CPED1基因在鸡胚胎干细胞向精原干细胞分化过程中的作用

发布时间：2018-04-25 21:47

  本文选题：CPED1 + 鸡　； 参考：《扬州大学》2017年硕士论文

【摘要】：精原干细胞(spermatogonial stem cells,SSCs)是动物体内重要的成体干细胞,能够将遗传物质传递给下一代,它既有胚胎干细胞的发育特性又能发育成单倍体生殖细胞。现今有大量研究表明精原干细胞可在体外被诱导为具有不同功能的细胞系,用于遗传修饰、疾病治疗和转基因动物制备等,因此对精原干细胞体外操作的研究尤为重要。其中将SSCs诱导形成精子一直是科学家们研究的热门话题,但如何大量获得SSCs并使其产生具有功能性的精子就成为科学家们亟需解决的科学问题。通过本课题组前期已完成的鸡雄性ESCs,雄性PGCs以及SSCs的转录组测序(RNA-Seq)的实验结果,分析得到了鸡雄性生殖细胞发生过程中基因动态表达的水平变化,有助于对该过程中重要作用的未知基因的发现。本实验研究的目的基因CPED1在SSCs的转录组测序中特异高表达并功能未知,因此有可能对SSCs分化具有重要作用,因此本实验同时利用基因过表达技术和CRISPR/Cas9技术实现对测序过程中发现的精原干细胞高表达基因CPED1功能的研究,为阐明生殖细胞发生及分化机制提供高效方法。本研究的主要内容如下：(1)基于本实验室前期RNA-Seq技术发现CPED1在ESCs向SSCs分化过程中存在显著差异表达。通过qPCR克隆如皋黄鸡CPED1编码序列,同时构建过表达载体pcDNA3.0-CPED1和敲除载体Cas9/gRNA。通过Fugene转染Cas9/gRNA载体,利用T7E1酶切、SSA活性检测、TA克隆测序和脱靶效率检测Cas9/gRNA载体敲除活性及脱靶情况。在DF-1细胞中通过T7E1酶切检测Cas9/gRNA载体的活性,酶切结果表明,通过条带灰度值估计Cas9/gRNA1载体、Cas9/gRNA2载体和Cas9/gR/A3载体活性分别为377、20%和30%%,Ca9/gR/A1载体敲除活性最佳;SSA活性检测结果表明gRNA1的荧光活性值与对照组相比增加了 2倍左右,活性最佳;TA克隆测序结果表明测序的8菌液样中有2管菌液样发生了突变,初步估计基因敲除率为25%左右;脱靶效率检测结果表明在DF-1细胞中无脱靶现象。同时实现了在ESCs中CPED1的定点敲除,敲除效率为25%。这一结果表明CRISPR/Cas9技术能稳定的在鸡DF-1细胞和ESCs上实现CPED1敲除。(2)分别将Cas9/gRNA1载体和pcDNA3.0-CPED1过表达载体通过Fugene转染ESCs,转染48小时候换RA诱导培养基培养12d。采用细胞形态学观察、间接免疫荧光检测、流式细胞分析和qRT-PCR等方法检测CPED1于敲除与过表达对ESCs向SSCs分化的影响,结果表明敲除CPED1的ESCs与正常RA诱导组相比,类胚体形成时间延长且停止向雄性生殖细胞分化;第12dRA诱导组与过表达组均能形成类SSCs,敲除组则无类SSCs形成。间接免疫荧光检测结果表明敲除CPED1导致体外诱导10d的ESCs分化生成的integrin α6和integrin β1双阳性类SSCs数量显著低于过表达组和RA诱导组。流式细胞分析结果表明RA诱导组、过表达组和敲除组中integrin α6阳性类SSCs数量比例分别为1.5%±0.163、1.8%%±0.294和0.9%±0.216,敲除组中阳性细胞率明显低于其它两组。12d qRT-PCR结果表明RA诱导组中目的基因CPED1和生殖相关基因Cvh、C-kit、Stra8、integrin α6和integrin β1的最高表达量分别为 1.72,1.62,1.74,2.01,2.36,2.42;过表达组中目的基因CPED1和生殖相关基因Cvh、C-kit、Strt8、integrinα6和integrin β1的最高表达量分别为2.68,1.71,1.95,2.46,2.67,2.78;敲除组中目的基因CPED1和生殖相关基因Cvh、C-kit、Stra8、integrin α6和integrin β1的最高表达量分别为 0.42,1.12,1.03,0.87,1.41,1.53;RA诱导组和过表达组中CPED1的表达量具有显著差异,敲除组中CPED1以及生殖相关基因Cvh、C-kit、、Stra8、integrin α6和integrin β1的表达量与RA诱导组和过表达组相比呈显著下调。在RA诱导ESCs向SSCs分化过程中,敲除CPED1抑制类SSCs形成,证明CPED1在调控家禽ESCs向SSCs分化过程中起重要作用。(3)取新鲜的受精鸡胚,利用PEI包裹Cas9/gRNA1载体和pcDNA3.0-CPED1过表达载体分别进行鸡胚注射,并设立正常孵化组与对照组。分别收集正常孵化至4.5d鸡胚生殖嵴样和18.5d睾丸样,采用石蜡切片及PAS染色、qRT-PCR和流式细胞分析等方法检测CPED1敲除和过表达对鸡雄性生殖细胞体内动态变化的影响。qRT-PCR检测结果显示4.5d的过表达组中生殖相关基因Cvh、Stra8和integrin α6的表达量分别为2.12,1.37,1.20,显著高于敲除组,CPED1表达量(1.07)极显著高于敲除组;18d的过表达组中的Nanog的表达量(0.32)显著低于对照组,integrin α6基因表达量(1.89)显著高于敲除组,CPED1表达量(2.09)极显著高于敲除组。4.5d鸡胚石蜡切片结果表明对照组中PGCs数量为(45±2.236)个,过表达组中PGCs数量为(41±1.699)个,敲除组中PGCs数量为(18±0.745)个,敲除组中含有的PGCs数量低于对照组和过表达组。同时对18d睾丸分离培养的SSCs进行流式细胞分析检测,结果表明对照组、过表达组和敲除组中integrin α6阳性细胞率分别为1.3%±0.141、1.6%±0.356和0.8%±0.245,敲除组中SSCs数量显著减少,对照组和过表达组中却无显著差异。结果证明CPED1对鸡胚干细胞向精原干细胞分化具有促进作用。
[Abstract]:Spermatogonial stem cells (SSCs) is an important adult stem cell in the animal. It can transmit genetic material to the next generation. It has both the developmental characteristics of embryonic stem cells and the development of haploid germ cells. There is a lot of research now that spermatogonial stem cells can be induced in vitro into different functional cell lines. The research on the operation of spermatogonial stem cells in vitro is particularly important for genetic modification, disease treatment and the preparation of genetically modified animals. It has been a hot topic for scientists to induce the formation of spermatozoa by SSCs, but how to obtain SSCs and make it produce functional sperm has become a science that scientists need to solve. Problems. Through the experimental results of the male ESCs, male PGCs and SSCs transcriptional sequence (RNA-Seq), the changes of gene dynamic expression during the development of the male reproductive cells of the chicken were analyzed, which was helpful to the discovery of the unknown genes that played an important role in the process. The target gene of this experiment was CPE. D1 is highly expressed in SSCs transcriptional sequence and has unknown function. Therefore, it may play an important role in the differentiation of SSCs. Therefore, this experiment also uses gene overexpression technology and CRISPR/Cas9 technology to study the function of high expression gene CPED1 of spermatogonial stem cells found in the sequencing process, in order to clarify the pathogenesis and differentiation machine of germ cells. The main contents of this study are as follows: (1) there is a significant difference in the expression of CPED1 during the differentiation of ESCs to SSCs in the pre laboratory RNA-Seq technology. The CPED1 coding sequence of Rugao yellow chicken was cloned by qPCR, and the expression vector pcDNA3.0-CPED1 and the knockout carrier Cas9/gRNA. were constructed by Fugene transfection Cas9/gRNA. Vector, using T7E1 enzyme cutting, SSA activity detection, TA cloning sequencing and miss efficiency detection of Cas9/gRNA vector knockout activity and miss target. The activity of Cas9/gRNA vector was detected by T7E1 enzyme digestion in DF-1 cells. The results showed that Cas9/gRNA1 vector was estimated by the gray value of strip and the activity of Cas9/gRNA2 carrier and Cas9/gR/A3 carrier was 377,2. 0% and 30%%, Ca9/gR/A1 carrier knockout activity was the best, SSA activity detection results showed that the fluorescence activity of gRNA1 increased about 2 times compared with the control group, and the activity was the best. The result of TA cloning sequencing results showed that there was a mutation of 2 tube samples in the sequence of 8 bacteria samples, and the initial estimated gene knockout rate was about 25%. There is no dislocation in DF-1 cells. At the same time, the targeted knockout of CPED1 in ESCs and the knockout efficiency of 25%. show that CRISPR/Cas9 technology can stabilize the CPED1 knockout on chicken DF-1 cells and ESCs. (2) the Cas9/gRNA1 carrier and pcDNA3.0-CPED1 overexpression vector pass through Fugene transfection ESCs, and the transfection 48 When transfection is induced to induce culture. Cell morphology observation, indirect immunofluorescence detection, flow cytometry and qRT-PCR were used to detect the effect of CPED1 on the differentiation of ESCs to SSCs by CPED1. The results showed that the ESCs of knockout CPED1 was longer than normal RA induction group and stopped to the male reproductive cells. 12dRA Both the induced group and the overexpressed group could form a class of SSCs, and the knockout group had no SSCs like formation. The results of indirect immunofluorescence showed that the number of integrin alpha 6 and integrin beta 1 positive class SSCs generated by the knockout of CPED1 in vitro induced the differentiation of 10d was significantly lower than that of the overexpressed and RA induced groups. The proportion of integrin alpha 6 positive SSCs in the group and the knockout group were 1.5% + 0.163,1.8%% + 0.294 and 0.9% + 0.216 respectively. The positive cell rate in the knockout group was significantly lower than the other two groups of.12d qRT-PCR results showed that the target gene CPED1 and the reproductive related gene Cvh, C-kit, Stra8, integrin alpha 6 and integrin beta 1 in the RA induction group were the highest levels of expression. The highest expression of the target gene CPED1 and reproductive related genes Cvh, C-kit, Strt8, integrin alpha 6 and integrin beta 1 in the overexpressed group was 2.68,1.71,1.95,2.46,2.67,2.78, and the highest expression of the target gene CPED1 and reproductive related genes Cvh, C-kit, Stra8, alpha 6 and C-kit beta 1 in the knockout group The amount of CPED1 expression in RA induced and overexpressed groups was significantly different. The expression of CPED1 and reproductive related genes Cvh, C-kit, Stra8, integrin alpha 6 and integrin beta 1 in the knockout group were significantly down regulated by RA induced and overexpressed groups. In addition to the formation of CPED1 inhibitory SSCs, CPED1 plays an important role in regulating the differentiation of poultry ESCs into SSCs. (3) take fresh fertilized chicken embryos, use PEI to encapsulate Cas9/gRNA1 vector and pcDNA3.0-CPED1 overexpression vector for chicken embryo injection respectively, and set up normal hatching group and control group, and collect normal hatching to 4.5d chicken embryo reproductive crest samples, respectively. The effects of CPED1 knockout and over expression on the dynamic changes of the male reproductive cells in the chickens were detected by paraffin section and PAS staining, qRT-PCR and flow cytometry. The results of.QRT-PCR detection showed that the expression of reproductive related genes Cvh, Stra8 and integrin 6 were 2.12,1.37,1.20, respectively, in the overexpressed group of 4.5d. The results showed that the expression of Cvh, Stra8 and integrin 6 were 2.12,1.37,1.20, respectively. The expression of CPED1 (1.07) was significantly higher than that in knockout group, and the expression of Nanog in the overexpression group of 18D was significantly lower than that of the control group. The expression of integrin alpha 6 gene (1.89) was significantly higher than that in the knockout group, and the CPED1 expression (2.09) was significantly higher than that of the knockout group.4.5d chicken embryo paraffin section, indicating that the number of PGCs in the control group was (45 + 2.236). The number of PGCs in the overexpression group was (41 + 1.699), and the number of PGCs in the knockout group was (18 + 0.745). The number of PGCs contained in the knockout group was lower than that of the control group and the overexpressed group. At the same time, the SSCs of the 18D testis isolated and cultured was detected by flow cytometry. The results showed that the ratio of integrin alpha 6 positive cells in the over expression group and the knockout group was in the control group. It was 1.3% + 0.141,1.6% + 0.356 and 0.8% + 0.245. The number of SSCs decreased significantly in the knockout group, but there was no significant difference between the control group and the overexpressed group. The results showed that CPED1 could promote the differentiation of chick embryonic stem cells to spermatogonial stem cells.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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