健脾疏肝抗毒方对三阴性乳腺癌异种移植瘤生长及转移的影响

发布时间：2018-11-02 08:09

【摘要】：研究目的：研究健脾疏肝抗毒方对三阴性乳腺癌荷瘤鼠抗肿瘤作用及其可能的机制。研究方法：应用Fugene HD Transfection Reagent转染乳腺癌MDA-MB-231细胞,经G418筛选获得稳定表达绿色荧光蛋白的乳腺癌细胞(MDA-MB-231-pAcGFP).流式细胞仪检测荧光细胞百分率。采用皮下接种法建立MDA-MB-231-pAcGFP细胞异种移植瘤裸鼠,观察移植瘤的生长情况,荧光成像技术观察各脏器转移瘤的数目和大小。应用免疫组化法检测不同空白组别：对照组(A组)、健脾疏肝抗毒方组(B组)、ADM3100(CXCR4抑制剂)组(C组)、健脾疏肝抗毒方联合ADM3100组(D组)肿瘤微环境中的肿瘤相关巨噬细胞、肿瘤相关成纤维细胞浸润,蛋白印迹方法检测各组肿瘤组织CXCR4/CXCL12轴相关分子的表达。研究结果：流式细胞仪检测乳腺癌细胞转染率高达96.5%,表达有绿色荧光蛋白的MDA-MB-231-pAcGFP细胞成瘤率为100%。对照组(A组)、健脾疏肝抗毒方组(B组)、CXCR4抑制剂组(C组)、健脾疏肝抗毒方联合CXCR4抑制剂组([D组)。瘤体平均重量分别为：1.30±0.11g,0.75g±0.12g,0.48g±0.09g,0.44g±0.07g,瘤体平均体积分别为：6.635±0.5cm3,4.017±0.466 cm3,2.664±0.7 cm3,2.323±0.492 cm3(P0.05).三组的抑瘤率分别为42.18%、62.95%、66.28%。A组、C组肝脏可见散在荧光转移结节,B组及D组肝脏未见有荧光结节。健脾疏肝抗毒方能显著抑制肿瘤微环境中肿瘤相关巨噬细胞、肿瘤相关成纤维细胞的浸润,抑制CXCR4/CXCL12轴相关分子CXCR4.CXCL12、HIFα, NF-κB,VEGF的表达,且与ADM3100联合后抑制作用显著增强。结论：健脾疏肝抗毒方可以通过改变肿瘤微环境,调节CXCR4、CXCL12轴相关分子的表达而发挥抑制乳腺癌荷瘤鼠异种移植瘤的生长的作用。
[Abstract]:Objective: to study the anti-tumor effect and possible mechanism of Jianpi Shugan Kangdu recipe on triple-negative breast cancer bearing mice. Methods: breast cancer cells (MDA-MB-231-pAcGFP) expressing green fluorescent protein (GFP) were obtained by G418 screening after transfection of MDA-MB-231 cells with Fugene HD Transfection Reagent. The percentage of fluorescent cells was detected by flow cytometry. Nude mice with xenotransplantation of MDA-MB-231-pAcGFP cells were established by subcutaneous inoculation. The growth of xenografts was observed and the number and size of metastatic tumors in various organs were observed by fluorescence imaging. Immunohistochemical method was used to detect different blank groups: control group (A group), Jianpi Shugan Kangdu prescription group (B group), ADM3100 (CXCR4 inhibitor) group (C group). Tumor-associated macrophages and tumor-associated fibroblasts infiltrated in tumor microenvironment in group D combined with Jianpi Shugan Kangdu recipe and ADM3100 group. Western blot method was used to detect the expression of CXCR4/CXCL12 axis related molecules in tumor tissues. Results: the transfection rate of breast cancer cells was 96. 5% by flow cytometry, and the tumor forming rate of MDA-MB-231-pAcGFP cells expressing green fluorescent protein was 100%. Control group (A group), Jianpi Shugan Kangdu prescription group (B group), CXCR4 inhibitor group (C group), Jianpi Shugan Kangdu prescription combined with CXCR4 inhibitor group (group D). The mean weight of tumor was 1.30 卤0.11g / g 0.75g 卤0.12g / g 0.48g 卤0.09g / L 0.44g 卤0.07g respectively, and the average volume of tumor was 6.635 卤0.5cm34.017 卤0.466 cm3,2.664 卤0.7 cm3,2.323 卤0.492 cm3 (P0.05). The tumor inhibition rates of the three groups were 42.18 and 62.95 and 66.28.A respectively. The liver of group C could be seen in fluorescent metastatic nodules, but there were no fluorescent nodules in the liver of group B and group D. Jianpi Shugan Kangdu prescription could significantly inhibit the infiltration of tumor-associated macrophages, tumor-associated fibroblasts, and the expression of CXCR4.CXCL12,HIF 伪 and NF- 魏 B in tumor microenvironment. In combination with ADM3100, the inhibitory effect was significantly enhanced. Conclusion: Jianpi Shugan Kangdu recipe can inhibit the growth of breast cancer xenotransplantation by changing the tumor microenvironment and regulating the expression of CXCR4,CXCL12 axis related molecules.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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