helq对生殖细胞分化影响的研究细胞生物学专业论文.docxVIP

AB AB STRACT According to the World Health Organization(WHO)assessment， the proportion of global infertility has accounted for 1 5％in the couples of child—bearing age．And the patients with male infertility have occupied about half of the total of human infertility patients．Therefore， establishing in vitro reproductive disease differentiation model is necessary to study the male infertility mechanism by using of stem cells combinationcombination withwith gene editingediting techniqtectmlques ．ItIt alsoalSO willW111 De 1nave very good application prospect． HELQ，named POLQ．1ike helicase，a DNA—dependent ATPase and DNA helicase，plays an important role in the process of repak DNA double．strand breaks(DSB)and inter-strand crosslink(ICL)．HELQ directly intact with RAD5 1 homologue RAD5 1 B／C／D，XRCC2 and the kinase ATR corresponding effect on DNA damage．Some researchers suggests the defect of HELQ result in testicular atrophy in male mice of seminiferous tubules structure，and the、number of spermatogonial stem cells reduced obviously，it appeared like the setroli cell only syndronie (scos)phenotype．So we supposed that I-mLQ may work in gonocyte cells stage or primordial germ cellS stage· In our experiment，we have obtained the knockout of HELQ gene in mice embryonic stem cell which Can stably growth and passage by using of CRISPR-cas9 gene editing techniques．Immunofluorescence staining showed that after knock out HZLQ，the expression of OCT4 and Nanog III 万方数据 have have no significant difference as compared with the control group Meanwhile，tetraploid compensation results also showed that the pluripotency of embryonic stem cells did not affected，and we have get the health mouse tetraploid offspring．At the same time，we use the HELQ knockout embryos stem cells in specific culture conditions to differentiate in vitro and eventually acquired the Day2 epiblast like cells． 一 。mmunofllmmunotlurescence — andand thethe real．timereal PCR staminz time PCR analysis．the results illustrated