支持细胞中促卵泡素信号通路研究进展

支持细胞在精子生成过程中起重要作用,支持细胞的数量决定睾丸大小、生精细胞数量以及最终生成精子数量。促卵泡素作为保证雄性生育能力的重要激素之一,通过与支持细胞上的促卵泡素受体结合,诱导5种信号途径,导致支持细胞中各类转录因子被激活,引起基因表达发生变化,从而保证生精细胞顺利发育成精子。文章主要围绕支持细胞中促卵泡素信号途径,及其对细胞发育过程中相关基因表达的影响等方面进行了综述。     

Development of transferred xenogeneic vole embryos in mouse uteri

An experimental model to study interspecific pregnancy using voles, Microtus arvalis, and green fluorescent protein gene‐induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene‐induced transgenic mice. The uteri containing xeno‐allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid‐Schiff‐positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.     

西门塔尔牛胰腺间充质干细胞原代培养及其多向分化潜能研究

旨在对西门塔尔牛胰腺间充质干细胞（pancreatic mesenchymal stem cells,PMSCs）进行原代培养并研究其体外分化潜能,为细胞疗法和组织工程学方面提供新的种子细胞。本研究从3月龄的西门塔尔牛胚胎中无菌分离胰腺组织,分别采用胶原酶消化法和组织块贴壁法分离PMSCs,进行原代培养;绘制第3代、第9代、第15代细胞生长曲线并测定群体倍增时间及克隆形成能力,采用免疫荧光检测干细胞表面标志物（CD29、CD44、CD73、CD90、CD34和CD45）,RT-PCR检测干细胞细胞表面标志物（CD29、CD44、CD73、CD90、CD106、CD166、CD34和CD45）;染色体核型分析检测其基因稳定性,通过向成脂、成骨、成软骨和成肝样细胞诱导分化,检测其多向分化潜能。结果表明,两种方法都可成功分离出PMSCs,细胞贴壁后形态均为长梭形,漩涡状生长,生长趋势呈典型的S形;第9代细胞群体倍增时间显著低于第15代而高于第3代（P<0.01）;第9代PMSCs克隆形成率显著低于第3代而显著高于第15代（P<0.05）;免疫荧光结果表明,PMSCs特异性表达CD29、CD44、CD73和CD90,RT-PCR结果显示PMSCs特异性表达CD29、CD44、CD73、CD90、CD106和CD166,未表达造血细胞表面标志物CD34和CD45,与国际细胞治疗学会组织干细胞委员会指定的MSCs表面标记物相对应;核型分析表明,PMSCs为正常二倍体（2n=60,XY）,染色体基因组未发生变异;特异性染色和RT-PCR结果表明,从西门塔尔牛体内获得的PMSCs可分化为脂肪细胞、骨细胞、软骨细胞和肝样细胞。本试验证实两种方法均可成功建立西门塔尔牛PMSCs体外分离培养体系,PMSCs具有活性好、增殖速度快的特点,与MSCs有相似的生物学特性和多项分化的潜能。可为组织工程学提供新的种子细胞。     

Wnt5a介导Wnt/Ca2+通路对BCG诱导牛原代肺泡上皮细胞自噬的调控作用

旨在探讨体外分离培养牛肺泡上皮细胞（bovine alveolar epithelial cells,BAECs）的方法及Wnt5a对牛结核分枝杆菌卡介苗（bacille Calmette-Guérin,BCG）感染BAECs细胞自噬的调控机制。试验选用酶联合消化法和机械刮刷法分离细胞,差速贴壁法纯化BAECs,免疫荧光染色检测上皮细胞标志物角蛋白14（cytokeratin 14,CK14）和角蛋白5（cytokeratin 5,CK5）的表达;BCG感染BAECs,并用Box-5抑制Wnt5a的表达,Western blot和免疫荧光染色检测自噬相关蛋白及非经典Wnt信号通路相关蛋白的表达。结果表明,采用酶联合消化法和机械刮刷法能够成功分离纯度较高的BAECs,细胞经CK14和CK5鉴定为阳性;BCG感染BAECs促进Wnt5a表达,增加细胞自噬,Box-5预处理下调BCG诱导的细胞自噬相关蛋白LC3II、P62、Atg7及Atg5的表达,且抑制非经典Wnt/Ca²⁺信号通路相关蛋白Wnt5a、CaMKII及NFAT的表达。综上,试验成功建立BAECs分离培养方法,BCG感染增加BAECs内Wnt5a表达和细胞自噬,抑制Wnt5a下调BCG诱导的BAECs细胞自噬,且Wnt5a是通过非经典Wnt/Ca²⁺信号通路调控BCG诱导的BAECs细胞自噬。     

犬造血干细胞移植的作用机制及应用研究进展

造血干细胞(hematopoietic stem cell, HSC)位于造血系统的顶端, 是造血系统中唯一具有多能性和自我更新能力的细胞, 可以分化为各种功能的血细胞, 维持血液系统的建立和动态平衡, 是目前研究最为透彻、临床应用最为成熟的成体干细胞。造血干细胞的这些重要特性, 使得基于造血干细胞在治病机制研究和临床应用中的研究取得了很大进展。以造血干细胞为基础的再生医学治疗是目前治疗恶性血液病和遗传病的首选方法, 如造血干细胞治疗犬白细胞黏附缺陷综合征、犬遗传性贫血、犬淋巴瘤、犬白细胞减少症、犬X连锁严重联合免疫缺陷病等, 但由于白细胞抗原匹配的供体稀缺、可获得的造血干细胞数量有限等原因, 无法满足临床需求, 因此, 如何通过体外培养获得满足临床需要的造血干细胞成为近年来学者们研究的热点问题。作者参照现有研究成果对造血干细胞的来源和体外分离培养、治病机制进行系统的描述, 并对造血干细胞移植治疗遗传性溶血性贫血、白细胞黏附缺陷综合征、淋巴瘤、白细胞减少症、X连锁严重联合免疫缺陷病的临床研究进行综述, 以期为今后将造血干细胞广泛应用于临床治疗提供参考依据。     

油酸和硬脂酸对奶牛乳腺上皮细胞中脂肪酸结合蛋白3表达和脂滴分泌的影响

为确定外源添加长链脂肪酸油酸和硬脂酸对奶牛乳腺上皮细胞中脂肪酸结合蛋白3（FABP3）表达的影响,本试验采用荧光定量RT-PCR检测乳脂合成相关基因的表达情况,确定油酸和硬脂酸的最佳浓度和培养时间分别为75 μmol/L 12 h和100 μmol/L 36 h。采用Western blotting和免疫荧光法检测添加油酸和硬脂酸后FABP3表达和脂滴分泌的情况,为进一步揭示FABP3在乳脂合成信号转导通路中的作用提供理论依据。     

Cell-cycle-dependent Colonization of Mouse Spermatogonial Stem Cells After Transplantation into Seminiferous Tubules

Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5–10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.     

Relationships between endometrial cytology and interval to first ovulation, and pregnancy in postpartum dairy cows in a single herd

The objectives were to investigate the relationships between endometrial cytology (EC) and interval from calving to first ovulation, and pregnancy in dairy cows, and that between uterine fluid and EC. On day 25 postpartum, 39 dairy cows were grouped based on EC, as having low (?8%) or high (>8%) polymorphonuclear cells (PMN), and the quantity of uterine fluid was assessed by ultrasound. The interval from calving to first ovulation was shorter in low, than in high PMN cows (32 vs. 45 d). A greater proportion of cows with uterine fluid had high PMN (64% vs. 21%), and the PMN increased from 14% to 34% as the quantity of uterine fluid increased. The mean interval from calving to ovulation was longer in primiparous cows with high PMN (49 d) compared to that of primiparous and multiparous cows with low PMN (28 and 29 d, respectively). Although the conception rate to first service at 92 d postpartum was not different between PMN groups, the cumulative pregnancy at 270 d tended to be higher in low than in high PMN (80% vs. 58%) multiparous cows. Also, cows that had uterine fluid on day 25 postpartum had a shorter interval from calving to pregnancy than those with no uterine fluid (161 vs. 208 d). In conclusion, combining transrectal ultrasonography with endometrial cytology on day 25 postpartum has diagnostic value in the assessment of uterine inflammation.     

Ultrastructure of Colonic Epithelial Cells in Vitamin E and Selenium Deficient Pigs

Pigs deficient in vitamin E and selenium had a decreased contrast of the intracellular membranes compared with pigs supplemented with vitamin E and selenium. Other common changes in the deficient animals included a reduced number of microvilli, with a short and irregular appearance, numerous swollen mitochondria with empty spaces and intercellular oedema. It is suggested that these morphological changes are attributed to the insufficient supply of vitamin E and selenium, though it is not excluded that particular intestinal factors may participate in the development of the lesions.     

ST贴壁细胞悬浮驯化及应用

为将ST贴壁细胞通过自主驯化,使其能在ST-S细胞无血清培养基中悬浮生长且能稳定传代,在摇瓶中实现高密度生长,并应用于猪伪狂犬病毒(PRV)悬浮培养,经ST-A低血清培养基适应培养,对一株贴壁的ST细胞进行了无血清的全悬浮驯化,并将PRV在悬浮细胞中连续盲传培养。结果显示:ST悬浮细胞能在无血清培养基中传代,培养48 h至第3代后,所能达到的最终细胞密度为3.00×10^(6 )cells/mL以上;PRV连续培养至第5代,毒价可达到109.0 TCID50/mL。结果表明,用专用培养基可使ST贴壁细胞实现悬浮驯化,并可应用于PRV悬浮培养。本研究为获得高密度ST悬浮细胞和提高PRV增殖效率奠定了技术基础。