乳痈方对4T1乳腺癌小鼠胸腺上皮细胞表型变化的作用及机制研究

Effects and mechanisms of Ruyong Formula on phenotypic changes of thymic epithelial cells in 4T1 breast cancer mice

目的:探讨乳痈方对4T1乳腺癌小鼠胸腺上皮细胞表型变化的作用及机制。方法:①选用BALB/c小鼠,建立4T1乳腺癌荷瘤小鼠模型。模型动物随机分为模型组、环磷酰胺(CTX,30 mg/kg)组和乳痈方低、中、高剂量(0.145,0.29,0.58 g/kg)组,每组4只。各组给予相应干预,连续20 d。末次给药后,采集小鼠胸腺、肺、脾、肿瘤、淋巴结组织,计算脏器指数和抑瘤率;观察肺表面结节情况,计算肺转移率。HE染色后光镜下观察胸腺形态学变化,免疫组化检测胸腺组织钙黏附蛋白E(E-cadherin)和波形纤维蛋白(Vimentin)的表达。②体外分离小鼠原代胸腺上皮细胞(TECs),通过转染SV40T基因构建永生化TECs(iTECs)并验证。采用不同浓度(0、25、50、100、200、400μg/ml)乳痈方提取液处理iTECs。细胞培养48 h后,MTT法检测细胞增殖率,结晶紫染色观察细胞增殖情况。将iTECs分为空白组、TGF-β1组(10 ng/ml TGF-β1)、乳痈方低剂量组(10 ng/ml TGF-β1+50μg/ml提取液)、乳痈方中剂量组(10 ng/ml TGF-β1+100μg/ml提取液)、乳痈方高剂量组(10 ng/ml TGF-β1+200μg/ml提取液)。先加入TGF-β1诱导48 h后,再给予相应浓度的乳痈方提取液干预24 h。免疫荧光和PCR检测细胞表型标志物E-cadherin、Vimentin、α-微管蛋白(α-tubulin)及相关转录因子Zeb 1、Snail 1的表达水平。建立Smad转染的iTECs,荧光素酶报告基因检测乳痈方对TGF-β1诱导的转染iTECs细胞Smad通路活化的影响。结果:①与模型组相比,乳痈方低、中、高剂量组的肿瘤指数均显著降低(P<0.05),抑瘤率分别为33.45%、31.43%和6.49%;肺转移总数以及直径>2 mm转移灶数均明显减少(P<0.05,P<0.01),肺转移率分别为75%、75%和100%。HE染色观察显示,模型组小鼠胸腺组织皮质和髄质交界模糊,细胞排列紊乱,皮质萎缩;乳痈方各剂量组小鼠的胸腺组织皮质区和髄质边界较明显,皮质区细胞总数较模型组显著增多(P<0.05,P<0.01),髄质区细胞总数较模型组显著减少(P<0.05,P<0.01)。免疫组化检测显示,乳痈方干预后,模型小鼠胸腺组织上皮细胞标记物E-cadherin阳性表达增多,间质细胞标记物Vimentin阳性表达减少。②成功构建iTECs,iTECs与TECs形态相似,且高表达SV40基因和CK5蛋白。MTT检测和结晶紫染色观察显示,100、200、400μg/ml乳痈方干预可促进iTECs增殖(P<0.01)。③TGF-β1诱导后,iTECs逐渐变为长梭形,E-cadherin表达和mRNA水平降低(P<0.05),Vimentin表达和mRNA水平上调(P<0.05),且相关基因Snail 1和Zeb 1的mRNA水平显著升高(P<0.01)。乳痈方干预可抑制TGF-β1诱导的细胞形态向长梭形变化,上调E-cadherin表达和mRNA水平(P<0.01),下调Vimentin表达和mRNA水平(P<0.05,P<0.01),且显著降低Snail 1和Zeb 1的mRNA水平(P<0.01)。④TGF-β1诱导可引起iTECs细胞Smad mRNA水平明显上调(P<0.01),不同剂量乳痈方作用均可使Smad mRNA水平显著下调(P<0.01),且低剂量组的信号通路活化程度较TGF-β1组明显降低(P<0.05)。结论:乳痈方可有效逆转乳腺癌小鼠胸腺上皮细胞的表型变化,其作用机制与抑制TGF-β1/Smad通路有关。

Objective: To study the effects and mechanisms of Ruyong Formula on the phenotypic changes of thymic epithelial cells in 4 T1 breast cancer mice. Methods:①The orthotopic transplantation model of 4 T1 breast cancer was made with BALB/c mice. The modeling mice were randomly divided into the model group, cyclophosphamide(CTX, 30 mg/kg) group and Ruyong Formula groups with low-, medium-and high-dose(0.145, 0.29, 0.58 g/kg), 4 mice in each group. Each group was treated with the corresponding intervention for 20 days. After the last administration, the thymus, lung, spleen, tumor and lymph node of the mice were collected, and the organ index and the tumor inhibition rate were calculated;the lung surface nodules were observed and the lung metastasis rate was calculated. The morphological changes of thymus were observed under light microscope after HE staining. The protein expressions of E-cadherin and Vimentin in thymus tissue were detected by immunohistochemistry. ②Mouse thymic epithelial cells(TECs) were isolated in vivo, and the immortalized TECs(iTECs) was established by infection with retrovirus carrying SV40 T gene. The iTECs were treated with the extract of Ruyong Formula at different concentrations(0, 25, 50, 100, 200, 400 μg/ml). After cell culture for 48 hours, the cell proliferation rate was detected by MTT assay, and the cell proliferation was observed by crystal violet staining. The iTECs were divided into the blank group, TGF-β1 group(only treated with 10 ng/ml TGF-β1) and Ruyong Formula groups with low-, medium-and high-dose(10 ng/ml TGF-β1 and 50, 100 or 200 μg/ml extract of Ruyong Formula). After TGF-β1 induction for 48 hours, the Ruyong Formula extract at the corresponding concentrations was added for 24 hours intervention. The expressions of E-cadherin, Vimentin, α-tubulin and related transcription factors Zeb 1 and Snail 1 were detected by immunofluorescence and PCR. The Smad-transfected iTECs were established, and the effect of Ruyong Formula on the activation of Smad pathway in TGF-β1 induced transfected iTECs was detected by luciferase reporter gene assay. Results: ①Compared with the model group, the tumor index was significantly decreased in the Ruyong Formula groups with low-, medium-and high-dose(P<0.05), and the tumor inhibition rates were 33.45%, 31.43% and 6.49% respectively;the total number of lung metastasis and the number of metastases with diameter more than 2 mm were significantly reduced(P<0.05, P<0.01), and the lung metastasis rates were 75%, 75% and 100%, respectively. The results of HE staining showed that the boundary of cortex and medulla of thymus tissue was indistinct in the model group, and the disorder of cell arrangement and the atrophy of cortex were found. In the Ruyong Formula groups with different doses, the boundary of cortex and medulla of thymus tissue was more obvious, and the cell numbers were significantly increased in the cortical area and significantly decreased in the medullary area compared with the model group(P<0.05, P<0.01). The results of immunohistochemistry showed that, after the treatment with Ruyong Formula, the positive expression of E-cadherin as epithelial cell marker was increased in the thymus tissue of the model mice, while the positive expression of Vimentin as interstitial cell marker was decreased. ②The iTECs were successfully constructed, its shape was similar to TECs, with high expressions of SV40 gene and CK5 protein. MTT assay and crystal violet staining showed that the intervention of Ruyong Formula at 100, 200, 400 μg/ml could promote the proliferation of iTECs(P<0.01). ③After induction of TGF-β1, iTECs gradually changed into long spindle shape, the expression and mRNA levels of E-cadherin were down-regulated(P<0.05), and the expression and mRNA levels of Vimentin were up-regulated(P<0.05), and the mRNA levels of Snail 1 and Zeb 1 were significantly increased(P<0.01). After treatment with Ruyong Formula, the morphological change of iTECs induced by TGF-β1 was inhibited, the expression and mRNA levels of E-cadherin were up-regulated(P<0.01), and the expression and mRNA levels of Vimentin were down-regulated(P<0.05, P<0.01), the mRNA levels of Snail 1 and Zeb 1 were significantly decreased(P<0.01). ④After induction of TGF-β1, the mRNA level of Smad was obviously up-regulated in iTECs(P<0.01). After treatment with Ruyong Formula at different doses, the mRNA level of Smad was significantly down-regulated(P<0.01), and the activation of Smad signaling pathway in the Ruyong Formula group with low-dose was weaker than that in the TGF-β1 group(P<0.05). Conclusion: Ruyong Formula can effectively reverse the phenotypic changes of thymic epithelial cells in mice with breast cancer, and its mechanism is related to the inhibition of TGF-β1/Smad pathway.