本文選題：雞原始生殖細(xì)胞　切入點(diǎn)：羊腎間充質(zhì)干細(xì)胞　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年碩士論文

【摘要】：本研究通過分離孵化5.5 d的雞胚胎性腺中的原始生殖細(xì)胞(Primordial Germ Cells,PGCs),建立體外培養(yǎng)體系,研究PGCs生物學(xué)特性及定向誘導(dǎo)精子發(fā)生,探討雞PGCs體外發(fā)育潛能;分離培養(yǎng)6周齡胎羊腎間充質(zhì)干細(xì)胞(Mesenchymal Stem Cells,MSCs),研究其生物學(xué)特性,并在體外誘導(dǎo)其減數(shù)分裂,為體外誘導(dǎo)干細(xì)胞精子發(fā)生提供理論依據(jù)。研究表明:1分離雞胚性腺組織,經(jīng)0.125%胰酶消化,以添加了10%胎牛血清、2%雞血清、1 mM丙酮酸鈉、2 mM L-谷氨酰胺、55μMβ巰基乙醇、10 ng/mL hSCF、10 units/mL LIF、20 ng/mL bFGF和10 ng/mL IGF的H-DMEM培養(yǎng)液進(jìn)行原代培養(yǎng)。培養(yǎng)24 h后可見部分PGCs粘附在性腺原基細(xì)胞上生長(zhǎng),48 h后PGCs克隆團(tuán)明顯增大增多,3 d后接種到經(jīng)10μg/mL絲裂霉素C處理過的雞成纖維細(xì)胞飼養(yǎng)層上培養(yǎng)。通過對(duì)比原代PGCs與性腺原基細(xì)胞共培養(yǎng)和在飼養(yǎng)層細(xì)胞上培養(yǎng)兩種方法,結(jié)果發(fā)現(xiàn),原代PGCs與共同來源的性腺原基細(xì)胞共培養(yǎng)能顯著提高PGCs克隆團(tuán)形成率。2本研究的培養(yǎng)體系中,PGCs可培養(yǎng)至第6代,其形態(tài)學(xué)上比周圍細(xì)胞大且胞質(zhì)飽滿,折光率高;經(jīng)PAS和AKP染色鑒定為陽性;RT-PCR鑒定克隆團(tuán)細(xì)胞表達(dá)CVH、BLIMP1、POUV和NANOG基因;免疫熒光鑒定克隆團(tuán)細(xì)胞表達(dá)SSEA-1、SSEA-3、BLIMP1、OCT4和SOX2。3 PGCs經(jīng)1μM維甲酸(Retinoic acid,RA)誘導(dǎo)24 h后可見精子樣細(xì)胞出現(xiàn),RT-PCR鑒定誘導(dǎo)后的細(xì)胞表達(dá)減數(shù)分裂和單倍體細(xì)胞特異基因SYCP1、BOULE、DAZL、STRA8、DMC1和ACR。結(jié)合干細(xì)胞因子共同誘導(dǎo)PGCs精子發(fā)生,可顯著提高SYCP1、BOULE和DMC1基因的表達(dá)量,提高單倍體細(xì)胞的誘導(dǎo)率。4分離腎組織,經(jīng)0.25%胰酶消化后,以添加10%胎牛血清的DMEM/F12進(jìn)行原代培養(yǎng)。24 h后換液去除未貼壁的細(xì)胞。細(xì)胞生長(zhǎng)至80%覆蓋皿底進(jìn)行傳代,傳3~4代后細(xì)胞可達(dá)到較高純度,經(jīng)流式分析細(xì)胞純度達(dá)到95%以上。經(jīng)RT-PCR和免疫熒光鑒定細(xì)胞表達(dá)OCT4、CD44、VIM、PAX2和FN1。生長(zhǎng)曲線分析MSCs的生長(zhǎng)經(jīng)歷潛伏期、對(duì)數(shù)生長(zhǎng)期、穩(wěn)定期和衰退期,呈典型的S形�？寺⌒纬赡芰Y(jié)果顯示P4代細(xì)胞克隆形成率為56.33%±2.52%,P10代為34.33%±3.06%,P16代為19.67%±2.08%。核型分析結(jié)果表明體外培養(yǎng)的羊腎MSCs染色體數(shù)目為2n=54條,95%以上的細(xì)胞核型均正常。5在體外對(duì)MSCs展開了多向分化潛能的研究,在相應(yīng)的誘導(dǎo)條件下,成功將MSCs誘導(dǎo)成脂肪樣細(xì)胞、肝樣細(xì)胞和軟骨樣細(xì)胞,經(jīng)特異性染色和RT-PCR鑒定誘導(dǎo)形成了目的細(xì)胞。6 RA與睪丸提取液都能誘導(dǎo)MSCs進(jìn)入減數(shù)分裂階段,通過RT-PCR檢測(cè)細(xì)胞表達(dá)DAZL、PRM1、TNP1、TNP2、DDX4、MLH1和SCYP3,免疫熒光鑒定細(xì)胞表達(dá)減數(shù)分裂和單倍體細(xì)胞特異標(biāo)記物DAZL、C-KIT、CTDSPL和ACR。睪丸提取液結(jié)合RA共同誘導(dǎo)能夠顯著提升單倍體細(xì)胞的誘導(dǎo)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% 鹵2.52%, 34.33% 鹵3.06% 鹵3.06% and 19.67% 鹵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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.2

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