人類胎兒睪丸組織異種移植模型的建立
發(fā)布時間：2019-06-01 17:35
【摘要】：目的：性別發(fā)育異常(Disorders of Sex Development, DSD)是指染色體性別與性腺性別或包括內(nèi)外生殖器在內(nèi)的表型性別的不一致,其中,男性性別分化發(fā)育幾乎完全依賴胚胎睪丸的激素分泌。睪丸源性生殖障礙綜合征(Testicular Dysgenesis Syndrome, TDS)是包含兒童期隱睪、尿道下裂和成年期睪丸腫瘤、精子數(shù)量不足等四種臨床極為常見疾病在內(nèi)的一種綜合癥。近幾十年來DSD和TDS發(fā)病率在不斷上升。目前觀點認(rèn)為這些異常源于胎兒時期睪丸受到環(huán)境內(nèi)分泌干擾因子干擾致未正常分化發(fā)育,但絕大多數(shù)研究證據(jù)來源于動物實驗。建立去勢裸鼠為受體的異種人類睪丸組織移植模型,將為探索人類DSD和TDS的發(fā)病機(jī)制提供證據(jù)等級更高的研究載體。 方法：切取10-13孕周非因疾病因素人工引產(chǎn)胎兒的性腺組織(n=12個),剪成約1mm3組織塊,移植入雄性去勢裸鼠背部皮下,移植9周左右移植物回收,測量移植物大小重量。收集21孕周的人工引產(chǎn)胎兒睪丸組織作為同齡對照標(biāo)本(n=1)。通過對照睪丸組織移植前后標(biāo)本,及同齡對照標(biāo)本的組織細(xì)胞形態(tài)學(xué)改變和免疫組織化學(xué)表現(xiàn),來觀察人類早期胎兒睪丸組織在異種異位的生長發(fā)育情況。 結(jié)果：移植時胎兒性腺組織標(biāo)本大小約1mm3、重約5mg,移植后取出的移植物最大重量為84.1mg。12例未成熟性腺組織移植后觀察到有11例在受體中繼續(xù)生長發(fā)育。其中7例為卵巢組織,4例為睪丸組織。共移植12個睪丸組織塊,回收9個組織塊,胚胎睪丸移植物的回收率為9/12(75%)。光鏡下移植物標(biāo)本顯示出正常的大體形態(tài),可辨認(rèn)出含有Sertoli細(xì)胞和生殖細(xì)胞的生精小管,由含有Leydig細(xì)胞的間質(zhì)細(xì)胞包繞。小管中無規(guī)則分布的原始生殖細(xì)胞和原始Sertoli細(xì)胞開始向基底膜部位遷移,其中部分原始生殖細(xì)胞已定位于基底膜處,并具有精原細(xì)胞的特征；Sertoli細(xì)胞也由幼稚狀態(tài)發(fā)育為具有豐富胞質(zhì)的成熟型細(xì)胞,有序排列在精原細(xì)胞周圍,橢圓形的細(xì)胞核較整齊地排列于近基底膜部位,核的長軸垂直于基底膜,其幕布樣胞質(zhì)將精原細(xì)胞包圍,形成壁龕樣結(jié)構(gòu)。 免疫組化顯示,移植時標(biāo)本中生精小管未完全形成,移植后生精小管形成并顯示出正常的形態(tài)。移植時Sertoli細(xì)胞(Vim染色顯示)已經(jīng)聚集,但未形成明顯的生殖細(xì)胞索；許多生殖細(xì)胞(OCT4染色顯示)開始聚集,多表達(dá)細(xì)胞全能性標(biāo)志物OCT4；未檢測到雄激素受體(AR)、抗苗勒管激素(AMH)、SMA表達(dá)。移植后,生殖細(xì)胞位于生精小管內(nèi),一些生殖細(xì)胞停止表達(dá)OCT4,被Sertoli細(xì)胞包繞,此時,生精小管外形成一層平滑肌樣細(xì)胞,為管周肌樣細(xì)胞(SMA染色顯示),此細(xì)胞在移植前對照標(biāo)本中不存在,同時可測到AR、AMH表達(dá),且與同齡對照標(biāo)本相似。移植時、移植后及同齡對照表本表達(dá)細(xì)胞增殖標(biāo)記物ki-67,移植后標(biāo)本Leydig細(xì)胞增殖較移植前標(biāo)本活躍。 結(jié)論：人類早期人工引產(chǎn)胎兒睪丸組織移植到免疫缺陷小鼠體內(nèi)后,睪丸組織可繼續(xù)正常生長發(fā)育,因此可作為研究人類性別分化發(fā)育和、DSD及TDS的發(fā)病機(jī)制的較好模型。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R-332
本文編號：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R-332
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 于潔,葉靜,張芳婷,萬匯涓,房家智,王玉潔,宗書東,蔡志明;胎兒睪丸組織異種移植后生精細(xì)胞發(fā)育初探[J];中華男科學(xué)雜志;2004年12期
，本文編號：2490455
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