大鼠A型精原细胞的体外培养

目的探讨大鼠A型精原细胞的分离、鉴定及体外培养的方法与技术。方法利用非连续密度梯度离心及差速贴壁法纯化A型精原细胞;用抗c-kit和抗TERT免疫组织化学法进行细胞鉴定;用含10%NBS的DMEM进行体外培养。结果Percoll分离法结合差速贴壁法最终平均大鼠的每个睾丸可得到0.614×106个A型精原细胞,锥虫蓝染色显示细胞的存活率为92.1%。c-kit和TERT免疫组织化学阳性细胞平均分别为(90.8±1.0)%和(91.7±1.2)%。在含10%NBS的DMEM条件下,A型精原细胞于培养96 h增殖达高峰。结论Percoll分离与差速贴壁法结合是纯化A型精原细胞的有效方法;c-kit和TERT免疫组织化学结合证实本实验所获得的细胞是A型精原细胞;大鼠A型精原细胞可以在体外进行增殖。     

温度对体外培养精原细胞增殖和凋亡的影响

目的:探讨精原细胞最适培养条件并观察环境温度对体外培养的精原细胞增殖和凋亡的影响.方法:将分离、富集的小鼠精原细胞分别置于不同温度条件下进行体外培养,观察其贴壁、增殖、形态学变化和存活时间;MTT 法检测精原细胞活性,Annexin V-FITC/PI 染色法检测精原细胞的凋亡.结果:32℃、34℃、37℃条件下均可以看到精原细胞的贴壁、分裂和增殖,39℃培养条件下,精原细胞极少贴壁,基本看不到细胞增殖,细胞很快发生凋亡.随着培养时间延长,精原细胞增殖形成的精原细胞克隆数和精原细胞存活时间明显不同,其中34℃条件下精原细胞增殖最多,存活时间最长.结论:温度对体外培养的精原细胞的增殖和凋亡有明显的影响,34℃为精原细胞体外培养的最适温度.     

锌指蛋白265在大鼠睾丸Sertoli细胞中的特异性表达

目的:研究锌指蛋白265(ZNF265)在大鼠睾丸Sertoli细胞中的特异性表达.方法:分离培养SD大鼠睾丸Sertoli细胞, 以免疫荧光染色、流式细胞术(FCM)检测ZNF265在Sertoli细胞的表达, 且抽提Sertoli细胞总RNA, 用PT-PCR法检测ZNF265的表达.结果:经免疫荧光染色, 实验组Sertoli细胞核核周呈现明亮特异荧光, 对照组未见特异性荧光;FCM检测结果显示实验组Sertoli细胞ZNF265的表达较对照组明显增高;RT-PCR扩增出目的条带ZNF265.结论:大鼠睾丸Sertoli细胞特异性表达ZNF265, 且多表达于Sertoli细胞核核周胞质.     

新生昆明小鼠睾丸支持细胞和精原细胞的共培养及支持细胞的作用

目的 分离、纯化、培养小鼠睾丸支持细胞,通过与精原细胞共培养,研究体外培养的支持细胞对精原细胞的影响.方法 用复合酶消化、密度梯度离心和差异贴壁法分离、纯化、培养新生小鼠睾丸支持细胞,以苏丹Ⅳ染色鉴定支持细胞;用丝裂霉素-C处理支持细胞后作为饲养层,与精原细胞共培养;以直接分离培养的精原细胞作对照.倒置相差显微镜观察,细胞培养第3天,四甲基偶氮唑盐(MTT)法测定精原细胞的增殖率;膜联蛋白V-异硫氰酸荧光素/磷脂酰肌醇(ANNEXIN-V-FITC/PI)染色;激光扫描共焦显微镜观察记录精原细胞的凋亡率,比较精原细胞克隆大小、存活时间、增殖率和凋亡率的不同. 结果 分离、纯化后经苏丹Ⅳ染色鉴定的小鼠支持细胞纯度可达95%以上,与支持细胞共培养的精原细胞的克隆大小、存活时间和细胞增殖率明显高于对照组,而凋亡鲻率则明显低于对照组.结论支持细胞可以促进精原细胞的增殖,抑制其凋亡.     

新生大鼠心肌telocytes在体外培养状态下的分裂方式

目的 探讨体外培养新生大鼠心肌telocytes的分裂方式。 方法 采用0.1%胶原酶I/0.125%胰蛋白酶联合消化法分离培养新生大鼠心肌telocytes,活细胞工作站动态捕捉和苏木素染色检测telocytes的分裂方式。 结果 酶联合消化法培养新生大鼠心肌telocytes纯度高、活力好,增殖迅速。无丝分裂时,胞核变大,以出芽方式生长,形成哑铃状细胞核,核芽逐渐拉伸并与母细胞分离。有丝分裂前中期,染色质缩短变粗为染色体,整齐地排列在赤道板处;分裂末期,染色体到达两极,子核已初步形成;胞质分裂期,胞质逐步拉开,形成两个子细胞。在有丝分裂不同时期,心肌telocytes均保持其原有的形态特征。 结论 体外培养心肌telocytes增殖时有丝分裂和无丝分裂两种方式并存。     

大鼠睾丸支持细胞的分离纯化与鉴定

目的　简化大鼠睾丸支持细胞的分离纯化方法 ,通过不同方法对Sertoli细胞进行形态学观察鉴定。方法　取鼠龄 16～ 2 2天的雄性Wistar大鼠睾丸 ,采用酶次第消化 ,培养过程中纯化Sertoli细胞 ,并用多种方法对Sertoli细胞进行鉴定。结果　大鼠睾丸支持细胞占培养细胞总数的 90 %以上。HE染色 ,Sertoli细胞突起很多 ,核仁清晰 ,在胞质中可见吞噬物和大小不等的空泡 ;甲基绿 派洛宁染色 ,Sertoli细胞富含RNA ;Feulgen染色和透射电镜 ,核仁周围可见卫星核小体。结论　本实验培养方法可获得更多、纯度高的Sertoli细胞 ,HE染色、甲基绿 派洛宁染色、Feulgen染色和透射电镜是鉴定Sertoli细胞的有效方法     

近似体腔温度不宜体外培养大鼠A型精原细胞

背景：温度会影响体外培养精原细胞的增殖与凋亡。 目的：观察不同体外培养温度对A型精原细胞细胞周期及增殖相关基因c-kit、CyclinD3的影响。 方法：通过两步酶消化法分离大鼠生精细胞,分别置于32,37 ℃条件下与支持细胞进行体外共培养,观察其形态及细胞数变化等生长特性;培养第8天采用非连续密度梯度离心、差异性贴壁分离富集A型精原细胞。 结果与结论：体外培养第1天,32,37 ℃组均可见悬浮的呈圆形或卵圆形的生精细胞和贴壁的呈长梭形或不规则形的支持细胞;从第4天开始,37 ℃组生精细胞数、生长状态逐渐降低,第8天时,37 ℃生精细胞数明显低于32 ℃组 (P < 0.05);体外培养第8天,32 ℃组处于S期A型精原细胞所占比例明显高于37 ℃组(P < 0.05),32 ℃组A型精原细胞c-kit、CyclinD3 mRNA及蛋白的相对表达量均高于37 ℃组(P < 0.05)。说明在37℃这种近似体腔的温度下体外培养生精细胞会抑制精原细胞的增殖。     

长白猪精原细胞的分离和纯化

目的　分离和纯化猪的精原细胞 ,从而对与人类具有高同源性的猪的精子发生进行研究。　方法　酶消化法制备 2月龄未成熟长白猪睾丸组织的单细胞悬液 ,以 2 %～ 4 %牛血清白蛋白 (BSA)连续梯度作为分离介质 ,采用重力沉降法并结合细胞贴壁培养的方法分离精原细胞。　结果　重力沉降法分离细胞后所获精原细胞纯度为 91% ,进一步经过贴壁培养纯化后 ,精原细胞纯度达到 94 2 %。　结论　该方法方便、快捷 ,分离效果好 ,能满足在分子水平研究精原细胞的需要。     

小鼠精原细胞的分离和纯化

目的　探讨小鼠精原细胞的分离纯化。　方法　用组合酶消化法制备 7～ 8d小鼠的生殖细胞悬液 ;用Percoll不连续密度梯度法分离精原细胞。　结果　所获细胞悬液内活细胞、死细胞及细胞团的百分比分别为90 .0 8%、9.92 %及 8.91% ;平均每个睾丸可获得 4.136× 10 5 个细胞 ;精原细胞主要分布于位于 2 7%～ 35 %间的Percoll梯度中 ,其超微结构与 7～ 8d小鼠睾丸切片内精原细胞的超微结构一致 ,经纯化后其纯度达 6 8.76 %。　结论　用组合酶消化、Percoll不连续密度梯度法分离的 7～ 8d小鼠的精原细胞能满足体外培养的需要     

大鼠牙髓细胞的分离培养及超微结构观察

目的 探讨牙髓细胞的培养方法和生长状况,观察其形态结构特征,为进一步研究牙齿的发育提供实验基础.方法 采用胶原酶消化分离的方法分离大鼠的牙髓细胞并在DMEM培养液中培养,运用透射和扫描电镜技术观察细胞的形态结构特征.结果 培养的牙髓细胞呈梭形,细胞生长状况良好,细胞器发育正常,并且有细胞增殖和分裂能力.结论 利用酶消化分离的方法分离培养的牙髓细胞,能够保持其生活状态及增殖分裂能力.     

Postnatal testis development, Sertoli cell proliferation and number of different spermatogonial types in C57BL/6J mice made transiently hypo- and hyperthyroidic during the neonatal period

The role of thyroid hormones in testis structure and function has been fairly well studied in laboratory rodents. However, there are no comprehensive data in the literature for mice regarding the effects of transiently induced neonatal hypo‐ and hyperthyroidism on testis and spermatogonial cell development from birth to adulthood. Our goals were to evaluate the effects of propylthiouracil (PTU) and triidothyronine (T3) on Sertoli cell proliferation/differentiation and to correlate these events with the evolution of the spermatogenic process, tubular lumen formation, blood vessel volume density, and size and number of different spermatogonial types. Although Sertoli cell maturation was accelerated or delayed, respectively, in T3‐ and PTU‐treated mice, the pace of the germ cell maturation was only slightly altered before puberty and the period of Sertoli cell proliferation was apparently not affected by the treatments. However, compared with controls, the total number of Sertoli cells per testis from 10 days of age to adulthood was significantly increased and decreased in PTU‐ and T3‐treated mice, respectively. In comparison to all other spermatogonia, type A₂ was the largest cell in all ages and groups investigated. The PTU‐treated mice had a significantly increased total number of undifferentiated spermatogonia as well as volume and percentage of vessels/capillaries, probably due to the higher number of Sertoli cells, particularly at 10 days of age. Taken together, our results suggest that neonatal hypothyroidism may be a valuable tool for studying spermatogonial biology as well as a means for providing more spermatogonial stem cells that could potentially be used for spermatogonial transplantation, thereby optimizing the efficiency of this technique when young mice are used as donors.     

枸杞多糖对精原干细胞体外增殖的影响

背景：精原干细胞移植对不育具有潜在的临床应用价值,体外建立精原干细胞的培养系统获得数量较多的精原干细胞,仍是目前研究中亟待解决的问题。 目的：观察枸杞多糖对精原干细胞体外增殖的影响。 方法：采用两步酶消化法获取出生4~6 d雄性C57BL/6小鼠睾丸Sertoli细胞与精原干细胞,将精原干细胞接种在Sertoli细胞饲养层上,再加入枸杞多糖或联合细胞因子添加到细胞培养液中。1周后以流式细胞仪检测细胞周期及细胞活性率,并检测各组精原干细胞GFRa-1、Thy-1、c-kit的阳性率。 结果与结论：单独加入枸杞多糖后精原干细胞数量明显增加,增殖明显,联合加入胶质细胞源性神经营养因子与白血病抑制因子精原干细胞增殖更加明显(P < 0.05)。并发现体外培养1周后的精原干细胞仍保持其睾丸组织内的精原干细胞特征,大多仍维持在未分化状态。表明在枸杞多糖或枸杞多糖联合胶质细胞源性神经营养因子及白血病抑制因子作用下,可促进精原干细胞体外增殖。     

大鼠睾丸支持细胞的分离、纯化和鉴定

目的 培养高纯度的大鼠睾丸支持细胞,并应用检测ABP mRNA原位杂交方法加以鉴定.方法 选用18～22d龄SD雄性大鼠睾丸,采用0.25%胰蛋白酶、0.1%透明质酸酶、 0.1%胶原酶三酶依次消化法分离支持细胞,置于32℃ 5% CO2的培养箱培养,48h后用20mmol/L Tris-HCl低渗处理培养细胞.培养1周后应用伊红染色、吖啶橙荧光染色、Feulgen染色对所培养的支持细胞进行鉴定,同时应用原位杂交检测ABP mRNA方法鉴定分离培养的支持细胞.结果 培养1周后所获培养的支持细胞纯度达95%以上.分离培养的支持细胞ABP mRNA表达阳性,其形态结构特征与用其他方法鉴定为支持细胞的形态结构特征一致.结论 采用三酶连续消化及低渗处理法分离培养的支持细胞纯度高,而且应用原位杂交方法检测ABP mRNA是一种新的、特异、有效的鉴定支持细胞及其功能的方法.     

Caspase activity inhibition delays programmed spermatogenic cell death in vitro

Programmed cell death or apoptosis was analyzed in rat Sertoli-spermatogonial cell cocultures prepared from 2-9 day old rats using time-lapse video microscopy, a cell viability fluorescence microscopy assay, immunocytochemical markers, and cell-permeable caspase inhibitory peptides with reversible and irreversible effects. We show that apoptosis can initially affect a single member of a spermatogonial cell cohort and that single non-viable spermatogonial cells can remain conjoined to viable spermatogonial cells. The integrity of the cytoskeletal F-actin network and the presence on Bcl-2 immunoreactivity are valuable markers of spermatogonial cell viability. Apoptotic bodies released into the culture medium are generally eliminated after culture medium replenishment; however, spermatogonial apoptotic cell remnants can be taken up by Sertoli cells, which are known to represent a phagocytic somatic population within the seminiferous epithelium. Cell permeable caspase-1 and caspase-4 inhibitory peptides with reversible and irreversible action were supplemented to a serum-free hormone-growth factor-supplemented medium. In the absence of the caspase inhibitory peptide, the viability of spermatogonial cells decreases gradually with time in coculture. However, the addition of caspase inhibitory peptides causes a significant accumulation of spermatogenic cells per unit surface area. Although inhibition of caspases, the executors of spermatogonial cell death, results in a substantial increase of spermatogonial cells in the cocultures, it remains to be determined what the differentiation potential of caspase-inhibited spermatogonial cell cohorts is.     

Effect of follicle-stimulating hormone and testosterone on colony formation of bovine spermatogonial stem cell

The complex process of spermatogenesis is regulated by various factors. In this study, the in vitro effects of follicle-stimulating hormone and testosterone on spermatogonial cell colony formation were investigated. Sertoli and spermatogonial cells were isolated from 3- to 5-month-old calves. Co-cultured Sertoli and spermatogonial cells were treated with follicle-stimulating hormone and testosterone in treatment groups before colony assay. Results indicated that follicle-stimulating hormone is the best factor for in vitro colonization of spermatogonial cells. In conclusion, using follicle-stimulating hormone provided proper bovine spermatogonial stem cell culture medium for in vitro study of these cells.     

睾丸局部感染时Sertoli细胞IL-1、IL-6、TGF-β和FasL的变化

目的：研究大鼠Sertoli细胞在抗感染中的免疫调节作用。方法：以溶脲脲原体(UU)和致病性大肠杆菌(E．coli)直接注入大鼠膀胱模拟上行性感染的途径，分别在1周、2周、3周处死大鼠，分离取得睾丸组织作组织切片观察病理变化及从大鼠睾丸组织中分离获得高纯度的Sertoli细胞，用免疫组化方法比较正常组与UU感染组、致病性大肠杆菌组之间：IL-1,IL-6,TGF-β和FasL表达的差异。结果：与正常组相比，UU和E .coli感染后，其IL-1分泌升高，IL-6分泌下降，TGF-β分泌升高，FasL表达也升高，结论：大鼠Sertoli细胞在抗感染免疫中，可通过IL-1，IL-6，TGF-β和FasL的表达来发挥免疫调节作用。     

分离的大鼠睾丸曲细精管体外重组的观察

将生后15天和20天大鼠的睾丸,去被膜后以胶原酶反复消化,使Sertoli细胞和生精细胞分离。以HamF-12无血清培养基培养子(35℃)5％CO_2温箱。经过19天的连续培养及光镜和电镜样品观察,发现分离的曲细精管上皮细胞经过贴壁生长期(培养后1～6天),形成单层培养细胞。再经索状生长期(培养后6～14天),由单层细胞变成致密的细胞索,并释放出有头尾结构的蝌蚪形精子样细胞。最后经管状生长期(培养第14天以后),细胞索发育成细胞团和类似曲细精管样的结构。光镜和电镜下见其中有管周细胞、Sertoli细胞和各级生精细胞。本文对分离的曲细精管上皮细胞在体外重组成小管样结构和体外精子发生,进行了讨论。     

INVESTIGATION OF CARCINOMA IN SITU CELLS OF TESTIS BY QUANTIFICATION OF ARGYROPHILIC NUCLEOLAR ORGANIZER REGION ASSOCIATED PROTEINS (AgNORs)

The silver staining which specifically stains argyrophilic proteins (AgNORs) in interphase nuclei was applied to paraffin sections of 24 testicular specimens with carcinoma in situ (CIS). AgNOR area per nucleus was quantified by a computerized image analyser. Significant quantitative differences were found between CIS, Sertoli cells, and spermatogonia ( P =0·0001), with median values of 10·3, 2·8, and 1·4 μm² in the three cell types, respectively. A Sertoli cell index (SCI), defined as the ratio between AgNORs in CIS or spermatogonia and Sertoli cells, was shown to be significant in the differential diagnosis of CIS cells from spermatogonia when 1·0 was used as the cut-off value (CIS>1; spermatogonia<1). Furthermore, CIS associated with non-seminoma was found to have a significantly higher level of AgNORs than CIS associated with pure seminoma ( P <0·01), indicating that subclonal variation in transformation potential might be present within morphologically identical CIS of the testis. It remains to be seen whether quantification of AgNORs in isolated CIS could be used to predict transformation of CIS into seminoma or non-seminoma.     

大鼠精原干细胞的筛选与培养

目的:建立大鼠精原干细胞的筛选和培养的方法体系。方法:采用改良的二步酶消化法分离大鼠睾丸细胞,用改进的差异贴壁分选法筛选大鼠精原干细胞,用添加了胶质细胞源神经营养因子(GDNF)、可溶性GFRα1和bFGF的DMEM/F12无血清培养基和大鼠胚胎成纤维细胞饲养层培养高度富集的大鼠精原干细胞,通过形态观察、标志基因的RT-PCR检测和免疫细胞化学分析鉴定培养细胞的干细胞活性。结果:我们的改良二步酶消化法和差异贴壁分选法能有效的分离和筛选大鼠精原干细胞,这些高度富集的精原干细胞能够存活20 d以上,形成较大的干细胞克隆,并表达精原干细胞的标志基因和具有干细胞活性。结论:成功建立了大鼠精原干细胞的分离、筛选和培养体系。