人类胎儿睾丸组织异种移植模型的建立
发布时间：2019-06-01 17:35
【摘要】：目的：性别发育异常(Disorders of Sex Development, DSD)是指染色体性别与性腺性别或包括内外生殖器在内的表型性别的不一致,其中,男性性别分化发育几乎完全依赖胚胎睾丸的激素分泌。睾丸源性生殖障碍综合征(Testicular Dysgenesis Syndrome, TDS)是包含儿童期隐睾、尿道下裂和成年期睾丸肿瘤、精子数量不足等四种临床极为常见疾病在内的一种综合症。近几十年来DSD和TDS发病率在不断上升。目前观点认为这些异常源于胎儿时期睾丸受到环境内分泌干扰因子干扰致未正常分化发育,但绝大多数研究证据来源于动物实验。建立去势裸鼠为受体的异种人类睾丸组织移植模型,将为探索人类DSD和TDS的发病机制提供证据等级更高的研究载体。 方法：切取10-13孕周非因疾病因素人工引产胎儿的性腺组织(n=12个),剪成约1mm3组织块,移植入雄性去势裸鼠背部皮下,移植9周左右移植物回收,测量移植物大小重量。收集21孕周的人工引产胎儿睾丸组织作为同龄对照标本(n=1)。通过对照睾丸组织移植前后标本,及同龄对照标本的组织细胞形态学改变和免疫组织化学表现,来观察人类早期胎儿睾丸组织在异种异位的生长发育情况。 结果：移植时胎儿性腺组织标本大小约1mm3、重约5mg,移植后取出的移植物最大重量为84.1mg。12例未成熟性腺组织移植后观察到有11例在受体中继续生长发育。其中7例为卵巢组织,4例为睾丸组织。共移植12个睾丸组织块,回收9个组织块,胚胎睾丸移植物的回收率为9/12(75%)。光镜下移植物标本显示出正常的大体形态,可辨认出含有Sertoli细胞和生殖细胞的生精小管,由含有Leydig细胞的间质细胞包绕。小管中无规则分布的原始生殖细胞和原始Sertoli细胞开始向基底膜部位迁移,其中部分原始生殖细胞已定位于基底膜处,并具有精原细胞的特征；Sertoli细胞也由幼稚状态发育为具有丰富胞质的成熟型细胞,有序排列在精原细胞周围,椭圆形的细胞核较整齐地排列于近基底膜部位,核的长轴垂直于基底膜,其幕布样胞质将精原细胞包围,形成壁龛样结构。 免疫组化显示,移植时标本中生精小管未完全形成,移植后生精小管形成并显示出正常的形态。移植时Sertoli细胞(Vim染色显示)已经聚集,但未形成明显的生殖细胞索；许多生殖细胞(OCT4染色显示)开始聚集,多表达细胞全能性标志物OCT4；未检测到雄激素受体(AR)、抗苗勒管激素(AMH)、SMA表达。移植后,生殖细胞位于生精小管内,一些生殖细胞停止表达OCT4,被Sertoli细胞包绕,此时,生精小管外形成一层平滑肌样细胞,为管周肌样细胞(SMA染色显示),此细胞在移植前对照标本中不存在,同时可测到AR、AMH表达,且与同龄对照标本相似。移植时、移植后及同龄对照表本表达细胞增殖标记物ki-67,移植后标本Leydig细胞增殖较移植前标本活跃。 结论：人类早期人工引产胎儿睾丸组织移植到免疫缺陷小鼠体内后,睾丸组织可继续正常生长发育,因此可作为研究人类性别分化发育和、DSD及TDS的发病机制的较好模型。
[Abstract]:Objective: The sex development (DSD) refers to the non-conformity of the sex of the sex with the sex of the gonad or the sex of the sex, including the internal and external genitals, among which, the development of male sex differentiation is almost entirely dependent on the hormone secretion of the testis. Testicular Dysgenesis Syndrome (TDS) is a syndrome that includes four types of clinically significant diseases such as childhood cryptorchidism, hypospadias, and adult testicular tumors. The incidence of DSD and TDS has been increasing in recent decades. The present point of view is that these abnormalities stem from the normal differentiation of the testis of the fetus during the fetal period, but most of the evidence is derived from animal experiments. In order to study the mechanism of human DSD and TDS, we will provide a higher level of research carrier to explore the pathogenesis of human DSD and TDS. Methods: The gonad tissue (n = 12) of the fetus with non-induced labor induced by the factors of 10-13 weeks was cut and the tissue mass of about 1 mm3 was cut. The graft was transplanted into the back of the male castrated nude mice, and the graft was recovered at the left and right after the transplantation for 9 weeks. The graft size was measured. Amount. The artificial induced labor induced by 21 weeks of pregnancy was used as the control specimen of the same age (n = 1). Methods: The growth and development of the testicular tissue of the fetus in the early stage of the human fetus were observed by the change of the cell morphology and the immunohistochemical expression of the specimen before and after the transplantation of the control testis tissue and the control specimens of the same age. Results: At the time of transplantation, the size of the fetal gonad tissue was about 1 mm3, the weight was about 5 mg, and the maximum weight of the graft was 84.1 mg. Long-developed. Of these,7 were ovarian, and 4 were clonorchiasis. Pills. A total of 12 testicular tissue blocks were transplanted,9 tissue blocks were recovered, and the recovery of the embryonic testis grafts was 9/12 (7 5%). The specimen of the graft under the light microscope showed a normal general form, which can identify the seminiferous tubules containing Sertoli cells and germ cells, which are made from the stroma containing Leydig cells. The non-regular distribution of the original germ cells and the original Sertoli cells in the small tube begin to migrate to the basement membrane site, where some of the original germ cells have been positioned at the basement membrane and have the characteristics of spermatogonial cells; the Sertoli cells are also developed from a naive state to a mature cytoplasmic maturation The nucleus of the ellipse is arranged close to the basement membrane, the long axis of the nucleus is perpendicular to the basement membrane, the cytoplasm of the nucleus of the nucleus is surrounded by the spermatogonia, and the niches are formed. The samples were not completely formed during the transplantation, and the seminiferous tubules were formed and displayed after the transplantation. Normal form. Sertoli cells (Vim staining showed) had been gathered at the time of the transplantation, but no significant germ cells were formed; many germ cells (OCT4 staining showed) began to gather, and the pluripotent marker OCT4 was expressed; no androgen receptor (AR), anti-mullerian hormone (AMH) was detected, SMA was expressed. After the transplantation, the germ cells were located in the spermatogenic cells, and some germ cells stopped expressing OCT4 and were surrounded by Sertoli cells. At this time, a layer of smooth muscle-like cells were formed outside the seminiferous tubules. The present invention does not exist, and the expression of AR and AMH can be detected at the same time, The cell proliferation marker ki-67 was expressed in the post-transplant and peer-to-peer look-up table, and the proliferation of Leydig cells was better after the transplantation. Conclusion: The tissue of the testis can continue to grow and grow normally after the human fetal testis tissue is transplanted into the immune deficient mice, so it can be used as the study on the development of human sex differentiation and the development of DSD and TDS.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332
本文编号：2490455
[Abstract]:Objective: The sex development (DSD) refers to the non-conformity of the sex of the sex with the sex of the gonad or the sex of the sex, including the internal and external genitals, among which, the development of male sex differentiation is almost entirely dependent on the hormone secretion of the testis. Testicular Dysgenesis Syndrome (TDS) is a syndrome that includes four types of clinically significant diseases such as childhood cryptorchidism, hypospadias, and adult testicular tumors. The incidence of DSD and TDS has been increasing in recent decades. The present point of view is that these abnormalities stem from the normal differentiation of the testis of the fetus during the fetal period, but most of the evidence is derived from animal experiments. In order to study the mechanism of human DSD and TDS, we will provide a higher level of research carrier to explore the pathogenesis of human DSD and TDS. Methods: The gonad tissue (n = 12) of the fetus with non-induced labor induced by the factors of 10-13 weeks was cut and the tissue mass of about 1 mm3 was cut. The graft was transplanted into the back of the male castrated nude mice, and the graft was recovered at the left and right after the transplantation for 9 weeks. The graft size was measured. Amount. The artificial induced labor induced by 21 weeks of pregnancy was used as the control specimen of the same age (n = 1). Methods: The growth and development of the testicular tissue of the fetus in the early stage of the human fetus were observed by the change of the cell morphology and the immunohistochemical expression of the specimen before and after the transplantation of the control testis tissue and the control specimens of the same age. Results: At the time of transplantation, the size of the fetal gonad tissue was about 1 mm3, the weight was about 5 mg, and the maximum weight of the graft was 84.1 mg. Long-developed. Of these,7 were ovarian, and 4 were clonorchiasis. Pills. A total of 12 testicular tissue blocks were transplanted,9 tissue blocks were recovered, and the recovery of the embryonic testis grafts was 9/12 (7 5%). The specimen of the graft under the light microscope showed a normal general form, which can identify the seminiferous tubules containing Sertoli cells and germ cells, which are made from the stroma containing Leydig cells. The non-regular distribution of the original germ cells and the original Sertoli cells in the small tube begin to migrate to the basement membrane site, where some of the original germ cells have been positioned at the basement membrane and have the characteristics of spermatogonial cells; the Sertoli cells are also developed from a naive state to a mature cytoplasmic maturation The nucleus of the ellipse is arranged close to the basement membrane, the long axis of the nucleus is perpendicular to the basement membrane, the cytoplasm of the nucleus of the nucleus is surrounded by the spermatogonia, and the niches are formed. The samples were not completely formed during the transplantation, and the seminiferous tubules were formed and displayed after the transplantation. Normal form. Sertoli cells (Vim staining showed) had been gathered at the time of the transplantation, but no significant germ cells were formed; many germ cells (OCT4 staining showed) began to gather, and the pluripotent marker OCT4 was expressed; no androgen receptor (AR), anti-mullerian hormone (AMH) was detected, SMA was expressed. After the transplantation, the germ cells were located in the spermatogenic cells, and some germ cells stopped expressing OCT4 and were surrounded by Sertoli cells. At this time, a layer of smooth muscle-like cells were formed outside the seminiferous tubules. The present invention does not exist, and the expression of AR and AMH can be detected at the same time, The cell proliferation marker ki-67 was expressed in the post-transplant and peer-to-peer look-up table, and the proliferation of Leydig cells was better after the transplantation. Conclusion: The tissue of the testis can continue to grow and grow normally after the human fetal testis tissue is transplanted into the immune deficient mice, so it can be used as the study on the development of human sex differentiation and the development of DSD and TDS.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332
【参考文献】
相关期刊论文 前1条
1 于洁,叶静,张芳婷,万汇涓,房家智,王玉洁,宗书东,蔡志明;胎儿睾丸组织异种移植后生精细胞发育初探[J];中华男科学杂志;2004年12期
，本文编号：2490455
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