Effect of Shugan Yishen Prescription on Tamoxifen-resistant Cell Line from Exosome-mediated Crosstalk in Breast Cancer Microenvironment

Sha-sha CUI; Wen-ping LU

doi:10.13422/j.cnki.syfjx.20212092

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Effect of Shugan Yishen Prescription on Tamoxifen-resistant Cell Line from Exosome-mediated Crosstalk in Breast Cancer Microenvironment

更新时间：2021-09-24

- Effect of Shugan Yishen Prescription on Tamoxifen-resistant Cell Line from Exosome-mediated Crosstalk in Breast Cancer Microenvironment
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 20, Pages: 72-79(2021)
- 作者机构：
  
  中国中医科学院广安门医院，北京 100053
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20212092
  CLC： R22;R242;R285.5;R2-031;R979.1
- Received：09 May 2021，
  
  Published Online：02 September 2021，
  
  Published：20 October 2021
- 稿件说明：
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崔莎莎,卢雯平.从乳腺癌微环境外泌体串扰角度探讨疏肝益肾方对他莫昔芬耐药细胞株的影响[J].中国实验方剂学杂志,2021,27(20):72-79.

CUI Sha-sha,LU Wen-ping.Effect of Shugan Yishen Prescription on Tamoxifen-resistant Cell Line from Exosome-mediated Crosstalk in Breast Cancer Microenvironment[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(20):72-79.
崔莎莎,卢雯平.从乳腺癌微环境外泌体串扰角度探讨疏肝益肾方对他莫昔芬耐药细胞株的影响[J].中国实验方剂学杂志,2021,27(20):72-79. DOI： 10.13422/j.cnki.syfjx.20212092.

CUI Sha-sha,LU Wen-ping.Effect of Shugan Yishen Prescription on Tamoxifen-resistant Cell Line from Exosome-mediated Crosstalk in Breast Cancer Microenvironment[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(20):72-79. DOI： 10.13422/j.cnki.syfjx.20212092.

摘要

目的

探讨疏肝益肾方（SGYS）通过干预乳腺癌微环境中外泌体的串扰对他莫昔芬（TAM）耐药细胞株LCC9的影响。

方法

分别制备空白组，TAM（2 mg·kg

-1

·d

-1

）组，SGYS（113.2 g·kg

-1

·d

-1

）组，联合组共4组含药血清；采用超速离心法提取LCC9细胞的外泌体，并应用透射电子显微镜，纳米粒子追踪技术及蛋白免疫印迹法（Western blot）对其进行鉴定。然后将收集的LCC9外泌体（LCC9-EXO）与骨髓间充质干细胞（BMMSCs）共培养，在共培养体系中分别加入10%的上述4组含药血清。共培养48 h后，提取BMMSCs的外泌体（BMMSCs-EXO），与LCC9细胞进一步孵育。应用荧光显微镜观察细胞对外泌体的摄取情况，细胞增殖与活性检测试剂盒（CCK-8）法检测各组含药血清对LCC9细胞增殖的影响，流式细胞仪分析各组含药血清对LCC9细胞凋亡的影响，Transwell观察各组含药血清对LCC9细胞迁移的影响，Western blot检测各组CD24，CD44，人类表皮生长因子受体2（HER2），雌激素受体

（ER

）蛋白的表达。

结果

荧光显微镜观察到LCC9-EXO可以被BMMSCs成功摄取，且BMMSCs-EXO可以被LCC9细胞成功摄取；CCK-8检测细胞增殖结果显示，与TAM组相比，SGYS组和联合组的细胞增殖能力在各时间段均明显降低（

0.05），联合组较SGYS组降低更明显（

0.05）；流式细胞仪检测凋亡结果显示，与TAM组相比，SGYS组和联合组的细胞凋亡水平明显升高（

0.05）；Transwell迁移结果显示，与TAM组相比，SGYS组与联合组的细胞迁移能力明显降低（

0.05）。Western blot结果显示，与TAM组相比，SGYS组与联合组的ER

和CD24表达量均明显上调（

0.05），HER2和CD44的表达量均明显下调（

0.05），且联合组对蛋白表达量的影响优于SGYS组（

0.05）。

结论

SGYS通过干预BMMSCs-EXO与LCC9-EXO串扰，从而逆转LCC9细胞的TAM耐药性。

Abstract

Objective

To explore the effect of Shugan Yishen prescription（SGYS） on the tamoxifen （TAM） -resistant cell line LCC9 by the intervention of exosome-mediated crosstalk in the breast cancer microenvironment.

Method

Four groups of serum were set up， specifically， a blank group， a TAM （2 mg·kg

-1

·d

-1

） group，an SGYS（113.2 g·kg

-1

·d

-1

） group，and a combination group. The exosomes of LCC9 cells were extracted by ultracentrifugation and identified by transmission electron microscopy （TEM），nanoparticle tracking analysis （NTA）， and Western blot. Then the collected LCC9 exosomes （LCC9-EXO） were co-cultured with bone marrow mesenchymal stem cells（BMMSCs），and 10% of the above four groups of serum were added to the co-culture system. After 48 hours of co-culture，the exosomes of BMMSCs （BMMSCs-EXO） were extracted and incubated with LCC9 cells. Fluorescence microscope was used to observe the uptake of exosomes by cells. Cell counting kit-8 （CCK-8） assay，flow cytometry， and Transwell assay were used to detect the effects of drug-containing serum in the four groups on the proliferation，apoptosis， and migration of LCC9 cells. Western blot was used to detect the protein expression of CD24，CD44，human epidermal growth factor 2（HER2）， and estrogen receptor

（ER

） in each group.

Result

Fluorescence microscope observed that LCC9-EXO could be taken up by BMMSCs，and BMMSCs-EXO could be taken up by LCC9 cells. CCK-8 assay revealed that compared with the TAM group，the SGYS group and the combination group showed reduced cell proliferation ability at each period （

0.05），especially the combination group，but no statistically significant difference between the SGYS group and the combination group was observed （

0.05）. Flow cytometry revealed that compared with the TAM group，the SGYS group and the combination group showed increased levels of apoptosis （

0.05）. Transwell assay revealed that compared with the TAM group，the SGYS group and the combination group showed decreased cell migration ability （

0.05）. Western blot revealed that compared with the TAM group，the SGYS group and the combination group showed up-regulated expression of ERα and CD24（

0.05），and down-regulated expression of HER2 and CD44 （

0.05）. The effect of the combination group on protein expression was superior to that of the SGYS group （

0.05）.

Conclusion

SGYS reverses the TAM resistance of LCC9 cells by interfering with the crosstalk between BMMSCs-EXO and LCC9-EXO.

关键词

Keywords

references

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