目的 探讨健脾化瘀方（人参、茯苓、白术、丹参等）通过 TGF-β1/Smad7 通路对肝细胞癌上皮间质转化 （EMT）及血管生成的调控作用。方法 （1）采用 BALB/C-nu 裸鼠建立 Hep3B 荷瘤小鼠模型，随机分为对照组 及健脾化瘀方低、中、高剂量组（3.844、7.689、15.378 g·kg-1·d-1），每组 5 只。灌胃给药，每日 1 次，连续给 药 21 d。（2）将 Hep3B 细胞分为空白组、造模组（10 ng·mL-1 TGF-β1）、低浓度组（10 ng·mL-1 TGF-β1+4 mg·mL-1 健脾化瘀方）、高浓度组（10 ng·mL-1 TGF-β1+6 mg·mL-1健脾化瘀方），TGF-β1 诱导 48 h 后更换为相应浓度的 健脾化瘀方完全培养液（0、4、6 mg·mL-1）继续培养 24 h。（3）将 HUVEC 细胞分为正常组、模型组、中药组、 模型加中药组。正常组细胞用不含 TGF-β1 的条件培养基培养；模型组细胞用含 TGF-β1 的条件培养基培养； 中药组细胞用含有 4 mg·mL-1健脾化瘀方但不含 TGF-β1 的条件培养基培养；模型加中药组细胞用含 4 mg·mL-1 健脾化瘀方且含 TGF-β1 的条件培养基培养。继续培养 24 h 后收集细胞进行后续实验。（4）计算 Hep3B 荷瘤裸 鼠皮下移植瘤的瘤质量指数和抑瘤率；采用免疫荧光法检测荷瘤裸鼠皮下移植瘤中 CD31 的表达；免疫组化法 检测荷瘤裸鼠皮下移植瘤的微血管密度；细胞划痕实验检测 Hep3B 细胞的迁移能力；Transwell 实验检测 Hep3B/HUVEC 细胞的侵袭能力；检测 HUVEC 细胞小管形成能力；Western Blot 法检测荷瘤裸鼠皮下移植瘤、 Hep3B 及 HUVEC 细胞中相关蛋白的表达水平。结果 （1）与对照组比较，健脾化瘀方中、高剂量组荷瘤裸鼠 皮下移植瘤质量及瘤质量指数显著降低（P＜0.01）；低、中、高剂量组荷瘤裸鼠皮下移植瘤中 CD31 的表达量 及微血管密度显著降低（P＜0.05，P＜0.01），VE-cadherin、EphA2、N-cadherin 蛋白表达显著下调（P＜0.05， P＜0.01），E-cadherin、Smad7 蛋白表达显著上调（P＜0.01）。（2）与空白组比较，造模组的 Hep3B 细胞的相对迁 移率明显升高（P＜0.05），侵袭细胞数显著增加（P＜0.01）；Vimentin、N-cadherin 蛋白表达显著上调（P＜ 0.01），E-cadherin、Smad7 蛋白表达显著下调（P＜0.01）。与造模组比较，健脾化瘀方低、高浓度组 Hep3B 细 胞的相对迁移率显著降低（P＜0.01），侵袭细胞数显著减少（P＜0.01）；Vimentin、N-cadherin 蛋白表达显著下 调（P＜0.01），E-cadherin、Smad7 蛋白表达显著上调（P＜0.01）。（3）与正常组比较，模型组 HUVEC 细胞形成的 小管分支点数显著增多（P＜0.01），小管分支长度及侵袭细胞数显著增加（P＜0.01）；VE-cadherin、EphA2 蛋白表达显著上调（P＜0.01），Smad7 蛋白表达显著下调（P＜0.01）。与模型组比较，模型加中药组 HUVEC 细 胞形成的小管分支点数显著减少（P＜0.01），小管分支长度显著缩短（P＜0.01），侵袭细胞数显著减少（P＜ 0.01）；VE-cadherin、EphA2 蛋白表达显著下调（P＜0.01），Smad7 蛋白表达显著上调（P＜0.01）。结论 健脾 化瘀方可明显抑制肝细胞癌的生长、侵袭与迁移，可能与通过调控 TGF-β1/Smad7 通路介导的 EMT 及血管生 成有关。

Objective To investigate the regulatory effect of Jianpi Huayu Prescription （Ginseng Radix et Rhizoma， Poria， Atractylodis Macrocephalae Rhizoma， Salviae Miltiorrhizae Radix et Rhizoma， etc.） on epithelialmesenchymal transition （EMT） and angiogenesis of hepatocellular carcinoma through TGF- β1/Smad7 pathway. Methods （1） Hep3B tumor-bearing mouse model was established by BALB/C-nu nude mice. The mice were randomly divided into control group and Jianpi Huayu Prescription low-，medium- and high- dose groups （3.844， 7.689，15.378 g·kg-1·d-1），with five mice in each group. Intragastric administration was performed once a day for 21 days. （2） Hep3B cells were divided into blank group，model group （10 ng·mL-1 TGF-β1），low-concentration group （10 ng·mL-1 TGF-β1 + 4 mg·mL-1 Jianpi Huayu Prescription），and high-concentration group （10 ng·mL-1 TGF-β1+6 mg·mL-1 Jianpi Huayu Prescription） . After 48 hours of TGF-β1 induction，the cells were replaced with the corresponding concentration of Jianpi Huayu Prescription complete culture medium （0， 4， 6 mg·mL-1） and cultured for 24 hours. （3） HUVEC cells were divided into normal group，model group，Chinese medicine group， model plus Chinese medicine group. The cells in the normal group were cultured with conditioned medium without TGF-β1；the cells in the model group were cultured with conditioned medium containing TGF-β1. The cells in the Chinese medicine group were cultured in conditioned medium containing 4 mg·mL-1 Jianpi Huayu Prescription without TGF- β1. The cells in the model plus Chinese medicine group were cultured with conditioned medium containing 4 mg·mL-1 Jianpi Huayu Prescription and TGF-β1. After 24 hours of continuous culture，the cells were collected for subsequent experiments. （4） The tumor mass index and tumor inhibition rate of subcutaneous transplantation tumor of Hep3B tumor-bearing nude mice were calculated. The expression of CD31 in subcutaneous xenografts of tumor-bearing nude mice was detected by immunofluorescence. The microvessel density of subcutaneous transplanted tumor in nude mice was detected by immunohistochemistry. The migration ability of Hep3B cells was detected by cell scratch test. Transwell assay was used to detect the invasion ability of Hep3B/HUVEC cells. The tube formation ability of HUVEC cells was detected. The expression levels of related proteins in subcutaneous transplanted tumors，Hep3B and HUVEC cells of tumor-bearing nude mice were detected by Western Blot. Results （1） Compared with the control group，the weight and tumor mass index of subcutaneous transplanted tumor in nude mice in the medium- and high- dose groups of Jianpi Huayu Prescription were significantly decreased （P＜0.01）. The expression of CD31 and microvessel density in subcutaneous transplanted tumors of nude mice in low- ， medium- and high- dose groups were significantly decreased （P＜0.05， P＜0.01）， the protein expressions of VE-cadherin， EphA2 and N-cadherin were significantly down-regulated （P＜0.05， P＜0.01）， and the protein expressions of E-cadherin and Smad7 was significantly up-regulated （P＜0.01）. （2） Compared with the blank group，the relative migration rate of Hep3B cells in the model group was significantly increased （P＜0.05），and the number of invasive cells was significantly increased （P＜0.01）. The protein expressions of Vimentin and N-cadherin were significantly up-regulated （P＜0.01），and the protein expressions of E-cadherin and Smad7 was significantly down-regulated （P＜0.01）. Compared with the model group，the relative migration rate of Hep3B cells in the lowand high- concentration groups of Jianpi Huayu Prescription was significantly decreased （P＜0.01）， and the number of invasive cells was significantly decreased （P＜0.01）. The protein expressions of Vimentin and N-cadherin was significantly down-regulated （P＜0.01）， and the protein expressions of E-cadherin and Smad7 were significantly up-regulated （P＜0.01）. （3） Compared with the normal group， the number of tubular branching points formed by HUVEC cells in the model group was significantly increased （P＜0.01）， the length of tubular branching was significantly increased （P＜0.01），and the number of invasive cells was significantly increased （P＜ 0.01）. The protein expressions of VE-cadherin and EphA2 were significantly up-regulated （P＜0.01）， and the protein expression of Smad7 was significantly down-regulated （P＜0.01）. Compared with the model group， the number of tubular branching points formed by HUVEC cells in the model plus Chinese medicine group was significantly reduced （P＜0.01），the length of tubular branching was significantly shortened （P＜0.01），and the number of invasive cells was significantly reduced （P＜0.01）. The protein expressions of VE-cadherin and EphA2 were significantly down-regulated （P＜0.01），and the protein expression of Smad7 was significantly up-regulated （P＜0.01）. Conclusion Jianpi Huayu Prescription can significantly inhibit the growth，invasion and migration of hepatocellular carcinoma，which may be related to the regulation of EMT and angiogenesis mediated by TGF- β1/ Smad7 pathway.

R285.5

国家自然科学基金面上项目（82274526）；广东省自然科学基金面上项目（2023A1515011069）；广州中医药大学“双一流”与高水平大学 学科后备人才培育项目（A1-2601-22-415-023）