摘要
作为性腺发育研究的重要动物模型,家禽提供了更直观、方便的研究途径。其中,家禽原始生殖细胞(primordial germ cell, PGC)的体外培养和调控的成功,为胚胎生殖细胞系的建立和保存,以及转基因家禽的研究提供了坚实的基础。然而,家禽PGC外源性调控的稳定性和产量仍需提高,以保证相关研究的顺利进行。维甲酸(retinoic acid, RA)作为维生素A的代谢产物,在哺乳动物生殖发育中的作用被逐渐发现,对多种细胞的增殖和细胞间粘附有着重要的调节作用。本实验以艾维因鸡胚为材料,分离出胚胎期PGC,经过原代和传代培养,对不同生长状态的细胞进行多能性指标的检测和观察,探讨了RA对PGC体外增殖和细胞间粘附的影响,以及部分胞内信号转导的机制。
1.鸡胚PGC原代和传代培养模型的建立
取培养至4d的鸡胚,在体视显微镜下用玻璃针取其生殖嵴,用0.25%胰蛋白酶-0.02% EDTA消化组织,得到的细胞在含5%胎牛血清的DMEM培养液中进行原代培养。培养液中添加了10ng/ml干细胞生长因子、10ng/ml白血病抑制因子、10ng/ml碱性成纤维生长因子、0.1mmol/Lβ-巯基乙醇、0.1mmol/L MEM非必需氨基酸、2mmol/L L-谷氨酰胺、100IU/ml青霉素和100ug/ml链霉素。对原代和传代后形成的细胞集落进行过碘酸雪夫氏反应(PAS)、SSEA-1和SSEA-3免疫细胞化学检测,同时利用RT-PCR检测干细胞多能性基因PouV、Nanog和Sox2的表达,均证实所形成的细胞集落是PGC。上述实验结果表明：PGC-体细胞原代及传代于饲养层培养的模型可以作为PGC增殖活性调节的研究。
2.鸡胚PGC在体内和体外培养时的形态
取孵化至5d的鸡胚,在体视显微镜下取出生殖嵴,经4%多聚甲醛固定,经常规脱水浸蜡,石蜡包埋,切片烤片后,进行SSEA-1免疫组化荧光染色,定位PGC及其存在状态。经证实大量PGC存在于生殖嵴中,以单个或多个粘附的形式存在,同时证明了体内PGC的多能性。
体外培养的PGC经定点观察其生长状况,确定PGC集落的形成过程。另外,在不同时间阶段经观察和SSEA-1免疫细胞荧光染色,以确定PGC集落生长的状况。结果显示：不同数量PGC的集落均有SSEA-1的表达。
3.维甲酸对鸡胚PGC增殖的调控
利用已建立的PGC培养模型,探讨了RA对体外培养的鸡胚PGC增殖的调节作用。在传代培养PGC的培养液中添加RA以及PKC信号途径的抑制剂H7分别或联合处理细胞,在不同时间点检测PGC的多能性基因的表达。通过PAS染色,观察并统计PGC集落的数量和面积的变化。同时,通过RT-PCR检测细胞周期蛋白基因CCND1和CCNE1、周期蛋白依赖性激酶CDK6和CDK2的表达,以确定RA相关通路对细胞增殖的影响。PAS染色统计,10-7-10-5mol/LRA对体外培养PGC的数量和集落面积有明显的提高效应(P<0.05)；同时能显著促进相关检细胞周期调控基因的表达。其中,10-6mol/LRA的效果最为明显。而在与H7联合处理组中,RA的效应明显减弱。此结果表明,RA (10-7-10-5mol/L)可促进鸡胚PGC的增殖,其中10-6mol/L浓度的RA作用效果最为明显,此效应可被10-6mol/L的H7所抑制。
4.RA对鸡胚PGC胞间粘附的影响
利用已建立的PGC培养模型,探讨RA对体外培养的鸡胚PGC胞间粘附的调节作用。在传代培养PGC的培养液中添加RA以及PKC信号途径的抑制剂H7分别或联合处理细胞。通过特定浓度的消化液消化PGC细胞团,并统计其集落数与单个细胞数,以计算其粘附系数。通过RT-PCR检测不同处理条件下粘附蛋白钙粘蛋白(E-钙粘蛋白)和连锁蛋白(α-和β-连锁蛋白)mRNA表达的变化。粘附系数的统计结果显示：10-7-10-5mol/LRA明显降PGC间的粘附系数(P<0.05),表明PGC胞间粘附性增强；同时能够明显促进E-钙粘蛋白、α-连锁蛋白和β-连锁蛋白的表达。而在H7联合处理组中,RA的效应明显减弱。此结果表明：RA(10-7-10-5mol/L)可促进鸡胚PGC的胞间粘附,其中以10-6mol/L浓度RA的作用效果最为明显,此效应可被10-6mol/L的H7所抑制。
以上实验结果表明：经SSEA-1鉴定,鸡胚生殖嵴内的PGC以单个或少量聚合的形式存在。从鸡胚生殖嵴分离的PGC原代和传代培养后,经PAS染色、SSEA-1和SSEA-3免疫细胞化学染色,及多能性基因PouV、Nanog和Sox2的RT-PCR检测,证实所培养的PGC具有干细胞的多能性。定点观察PGC的体外生长情况的结果证明,在培养的初期PGC集落的形成方式主要是聚集；之后主要是PGC集落细胞的自身分裂增殖。对体外培养不同时期PGC的SSEA-1染色说明多个PGC倾向以细胞间粘附的形式生长。在此模型的基础上,通过细胞集落数量和面积的统计,以及细胞周期调控基因CCND1/CDK6、CCNE1/CDK2 mRNA的表达变化结果说明,10-7-10-5mol/L的RA能促进鸡胚PGC的有丝分裂,从而促进其增殖。同时,PKC途径的抑制剂H7可减弱RA的促增殖作用。另外,通过粘附系数的统计,以及粘附蛋白基因表达变化检测表明,10-7-10-5mol/L的RA可增强PGC的胞间粘附。同时,PKC途径的抑制剂H7能够明显减弱RA的促粘附作用。以上结果提示,RA可能通过PKC信号途径促进鸡胚PGC的增殖和胞间粘附。这些结果有助于鸡胚PGC增殖调控的研究,并有利于PGC的体外大量扩增,为嵌合体和转基因家禽的制备提供实验平台。
PGCs, as precursor cells developing into ova and spermatozoa in embryonic stage, are model type cells for genome research and transgenic application in a variety of vertebrates. Recent years, the limit of great number and stability of cultured embryonic PGCs made the research of avian PGCs attenuated. Coordinating the proliferation and aggregation of cultured PGCs is a crucial method to get enough and survival cells for various studies.
Intercellular connection plays an important role in male stem spermatogonium, spermatogonium, spermatocytes, and the female oogonia. In the mouse, PGCs connect to each other in the process of migrating to genital ridge until forming cell colonies. Proliferation and cellular aggregation were both crucial features on survival and self renewal of primordial germ cells (PGC). Adhesive proteins play pivotal roles in cell-cell adhesion and signal exchanges under the influence of many cytokines, growth factors and bioactive metabolites such as retinoic acid (RA). RA is a metabolite of vitamin A with essential biological activity. For many types of cells, RA plays an important role in regulating cell proliferation and differentiation. RA synthesis and signaling play an important role during early organogenesis.
In this study, chicken PGC culture system was adopted to evaluate the effect of RA on germ cell aggregation by demonstration of the changes in expression of E-cadherin andα/βcatenins mRNAs. At the same time, the change in cell proliferation was also detected by the colony number and area of PGC aggregates. The results will facilitate to elucidate the regulation of PGCs proliferation by cell-cell adhesion.
PGCs were isolated from the genital ridge of 4-day chicken embryos and cultured on chicken embryonic fibroblast feeder. Cells were primary seeded at 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS) and 10 ng/ml LIF in 6-well culture plates. Cells were cultured at 39℃in a water-saturated atmosphere of 5% CO2. After 48h culture, PGC colonies were picked up and dispersed, and then subcultured on CEF in medium supplemented with 0.5% FCS. Subcultured PGCs were treated with RA (10-7-10"5 M) along and in combinations with PKC inhibitor H7 (10-7-10-5 M) for 24 h, respectively.
After culture, the PGCs proliferated from single cells to double or more cells. These cells colonized together to become cell aggregates, finally form compact PGCs colonies. PGCs were detected by SSEA-1 immunofluorescence and the nuclei were stained with DAPI Cultured PGCs colonies were also identified by SSEA-1/3 immunocytochemical staining with brown color. Slices of genital ridge from 5.5d chicken embryo were stained by SSEA-1 immunofluorescence method, with significant cell-surface red fluorescence for chicken PGCs clusters. In addition, expression of several pluripotency-associated genes including POU5F1, NANOG and SOX2 mRNA in PGCs were examined in cultures PGCs after treatment with 10-6 M RA for 24h. There was no significant difference in the mRNA abundance between the treated cells and the control.
Adhesion index and expression of E-cadherin, a-catenin andβ-catenin of PGCs were determined to detected impact of RA on PGC adhesion. RA (10-7 M) reduced the adhesion index from 0.67 to 0.60 with 10% decrease, while in 10-6 M RA-treated cells, the adhesion index was reduced to 0.37 with 45% decrease with significant difference. No further decrease in the adhesion index was achieved after higher RA treatment (10-5 M). At the same time, the mRNA expression of relative adhesion proteins was altered. Compared with the control group, after treatment with RA at 10-7,10-6 and 10-5 M, the increase of E-cadherin mRNA expression were increase at 5%(P> 0.05), 18% and 21%(P< 0.05). The a-catenin mRNA was increased at 8%,22% and 24%, andβ-catenin was increased at 7%,25% and 28.5%(P< 0.05).
Though the PKC inhibitor H7 at 10-7 M imposed no significant effect on RA-stimulated reduction of adhesion index of PGCs, higher H7 at 10-6 and 10-5M significantly attenuated the RA-elicited PGC aggregation. Simultaneous treatment of H7 at 10-6 and 10"5M remarkably attenuated the effect of RA on mRNA expression of adhesive proteins E-cadherin,α-catenin andβ-catenin. After treatment with RA at 10-7-10-5M for 24 h, the number and area of PGCs colonies was increased in a dose-dependent manner. However, the RA-elicited PGC proliferation was reduced by the combined treatment of H7. Meanwhile, the three-dimensional cell morphology of PGC colonies was weakened by H7.
To further confirm the effect of RA on PGC proliferation, we examined the effect of RA on mRNA expression of cyclins CCND1 and CCNE1, cyclin-dependent kinase 6 (CDK6), CDK2 which are considered to be critical factors in G1-S progression in cell cycle.. Treatment with RA at 10"6 M increased the mRNA expression of all four genes significantly. (P<0.05). This effect was hindered by combined H7 treatment.
In conclusion, proliferating effect of RA was revealed in cultured chicken PGCs. This stimulating action involves the increased expression of the adhesive proteins E-cadherin andα/βcatenins. RA-elicited effect was attenuated by PKC inhibition. Increased cell proliferation is accompanied with elevated mRNA expression of cyclins CCND1 and CCNE1, CDKs 6 and 2. These results indicated that RA promoted PGCs proliferation and strengthened aggregation of PGCs via E-cadherin andα/βcatenins expression through PKC signaling pathway in chicken PGCs.
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