诱导原始生殖细胞和肾间充质干细胞减数分裂研究

发布时间：2018-03-23 18:23

本文选题：鸡原始生殖细胞　切入点：羊肾间充质干细胞　出处：《中国农业科学院》2016年硕士论文

【摘要】：本研究通过分离孵化5.5 d的鸡胚胎性腺中的原始生殖细胞(Primordial Germ Cells,PGCs),建立体外培养体系,研究PGCs生物学特性及定向诱导精子发生,探讨鸡PGCs体外发育潜能;分离培养6周龄胎羊肾间充质干细胞(Mesenchymal Stem Cells,MSCs),研究其生物学特性,并在体外诱导其减数分裂,为体外诱导干细胞精子发生提供理论依据。研究表明:1分离鸡胚性腺组织,经0.125%胰酶消化,以添加了10%胎牛血清、2%鸡血清、1 mM丙酮酸钠、2 mM L-谷氨酰胺、55μMβ巯基乙醇、10 ng/mL hSCF、10 units/mL LIF、20 ng/mL bFGF和10 ng/mL IGF的H-DMEM培养液进行原代培养。培养24 h后可见部分PGCs粘附在性腺原基细胞上生长,48 h后PGCs克隆团明显增大增多,3 d后接种到经10μg/mL丝裂霉素C处理过的鸡成纤维细胞饲养层上培养。通过对比原代PGCs与性腺原基细胞共培养和在饲养层细胞上培养两种方法,结果发现,原代PGCs与共同来源的性腺原基细胞共培养能显著提高PGCs克隆团形成率。2本研究的培养体系中,PGCs可培养至第6代,其形态学上比周围细胞大且胞质饱满,折光率高;经PAS和AKP染色鉴定为阳性;RT-PCR鉴定克隆团细胞表达CVH、BLIMP1、POUV和NANOG基因;免疫荧光鉴定克隆团细胞表达SSEA-1、SSEA-3、BLIMP1、OCT4和SOX2。3 PGCs经1μM维甲酸(Retinoic acid,RA)诱导24 h后可见精子样细胞出现,RT-PCR鉴定诱导后的细胞表达减数分裂和单倍体细胞特异基因SYCP1、BOULE、DAZL、STRA8、DMC1和ACR。结合干细胞因子共同诱导PGCs精子发生,可显著提高SYCP1、BOULE和DMC1基因的表达量,提高单倍体细胞的诱导率。4分离肾组织,经0.25%胰酶消化后,以添加10%胎牛血清的DMEM/F12进行原代培养。24 h后换液去除未贴壁的细胞。细胞生长至80%覆盖皿底进行传代,传3~4代后细胞可达到较高纯度,经流式分析细胞纯度达到95%以上。经RT-PCR和免疫荧光鉴定细胞表达OCT4、CD44、VIM、PAX2和FN1。生长曲线分析MSCs的生长经历潜伏期、对数生长期、稳定期和衰退期,呈典型的S形。克隆形成能力结果显示P4代细胞克隆形成率为56.33%±2.52%,P10代为34.33%±3.06%,P16代为19.67%±2.08%。核型分析结果表明体外培养的羊肾MSCs染色体数目为2n=54条,95%以上的细胞核型均正常。5在体外对MSCs展开了多向分化潜能的研究,在相应的诱导条件下,成功将MSCs诱导成脂肪样细胞、肝样细胞和软骨样细胞,经特异性染色和RT-PCR鉴定诱导形成了目的细胞。6 RA与睾丸提取液都能诱导MSCs进入减数分裂阶段,通过RT-PCR检测细胞表达DAZL、PRM1、TNP1、TNP2、DDX4、MLH1和SCYP3,免疫荧光鉴定细胞表达减数分裂和单倍体细胞特异标记物DAZL、C-KIT、CTDSPL和ACR。睾丸提取液结合RA共同诱导能够显著提升单倍体细胞的诱导效率。
[Abstract]:In this study, primordial Germ cells of primordial Germ cells were isolated from the gonads of chicken embryos hatched for 5.5 days. The in vitro culture system was established to study the biological characteristics of PGCs and induce spermatogenesis, and to explore the developmental potential of PGCs in vitro. Mesenchymal Stem cells (MSCs) were isolated from fetal sheep kidney mesenchymal stem cells at 6 weeks of age, their biological characteristics were studied, and the meiosis was induced in vitro, which provided a theoretical basis for inducing stem cell spermatogenesis in vitro. Digested by 0.125% trypsin, The primary culture was carried out in H-DMEM medium supplemented with 10% fetal bovine serum and 2% chicken serum 1 mm sodium pyruvate 2 mm L- Glutamide 55 渭 M 尾 -mercaptoethanol for 10 ng/mL hSCFN 10 units/mL LIF-20 ng/mL bFGF and 10 ng/mL IGF. After 24 h culture, some PGCs adherent to the progonadal gland was observed. After 48 h of growth on the basal cells, the PGCs clones were obviously enlarged and increased, and then inoculated into the feeder layer of chicken fibroblasts treated with 10 渭 g/mL mitomycin C for 3 days. By comparing the primary PGCs and gonadal primordial cells co-culture and feeding in the culture. There are two ways to culture a layer of cells, The results showed that the co-culture of the primary PGCs and the co-derived gonadal primordium cells could significantly increase the colony formation rate of PGCs clone. 2. In this culture system, the PGCs clones could be cultured to the sixth generation, which was larger in morphology than that in the peripheral cells, and the cytoplasm was plump and the refractive index was higher. PAS and AKP staining were used to identify the expression of CVHN BLIMP1POUV and NANOG genes in the cloned cells by reverse transcription-polymerase chain reaction (RT-PCR). The expression of SSEA-1OCT4 and SOX2.3 PGCs were identified by immunofluorescence. After induction with 1 渭 M retinoic acid (Retinoic acidido RAA) for 24 h, the spermatocyte-like cells were identified to express meiosis and haploid cell specific gene SYCP1BOULEDAZL straz8DMC1 and ACR1, which were identified by reverse transcription-polymerase chain reaction (RT-PCR). PGCs spermatogenesis was induced by cytokines. It could significantly increase the expression of SYCP1 and DMC1 genes, and increase the induction rate of haploid cells. 4. The isolated kidney tissue was digested by 0.25% trypsin. DMEM/F12 supplemented with 10% fetal bovine serum was used in primary culture for 24 h to remove unattached cells. The cells grew to the bottom of 80% covering dish for passage, and the cells reached a high purity after 3 ~ 4 passages. The purity of the cells was over 95% by flow cytometry. The expression of OCT4CD4VIMX _ 2 and FN _ 1 was identified by RT-PCR and immunofluorescence. The growth curve was used to analyze the growth latency, logarithmic growth period, stable phase and decline phase of MSCs. The clone forming rate of P4 passage cells was 56.33% 卤2.52%, 34.33% 卤3.06% 卤3.06% and 19.67% 卤2.08.The karyotype analysis showed that the number of MSCs chromosomes in sheep kidney cultured in vitro was 95% of 2n=54 strips and 95% of them were normal. The multidirectional differentiation potential of MSCs was studied in vitro. Under the corresponding induction conditions, MSCs was successfully induced into adipoid cells, hepatoid cells and chondroid cells. By specific staining and RT-PCR identification, the target cells, 6.RA and testicular extract could induce MSCs to enter the meiosis stage. RT-PCR was used to detect the expression of DAZL1, TNP1, TNP2, DDX4, MLH1 and SCYP3, and immunofluorescence was used to identify the expression of meiosis and haploid cell specific markers DAZLC- KITT CTDSPL and ACR.Testis extract combined with RA could significantly enhance the induction efficiency of haploid cells.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.2

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