Pregnancy influences the proliferation and hepatic differentiation of rat adipose-derived stem cells

doi:10.3969/j.issn.2095-4344.2017.33.007

Abstract

Abstract:

BACKGROUND: Pregnancy exhibits a significant association with stem cell activity. However, there is yet no report on the effects of pregnancy on adipose-derived stem cells.
OBJECTIVE: To investigate the effect of pregnancy on the proliferation and differentiation of rat adipose-derived stem cells into hepatocyte-like cells.
METHODS: Adipose-derived stem cells were isolated and cultured from normal and pregnant Sprague-Dawley rats, and identified. The cell proliferation was detected by cell counting kit-8. All harvested adipose-derived stem cells were cultured in hepatocyte-induced medium and induced to differentiate into hepatocyte-like cells. Then the expressions of hepatocyte markers were detected at 0, 7, 14, 21 days of induction by fluorescent quantitative PCR; albumin secretion and urea synthesis were detected by using ELISA method.
RESULTS AND CONCLUSION: Adipose-derived stem cells were successfully isolated from normal and pregnant rats, and CD45 negative staining and CD44/CD90 positive staining were observed, which was consistent with the characteristics of stem cells. Compared to the normal cells, adipose-derived stem cells from pregnant rats showed faster proliferation as well as osteogenic, adipogenic and hepatocyte-like differentiation, with the better liver function of hepatocyte-like cells. To conclude, pregnancy can promote the proliferation and differentiation of adipose-derived stem cells, which may be related to the estrogen level.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Adipose Tissue, Mesenchymal Stem Cells, Pregnancy, Cell Proliferation, Cell Differentiation, Hepatocytes, Tissue Engineering

CLC Number:

R394.2

Li Jun-nan, Chen Jia-yong, Zhang Yi, Lu Xiao-tao. Pregnancy influences the proliferation and hepatic differentiation of rat adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5287-5292.

Figures/Tables 2

2.1 脂肪干细胞分离、培养与鉴定原代大鼠脂肪干细胞最开始呈圆形，0.5-1.0 h内开始贴壁，在2-4 h后基本完成贴壁。传代后培养24 h可见脂肪干细胞贴壁生长，细胞形态伸展为三角形、多角形，最终逐渐变为典型的短梭形(图1)。取第3代脂肪干细胞进行鉴定，结果表明，妊娠大鼠来源脂肪干细胞符合干细胞表面分子标志物特性(CD44+、CD45-、CD90+)(图2)。成骨诱导21 d后茜素红染色和成脂诱导7 d后油红O染色结果表明分离培养的脂肪干细胞具有多向分化潜力(图3)。 2.2 正常大鼠来源和妊娠大鼠来源脂肪干细胞增殖情况通过CCK8法对正常大鼠脂肪干细胞和妊娠大鼠脂肪干细胞的生长趋势进行了对比分析(图4)。通过生长曲线及统计结果可以看出，大鼠脂肪干细胞在第1次传代培养后的前2 d，处于细胞生长潜伏期，生长比较缓慢；随后开始进入对数生长期，表现为生长曲线一直呈现上升趋势，在第5天时达到最多数量。妊娠大鼠脂肪干细胞从第1次传代开始，同样经历了一个24 h的缓慢生长期，随后第3，4，5天，妊娠大鼠脂肪干细胞的增殖速度高于正常大鼠脂肪干细胞，差异均有显著性意义。 2.3 正常大鼠和妊娠大鼠来源脂肪干细胞的成肝分化能力在诱导液的作用下，正常大鼠脂肪干细胞的增殖速度明显放缓。诱导3 d后，脂肪干细胞开始收缩，并逐步转化为不规则的多边形态。随着成肝诱导时间的延长，这种不规则多边形细胞数量也不断增加(图5A-B)。诱导14 d后，部分脂肪干细胞转化为正常贴壁的不规则多边形态或具有上皮形态的细胞，其与实验室常用的大鼠肝细胞相似(图5C)。与正常大鼠脂肪干细胞相比较，妊娠大鼠脂肪干细胞的增殖速度并未受到成肝诱导液影响。诱导3 d后，妊娠大鼠脂肪干细胞的细胞学形态同样开始出现变化。诱导7 d和14 d时，大部分细胞开始逐渐变化为类肝细胞形态(图5E-F)。 2.4 正常大鼠和妊娠大鼠来源脂肪干细胞成肝分化后肝细胞标志因子的表达见图6。正常大鼠来源脂肪干细胞在诱导第7天检测到AFP和CK18的表达，但未检测到ALB的表达；诱导第14天开始检测到ALB的表达，诱导第21天时检测到TAT和G-6-P的表达；AFP的表达在第21天时明显降低。与正常大鼠来源的脂肪干细胞相比，妊娠大鼠来源的脂肪干细胞在诱导第7天检测到AFP、CK18和ALB的表达，但ALB的表达水平较低；诱导第14天开始AFP、ALB和CK18的表达水平均显著升高，且均高于正常大鼠来源脂肪干细胞的表达水平，差异有显著性意义。诱导第21天时检测到TAT和G-6-P的表达，且均高于正常大鼠来源脂肪干细胞的表达水平，差异有显著性意义。诱导第21天时妊娠大鼠来源脂肪干细胞中AFP的表达水平与第14天时基本一致，未见到明显降低，差异无显著性意义。 2.5 正常大鼠和妊娠大鼠来源脂肪干细胞成肝分化后类肝细胞的功能为进一步检测脂肪干细胞分化成的类肝样细胞是否具有成熟肝细胞的常规功能，对诱导后的分化细胞进行了白蛋白和尿素分泌能力检测。结果发现，与未诱导前二者的分泌水平相比，正常大鼠和妊娠大鼠来源脂肪干细胞诱导分化成的类肝样细胞均能够正常分泌白蛋白，也能够利用培养液中的HN4Cl合成尿素。其中正常大鼠来源脂肪干细胞在诱导21 d后，白蛋白分泌较第0，7，14天时显著升高，差异有显著性意义；而妊娠大鼠来源脂肪干细胞在第14天时的白蛋白分泌水平显著升高，在第21天时持续升高，差异均有显著性意义(P均< 0.05)，见图7A。正常大鼠和妊娠大鼠来源脂肪干细胞在诱导后第7天时均开始展示尿素合成能力，随后尿素水平不断升高，并在第21天时达到顶峰，正常大鼠和妊娠大鼠来源脂肪干细胞诱导后的尿素合成能力差异无显著性意义(P均> 0.05)，见图7B。"

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