一种简便的机械法分离绵羊睾丸间质细胞的研究

本文旨在探寻一种取代酶消化法分离绵羊睾丸间质细胞的简便新方法。首先将绵羊睾丸被膜剥离,然后将睾丸切块,采用机械震荡的方法使间质细胞自动脱落,然后用差异贴壁法进一步纯化间质细胞。试验结果表明,该方法分离绵羊睾丸间质细胞省时省力,睾丸间质细胞存活率与纯度都较高。     

成年小鼠睾丸间质细胞的分离、鉴定及功能检测

为建立成年小鼠睾丸间质细胞分离纯化方法,并对分离纯化的睾丸间质细胞进行睾酮分泌功能检测,对成年小鼠睾丸组织进行Ⅰ型胶原酶消化、Percoll分离液密度梯度离心,分离成年小鼠睾丸间质细胞.细胞纯度用3β-羟类固醇脱氢酶(3β-hydroxy-steroid-dehydrogenase,3β-HSD)染色鉴定.将分离纯化的睾丸间质细胞进行体外培养,在基础睾酮和人绒毛膜促性腺激素(hCG)刺激培养条件下,用放射免疫分析法对培养上清中的睾酮含量进行检测.结果显示,通过该方法能获得高纯度成年小鼠睾丸间质细胞(＞95％),培养的睾丸间质细胞具有分泌基础睾酮以及对hCG刺激反应的能力.提示采用该方法对成年小鼠睾丸间质细胞进行分离纯化具有高效性、可用性,通过该方法获得的睾丸间质细胞是体外研究药物对睾酮分泌功能影响的良好模型.     

小鼠睾丸间质细胞的分离纯化与体外培养的研究

为了寻求一种快速、高效的体外分离、纯化小鼠睾丸间质细胞的方法,试验采用两种酶(胶原酶Ⅳ和0.25%胰蛋白酶)消化法和6个梯度(70%、65%、60%、55%、50%、45%)的Percoll液对小鼠睾丸间质细胞进行分离、纯化,对分离的细胞进行细胞学观察、台盼蓝染色、3β-羟基类固醇脱氢酶(3β-HSD)染色,采用物理方法、胶原酶Ⅳ+0.25%胰蛋白酶和柠檬酸钠+KCl对睾丸间质细胞进行消化传代培养。结果表明:胶原酶Ⅳ和0.25%胰蛋白酶限时消化能够获得较高活率(90%)的小鼠睾丸间质细胞;经Percoll细胞分离液分离的细胞纯度高达86%,且在34℃、5%CO2条件下培养的细胞生长良好。     

半胱亚磺酸脱羧酶和牛磺酸转运体mRNA在成年小鼠睾丸间质细胞的表达

为检测成年小鼠睾丸间质细胞是否存在牛磺酸生物合成关键酶半胱亚磺酸脱羧酶(CSD)和牛磺酸转运体(TauT)mRNA的表达,通过Percoll分离液分离纯化成年小鼠睾丸间质细胞,运用RT-PCR方法测定睾丸间质细胞中CSD和TauT mRNA表达情况。结果显示,成年小鼠睾丸间质细胞存在CSD和TauT mRNA表达。说明成年小鼠睾丸间质细胞能通过CSD途径合成牛磺酸以及睾丸间质细胞可能存在牛磺酸的转运活动。     

家兔睾丸间质细胞的分离纯化及原代培养

为了研究家兔睾丸间质细胞(Leydigcell,LC)的分离纯化程序及体外生长特点,试验采用4种不同消化程序对家兔睾丸组织进行消化,percoll梯度分离出兔LC,并进行体外培养。结果表明:经4℃保存和34℃孵育,0.5mg/mLⅠ型胶原酶消化40min获得的细胞数最多,经percoll梯度分离的细胞纯度达92%;34℃、5%CO2条件下细胞生长良好,呈现梭形和不规则形2种形状。     

哺乳动物睾丸间质细胞的分离及体外培养

作者综述了哺乳动物睾丸间质细胞体外培养的研究现状、程序及方法并就间质细胞的形态特征、功能、分离和鉴定结合自己的试验体会进行了逐一论述,为建立睾丸间质细胞体外培养体系提供参考。     

亚硒酸钠对绵羊睾丸间质细胞体外增殖和凋亡的影响

本实验旨在研究硒对绵羊睾丸间质细胞体外增殖与凋亡的影响.使用不同浓度的亚硒酸钠(0、2、4、6、8μmol/L)处理绵羊睾丸间质细胞,其中0μmol/L亚硒酸钠为对照组,CCK-8法检测细胞增殖,定量PCR(Quantitative Real-Time PCR,qRT-PCR)检测细胞周期及凋亡相关基因的表达情况,We...     

绵羊精原干细胞的分离纯化与鉴定

旨在探究一种简便高效的绵羊精原干细胞(SSCs)分离纯化方法,为后续研究SSCs奠定基础。本实验采用两步酶消化法对4月龄的绵羊睾丸进行消化处理,获得单细胞悬液。将睾丸细胞接种于0.2%的明胶预处理培养皿中,根据各细胞贴壁能力和速度的不同将SSCs纯化,并在纯化后用SSCs特异标记分子PLZF进行免疫荧光鉴定。结果表明,经免疫荧光染色得出存在PLZF阳性信号。这说明采用两步酶消化和差异贴壁法可以得到纯度较高的SSCs。     

维生素E对绵羊原代睾丸间质细胞睾酮合成的影响

本试验旨在研究维生素E对绵羊原代睾丸间质细胞睾酮合成的影响。选择2月龄杜×寒杂交公羔,屠宰后取睾丸组织。分离绵羊睾丸间质细胞,随机分为4个处理组,每个处理组3皿,培养基内添加不同浓度维生素E(0、40、80、160μg/m L)。培养结束后,测定培养基上清睾酮含量,将细胞裂解测定睾酮合成相关酶3β-羟基类固醇脱氢酶(3β-HSD)、17β-羟基类固醇脱氢酶(17β-HSD)、胆固醇侧链裂解酶(P450scc)、17α-羟化酶(CYP17)以及睾酮合成相关基因3β-HSD、17β-HSD、P450scc、CYP17、类固醇合成急性调节蛋白(St AR)基因相对表达量。结果表明:与对照组相比,添加40μg/m L维生素E有提高绵羊睾丸间质细胞睾酮分泌量的趋势(P=0.061),添加40μg/m L维生素E能显著提高P450scc和3β-HSD酶含量(P0.05),P450scc m RNA与3β-HSD m RNA相对表达量也显著提高(P0.05)。     

猪睾丸间质细胞的分离及培养

为丰富体细胞核移植的供体细胞种类,本研究对猪睾丸间质细胞的分离和培养方法进行了系统研究。运用酶消化法和钢网过滤筛选法获得的猪睾丸细胞,呈现圆形、细胞体积较大,培养4~6 h后开始贴壁;培养48 h后,贴壁增殖;培养3~5 d后可形成单层细胞。这一研究结果为研究和建立睾丸间质细胞体外培养体系提供技术方法和试验依据。     

成年公兔睾丸支持细胞的分离纯化及鉴定

为了寻求一种从成年公兔睾丸获得大量高纯度、高活率支持细胞的方法，本试验采用了胶原酶、胰蛋白酶和透明质酸酶联合消化法，从成年公兔生精小管分离出支持细胞，通过Tris-HCl低渗处理和选择性贴壁培养对所得的支持细胞进行纯化，并采用形态学和HE染色鉴定。最终获得了大量较高纯度（78.3%）和高活率(90.7%)的支持细胞。     

3种分离小鼠腹腔肥大细胞方法的研究

In order to get mast cells in mice abdominal cavity,we used three Percoll gradient separation methods to extract mast cells from mice abdominal cavity,and counted cell viability, cell purity and the total number of cells.The 3 mL Percoll gradient separation liquids in the centrifugal tube from top to bottom were divided into 4 layers to analyze the dynamic distribution of mast cells.The results showed that in the method Ⅰ,the cell viability, cell purity and the total number of cells were (83.51±14.00)%，(69.04±11.75)% and（10.60±5.20）×10⁵ /mL,respectively；in the method Ⅱ,the cell viability, cell purity and the total number of cells were (85.71±9.23)%，(87.10±3.93)% and（11.64±5.73）×10⁵ /mL,respectively；in the method Ⅲ,the cell viability, cell purity and the total number of cells were (72.25±24.81)%，(68.34±10.20)% and（8.87±5.18）×10⁵ /mL,respectively.The cell viability and total number of cells in the three groups were not significantly different(P＞0.05).The cell purity of method Ⅱ was extremely significantly higher than that of methods Ⅰ and Ⅲ (Ρ＜0.01). The dynamic distribution of mast cells in the Percoll gradient separation liquid was that the cell viabilities of 3,4 layers were extremely significantly higher than 1 layer (Ρ＜0.01),significantly higher than 2 layer (Ρ＜0.05); the cell purity of 3 layer was extremely significantly higher than 1,4 layers (Ρ＜0.01), significantly higher than 2 layer (Ρ＜0.05); the number of cells of the 3,4 layers were extremely significantly higher than 1,2 layers (Ρ＜0.01). Therefore, adopting the method Ⅱ, layer 3 of extraction, the middle and lower 0.5 mL, which was close to the interface parts in 30%∶ 80% of the Percoll gradient separation liquid, the separation of mast cells of mice peritoneal cavity was best.     

Cry1基因在雄性绵羊生殖轴系的表达与定位

隐花色素1(Cryptochrome 1,Cry1)基因作为生物钟调控环路的负调控因子,对生物节律的稳定发挥着重要作用,因此研究Cry1基因在哺乳动物生殖轴系的表达对揭示哺乳动物季节性繁殖的调控机理有着重要意义。本实验应用qPCR和免疫组织化学等方法研究了Cry1基因在雄性绵羊生殖轴系(松果体、下丘脑、垂体、睾丸和附睾)的分布和表达情况,并通过生物信息学分析对Cry1蛋白的结构做了预测。结果显示:在绵羊生殖轴系各组织中均有Cry1表达,其中在下丘脑中的表达最高,睾丸次之,各组数据间差异显著。免疫组化结果显示Cry1蛋白在松果体和下丘脑中成弥散性表达,胞膜、胞质及胞核均有阳性表达。在垂体上Cry1蛋白主要位于毛细血管的管壁细胞和血管周围的腺细胞上。Cry1蛋白还在睾丸组织的基膜和间质细胞,以及附睾管腔上皮细胞、管周肌样细胞和腔面精子上呈阳性表达。生物信息学分析发现Cry1蛋白无信号肽信息和跨膜结构,主要位于细胞质和细胞核中,与牛、鹿的亲缘关系最近。Cry1基因参与了绵羊的繁殖,为生物钟调控哺乳动物的季节性繁殖提供了一定的研究基础。     

蒙古马睾丸支持细胞的体外分离培养与鉴定

为研究一种简单快速分离蒙古马睾丸支持细胞并保证其活性的基本方法,本试验采集2岁蒙古马睾丸组织于低温环境下进行机械分离,将1~3 g睾丸组织剪碎,采用重力沉淀法去除游离的红细胞和间质细胞;使用0.1%胶原酶Ⅳ和0.25%胰蛋白酶+EDTA逐步进行组织消化,并用含有10%胎牛血清的培养基进行细胞培养;在接种后采用差异贴壁法分离纯化支持细胞,使用Tris-HCl低渗法去除杂质细胞,并采用碱性磷酸酶（AKP）染色、HE染色、实时荧光定量PCR方法进行鉴定。结果显示,支持细胞贴壁性能较强,培养24 h后大多数细胞开始贴壁,细胞形状呈椭圆形;培养48 h后胞质增多,折光性变强;培养3~4 d细胞胞质展开紧密连接,此时细胞呈三角形,有明显的核仁,培养6~7 d细胞生长状态进入稳定期。实时荧光定量PCR结果显示,GATA4和GDNF基因在培养细胞中极显著表达（P<0.01）,AKP染色支持细胞呈阴性表达,表明分离培养的细胞确为支持细胞。本试验使用机械分离法与两步酶消化法处理组织,可快速高效地获得睾丸支持细胞,成功构建了蒙古马睾丸支持细胞体外培养方法。     

Isolation,Culture and Identification of Testis Sertoli Cells in Mongolian Horses in vitro

To study a simple and rapid separation of testis Sertoli cells in Mongolian horses and ensure their activity,the testis tissue of two-year-old Mongolian horses was mechanically isolated under a low temperature environment in this experiment,and 1-3 g of testis tissue was chopped,the free red blood cells and interstitial cells were removed by gravity precipitation method.0.1% collagenase Ⅳ and 0.25% trypsin-EDTA were used for tissue digestion, and cells were cultured in medium containing 10% fetal bovine serum.After inoculation,the purified Sertoli cells were isolated and purified by differential sticking method,and the impurity cells were removed by Tris-HCl infiltration method,and were identified by alkaline phosphatase (AKP) staining,HE staining and Real-time quantitative PCR.The results showed that most of the cells began to adhere to the wall after culture 24 h,and the shape of the cells was oval.After culture 48 h,the cytoplasm increased and the refraction became stronger.After culture 3-4 d,the cytoplasm of the cells expanded into tight junction.At this time,the cells were triangular with obvious nucleoli.After culture 6-7 d,the cells entered the stable phase.Real-time quantitative PCR results showed that the expression of GATA4 and GDNF genes were extremely significantly expressed in cultured cells (P<0.01).AKP staining supported the negative expression in Sertoli cells,indicating that the isolated cultured cells were indeed Sertoli cells.In this experiment,tissue was treated by mechanical separation and two-step enzyme digestion,and testicular support cells were obtained quickly and efficiently,the method of culturing Sertoli cells in Mongolian horses testis in vitro was successfully constructed.     

细毛羊毛囊干细胞分离培养方法比较研究

试验旨在对细毛羊毛囊干细胞的分离培养方法进行初步研究,建立细毛羊毛囊干细胞体外培养体系。采用两步酶消化法、机械分离法和两步酶消化法+机械分离法3种方法分离培养细毛羊毛囊干细胞,通过测定毛囊干细胞的数量、克隆形成率及毛囊干细胞的增殖能力等综合评估3种培养方法的优劣,寻求既能方便获得大量毛囊干细胞,又能减少其分化的理想培养条件。倒置显微镜下观察3种培养方法获得的细胞均为铺路石状,均表达角蛋白K19和整合素β1,表明成功获得细毛羊毛囊干细胞,建立体外培养体系。采用"两步酶消化法和机械分离法"相结合的方法获得毛囊干细胞体外培养效果最优。     

绵羊睾丸差异基因及蛋白质互作网络关系研究

[目的] 整合分析多个绵羊睾丸转录组数据集,揭示绵羊睾丸差异基因及蛋白质互作网络关系,以期探索影响绵羊精子生成的关键基因,为绵羊的繁殖提供理论参考。[方法] 对74个绵羊睾丸转录组进行生物信息学分析,通过limma软件包进行差异基因分析,使用WGCNA构建加权绵羊睾丸差异基因共表达网络,并通过MCODE计算网络中重要基因;利用Metascape进行功能富集分析,利用Cytoscape插件AutoAnnotate识别基因集簇。[结果] 最终筛选到11 884个基因,构建237 366对蛋白质互作关系;对2 058个差异基因构建蛋白质互作网络,产生46 169对蛋白质互作关系,确定了4个得分最高的基因集合。对2 058个差异基因构建加权共表达网络,共获得7个模块,其中Blue模块内有929个基因,功能富集分析发现显著富集于雄性配子产生、繁殖、精子发生、鞭毛运动和AMPK信号通路等,且富集结果高度连接并聚成一个完整的网络,最终确定了25个参与精子发生和精子细胞发育过程的关键基因。[结论] 本研究揭示了绵羊睾丸表达基因的蛋白质互作网络和多维差异基因的蛋白质互作网络,最终找到与睾丸精子发生相关且有互作关系的25个基因,为绵羊繁殖研究提供理论依据。     

脂多糖对胚胎附植期Toll样受体4及相关免疫因子的影响

试验旨在探讨脂多糖（LPS）对绵羊胚胎附植期Toll样受体4（TLR4）及相关免疫因子表达的影响。以构建好的pcDNA3.1-TLR4过表达载体转染绵羊子宫内膜基质细胞,运用Western blotting技术鉴定细胞转染效果,然后用1 μg/L LPS刺激转染后的子宫内膜基质细胞,建立内膜基质细胞炎症模型。将经LPS处理的细胞分别培养12、24、48、72 h,采用实时荧光定量PCR技术和Western blotting法检测内膜基质细胞中TLR4 mRNA及蛋白表达量,以及免疫因子IL-1β和IL-6的mRNA表达水平,并以未经LPS处理的细胞作为对照。结果显示,与对照组相比,LPS促进了免疫因子IL-1β、IL-6的释放量,但随LPS作用时间的延长,细胞中IL-6的表达量逐渐下降,而IL-1β的表达量逐渐升高,使得Th1/Th2偏向不利于妊娠的Th1方向表达;TLR4 mRNA相对表达量在12、24、72 h均显著高于对照组（P<0.05）,48 h时极显著高于对照组（P<0.01）,且LPS处理后的细胞TLR4蛋白表达量也始终高于对照组。综上所述,pcDNA3.1-TLR4过表达载体成功转入绵羊子宫内膜基质细胞;LPS有效激活了子宫内膜细胞中TLR4信号通路,并促进了下游因子的表达;子宫蜕膜组织中TLR4受体蛋白对胚胎附植早期妊娠微环境的平衡维持也起到了重要作用。     

Transduction and Transplantation of Spermatogonia into the Testis of Ram Lambs through the Extra-testicular Rete

Spermatogonial transplantation will provide a new way to study spermatogenesis in domestic animals, disseminate male genetics and produce transgenic animals, if efficiency can be improved. We evaluated a 'surgical' method for transplanting donor cells into testes of ram lambs, where the head of the epididymis is reflected, and a catheter introduced into the extra-testicular rete testis. We also tested transduction of ram spermatogonia with a lentiviral (LV) vector as a means to identify permanent colonization, and introduce genes into donor cells. Eight ram lambs, 11- to 13-week olds, were the recipients: in five, spermatogonia were injected into one testis, and the contralateral testis was an un-manipulated control: in two, spermatogonia were injected into one testis and the contralateral was sham-injected: in one, both testes were injected. Six lambs received spermatogonia labelled with a cell-tracking dye and these were collected 1 or 2 weeks after transplantation; three lambs received spermatogonia transduced with a LV vector driving the expression of enhanced Green Fluorescence Protein and these were collected after 2 months. Donor cells were detected by immunohistochemistry in tubules of seven of nine recipient testes. Approximately 22% of tubule cross-sections contained donor cells immediately after transplantation, and 0.2% contained virally transduced cells 2 months after transplantation. The onset of spermatogenesis was delayed, and there were lesions in both injected and sham-injected testes. Despite the effects of the surgery, elongated spermatids were present in one recipient testis 2 months after surgery. The results suggest that, after modifying the surgical and transduction techniques, this approach will be a means to produce good colonization by donor spermatogonia in sheep testes.