need to develop a method by which large and purified populations of spermatids can be isolated, not only for the immediate requirement but also for cryoproservation for subsequent treatment cycles.In the first trial, combination of enzymatic digestion was used to prepare suspensions of spermatogenic cells from adult mouse testis, and then a modified discontinuous Percoll gradient centrifugation method (15%,22%,30%,40%,50%,60%) was introduced to isolate spermatids from the cellular suspensions. The content of spermatids in each isolated fraction by Percoll method was determined by morphology (Wright-Giemsa stain) and flow cytometry analysis, and the viability of spermatogenic cells was assessed by using eosin Y exclusion test. As a result, more than 97% of the testicular cells remained their viability after enzymatic digestion. Of the six fractions resulted from Percoll centrifugation, the 22% fraction contained mostly spermatids (mean 86.7%, P<0.05) and cell viability was more than 85.5%. While in the 30% fraction, immature spermatogenic cells were present, and more than 92% of the cells remained their viability. So a lagre populations of relatively purified spermatids can be isolated from mouse testis by enzymatic digestion combined with discontinuous Percoll gradient centrifugation method.Whereafter a total of fifteen mouse oocytes were injected with round spermatids and seventeen with elongated spermatids. By three days after injection, the number of embryos which had cleaved to the 2-cell stage was 0 with round spermatids and mree(17.6%) with elongated spermatids.In the second trial, this modified discontinuous Percoll gradient centrifugation method was introduced to isolate spermatids from the semen of fifteen male infertile patients. Then the effect was identified by Wright-Giemsa stain, flow cytometry analysis, immunocytochemistry and fluorescence in situ hybridization (FISH). Similary, the 22% Percoll fraction contained mostly haploid cells [(91.85 ? 5.18)%](P<0.005) and the mean density in this fraction was (1.010 ? 0.786) x 105/ml.
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